PREFACE TO VOLUME 41

The comprehensive profiling of drug substances and pharmaceutical excipi-

ents as to their physical and analytical characteristics remains essential to all
phases of pharmaceutical development, and such profiles are of immeasurable
importance to workers in the field. Consequently, the compilation and
publication of comprehensive summaries of physical and chemical data,
analytical methods, routes of compound preparation, degradation pathways,
uses and applications, etc. have always been and will continue to be a vital
function to both academia and industry.
As the science of pharmaceutics grows and matures, the need for infor-
mation similarly expands along new fronts, and this growth causes an equiv-
alent growth in the repository sources where investigators find the
information they need. The content of the Profiles series continues to
respond and expand to meet this need, and so chapters are published that
fall into one or more of the following main categories:
1. Comprehensive profiles of a drug substance or excipient
2. Physical characterization of a drug substance or excipient
3. Analytical methods for a drug substance or excipient
4. Detailed discussions of the clinical uses, pharmacology, pharmacokinet-
ics, safety, or toxicity of a drug substance or excipient
5. Reviews of methodology useful for the characterization of drug sub-
stances or excipients
6. Annual reviews of areas of importance to pharmaceutical scientists
The current volume contains comprehensive profiles of bupropion hydro-
chloride, calcium carbonate, carbamazepine, dacarbazine, and pioglitazone.
Particular attention should be drawn to the extraordinarily comprehensive
profiles on calcium carbonate and carbamazepine, and those chapter authors
are to be congratulated on the depth of their research.
As always, I welcome communications from anyone in the pharmaceu-
tical community who might want to provide an opinion or a contribution.
HARRY G. BRITTAIN
Editor, Profiles of Drug Substances,
Excipients, and Related Methodology
hbrittain@centerpharmphysics.com

vii
Contributing Editor
ABDULLAH A. AL-BADR
ABDULRAHMAN AL-MAJED

Founding Editor
KLAUS FLOREY
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 CHAPTER THREE

Carbamazepine
S.T. Alrashood
King Saud University, Riyadh, Saudi Arabia

Contents
1. Description 133
1.1 Nomenclature 133
1.2 Formulae 134
1.3 Elemental Analysis 134
1.4 Appearance 134
1.5 Uses and Applications 134
2. Methods of Preparations 137
3. Physical Characteristics 137
3.1 Solubility Characteristics 137
3.2 Thermal Method of Analysis 137
3.3 Spectroscopy 137
4. Methods of Analysis 142
4.1 Compendial Methods 142
4.2 Reported Methods of Analysis 147
5. Biological Assay 213
6. Stability 229
7. Pharmacokinetics, Metabolism, and Excretion 237
7.1 Pharmacokinetics 237
7.2 Metabolism 263
7.3 Excretion 267
8. Pharmacology 270
9. Toxicity 284
References 302

1. DESCRIPTION
1.1 Nomenclature
1.1.1 Systematic Chemical Names
5H-Dibenz[b, f]azepine-5-carboxamide.

Profiles of Drug Substances, Excipients, and Related Methodology, Volume 41 # 2016 Elsevier Inc. 133
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/bs.podrm.2015.11.001
134 S.T. Alrashood

1.1.2 Nonproprietary Names

Carbamazepine.

1.1.3 Proprietary Names

Carbamazepine; Biston; Finlepsin; Stazepine; Tegretal; Telesmin; Timonil.

1.2 Formulae
1.2.1 Empirical Formula, Molecular Weight, CAS Number

Carbamazepine C15H12N2O 298-46-4

1.2.2 Structural Formula

O
NH2
N

1.3 Elemental Analysis

Carbamazepine C, 76.25% H, 5.12% N, 11.86% O, 6.77%

1.4 Appearance
Carbamazepine. A white or almost white crystalline powder. It exhibits
polymorphism [16].

1.5 Uses and Applications

Carbamazepine is typically used for the treatment of seizure disorders
and neuropathic pain. It is used off-label as a second-line treatment for
bipolar disorder and in combination with an antipsychotic in some cases
of schizophrenia when treatment with a conventional antipsychotic alone
has failed. In the United States, the FDA-approved medical uses are
epilepsy (including partial seizures, generalized tonicclonic seizures, and
mixed seizures), trigeminal neuralgia, and manic and mixed episodes of
bipolar disorder. It is unclear if there is a significant difference in effectiveness
between controlled-release and immediate-release formulations in epilepsy.
Controlled release forms might, however, have lower risks of side effects.
Other uses may include attention deficit hyperactivity disorder, schizo-
phrenia, phantom limb syndrome, complex regional pain syndrome, bor-
derline personality disorder, and posttraumatic stress disorder [7,8]. Some
Carbamazepine 135

antiepileptic medicines have a place in the treatment of neuropathic pain

(pain due to nerve damage).
This updated review considers the treatment of chronic neuropathic pain
and fibromyalgia only and adds no new studies. The update uses higher stan-
dards of evidence than the earlier review, which results in the exclusion of five
studies that were previously included [9]. In order to assess the analgesic effi-
cacy of carbamazepine in the treatment of chronic neuropathic pain and fibro-
myalgia, and to evaluate adverse events reported in the studies, the authors
searched for relevant studies in MEDLINE, EMBASE, and CENTRAL up
to Feb. 2014. Additional studies were sought from clinical trials databases
and the reference list of retrieved articles and reviews. Randomized, dou-
ble-blind, active, or placebo-controlled trials (RCTs) investigating the use
of carbamazepine (CBZ) (any dose, by any route, and for at least 2 weeks
duration) for the treatment of chronic neuropathic pain or fibromyalgia, with
at least 10 participants per treatment group. Participants were adults aged 18
and over. Two study authors independently extracted data on efficacy, adverse
events, and withdrawals and examined issues of study quality.
Numbers needed to treat for an additional beneficial effect or harmful
effect with 95% confidence intervals (CIs) were calculated from dichoto-
mous data and performed analysis using three tiers of evidence: First tier evi-
dence derived from data meeting current best standards and subject to
minimal risk of bias (outcome equivalent to substantial pain intensity reduc-
tion, intention-to-treat analysis without imputation for dropouts, at least
200 participants in the comparison, at least 8 weeks duration, parallel
design), second tier from data that failed to meet one or more of these criteria
and were considered at some risk of bias but with adequate numbers in the
comparison, and third tier from data involving small numbers of participants
that were considered very likely to be biased or used outcomes of limited
clinical utility, or both. Ten included studies (11 publications) enrolled
480 participants with trigeminal neuralgia, diabetic neuropathy, and post-
stroke pain. Nine studies used a crossover design, and one a parallel group
design. Most of the studies were of short duration, lasting 4 weeks or less. No
study provided first or second tier evidence for an efficacy outcome.
Using third tier evidence, carbamazepine generally provided better pain
relief than placebo in the three conditions studied, with some indication of
pain improvement over mainly the short term, but with poorly defined out-
comes, incomplete reporting, and in small numbers of participants. There
were too few data in studies comparing carbamazepine with active compar-
ators to draw any conclusions. In four studies, 65% (113/173) of participants
experienced at least one adverse event with carbamazepine, and 27% (47/173)
136 S.T. Alrashood

with placebo; for every five participants treated, two experienced an adverse
event who would not have done so with placebo. In eight studies, 3% (8/268)
of participants withdrew due to adverse events with carbamazepine, and none
(0/255) with placebo. Serious adverse events were not reported consistently;
rashes were associated with carbamazepine. Four deaths occurred in patients
on carbamazepine, with no obvious drug association.
It was concluded that carbamazepine is probably effective in some people
with chronic neuropathic pain, but with caveats. No trial was longer than 4
weeks, had good reporting quality, nor used outcomes equivalent to sub-
stantial clinical benefit. In these circumstances, caution is needed in interpre-
tation, and meaningful comparison with other interventions is not possible.
The management of bipolar disorder has seen significant evolution in
terms of the number of treatment options now approved for both the acutely
manic phase and the maintenance stages of the illness. In addition, new for-
mulations of traditional agents are available for clinicians to use in their treat-
ment approach. One such example is carbamazepine, which has approval by
the US Food and Drug Administration for the treatment of acute and mixed
mania in an extended-release formulation that uses a three-bead delivery sys-
tem. Although the parent compound has been available for decades, its
approval for bipolar disorder is recent despite numerous clinical trials that
have supported its use in both the acute and maintenance phases of bipolar
disorder. Advantages of the new formulation include less fluctuation in
plasma concentration and, in general, improved tolerability. However,
issues remain with regard to cytochrome P450 drug-related interactions
and the need for therapeutic drug monitoring (TDM) (eg, drug concentra-
tions, epoxide metabolite concentrations, hematology, and liver function
tests) as part of the treatment and monitoring process. We review the current
body of the literature describing the use of carbamazepine in bipolar disorder
during both the acute and maintenance phases of the disorder, including tri-
als of both monotherapy and combination therapy, as well as findings from
trials that included patients with rapid cycling and mixed episodes [10].
Good evidence now exists for a therapeutic action of CBZ both in acute
mania and in the prophylaxis of manic depression. Comparison with other
existing therapies and incidence of adverse effects in psychiatric patients also
deserve more clinical research. This paper reviews these clinical issues and
then outlines what is known about the pharmacology of CBZ, in particular
comparing it to lithium, the drug with the most similar clinical spectrum of
action in psychiatry [11].
Clinical use of carbamazepine is reviewed from the literature.
Antidepressive, antimanic, and prophylactic uses in bipolar and unipolar
Carbamazepine 137

disorders are explained through six tables. The authors also arise clinical
results observed in personality disorders. Prophylactic and antimanic activ-
ities are widely commented. Based on this review of the literature, although
clinical efficacy in depression is more debated. Carbamazepine uses in per-
sonality disorders and schizoaffective patients seem to be promising. More-
over, the authors reported 25 clinical observations and attempt to draw some
conclusions for the clinical use of carbamazepine [12].

2. METHODS OF PREPARATIONS
Carbamazepine, 5H-dibenz[b, f]azepine-5-carboxamide, is synthe-
sized by reacting 5H-dibenz[b, f]azepine and phosgene, which forms 5-
chlorcarboxy-5H-dibenz[b, f]azepine, and its subsequent reaction with
ammonia to give the desired carbamazepine [13].
An alternative method of synthesis is the direct reaction of 5H-dibenz
[b, f]azepine with potassium cyanate [14].

3. PHYSICAL CHARACTERISTICS
3.1 Solubility Characteristics
Carbamazepine is very slightly soluble in water, is sparingly soluble in alco-
hol and acetone, and is freely soluble in dichloromethane.

3.2 Thermal Method of Analysis

3.2.1 Melting Behavior
Carbamazepine melts at 189193C.

3.3 Spectroscopy
3.3.1 UV Spectroscopy
The UV absorption spectrum of carbamazepine in methanol shown in Fig. 1
was recorded using Shimadzu UVvis Spectrometer 1601 PC. The com-
pound exhibited maxima at 288 and 259 nm. Clarke reported the following:
methanol237 and 285 nm (A 1%, 1 cm 490) [1].

3.3.2 Vibrational Spectroscopy

The FT-infrared absorption spectrum of carbamazepine was obtained in
a KBr pellet using a Perkin-Elmer FT-infrared spectrophotometer. FT-
infrared spectrum is shown in Fig. 2, where the principal peaks are observed
at 3465, 3157, 1675, 1604, 1594, 1488, 1381, 1307, 870, 800, 762, and
724 cm1.
138 S.T. Alrashood

Figure 1 The UV absorption spectrum of carbamazepine.

98.2

95

90
3280.49

85
3157.36 1436.21
1462.27
3465.11 1113.24
80 1245.45
1307.55 870.09

%T 75 1488.75

1604.75
1594.17
70
800.77
724.35

65 1675.33
1381.41

60
762.07

55
53.0
4000.0 3000 2000 1500 1000 650.0
cm1

Figure 2 The FT-infrared absorption spectrum of carbamazepine.

3.3.2.1 1H NMR Spectra

The proton nuclear resonance (1H NMR) spectra of carbamazepine were
obtained using a Bruker instrument operating at 500 MHz. Standard Bruker
software was used to execute the recording of the 1D and 2D spectra. The
sample was dissolved in DMSO-d6 and all resonance bands were referenced
Carbamazepine 139

5.545

2.494

2.485
7.451
7.431
7.424
7.417
7.413
7.343
7.338
7.329
7.323
7.317

6.984

3.337

2.489

2.480
7.455

7.310
7.303

2.498
11 10 9 8 7 6 5 4 3 2 ppm
16.54

11.56
49.14
15.92

13.05

7.74
Figure 3 1H NMR spectrum of carbamazepine.

to tetramethylsilane (TMS) as internal standard. The entire proton spectra

are shown in Figs. 3 and 4. A singlet resonates at 5.54 representing the
two protons of the amino group. An additional singlet which resonates at
6.99 ppm is assigned to the olefinic protons at positions 10 and 11. The
two multiplets which resonate at 7.307.34 and 7.417.43 ppm are
assigned to the aromatic protons of the two phenyl rings.

13
3.3.2.2 C NMR Spectra
A noise-modulated, broadband decoupling 13C NMR spectrum (Fig. 5)
showed 11 carbon absorptions in accordance with what is anticipated for
the structure of carbamazepine. Carbon resonance bands at 127.1,
129.0, 129.2, 129.3, 129.8, 130.3, 131.0, and 134.8 ppm account for the
CH functions. A carbon band at 140.6 ppm represents the ethylene car-
bons. The carbonyl carbon resonates at 156.3 ppm.
A DEPT experiment (Fig. 6) permitted the identification and confirma-
tion of the methyl and methine carbons. Another confirmation was obtained
through the HSQC experiment (Fig. 7).
 ppm

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 ppm

Figure 4 COSY experiment of carbamazepine.

156.31

140.59
134.78
130.27
129.27
129.14
129.06
127.17

40.18
39.97
39.76
39.55
39.34
39.13
38.93

200 180 160 140 120 100 80 60 40 20 0 ppm

13
Figure 5 C NMR spectrum of carbamazepine.
 130.28
129.27
129.14
129.06
127.17

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Figure 6 DEPT-135 experiment of carbamazepine.

ppm

20

40

60

80

100

120

140

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 ppm

Figure 7 HSQC experiment of carbamazepine.

142 S.T. Alrashood

4. METHODS OF ANALYSIS
4.1 Compendial Methods
4.1.1 British Pharmacopoeial Methods
Carbamazepine contains not less than 98.0% and not more than the equiv-
alent of 102.0% of 5H-dibenz[b, f]azepine-5-carboxamide, calculated with
reference to the dried substance.

4.1.1.1 Characters
White or almost white, crystalline powder, very slightly soluble in water,
freely soluble in methylene chloride, sparingly soluble in acetone and in eth-
anol (96%). It shows polymorphism (5.9). The acceptable crystalline form
corresponds to carbamazepine CRS.

4.1.1.2 Identification
A. Melting point (2.2.14): 189193C.
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: carbamazepine CRS.
Preparation: examine the substances as disks without prior treatment.

4.1.1.3 Tests
Acidity or alkalinity
To 1.0 g add 20 mL of carbon dioxide-free water R and shake for 15 min and
filter. To 10 mL of the filtrate add 0.05 mL of phenolphthalein solution R1
and 0.5 mL of 0.01 M sodium hydroxide; the solution is red. Add 1.0 L of
0.01 M hydrochloric acid; the solution is colorless. Add 0.15 mL of methyl
red solution R; the solution is red.

4.1.1.4 Related Substances

Liquid chromatography
Test solution (a). Dissolve 60.0 mg of the substance to be examined in
methanol R2 and dilute to 20.0 mL with the same solvent. Sonicate.
Dilute 10.0 mL of this solution to 20.0 mL with water R.
Test solution (b). Dilute 10.0 mL of test solution (a) to 50.0 mL with a
mixture of equal volumes of methanol R2 and water R.
Reference solution (a). Dissolve 7.5 mg of carbamazepine CRS, 7.5 mg of car-
bamazepine impurity A CRS, and 7.5 mg of iminodibenzyl R (impurity E)
in methanol R2 and dilute to 100.0 mL with the same solvent. Dilute
1.0 mL of this solution to 50.0 mL with a mixture of equal volumes
of methanol R2 and water R.
Carbamazepine 143

Reference solution (b). Dissolve 60.0 mg of carbamazepine CRS in methanol

R2 and dilute to 20.0 mL with the same solvent. Sonicate. Dilute 5.0 mL
of this solution to 50.0 mL with a mixture of equal volumes of methanol
R2 and water R.
Column:
size: l 0.25 m, 4.6 mm;
stationary phase: nitrile silica gel for chromatography R1 (10 m).
Mobile phase tetrahydrofuran R, methanol R2, water R (3:12:85, v/v/v); to
1000 mL of this solution add 0.2 mL of anhydrous formic acid R and 0.5 mL
of triethylamine R.
Flow rate: 2.0 mL/min.
Detection: a spectrophotometer at 230 nm.
Injection: 20 L of test solution (a) and reference solution (a).
Run time: six times the retention time of carbamazepine.
Relative retention With reference to carbamazepine (retention
time about 10 min): impurity A about 0.9; impurity E about 5.1.
System suitability
resolution: minimum 1.7 between the peaks due to carbamazepine and
impurity A in the chromatogram obtained with reference solution (a).
Limits:
impurities A and E: for each impurity, not more than the area of the
corresponding peak in the chromatogram obtained with reference
solution (a) (0.1%);
unspecified impurities: not more than the area of the peak due to carba-
mazepine in the chromatogram obtained with reference solution (a)
(0.10%);
total: not more than five times the area of the peak due to carbamaz-
epine in the chromatogram obtained with reference solution (a)
(0.5%);
disregard limit: 0.5 times the area of the peak due to carbamazepine in
the chromatogram obtained with reference solution (a) (0.05%).

Chlorides
Maximum 140 ppm.
Suspend 0.715 g in 20 mL of water R and boil for 10 min. Cool and dilute
to 20 mL with water R. Filter through a membrane filter (nominal pore
size: 0.8 m). Dilute 10 mL of the filtrate to 15 mL with water R. This
solution complies with the limit test for chlorides.
Heavy metals
Maximum 20 ppm.
144 S.T. Alrashood

1.0 g complies with test C. Prepare the reference solution using 2 mL of

lead standard solution (10 ppm Pb) R.
Loss on drying
When carbamazepine is tested according to the general procedure not
more than 0.5%, determined on 1.000 g by drying in an oven at 105C
for 2 h.
Sulfated ash
When carbamazepine is tested according to the general procedure not
more than 0.1%, determined on 1.0 g.
Assay
Liquid chromatography (LC) as described in the test for related sub-
stances using injection test solution (b) and reference solution (b).
System suitability:
repeatability: reference solution (b).
Calculate the percentage content m/m of dried substance.
Storage
In an airtight container.
Impurities
Specified impurities A, E.
Other detectable impurities (the following substances would, if present at a
sufficient level, be detected by one or other of the tests in the mono-
graph. They are limited by the general acceptance criterion for other/
unspecified impurities and/or by the general monograph).
Substances for pharmaceutical use (2034).
It is therefore not necessary to identify these impurities for demonstration of
compliance:
A. 10,11-dihydro-5H-dibenzo[b, f]azepine-5-carboxamide(10,11-
dihydro-carbamazepine),
B. 9-methylacridine,
C. (5H-dibenzo[b, f]azepin-5-ylcarbonyl)urea(N-
carbamoylcarbamazepine),
D. 5H-dibenzo[b, f]azepine (iminostilbene),
E. 10,11-dihydro-5H-dibenzo[b, f]azepine (iminodibenzyl),
F. 5H-dibenzo[b, f]azepine-5-carbonyl chloride (5-chlorocarbony
liminostilbene).

4.1.2 European Pharmacopoeial Methods

Definition
5H-Dibenzo[b, f]azepine-5-carboxamide.
Content: 98.0102.0% (dried substance).
Carbamazepine 145

Characters
Appearance: white or almost white crystalline powder.
Solubility: very slightly soluble in water, freely soluble in methylene chlo-
ride, and sparingly soluble in acetone and in alcohol.
It shows polymorphism; the acceptable crystalline form corresponds to
carbamazepine CRS.
Identification
A. Melting point (2.2.14): 189193C.
B. Infrared absorption spectrophotometry.
Comparison: carbamazepine CRS.
Preparation: examine the substances as disks without prior treatment.
Tests
Acidity or alkalinity. To 1.0 g add 20 mL of carbon dioxide-free water R,
shake for 15 min, and filter. To 10 mL of the filtrate add 0.05 mL of phe-
nolphthalein solution R1 and 0.5 mL of 0.01 M sodium hydroxide; the solu-
tion is red. Add 1.0 mL of 0.01 M hydrochloric acid; the solution is
colorless. Add 0.15 mL of methyl red solution R; the solution is red.

4.1.2.1 Related Substances

Liquid chromatography
Test solution (a). Dissolve 0.150 g of the substance to be examined in
methanol R2 and dilute to 50.0 mL with the same solvent. Sonicate.
Dilute 10.0 mL of this solution to 20.0 mL with water R.
Test solution (b). Dilute 10.0 mL of test solution (a) to 50.0 mL with a
mixture of equal volumes of methanol R2 and water R.
Reference solution (a). Dissolve 7.5 mg of carbamazepine CRS, 7.5 mg of car-
bamazepine impurity A CRS, and 7.5 mg of iminodibenzyl R (impurity E)
in methanol R2 and dilute to 100.0 mL with the same solvent. Dilute
1.0 mL of this solution to 50.0 mL with a mixture of equal volumes of
methanol R2 and water R.
Reference solution (b). Dissolve 0.150 g of carbamazepine CRS in methanol
R2 and dilute to 50.0 mL with the same solvent. Dilute 5.0 mL of this
solution to 50.0 mL with a mixture of equal volumes of methanol R2
and water R.
Column:
size: l 0.25 m, 4.6 mm,
stationary phase: nitrile silica gel for chromatography R1 (10 m).
Mobile phase: tetrahydrofuran R, methanol R2, water R (3:12:85, v/v/v). To
1000 mL of this solution add 0.2 mL of anhydrous formic acid R and 0.5 mL
of triethylamine R.
146 S.T. Alrashood

Flow rate: 2.0 mL/min.

Detection: a spectrophotometer at 230 nm.
Injection: 20 L; inject test solution (a) and reference solution (a).
Run time: six times the retention time of carbamazepine which is about
10 min.
Relative retention with reference to carbamazepine: impurity B about
0.7; impurity A about 0.9; impurity C about 1.6; impurity D about
3.5; impurity E about 5.1.
System suitability:
resolution: minimum of 1.7 between the peaks due to carbamazepine
and impurity A in the chromatogram obtained with reference solu-
tion (a).
Limits:
impurity A: not more than the area of the corresponding peak in the
chromatogram obtained with reference solution (a) (0.1%),
impurity E: not more than the area of the corresponding peak in the
chromatogram obtained with reference solution (a) (0.1%),
any other impurity: not more than the area of the peak due to carbamaz-
epine in the chromatogram obtained with reference solution (a)
(0.1%),
total: not more than five times the area of the peak due to carbamazepine
in the chromatogram obtained with reference solution (a) (0.5%),
disregard limit: 0.5 times the area of the peak due to carbamazepine in
the chromatogram obtained with reference solution (a) (0.05%).

Chlorides
maximum 140 ppm.
Suspend 0.715 g in 20 mL of water R and boil for 10 min. Cool and dilute
to 20 mL with water R. Filter through a membrane filter (nominal pore
size: 0.8 m). Dilute 10 mL of the filtrate to 15 mL with water R. This
solution complies with the limit test for chlorides.
Heavy metals
maximum 20 ppm.
1.0 g complies with limit test C. Prepare the standard using 2 mL of
lead standard solution (10 ppm Pb) R.
Loss on drying
maximum 0.5%,
determined on 1.000 g by drying in an oven at 100105C for 2 h.
Sulfated ash
maximum 0.1%, determined on 1.0 g.
Carbamazepine 147

Assay
Liquid chromatography as described in the test for related substances.
Injection: test solution (b) and reference solution (b).
System suitability:
repeatability: reference solution (b).
Calculate the percentage content m/m of dried substance.
Storage
In an airtight container.
Impurities
Specified impurities: A, B, C, D, E.
Other detectable impurities: F.
A. 10,11-dihydro-5H-dibenzo[b, f]azepine-5-carboxamide
(10,11-dihydrocarbamazepine),
B. 9-methylacridine,
C. (5H-dibenzo[b, f]azepin-5-ylcarbonyl)urea
(N-carbamoylcarbamazepine),
D. 5H-dibenzo[b, f]azepine (iminostilbene),
E. 10,11-dihydro-5H-dibenzo[b, f]azepine(iminodibenzyl),
F. 5H-dibenzo[b, f]azepine-5-carbonyl
chloride(5-chlorocarbonyliminostilbene).

4.2 Reported Methods of Analysis

4.2.1 Ultraviolet and Visible Spectrometric Methods
4.2.1.1 Ultraviolet Methods
A rapid, selective, and sensitive UPLC-UV method was developed and val-
idated for the quantitative analysis of carbamazepine and its epoxide metab-
olite in rat plasma. A relatively small volume of plasma sample (200 L) is
required for the described analytical method. The method includes simple
protein precipitation, liquidliquid extraction, evaporation, and reconstitu-
tion steps. Samples were separated on a Waters Acquity UPLC BEH C18
column (1.7 m, 2.1 mm 100 mm) with a gradient mobile phase consisted
of 60:40 going to 40:60 (v/v) wateracetonitrile at a flow rate of 0.5 mL/
min. The total run time was as low as 6 min, representing a significant
improvement in comparison to existing methods. Excellent linearity
(r2 > 0.999) was achieved over a wide concentration range. Close to com-
plete recovery, short analysis time, high stability, accuracy, precision and
reproducibility, and low limit of quantitation were demonstrated. Finally,
it was successfully applied this analytical method to a preclinical oral phar-
macokinetic study, revealing the plasma profiles of both carbamazepine and
carbamazepine-10,11-epoxide (CBZ-E) following oral administration of
148 S.T. Alrashood

carbamazepine to rats. The advantages demonstrated in this work make this

analytical method both time- and cost-efficient approach for drug and
metabolite monitoring in the preclinical/clinical laboratory [15].
The occurrence and removal of six pharmaceuticals and personal care
products (PPCPs) including caffeine (CF), N,N-diethyl-meta-toluamide
(DEET), carbamazepine, metoprolol, trimethoprim (TMP), and sulpiride in
a municipal wastewater treatment plant (WWTP) in Shanghai, China were
studied in Jan. 2013; besides, grab samples of the influent were also taken every
6 h, to investigate the daily fluctuation of the wastewater influent. The results
showed the concentrations of the investigated PPCPs ranged from 17 to
11,400 ng/L in the WWTP. A low variability of the PPCP concentrations
in the wastewater influent throughout the day was observed, with the relative
standard deviations (RSDs) less than 25% for most samples. However, for
TMP and CF, the slight daily fluctuation still reflected their consumption pat-
terns. All the target compounds except CF and DEET exhibited poor removal
efficiencies (<40%) by biological treatment process, probably due to the low
temperature in the bioreactor, which was unfavorable for activated sludge.
While for the two biodegradable PPCPs, CF and DEET, the anaerobic and
oxic tank made contributions to their removal, the anoxic tank had a negative
effect to their elimination. The tertiary UV treatment removed the investi-
gated PPCPs by 538%, representing a crucial polishing step to compensate
for the poor removal by the biological treatment process in winter [16].
Water disinfection technologies based on ultraviolet (UV) radiations
emitted by light-emitting diodes (LED), as a wastewater tertiary treatment,
have been shown to be promising for water reuse. The fate of two ubiqui-
tous pollutants, carbamazepine and anthracene, was assessed in soil watered
with either UV-LED-treated wastewaters or irrigation water. After 3
months, anthracene and carbamazepine were transformed two and three
times faster, respectively, in soils watered with UV-LED wastewater than
in soils watered with tap water (probably because of the addition of organic
matter by the effluent). Laccase activity was induced in the presence of the
pollutants, and anthraquinone was found as anthracene product of oxidation
by laccases. Moreover, the addition of these pollutants into soil did not affect
the functional diversity of autochthonous microbial communities assessed by
Ecolog plates. Cellulase, protease, and urease activities increased in soils
watered with UV-LED-treated wastewaters (UV-LEDWW), showing
transformation of organic matter from the effluent and lipase activity
increased by anthracene addition, confirming the potential role of these
enzymes as indicators of hydrocarbon contamination [17].
Carbamazepine 149

A sensitive and fast high-performance liquid chromatographic (HPLC)

method coupled with UV detection is herein reported for the simultaneous
determination of human plasma concentration of six antiepileptic drugs
(AEDs) frequently used in clinical practice [phenobarbital (PB), primidone
(PRM), phenytoin (PHT), CBZ, lamotrigine (LTG), oxcarbazepine
(OXC)] and some of their main metabolites, CBZ-E, 10,11-trans-
dihydroxy-10,11-dihydro-carbamazepine (trans-diol), and licarbazepine
(Lic). Sample preparation consisted of a deproteinization step with methanol
followed by a solid-phase extraction (SPE) procedure. Chromatographic
separation was achieved in approximately 15 min on a reversed-phase C18
column using a mobile phase composed by watermethanolacetonitrile
triethylamine (68.7:25:6:0.3, v/v/v/v; pH 6.5) pumped isocratically at
1.0 mL/min. The detector was set at 237 nm. Calibration curves were linear
with regression coefficients greater than 0.992 over the concentration ranges
of 0.25100 g/mL for PB, 0.450 g/mL for PRM, 0.550 g/mL for
PHT, 0.150 g/mL for CBZ, LTG, and CBZ-E, 0.125 g/mL for
OXC, 0.2510 g/mL for trans-diol, and 0.1580 g/mL for Lic. Inter-
and intraday imprecision did not exceed 12.15% and inaccuracy was within
14.91%. Absolute mean recoveries ranged from 78.49% to 101.04% and
no interferences were observed at the retention times of the analytes and inter-
nal standard (ketoprofen). This bioanalytical method was successfully applied
to real plasma samples from epileptic patients, and it seems to be a suitable tool
for routine TDM and also to support other clinical pharmacokinetic-based
studies [18].
The presence and elimination of 25 emerging contaminants in 2 full-
scale Spanish wastewater treatment plants was studied. The tertiary treat-
ment systems consisted of coagulation, flocculation lamellar settlement,
and filtration (pulsed-bed sand filters) units, and disinfection was carried
out by medium pressure UV light lamps and chlorination. Diclofenac and
carbamazepine were found to be the emerging contaminants with the
highest concentrations in secondary effluents. Photodegradable emerging
contaminants (eg, ketoprofen, triclosan, and diclofenac) were removed by
filtrationUV light radiationchlorination, whereas most hydrophobic
compounds (eg, galaxolide and tonalide) were eliminated by coagulation
flocculation followed by lamellar clarification, a unit in which a seasonal
trend was observed. Overall mass removal efficiency was about 60%. 1-
(8-Chlorocarbazolyl) acetic acid, an intermediate product of the
photodegradation of diclofenac, was detected after filtrationUV chlorina-
tion, but not after coagulationflocculation and lamellar clarification. This
150 S.T. Alrashood

study demonstrated potential for general applicability of two established ter-

tiary treatment systems to eliminate emerging contaminants [19].
To augment the removal of pharmaceuticals, different conventional and
alternative wastewater treatment processes and their combinations were
investigated. The efficiency of (1) two distinct laboratory scale biological
processes: suspended activated sludge and attached-growth biomass, (2) a
combined hydrodynamic cavitationhydrogen peroxide process, and (3)
UV treatment was tested. Five pharmaceuticals were chosen including ibu-
profen, naproxen, ketoprofen, carbamazepine, and diclofenac, and an active
metabolite of the lipid-regulating agent clofibric acid. Biological treatment
efficiency was evaluated using lab-scale suspended activated sludge and
moving bed biofilm flow-through reactors, which were operated under
identical conditions in respect to hydraulic retention time, working volume,
concentration of added pharmaceuticals, and synthetic wastewater compo-
sition. The suspended activated sludge process showed poor and inconsistent
removal of clofibric acid, carbamazepine, and diclofenac, while ibuprofen,
naproxen, and ketoprofen yielded over 74% removal. Moving bed biofilm
reactors were filled with two different types of carriers, ie, Kaldnes K1 and
Mutag BioChip, and resulted in higher removal efficiencies for ibuprofen
and diclofenac. Augmentation and consistency in the removal of diclofenac
were observed in reactors using Mutag BioChip carriers (85  10%) com-
pared to reactors using Kaldnes carriers and suspended activated sludge
(74  22% and 48  19%, respectively). To enhance the removal of pharma-
ceuticals, hydrodynamic cavitation with hydrogen peroxide process was
evaluated and optimal conditions for removal were established regarding
the duration of cavitation, amount of added hydrogen peroxide, and initial
pressure, all of which influence the efficiency of the process. Optimal param-
eters resulted in removal efficiencies between 3% and 70%. Coupling the
attached-growth biomass biological treatment, hydrodynamic cavitation/
hydrogen peroxide process and UV treatment resulted in removal efficien-
cies of >90% for clofibric acid and >98% for carbamazepine and diclofenac,
while the remaining compounds were reduced to levels below the LOD.
For ibuprofen, naproxen, ketoprofen, and diclofenac, the highest contribu-
tion to overall removal was attributed to biological treatment, for clofibric
acid UV treatment was the most efficient, while for carbamazepine, hydro-
dynamic cavitation/hydrogen peroxide process and UV treatment were
equally efficient [20].
Carbamazepine is one of the most persistent pharmaceutical compounds
in wastewater effluents due to its resistance to biodegradation-based
Carbamazepine 151

conventional treatment. Advanced oxidation can efficiently degrade carba-

mazepine, but the toxicity and persistence of the oxidation products may be
more relevant than the parent. This study sets out to determine whether the
products of advanced oxidation of carbamazepine can be biotransformed
and ultimately mineralized by developing a novel methodology to assess
these sequential treatment processes. The methodology traces the transfor-
mation products of the 14C-labeled carbamazepine during UV/hydrogen
peroxide advanced oxidation and subsequent biotransformation by mixed,
undefined cultures using liquid scintillation counting and LC with radioac-
tivity, mass spectrometry (MS), and UV detectors. The results show that the
oxidation by-products of carbamazepine containing a hydroxyl or carbonyl
group can be fully mineralized by a mixed bacterial inoculum. A tertiary
treatment approach that includes oxidation and biotransformation has the
potential to synergistically mineralize persistent pharmaceutical compounds
in wastewater treatment plant effluents. The methodology developed for
this study can be applied to assess the mineralization potential of other per-
sistent organic contaminants [21].
An isocratic simple rapid assay has been developed and validated for the
determination of CBZ in both solution form and rabbit plasma using
propylparaben as an internal standard. The assay was performed using a
-Bondapak C18 (150 mm  4.6 mm I.D.) with a mobile phase consisting
of methanol and water (50:50), and the flow rate was 1 mL/min and UV
detection at 285 nm. The method was found to be specific for CBZ, and
no interfering peaks were observed with an overall analytical run time of
15 min. Accuracy reported as % recovery was found to be 98.37
100.45% and 97.53103.58% for inter- and intraday accuracies, respectively.
Interday precision (reproducibility) was found to be 0.532.75% RSD,
while intraday precision (repeatability) was found to be 1.063.7% RSD
for the samples studied. The calibration curve was found to be linear with
the equation y 0.2847x + 0.0138, with a correlation coefficient of 0.9999
(R2) over a concentration range of 0.540 g/mL. The limit of quantitation
was the lowest concentration. The method is simple, rapid, and does not
require any preliminary treatment of the sample. The method was fully val-
idated [22].
Pharmaceutical compounds have been detected in freshwater for several
decades. Once they enter the aquatic ecosystem, they may be transformed
abiotically (ie, photolysis) or biotically (ie, microbial activity). To assess
the influence of pharmaceuticals on microbial growth, basal salt media
amended with seven pharmaceutical treatments (acetaminophen, caffeine,
152 S.T. Alrashood

carbamazepine, cotinine, ibuprofen, sulfamethoxazole, and a no pharma-

ceutical control) were inoculated with stream sediment. The seven pharma-
ceutical treatments were then placed in five different culture environments
that included both temperature treatments of 4, 25, and 37C and light treat-
ments of continuous UV-A or UV-B exposure. Microbial growth in the
basal salt media was quantified as absorbance (OD(550)) at 7, 14, 21, 31,
and 48 days following inoculation. Microbial growth was significantly
influenced by pharmaceutical treatments (P < 0.01) and incubation treat-
ments (P < 0.01). Colonial morphology of the microbial communities post-
incubation identified the selection of microbial and fungal species with
exposure to caffeine, cotinine, and ibuprofen at 37C; acetaminophen, caf-
feine, and cotinine at 25C; and carbamazepine exposed to continuous UV-
A. Bacillus and coccus cellular arrangements (1000 magnification) were
consistently observed across incubation treatments for each pharmaceutical
treatment although carbamazepine and ibuprofen exposures incubated at
25C also selected spiral-shaped bacteria. These data indicate that stream
sediment microbial communities are influenced by pharmaceuticals though
physiochemical characteristics of the environment may dictate microbial
response [23].
UV/chlorine (UV/HOCl and UV/ClO2) advanced oxidation processes
(AOPs) were assessed with varying process layout and compared to the state-
of-the-art UV/H2O2 AOP. The process comparison focused on the eco-
nomical and energy saving potential of the UV/chlorine AOP. Therefore,
the experiments were performed at the technical scale (250 L/h continuous
flow reactor), and at process energies, oxidant and model contaminant con-
centrations expected in full-scale reference plants. As model compounds, the
emerging contaminants (ECs), desethylatrazine, sulfamethoxazole, carba-
mazepine, diclofenac, benzotriazole, tolyltriazole, iopamidole, and 17-
ethinylestradiol (EE2), were degraded at initial compound concentrations
of 1 g/L in tap water and matrixes with increased organic load (46 mg/
L DOC). UV/chlorine AOP organic by-product forming potential was
assessed for trihalomethanes and N-nitrosodimethylamine. A process design
was evaluated which can considerably reduce process costs, energy con-
sumption, and by-product generation from UV/HOCl AOPs [24].
The Caco-2 cell line has been used as a model to predict the in vitro per-
meability of the human intestinal barrier. The predictive potential of the
assay relies on an appropriate in-house validation of the method. The objec-
tive of this study was to develop a single HPLC-UV method for the iden-
tification and quantitation of marker drugs and to determine the suitability of
Carbamazepine 153

the Caco-2 cell permeability assay. A simple chromatographic method was

developed for the simultaneous determination of both passively (proprano-
lol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively trans-
ported drugs (vinblastine and verapamil). Separation was achieved on a C18
column with step-gradient elution (acetonitrile and aqueous solution of
ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection
at 275 nm during the total run time of 35 min. The method was validated
and found to be specific, linear, precise, and accurate. This chromatographic
system can be readily used on a routine basis and its utilization can be
extended to other permeability models. The results obtained in the
Caco-2 bidirectional transport experiments confirmed the validity of the
assay, given that high- and low-permeability profiles were identified, and
P-glycoprotein (P-gp) functionality was established [25].
For the first time, a selective and sensitive chiral HPLC-UV method
was developed and fully validated for the simultaneous quantification
of eslicarbazepine acetate (ESL), CBZ, S-licarbazepine (S-Lic), R-
licarbazepine (R-Lic), OXC, and CBZ-E, in mouse plasma and brain
homogenate supernatant. After the addition of chloramphenicol as the inter-
nal standard, samples were processed using an SPE procedure. The chiral
chromatographic analysis was carried out on a LiChroCART 250-4
ChiraDex column, employing a mobile phase of water and methanol
(88:12, v/v) pumped at 0.9 mL/min and the UV detector set at 235 nm.
The assay was linear (r2  0.995) for ESL, CBZ, OXC, S-Lic, R-Lic,
and CBZ-E in the range of, respectively, 0.24, 0.430, 0.160, 0.260,
0.260, and 0.230 g/mL, in plasma, and of 0.061.5 g/mL for ESL,
0.1215 g/mL for CBZ and CBZ-E, and 0.0615 g/mL for OXC and
both licarbazepine (Lic) enantiomers in brain homogenate supernatant.
The overall precision was within 8.71% and accuracy ranged from
7.55% to 8.97%. The recoveries of all the compounds were over
92.1%. Afterward, the application of the method was demonstrated using
real plasma and brain samples obtained from mice administered simulta-
neously with ESL and CBZ [26].
Capillary electrophoresis (CE) offers a fast and cost-effective alternative
analytical technique to liquid chromatography/tandem mass spectrometry
(LC-MS/MS) for separation and quantitation of many PPCP compounds
in wastewater. In this study, we have developed a method that can simul-
taneously analyze eight different PPCP compounds in untreated wastewater
(ibuprofen, triclosan, carbamazepine, caffeine, acetaminophen, sulfameth-
oxazole, trimethoprim, and lincomycin), using CE with UV detection
154 S.T. Alrashood

(CE-UV). The method detection limit (MDL) ranged from 1.6 to 68.7 ppb
through SPE. The standard limit of quantification (LOQ) ranged from 0.63
to 7.72 ppm. Factors affecting separation and quantification of PPCPs, such
as pH, electrophoretic potential, buffer strength, buffer type, and additives,
were investigated and optimized. Water samples from two different waste-
water treatment plants were collected and analyzed. The results obtained
were comparable with those of LC-MS/MS. The technique developed in
this study provides a low cost, simple, fast, and relatively sensitive method
for determination of various PPCPs in wastewater samples for PPCP screen-
ing [27].
A methodology for the simultaneous determination of six control
analytes, including carbamazepine, desipramine, guanabenz, methotrexate,
propranolol, and warfarin, was developed and validated utilizing reversed-
phase HPLC with UV detection for high-throughput analysis for permeabil-
ity assessment. The analytes were separated on Agilent Zorbax SB-C18
(50 mm  4.6 mm I.D., 5 m) with a gradient mobile phase consisting of
water (containing 1% isopropyl alcohol and 0.01% heptafluorobutyric acid)
and acetonitrile (containing 1% isopropyl alcohol and 0.01%
heptafluorobutyric acid). The flow rate was 2.0 mL/min and the eluent
was monitored at 280 nm. A linear response was found for all six analytes
over a broad concentration range (1.00200 M). The correlation coeffi-
cient for each analyte was greater than 0.999. The limit of detection
(LOD) and limit of quantitation were 0.03 and 0.10 M, 0.10 and
0.30 M, 0.05 and 0.15 M, 0.03 and 0.10 M, 0.05 and 0.15 M, and
0.10 and 0.30 M for carbamazepine, desipramine, guanabenz, methotrex-
ate, propranolol, and warfarin, respectively. The optimized method was fur-
ther successfully applied to high-throughput analysis for parallel artificial
permeability assay [28].
For the first time, a simple, selective, and accurate HPLC method with
UV detection was developed and validated to quantify simultaneously three
structurally related AEDs: carbamazepine, oxcarbazepine, and the recently
launched ESL and their main metabolites, CBZ-E, 10,11-trans-
dihydroxy-10,11-dihydro-carbamazepine, and licarbazepine. The method
involves an SPE and a reversed-phase C18 column with 5 cm length.
The mobile phase consisting of water, methanol, and acetonitrile in the ratio
64:30:6 was selected as the best one and pumped at 1 mL/min at 40C. The
use of this recent column and an aqueous mobile phase instead of buffers give
several advantages over the method herein developed; namely the fact that
the chromatographic analysis takes only 9 min. The method was validated
Carbamazepine 155

according to the guidelines of the Food and Drug Administration, showing

to be accurate (bias within 12%), precise (coefficient variation <9%),
selective, and linear (r2 > 0.997) over the concentration range of 0.05
30 g/mL for carbamazepine; 0.0520 g/mL for oxcarbazepine; 0.15
4 g/mL for ESL; 0.130 g/mL for CBZ-E; 0.110 g/mL for 10,11-
trans-dihydroxy-10,11-dihydro-carbamazepine; and 0.160 g/mL for
licarbazepine. It was also shown that this method can adequately be used
for the TDM of the considered AEDs, carbamazepine, oxcarbazepine,
ESL, and their metabolites [29].
An implementation of a method allowing the newer AEDs rufinamide
(RFN) and zonisamide (ZNS) to be simultaneously determined with LTG,
oxcarbazepines (OXC) main active metabolite monohydroxycar-
bamazepine (MHD), and felbamate (FBM) in plasma of patients with epi-
lepsy using HPLC with UV detection was presented. Plasma samples
(250 L) were deproteinized by 1 mL acetonitrile spiked with citalopram
as internal standard (IS). HPLC analysis was carried out on a Synergi
4 m Hydro-RP, 250 mm  4.6 mm I.D. column. The mobile phase was
a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), ace-
tonitrile, and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of
0.8 mL/min. The UV detector was set at 210 nm. The chromatographic
run lasted 19 min. Commonly coprescribed AEDs did not interfere with
the assay. Calibration curves were linear for both AEDs over a range of
240 g/mL for RFN and 280 g/mL for ZNS. The limit of quantitation
was 2 g/mL for both analytes and the absolute recovery ranged from 97%
to 103% for RFN, ZNS, and the IS. Intra- and interassay precision and accu-
racy were lower than 10% at all tested concentrations. This study describes
the first simple and validated method for RFN determination in plasma of
patients with epilepsy. By grouping different new AEDs in the same assay,
the method can be advantageous for TDM [30].
Advanced oxidation treatment using low-pressure UV light coupled
with hydrogen peroxide (UV/H2O2) was evaluated for the oxidation of
six pharmaceuticals in three wastewater effluents. The removal of these
six pharmaceuticals (meprobamate, carbamazepine, dilantin, atenolol,
primidone, and trimethoprim) varied between no observed removal and
>90%. The role of the water quality (ie, alkalinity, nitrite, and specifically
effluent organic matter (EfOM)) on hydroxyl radical (OH) exposure was
evaluated and used to explain the differences in pharmaceutical removal
between the three wastewaters. Results indicated that the efficacy of
UV/H2O2 treatment for the removal of pharmaceuticals from wastewater
156 S.T. Alrashood

was a function of not only the concentration of EfOM but also its inherent
reactivity toward OH. The removal of pharmaceuticals also correlated with
reductions in UV absorbance at 254 nm (UV254), which offers utilities a
surrogate to assess pharmaceutical removal efficiency during UV/H2O2
treatment [31].
The reduction of UV absorbance at 254 nm (UV254) and true color was
identified as appropriate surrogates to assess the oxidation of six pharmaceu-
ticals (ie, carbamazepine, meprobamate, dilantin, primidone, atenolol, and
iopromide) during ozonation of wastewater. Three tertiary-treated waste-
waters were evaluated during oxidation with ozone (O3) and O3 coupled
with hydrogen peroxide (O3/H2O2). The correlation between pharmaceu-
tical oxidation and removal of UV254 was dependent upon the reactivity of
each specific compound toward ozone, as measured by the second-order
rate constant (k0 (O3)). Oxidation of compounds with k0 (O3) > 103 M1/s
correlated well (R2 > 0.73) with UV254 reduction between 0% and 50%.
Oxidation of compounds with apparent k0 (O3) < 10 M1/s resulted primar-
ily from hydroxyl radicals and correlated well (R2 > 0.80) with the UV254
reduction of 1585%. The removal of true color also correlated well
(R2 > 0.85) with the oxidation of pharmaceuticals during the ozonation
of two wastewaters. These correlations demonstrate that UV254 reduction
and true color removal may be used as surrogates to evaluate pharmaceutical
oxidation in the presence or absence of dissolved ozone residual during
advanced wastewater treatment with O3 or O3/H2O2. The use of online
UV254 measurements would allow wastewater utilities to optimize the
ozone dose required to meet their specific treatment objectives [32].
The application of sonolysis (US) for remediation of wastewater is an area
of increasing interest. The aim of this study was to evaluate the ultrasonic
(US) process on the degradation of pharmaceuticals (diclofenac (DCF),
amoxicillin (AMX), CBZ) in single solutions and also in three mixtures
spiked in urban wastewater effluent. Several operating conditions, such as
power density (25100 W/L), initial substrate concentrations (2.5
10 mg/L), initial solution pH (311), and air sparging, were varied for
the evaluation and understanding of the process. The degradation (as assessed
by measuring UV absorbance), the generation of hydroxyl radicals (as
assessed measuring H2O2 concentration), the mineralization (in terms of
TOC (total organic carbon) and COD removal), and the aerobic biodegrad-
ability (as assessed by the BOD(5)/COD ratio) were monitored during son-
ication. Ecotoxicity to Daphnia magna, Pseudokirchneriella subcapitata, and
Lepidium sativum before and after treatment was also evaluated. It was found
Carbamazepine 157

that the pharmaceutical conversion is enhanced at increased applied power

densities, acidic conditions, and in the presence of dissolved air. The reaction
rate increases with increasing initial concentration of single pharmaceuticals
but it remains constant in the mixtures, indicating different kinetic regimes
(ie, first and zero order, respectively). Mineralization is a slow process as
reaction by-products are more stable than pharmaceuticals to total oxida-
tion; nonetheless, they are also more readily biodegradable. The toxicity
of the wastewater samples before and after contamination with pharma-
ceuticals both in mixtures and in single substance-containing solutions
was observed more severely on P. subcapitata, a fact that raises concerns in
regard to the discharge of such effluents. D. magna displayed less sensitivity
compared to P. subcapitata because it belongs in a lower taxonomic species
than D. magna. The germination index of L. sativum in the presence of the
drugs mixture was stimulated instead of inducing any toxicity effect and this
might be attributed to the fact that the sample, laden with very low drug
concentrations, was able to act as a provider of additional nutrient elements
[33].
An HPLC assay using UV detection is described for the simultaneous
measurement of the newer generation antiepileptic medications lamo-
trigine, oxcarbazepine (parent drug and active metabolite 10- hydro-
xycarbazepine), and ZNS. Detection of all four compounds can be done
at 230 nm; however, there is a potential interference with ZNS in patients
on clonazepam therapy. Therefore, the method uses dual wavelength detec-
tion: 230 nm for oxcarbazepine and 10-hydroxycarbazepine and 270 nm for
lamotrigine and ZNS. In addition, a simple gas chromatography method
using a nitrogenphosphorus detector is described for the measurement
of levetiracetam, another of the recently approved antiepileptic medications.
For both methods, limits of quantitation, linearities, accuracies, and impre-
cisions cover the therapeutic range for drug monitoring of patients. A wide
variety of clinical drugs, including other antiepileptic drugs, do not interfere
with these assays. These procedures would be of special interest to clinical
laboratories, particularly due to the limited availability of immunoassays
for newer generation antiepileptic medications and that therapeutic uses
of these drugs are expanding beyond epilepsy to other neurologic and psy-
chiatric disorders [34].
Herein is reported, for the first time, a simple and reliable chiral reversed-
phase liquid chromatographic (RPLC) method coupled to UV detection for
simultaneous determination of ESL and its metabolites, S-licarbazepine (S-
LC), R-licarbazepine (R-LC), and OXC, in mouse plasma and brain, liver,
158 S.T. Alrashood

and kidney tissue homogenates. All analytes and the internal standard were
extracted from plasma and tissue homogenates by an SPE procedure using
Waters Oasis hydrophiliclipophilic balance cartridges. The chromato-
graphic separation was performed by isocratic elution with water/methanol
(88:12, v/v), pumped at a flow rate of 0.7 mL/min, on a LichroCART
250-4 ChiraDex (beta-cyclodextrin, 5 m) column at 30C. The UV detec-
tor was set at 225 nm. Calibration curves were linear (r2  0.996) in the
ranges 0.48 , 0.11.5, and 0.12 g/mL for ESL and OXC and in the
ranges 0.480, 0.115, and 0.120 g/mL for R-LC and S-LC in plasma,
brain, and liver/kidney homogenates, respectively. The overall precision
not exceeded 11.6% (%CV) and the accuracy ranged from 3.79% to
3.84% (%bias), considering all analytes in all matrices. Hence, this method
will be a useful tool to characterize the pharmacokinetic disposition of
ESL in mice [35].
ESL (BIA 2-093) is a novel central nervous system (CNS) drug under-
going clinical phase III trials for epilepsy and phase II trials for bipolar dis-
order. A simple and reliable chiral reversed-phase HPLC-UV method was
developed and validated for the simultaneous determination of ESL,
oxcarbazepine, S-licarbazepine, and R-licarbazepine in human plasma.
The analytes and internal standard were extracted from plasma by an SPE
using Waters Oasis HLB cartridges. Chromatographic separation was
achieved by isocratic elution with watermethanol (88:12, v/v), at a flow
rate of 0.7 mL/min, on a LichroCART 250-4 ChiraDex (beta-cyclodex-
trin, 5 m) column at 30C. All compounds were detected at 225 nm. Cal-
ibration curves were linear over the range 0.48 g/mL for ESL and
oxcarbazepine, and 0.480 g/mL for each licarbazepine enantiomer.
The overall intra- and interday precision and accuracy did not exceed
15%. Mean relative recoveries varied from 94.00% to 102.23% and the
LOQ of the assay was 0.4 g/mL for all compounds. This method seems
to be a useful tool for clinical research and TDM of ESL and its metabolites
S-licarbazepine, R-licarbazepine, and oxcarbazepine [36].
This article describes a rapid HPLC method for the measurement of
the primary metabolite of oxcarbazepine. Following a simple precipitation
step, 10,11-dihydro-10-hydroxy-5H-dibenzo(b, f )azepine-5-carboxamide is
quantitated (560 g/mL) by analysis on an HPLC-UV system. The instru-
ment time is less than 5 min per injection, an improvement over most pub-
lished methods. The assays limit of quantitation, linearity, imprecision, and
accuracy adequately cover the therapeutic range for appropriate patient
monitoring. In comparison to other published methods, this procedure
Carbamazepine 159

would be of interest to clinical laboratories because it employs a precipitation

step for sample preparation, instead of conventional yet time-consuming
SPE [37].
In the present report, carbamazepine is determined on serum samples of
real patients by a procedure completely assisted by chemometric tools. First,
a response surface methodology based on a mixture design was applied in
order to select the best conditions for the extraction step. Finally, partial least
squares multivariate calibration (PLS-1) was applied to second derivative
UV spectra, eliminating a shift baseline effect that originated in the extrac-
tion procedure. The performance assessment included: (a) a three-level pre-
cision study, (b) a recovery study analyzing spiked samples, and (c) a method
comparison with HPLC and fluorescence polarization immunoassay (FPIA)
applied on real patient samples. The obtained results show the potentiality of
the presently studied methodology for the monitoring of patients treated
with this anticonvulsant [38].
The UV/H2O2-induced degradation of carbamazepine, a worldwide
used AED, recently found as contaminant in many municipal sewage treat-
ment plant (STP) effluents and other aquatic environments, is investigated.
The oxidation treatment caused an effective removal of the drug. At com-
plete abatement of the substrate after 4 min treatment, a 35% value of
removed TOC was obtained. A kinetic constant of (2.05  0.14)  109 L/
mol/s was determined for OH radical attack to carbamazepine in the
UV/H2O2 process. Preparative TLC of the reaction mixture led to the iso-
lation of acridine-9-carboxaldehyde as a reaction intermediate. HPLC and
GC/MS analysis indicated the formation of small amounts of acridine, sali-
cylic acid, catechol, and anthranilic acid among the reaction products.
Under the same reaction conditions, synthetically prepared 10,11-epoxy
carbamazepine was easily degraded to acridine as the main product,
suggesting that this epoxide is a likely intermediate in the oxidative conver-
sion of carbamazepine to acridine. Under sunlight irradiation, carbamaze-
pine in water underwent slow degradation to afford likewise acridine as
the main product. In view of the mutagenic properties of acridine, these
results would raise important issues concerning the possible environmental
impact of carbamazepine release through domestic wastewaters and support
the importance of prolonged oxidation treatments to ensure complete deg-
radation of aromatic intermediates [39].
Nateglinide (NA) is a novel oral mealtime glucose regulator, recently
approved for the treatment of type II diabetes mellitus. To facilitate clinical
studies investigating the dependence of NA elimination on the genotype of
160 S.T. Alrashood

cytochrome P450 isoenzymes, a rapid HPLC method for determination of

NA in human plasma samples was developed. The validated limit of quan-
titation (LOQ) of 0.1 g/mL is low enough to allow determination of phar-
macokinetic parameters of the substance. The intra-assay coefficients of
variation (CV) ranged from 1.6% to 12.9% at NA concentrations of 0.5
7.5 g/mL. The interassay variation for the same plasma concentrations
ranged from 3.8% to 8.4%. The calibration was linear in the range of
0.120 g/mL. For the quantitation of NA, only 50 L of plasma was
needed. Following protein precipitation in human plasma, the samples were
separated by isocratic reversed-phase HPLC and analyzed using UV detec-
tion at 210 nm. Sample preparation time and analysis time are both short and
allow rapid analysis of large sample sets [40].
An isocratic LC assay using a microcolumn (800 m I.D.) coupled to a U-
shaped optical flow cell (cell volume 70 nL; optical path length 8 mm) for high-
sensitivity UV absorbance is described for the detection of oxcarbazepine and its
major and active metabolite, 10,11-dihydro-10-hydroxycarbamazepine in
microdialysates. Using the combination microcolumn-capillary UV detector,
a 10-fold increase in sensitivity was obtained resulting in an LOD of 10 pg/
10 L. This assay is sufficiently sensitive to allow quantification of drug and
metabolite in 10-L aliquots of rat blood and hippocampus microdialysates,
using CBZ-E as external standard [41].
The use of stable isotope-labeled tracer compounds is the safest and most
effective method to perform many steady-state pharmacokinetic and drug
interaction studies. A method by which the heavily deuterated 2H10 analogs
of carbamazepine (2H10 CBZ) and phenytoin (2H10 PHT) can be chromato-
graphically separated by HPLC from unlabeled CBZ and PHT was
described. All compounds are quantitated against an internal standard (IS)
(10,11-dihydrocarbamazepine) and measured using conventional UV
detection rather than MS. Baseline resolution of extracted serum containing
2
H10 CBZ, CBZ, 2H10 PHT, PHT, and IS is achieved on a heated (55C)
25 cm  4.6 mm BioAnalytical Systems Phase II 5 m ODS column with an
isocratic mobile phase consisting of water-acetonitrile-tetrahydrofuran
(80:16:4, v/v/v) at 1.2 mL/min. Eluting compounds were monitored at a
UV wavelength of 214 nm. Calculated resolution of 2H10 CBZ from
CBZ and of 2H10 PHT from PHT were 1.3. Serum standard curves were
linear (R greater than or equal to 0.999) over a range of 0.514 g/mL
for 2H10 CBZ, 0.520 g/mL for CBZ, 0.520 g/mL for 2H10 PHT,
and 0.530 g/mL for PHT. Within-day percent RSDs (precision) were
less than 6% in all cases [42].
Carbamazepine 161

The UV spectrum of chlorpromazine undergoes a red shift in the pres-

ence of vesicles of biological membranes or phospholipids, triglycerides,
serum lipoproteins, or fatty acids. The resulting difference spectrum has
two positive peaks at about 260 and 320 nm and two negative peaks at
250 and 290 nm. This interaction signal, which was elicited in the presence
of as little as 3 M oleic acid, was dependent on the concentrations of both
ligand and binder. It was abolished by 8 M urea, diminished by temperature
increase up to 70C, but not changed by varying the ionic strength from 0 to
0.5. The chlorpromazinetriglyceride interaction signal was strongly
enhanced with pH increasing from 6 to 10. The signal was only obtained
with ligands fulfilling specific structural requirements, eg, phenothiazines
and most iminostilbenes, but not carbamazepine, imipramine, and amitrip-
tyline [43].

4.2.1.2 Visible Spectrometric Methods

CBZ and PHT are two AEDs which are used simultaneously. In this paper, a
PLS calibration method is described for the simultaneous spectrophotomet-
ric determination of CBZ and PHT in plasma. Standard binary mixtures of
CBZ and PHT have been resolved by application of PLS-1 to their UV
spectra. Then, the binary standard solutions, spiked to plasma, were pre-
pared, and after the extraction of the drugs, their corresponding UV spec-
trum was analyzed by PLS regression to calculate the concentration of drugs
in unknown plasma. A leave-one-out cross-validation procedure was
employed to find the optimum numbers of latent variables using PRESS.
An HPLC method was also applied for simultaneous determination of
two drugs in the plasma and in methanol. The mean recoveries obtained
by PLS were 98.4 and 98.2 for CBZ and PHT and those obtained by HPLC
were 100.1 and 101.7, respectively. Although the HPLC method showed
better performance than PLS, it was found that the results obtained by
PLS were comparable with those obtained by HPLC method [44].
Two spectrophotometric methods are proposed for the assay of OXC in
bulk and dosage forms using FolinCiocalteus phenol reagent (FCP) and 3-
methyl-2-benzothiazolinone hydrazine hydrochloride (MBTH) as reagents.
The first method involves addition of FCP reagent to OXC in alkaline
medium followed by measurement of absorbance at 760 nm (method A),
and the other involves addition of a fixed volume of MBTH after treatment
of OXC with ferric chloride and measurement of absorbance at 456 nm
(method B). In both methods, the amount of chromogen formed corre-
sponds to the amount of OXC and the measured absorbance was found
162 S.T. Alrashood

to increase linearly with the concentration of OXC, which is corroborated

by the correlation coefficients of 0.9985 and 0.9984 for methods A and B,
respectively. The systems obey Beers law for 530 and 1050 g/mL for
methods A and B, respectively. The apparent molar absorptivity was calcu-
lated to be 8.06  103 and 3.126  103 L/mol/cm for methods A and B,
respectively. The LOD and LOQ were calculated to be 1.6 and 5 g/mL
for method A and 3 and 10 g/mL for method B. The inter- and intraday
imprecisions of the methods were found to be in the range of 1.11.7% and
0.91.1% for method A, and 1.11.9% and 0.60.9% for method B. The
accuracy ranged between 98.999.7% and 99.3100.1% for methods A
and B, respectively. No interference was observed from common pharma-
ceutical excipients. The methods were successfully applied to the assay of
OXC in tablet preparations [45].
CBZ undergoes enzyme biotransformation through epoxidation with
the formation of its metabolite, CBZ-E. A simple chemometrics-assisted
spectrophotometric method has been proposed for simultaneous determina-
tion of CBZ and CBZ-E in plasma. A liquid extraction procedure was oper-
ated to separate the analytes from plasma, and the UV absorbance spectra of
the resultant solutions were subjected to PLS regression. The optimum
number of PLS latent variables was selected according to the PRESS values
of leave-one-out cross-validation. An HPLC method was also employed for
comparison. The respective mean recoveries for analysis of CBZ and CBZ-
E in synthetic mixtures were 102.57 (0.25)% and 103.00 (0.09)% for PLS
and 99.40 (0.15)% and 102.20 (0.02)%. The concentrations of CBZ and
CBZ-E were also determined in five patients using the PLS and HPLC
methods. The results showed that the data obtained by PLS were compara-
ble with those obtained by HPLC method [46].
A selective and sensitive method was developed for the determination of
the anticonvulsants vigabatrin (I) (CAS 60643-86-9) and gabapentin (II)
(CAS 60142-96-3). The method is based on the condensation of the drugs
through their amino groups with acetylacetone and formaldehyde according
to the Hantzsch reaction yielding the highly fluorescent dihydropyridine
derivatives. The yellowish-orange color was also measured spectrophoto-
metrically at 410 and 415 nm for I and II, respectively. The absorbance
concentration plots were rectilinear over the ranges 1070 and 20
140 g/mL for I and II, respectively. As for the fluorescenceconcentration
plots, they were linear over the ranges 0.510 and 2.520 g/mL with min-
imum detection limits (S/N 2) of 0.05 g/mL (2.1  108 mol/L) and
0.1 g/mL (5.8  107 mol/L) for I and II, respectively. The
Carbamazepine 163

spectrophotometric method was applied to the determination of I and II

in their tablets. The percentage recoveries SD (n 6) were 99.45  0.13
and 98.05  0.53, respectively. The spectrofluorimetric method was suc-
cessfully applied to the determination of I and II in spiked human urine
and plasma. The % recoveries SD (n 5) were 98.77  0.29 and
98.39  0.53 for urine and 99.32  0.74 and 98.90  0.96 for plasma, for I
and II, respectively. No interference was encountered with the
coadministered drugs: valproic acid (CAS 99-66-1), diphenylhydantoin
(CAS 57-41-0), phenobarbital (CAS 50-06-6), carbamazepine (CAS
298-46-4), clonazepam (CAS 1622-61-3), clobazam (CAS 22316-47-8),
or cimetidine (CAS 51481-61-9). A proposal of the reaction pathway is
suggested. The advantages of the proposed methods over existing method
are discussed [47].
A near-infrared (IR) spectrophotometer, integrating optics, and parallel-
vector supercomputer are employed to develop a mathematical model that
predicts the dissolution rate of individual intact tablets from near-IR spectra
(r2 0.985). Each tablet can be analyzed nondestructively by the spectro-
photometer in less than 1 min. The model permits hundreds of near-IR
wavelengths to be used in the determination of dissolution rate, leading
to increased accuracy [48].
A 0.5-mL sample of serum, containing different AEDs, singly or in com-
bination, was made alkaline, overlayered with isooctane, and steamed in the
presence of KMnO4. The spectra of oxidized products in the organic layer
were recorded in the UV range. Oxidized phenobarbitone and primidone
show no absorption peak; diazepam a delta-max at 228 nm; phenytoin at
247 nm; and carbamazepine at 247 and 372 nm. Consequently, phenobar-
bitone and diazepam do not interfere in phenytoin quantitation, but carba-
mazepine does. The contribution of carbamazepine at 247 nm was
calculated from the absorption at 372 nm and the ratio of its molar extinction
coefficients at the two wave lengths. This was subtracted from the total A247
values to get the actual values due to phenytoin. Thus, a method for simul-
taneous analysis of carbamazepine and phenytoin in a single sample has been
developed [49].
Carbamazepine and 5,5-diphenylhydantoin are simultaneously ex-
tracted from 100 to 200 L blood with 1,2-dichloroethane. 5,5-Diphenyl-
hydantoin is removed by a one-step wash into alkali. The dichloroethane
is further washed with acid and then evaporated to dryness. 5,5-Diphenyl-
hydantoin is determined in the alkali washing by a benzophenone
procedure; carbamazepine is determined in the dried residue by the
164 S.T. Alrashood

9-methylacridine procedure described earlier. The combined method is

rapid, reliable, and has a detection threshold of less than 0.1 mg/100 mL
for each drug [50].
An UV spectrophotometric procedure for the microdetermination
of carbamazepine in blood is described which is based on the original 9-
methylacridine method proposed by K.H. Beyer, K. Klinge, Arzneim. For-
sch. 19 (1969) 17591760). Carbamazepine is extracted from blood with
dichloromethane, which is then washed with alkali and acid. An aliquot
of the extractant is evaporated to dryness and the residue heated briefly with
hydrochloric acid at 150C. Following removal of nonspecific interference
with n-heptane, the absorbance of the acid-catalyzed rearrangement product
(9-methylacridine) is determined at 258 nm. The resulting procedure is
rapid, reliable, sensitive, and specific. It requires 100200 L sample for a
single estimation and has a detection threshold of less than 0.1 mg/
100 mL. It is concluded that the method is suitable for routine clinical
use [51].
A specific direct gas chromatographic method to determine carbamaze-
pine and, semiquantitatively, 10,11-epoxy carbamazepine in serum is
described. The average recovery of carbamazepine is 98%, and the error
on duplicate determination is 4%. The method is compared with
Herrmanns classic spectrophotometric method. In material of 103 patients,
the mean serum concentration of carbamazepine was 25.5  12.8 mol/L
with GLC and 23.0  12.6 mol/L with spectrophotometry. The difference
was highly significant. The blood sample volume is 1/10th of that needed in
spectrophotometry [52].

4.2.2 Spectrofluorimetric and Chemiluminescence Methods

4.2.2.1 Spectrofluorimetric Methods
Upon online photochemical reaction, CBZ can be converted to a strong
fluorescent compound which has a maximum emission wavelength of
478 nm and maximum excitation wavelength of 254 nm. Acidity of reaction
medium and the acid type were found to be critical for the online photo-
chemically induced fluorescence, dilute hydrochloric acid being the most
suitable. Based on these observations, a flow injection photochemical spec-
trofluorimetric approach for determination of the drug was developed. At
optimized conditions, a detection limit of 0.08 ng/mL CBZ was achieved
at the sampling rate of 80 h1. Eleven determinations of a 100 ng/mL
CBZ standard solution gave an RSD of 0.45%. A linear calibration curve
was obtained in the CBZ concentration range of 2250 ng/mL. The
Carbamazepine 165

developed method was successfully applied to assay the CBZ contents in

pharmaceutical tablets [53].

4.2.2.2 Chemiluminescence Methods

A photochemically induced fluorescence system combined with second-
order chemometric analysis for the determination of the anticonvulsant
CBZ is presented. CBZ is a widely used drug for the treatment of epilepsy
and is included in the group of emerging contaminant present in the aquatic
environment. CBZ is not fluorescent in solution but can be converted into a
fluorescent compound through a photochemical reaction in a strong acid
medium. The determination is carried out by measuring excitationemission
photoinduced fluorescence matrices of the products formed upon UV light
irradiation in a laboratory-constructed reactor constituted by two simple
4 W germicidal tubes. Working conditions related to both the reaction
medium and the photoreactor geometry are optimized by an experimental
design. The developed approach enabled the determination of CBZ at trace
levels without the necessity of applying separation steps, and in the presence of
uncalibrated interferences which also display photoinduced fluorescence and
may be potentially present in the investigated samples. Different second-order
algorithms were tested and successful resolution was achieved using MCR-
ALS. The study is employed for the discussion of the scopes and yields of each
of the applied second-order chemometric tools. The quality of the proposed
method is probed through the determination of the studied emerging
pollutant in both environmental and drinking water samples. After a
preconcentration step on a C18 membrane using 50.0 mL of real water sam-
ples, a prediction relative error of 2% and LOD and LOQ of 0.2 and 0.6 ng/
mL were, respectively, obtained [54].
A novel, sensitive, and rapid CL method coupled with HPLC separation
for the determination of carbamazepine is described. The method was
based on the fact that carbamazepine could significantly enhance the chemi-
luminescence of the reaction of cerium sulfate and tris(2,2-bipyridyl) ruthe-
nium(II) in the presence of acid. The chromatographic separation was
performed on a Kromasil (Sigma-Aldrich) TM RP-C18 column (I.D.
150 mm  4.6 mm, particle size: 5 m, pore size: 100 A) with a mobile
phase consisting of methanolwaterglacial acetic acid (70:29:1, v/v/v) at
a flow rate of 1.0 mL/min, and the total analysis time was within 650 s.
Under optimal conditions, CL intensity was linear for carbamazepine in
the range 2.0  1084.0  105 g/mL, with a detection limit of
6.0  109 g/mL (S/N 3) and the relative standard detection was 2.5%
166 S.T. Alrashood

for 2.0  106 g/mL (n 11). This method was successfully applied to the
analysis of carbamazepine in human urine and serum samples. The possible
mechanism of the CL reaction is also discussed briefly [55].
Carbamazepine is a first-choice AED for the treatment of simple and
complex partial seizures. The use of an established therapeutic range for car-
bamazepine concentration is limited by the presence of CBZ-E, its active
metabolite that significantly contributes to the efficacy and toxicity and is
not routinely measured and accounted for. This article describes the devel-
opment of an HPLC method for determination of CBZ and CBZ-E in
serum and compares it with chemiluminescence immunoassay to evaluate
the importance of considering the active metabolite in therapeutic strategies.
Methods: The procedure involves protein precipitation, separation on a
reversed-phase column, and UV detection. The analytical procedure proved
to be sensitive, selective, precise, accurate, and linear (regression coefficients
>0.999) in the range of 0.525.0 and 0.110.0 g/mL for quantification of
CBZ and CBZ-E, respectively. For the comparison between methods,
serum samples of 75 patients using the medication were evaluated. Results:
The Pearson correlation coefficient showed that the carbamazepine concen-
trations measured by HPLC are significantly higher than those obtained by
immunoassay (mean difference (MD) of 1.07 g/mL, 95% limits of agree-
ment from 0.65 to 2.80 g/mL). It was found that this difference may be
decisive for the therapy. In some cases, this may affect the individual dosage
adjustment and subsequent treatment [56].
A new chemiluminescence method for the determination of CBZ has
been developed. The method is based on the chemiluminescence produced
in the reaction of tris(2,20 -bipyridine)ruthenium(III) and CBZ in an acidic
medium. The chemiluminescence intensity was enhanced by organic sol-
vents in the reaction system. Under the optimum experimental conditions,
the calibration curve was linear over the range 4.0  1038.6 107 mol/L
for CBZ. The detection limit (S/N 3) was 2.5  107 mol/L and the RSD
of six replicate measurements was 2.6% for 4.0  104 mol/L of CBZ. The
possible reaction mechanism was also discussed. The chemiluminescence
method was successfully applied to assay the CBZ contents in pharmaceu-
tical tablets [57].

4.2.3 Polarographic Methods

The electrochemical characteristics of CBZ have been studied. In a solution
containing 0.40 mol/L NaOH, 0.15 mol/L KNO3, and 0.0020% sodium
lauryl sulfate (SLS), CBZ gives a sensitive polarographic wave which can
Carbamazepine 167

be used to determine trace amounts of the drug, the detection limit being
1.0  106 mol/L. The cyclic voltammetric data show that SLS promotes
adsorption of CBZ at the mercury electrode [58].
The quantitative determination of the impurity 10-bromocarbamazepine
(caused by manufacturing method) in the drug carbamazepine is possible by
using their cathodic two-electron debromination at a dropping mercury elec-
trode in tetraethylammonium perchlorate/acetonitrile or 80% aqueous meth-
anol as supporting electrolyte. Direct current polarographic (dcp) and
differential pulse polarographic (dpp) methods are described which can be
used in process control and quality control of the drug production. These
analytic methods allow to determine 10-bromocarbamazepine in carbamaz-
epine up to a limiting concentration of 3  105 mol/L (100 ppm bromine;
dcp) and of 3  106 mol/L (10 ppm bromine; dpp). On the basis of elec-
troanalytical results, the mechanism of the polarographic reduction of 10-
bromocarbamazepine is discussed [59].

4.2.4 Voltammetric Methods

A multiwalled carbon nanotube film-coated glassy carbon electrode (GCE)
was used for the voltammetric determination of CBZ. The results showed
that this simple modified electrode exhibited excellent electrocatalytic activ-
ity toward the oxidation of CBZ. The voltammetric response of CBZ at this
film-modified electrode increased significantly when compared with that at
a bare GCE and the sensor response was reproducible. The proposed
method was applied to the quantification of CBZ in wastewater samples,
collected in a municipal wastewater treatment plant, and in pharmaceutical
formulations. The developed methodology yields results in accord with
those obtained by chromatographic techniques commonly used in the quan-
tification of pharmaceutical compounds in real samples. Good recoveries
have been obtained and the LOD and LOQ (40 and 140 nM, respectively)
are among the lowest that have been reported to date for this pharmaceutical
compound using voltammetric techniques [60].

4.2.5 Chromatographic Methods

4.2.5.1 Thin-Layer Chromatography
A high-throughput method was developed for screening antidepressants in
blood by automated SPE and LC with high-resolution quadrupole-time-of-
flight mass spectrometry (ASPE-LC-Q-TOF/MS). The samples were
cleaned up by an HLB SPE cartridge and analyzed by LC-Q-TOF/MS
under electrospray ionization (ESI) mode with scanning range of m/z
168 S.T. Alrashood

501000 Da. The chromatographic separation was performed on an Agilent

Eclipse Plus C18 column (50 mm  2.1 mm, 1.8 m) with gradient elution
using methanol and 5 mmol/L ammonium formate aqueous solution (con-
taining 0.2% formic acid) as mobile phases. Rapid screening and confirma-
tion can be achieved using MS matching scores, deviation of retention time,
measured mass, isotopic abundance matching scores, isotope space matching
scores, and MS/MS matching scores. The quantitative analysis was carried
out by correlating the extracting peak area with accurate mass. Good line-
arities were observed in the range of 1500 g/L with the correlation coef-
ficients from 0.997 6 to 0.9997. The LODs were 0.010.5 g/L. The spiked
recoveries were 79.696.4% with the RSDs of 4.16.4%. The result screen-
ing database was built using Agilent MassHunter PCDL Manager Software
and then used for the analysis of spiked samples. MS matching scores, iso-
topic abundance matching scores, isotope space matching scores (all >95
points), and MS/MS matching scores (>70 points) were applied to identify
the analytes. The results showed that all the spiked antidepressants could be
correctly identified with low deviation of retention time (<0.1 min) and
mass (<1 mDa). The developed method was further applied for the analysis
of poisoning cases, and amitriptyline, carbamazepine, and doxepin were
detected. In brief, the method is rapid, sensitive, simple, reliable, and suitable
for the screening and confirmation of antidepressants in forensic and clinical
analytical toxicology [61].
The occurrence of CBZ and its metabolites in German and Portuguese
wastewater was investigated. A total of 46 samples from influent and
effluent wastewater were analyzed by LC tandem mass spectrometry.
The five metabolites 10,11-dihydro-10,11-dihydroxy-CBZ (DiOH-CBZ),
10,11-dihydro-10-hydroxy-CBZ (10-OH-CBZ), 10,11-epoxy-10,11-
dihydro-CBZ, 2-hydroxy-CBZ, and 3-hydroxy-CBZ were very persistent
with little to no removal during wastewater treatment. The highest concen-
trations were found for CBZ, DiOH-CBZ, and 10-OH-CBZ, with up to
5.0, 4.8, and 1.1 g/L, respectively. Furthermore, the related pharmaceutical
oxcarbazepine and the metabolites 9-hydroxymethyl-10-carbamoylacridan,
1-hydroxy-CBZ (1-OH-CBZ), and 4-hydroxy-CBZ (4-OH-CBZ) were
detected. Explicit care was taken to achieve a good chromatographic separa-
tion of the numerous isomers that were difficult to distinguish by MS alone. A
phenylether stationary phase provided the best separation. In combination
with high-resolution MS and hydrogendeuterium exchange, this LC col-
umn enabled us to identify 1-OH-CBZ and 4-OH-CBZ in wastewater
for the first time [62].
Carbamazepine 169

In this study, the separation of a variety of mixtures of drugs, metabolites,

and related analogs including representatives of the carbamazepine, methyl-
ated xanthine, steroid hormone, nicotine, and morphine families using sev-
eral automated chromatographic method development screening systems
including ultra-HPLC, coreshell HPLC, achiral supercritical fluid chroma-
tography (SFC), and chiral SFC was investigated. Of the 138 column and
mobile phase combinations examined for each mixture, a few chromato-
graphic conditions afford the best overall performance, with a single achiral
SFC method (4.6 mm  250 mm, 3.0 m GreenSep Ethyl Pyridine, 25 mM
isobutylamine in methanol/CO2) affording good separation for all samples.
Four of these mixtures were also resolved by achiral SFC on the Luna HILIC
and chiral SFC Chiralpak IB columns using methanol or ethanol with
25 mM isobutylamine as polar modifiers. Modifications of standard chroma-
tography screening conditions afforded fast separation methods (from 1 to
5 min) for baseline resolution of all components of each of these challenging
sets of closely related compounds [63].
A method for the simultaneous determination of the antiepileptic drugs,
phenobarbital (PHB), phenytoin (PTN), carbamazepine (CBZ), primidone
(PRM), and oxcarbazepine (OXC) in human plasma and urine samples by
using microextraction in a packed syringe as the sample preparation method
connected with LC/UV (MEPS/LC/UV) is described. Microextraction in
a packed syringe (MEPS) is a new miniaturized, SPE technique that can be
connected online to gas or liquid chromatography without any modifica-
tions. In MEPS, approximately 1 mg of the solid packing material is inserted
into a syringe (100250 L) as a plug. Sample preparation takes place on the
packed bed. The bed can be coated to provide selective and suitable sam-
pling conditions. The new method is very promising, easy to use, fully auto-
mated, inexpensive, and quick. The standard curves were obtained within
the concentration range of 1500 ng/mL in both plasma and urine samples.
The results showed high correlation coefficients (R2 > 0.988) for all of the
analytes within the calibration range. The extraction recovery was found to
be between 88.56% and 99.38%. The LOQ was found to be between 0.132
and 1.956 ng/mL. The precision (RSD) values of quality control samples
(QC) had a maximum deviation of 4.9%. A comparison of the detection
limits with similar methods indicates high sensitivity of this method. The
method is applied for the analysis of these drugs in real urine and plasma sam-
ples of epileptic patients [64].
The purpose of this study was to develop and validate a sensitive and
specific enantioselective LC-MS/MS method, for the simultaneous
170 S.T. Alrashood

quantification of ESL, eslicarbazepine (S-Lic), OXC, and R-licarbazepine

(R-Lic) in human plasma. Analytes were extracted from human plasma using
SPE and the chromatographic separation was achieved using a mobile phase
of 80% n-hexane and 20% ethanol/isopropyl alcohol (66.7:33.3, v/v). A
Daicel CHIRALCEL OD-H column (5 m, 50 mm  4.6 mm) was used
with a flow rate of 0.8 mL/min and a run time of 8 min. ESL, S-Lic, R-
Lic, OXC, and the internal standard, 10,11-dihydrocarbamazepine, were
quantified by positive ion electrospray ionization MS. The method was fully
validated, demonstrating acceptable accuracy, precision, linearity, and spec-
ificity in accordance with FDA regulations for the validation of bioanalytical
methods. Linearity was proven over the range of 50.01000.0 ng/mL for
ESL and OXC and over the range of 50.025,000.0 ng/mL for S-Lic and
R-Lic. The intra- and interday coefficient of variation in plasma was less than
9.7% for ESL, 6.0% for OXC, 7.7% for S-Lic, and less than 12.6% for R-Lic.
The accuracy was between 98.7% and 107.2% for all the compounds quan-
tified. The lower LOQ (LLOQ) was 50.0 ng/mL for ESL, S-Lic, OXC, and
R-Lic in human plasma. The short-term stability in plasma, freezethaw
stability in plasma, frozen long-term stability in plasma, autosampler stability,
and stock solution stability all met acceptance criteria. The human plasma
samples, collected from eight volunteers, showed that this method can be
used for therapeutic monitoring of ESL and its metabolites in humans treated
with ESL [65].
Recently, in silico models have been developed to predict drug pharma-
cokinetics. However, before application, they must be validated and, for
that, information about structurally similar reference compounds is required.
A chiral LC method with ultraviolet detection (LC-UV) was developed
and validated for the simultaneous quantification of BIA 2-024, BIA 2-
059, BIA 2-265, oxcarbazepine, eslicarbazepine (S-licarbazepine), and R-
licarbazepine in mouse plasma and brain. Compounds were extracted by
a selective SPE procedure, and their chromatographic separation was
achieved on a LiChroCART 250-4 ChiraDex column using a mobile phase
of watermethanol (92:8, v/v) pumped at 0.7 mL/min. The UV detector
was set at 235 nm. Calibration curves were linear (r2  0.996) over the con-
centration ranges of 0.230 g/mL for oxcarbazepine, eslicarbazepine, and
R-licarbazepine; 0.260 g/mL for the remaining compounds in plasma;
and 0.0615 g/mL for all the analytes in brain homogenate. Taking into
account all analytes at these concentration ranges in both matrices, the over-
all precision did not exceed 9.09%, and the accuracy was within 14.3%.
This LC-UV method is suitable for carrying out pharmacokinetic studies
Carbamazepine 171

with these compounds in mouse in order to obtain a better picture of their

metabolic pathways and biodistribution [66].
Two impurities were detected in the HPLC analysis of crude carbamaz-
epine active pharmaceutical ingredient. One of the impurities of the order of
0.5% was found to be unknown and has not been reported previously. An
LCMS compatible reversed-phase isocratic method was developed and
tandem MS was performed using ESI source and ion trap mass analyzer. Iso-
lation of unknown impurity was performed by semipreparative HPLC
followed by characterization using nuclear magnetic resonance (NMR)
spectroscopy, infrared spectroscopy (FT-IR), and elemental analysis
(CHNS) confirmed its structure as tetrabenzo[b, f,b0 f0 ]azepino[40 ,50 :4,5]
thieno[2,3-d]azepine-3,9-dicarboxamide. A plausible mechanism for the
formation of this impurity is proposed [67].
A rapid and reliable analytical method suitable for the simulta-
neous determination of the AED, oxcarbazepine, and its metabolites in
human plasma and saliva by means of LC with diode array detec-
tion (DAD) has been developed. Oxcarbazepine and its metabolites
(10,11-dihydro-10-hydroxycarbamazepine, trans-10,11-dihydro-10,11-
dihydroxycarbamazepine, and 3-hydroxycarbamazepine) were baseline sep-
arated within 6.5 min on a reversed-phase C18 column with a phosphate
bufferacetonitriletriethylamine mixture as the mobile phase. The DAD
detector was set at 240 nm. A sample preparation method for biological sam-
ples using a microextraction by packed sorbent technique has been
implemented, employing a C18 sorbent inserted into a microvolume
syringe and using only a small volume (25 L) of plasma or saliva. The
extraction yield values were satisfactory for all analytes (>86.5%) as well
as the precision data, which were always in the low percentage of RSD
values (<4.6%). The method was successfully applied to both plasma and
saliva samples drawn from psychiatric and neurological patients undergoing
treatment with oxcarbazepine (Tolep) tablets [68].
An instrumental planar chromatographic (HPTLC) method for quanti-
fication of carbamazepine in human serum was developed using liquidliq-
uid extraction with dichloromethane, fluorescence activation with
perchloric acid 60%/ethanol/water (1:1:1, v/v/v), and fluorescence detec-
tion. Planar chromatographic separation was performed on precoated silica-
gel F254 HPTLC plates using a mixture of ethyl acetate/toluene/methanol/
acetic acid glacial (5:4:0.5:0.5, v/v/v) as mobile phase. Densitometric detec-
tion was done at 366 nm. The method was validated for linearity, precision,
and accuracy. Linear calibration curves in the range of 3 and 20 ng/L
172 S.T. Alrashood

showed correlation coefficient of 0.998. The intra- and interassay precision,

expressed as the RSD, were in the range of 0.411.24% (n 3) and 2.17
3.17% (n 9), respectively. The LOD was 0.19 ng, and the LOQ was
0.57 ng. Accuracy, calculated as percentage recovery, was between
98.98% and 101.96%, with an RSD not higher than 1.52%. The method
was selective for the active principle tested. In conclusion, the method is use-
ful for quantitative determination of carbamazepine in human serum [69].
The present paper describes the development of a stability indicating
RPLC method for oxcarbazepine in the presence of its impurities and deg-
radation products generated from forced decomposition studies. The drug
substance was subjected to stress conditions of hydrolysis, oxidation, photol-
ysis, and thermal degradation. The degradation of oxcarbazepine was
observed under base hydrolysis. The drug was found to be stable to other
stress conditions attempted. Successful separation of the drug from the syn-
thetic impurities and degradation product formed under stress conditions
was achieved on a C18 column using mixture of aqueous 0.02 M potassium
dihydrogen phosphateacetonitrilemethanol (45:35:20, v/v/v) as mobile
phase. The developed HPLC method was validated with respect to linearity,
accuracy, precision, specificity, and robustness. The developed HPLC
method to determine the related substances and assay determination of
oxcarbazepine can be used to evaluate the quality of regular production sam-
ples. It can be also used to test the stability samples of oxcarbazepine [70].
Recent studies with carbamazepine on human serum albumin (HSA)
columns have noted an appreciable degree of nonspecific binding on
supports prepared by the Schiff base immobilization method. This work
examines an alternative immobilization method for HSA based on N-
hydroxysuccinimide (NHS)-activated silica. This support was prepared by
reacting HPLC-grade silica directly with disuccinimidyl carbonate. The
resulting material was compared to an HSA support prepared by the Schiff
base method in terms of its activity for carbamazepine and nonspecific inter-
actions with this drug. When examined by frontal analysis, both supports
gave comparable association equilibrium constants for carbamazepine inter-
actions with HSA ((0.530.55)  104 M1 at 37C). However, columns
prepared by the Schiff base method gave greater nonspecific binding. These
columns, as well as control columns prepared using the carbonyldiimidazole
immobilization method, were also evaluated for their nonspecific binding to
a variety of other solutes known to interact with HSA. From these results, it
was concluded that the NHS method was an attractive alternative to the
Schiff base technique in the preparation of immobilized HSA for HPLC-
Carbamazepine 173

based binding studies for carbamazepine. However, it was also noted that
nonspecific binding varies from one drug to the next in these immobilization
methods, indicating that such properties should be evaluated on a case-by-
case basis in the use and development of HSA columns for binding studies
[71].
An LC-MS/MS method for the simultaneous determination of carba-
mazepine and its main metabolite CBZ-E in rat plasma is described. The
method consists of a liquidliquid extraction procedure and electrospray
LC-MS/MS analysis. The chromatographic separation was achieved within
5 min using a C(8) (150 mm  2.1 mm) 5 m column with a mobile phase
composed of water/acetonitrile/acetic acid (69.5:30:0.5, v/v/v) at a flow
rate of 0.4 mL/min. d10-Carbamazepine is used as the internal standard
for all compounds. Analytes were determined by ESI tandem mass spec-
trometry in the positive ion mode using selected reaction monitoring. Car-
bamazepine was monitored by scanning m/z 237194, CBZ-E by m/z 253
210, and d10-carbamazepine by m/z 247204. The lower limit of quantita-
tion (LLOQ) is 5 ng/mL for each analyte, based on 0.1 -mL aliquots of
rat plasma. The extraction recovery of analytes from rat plasma was over
87%. Intra- and interday assay coefficients of variations were in the
range of 2.69.5% and 4.09.6%, respectively. Linearity is observed over
the range of 52000 ng/mL. This method was used for pharmacokinetic
studies of CBZ and CBZ-E in response to two different blood sampling
techniques (ie, manual sampling vs automated sampling) in the rat. Several
differences between the two sampling techniques suggest that the method of
blood collection needs to be considered in the evaluation of pharmacoki-
netic data [72].
A sensitive method for the determination of CBZ and CBZ-E in plasma is
described, using HPLC separation with tandem MS. Samples were purified
using liquidliquid extraction and separated on a Phenomenex Luna C18
5 m 150 mm  2 mm column with a mobile phase consisting of acetonitrile,
methanol, and formic acid (0.1%) (10:70:20, v/v/v). Detection was performed
by a Micromass Quattro Ultima mass spectrometer in the MRM mode
(LC-MS/MS) using ESI, monitoring the transition of the protonated molec-
ular ion for carbamazepine at m/z 237.05 and CBZ-E at m/z 253.09 to the
predominant ions of m/z 194.09 and 180.04, respectively. The mean recovery
was 95% for CBZ and 101% for CBZ-E, with an LLOQ of 0.722 ng/mL for
CBZ and 5.15 ng/mL for CBZ-E, when using 0.5 mL plasma. This high-
throughput method was used to quantify 230 samples per day and is sufficiently
sensitive to be employed in pharmacokinetic studies [73].
174 S.T. Alrashood

A simple, rapid, sensitive, and reproducible HPLC method for simulta-

neous determination of the AEDs (ethosuximide, primidone, lamotrigine,
phenobarbital, phenytoin, and carbamazepine) and two metabolites (carba-
mazepine-diol and carbamazepine epoxide) in human plasma is described.
The procedure involves extraction of the drugs from human plasma
(100 L) with ether using 9-hydroxymethyl-10-carbamyl acridan as an
internal standard. The extract was evaporated and reconstituted in mobile
phase and then injected onto the chromatograph. The drugs and the internal
standard were eluted from a Supelcosil LC-18 stainless-steel column at
ambient temperature with a mobile phase consisting of a 0.01 M phosphate
buffermethanolacetonitrile (65:18:17, v/v/v) adjusted to a pH of 7.5 with
phosphoric acid and a flow rate of 1 mL/min. The effluent was monitored at
220 nm. Quantitation was achieved by using peak area ratio of each drug to
the internal standard. The intra- and interassay coefficients of variation (CV)
ranged from 2.43% to 6.25% and from 3.02% to 5.85%, respectively. The
absolute (extraction) and relative (analytical) recoveries for the drugs ranged
from 70.7% to 104.4% and from 88.3% to 106.1%, respectively. Stability
tests showed that the drugs were stable in plasma for at least 4 weeks when
stored at 20C. The method was applied clinically for monitoring the
AEDs in epileptic patients [74].
Drug toxicological screening is commonly used as a diagnostic tool in
patients with suspected toxic ingestion. False-positive results due to cross-
reactive compounds in drug assays may lead to misdiagnosis and mis-
management, especially when child abuse is suspected. Two patients with
history of ingestion of carbamazepine were tested positive on screening with
the tricyclic antidepressant immunoassay. The immunoassays known cross-
reactivity for carbamazepine is reportedly as low as 0.3%. Plasma samples of
our patients were initially considered positive for tricyclic antidepressants
because the cross-reaction of carbamazepine gave tricyclic antidepressant
concentrations as imipramine equivalent sufficiently above the assay cutoff
point (20 ng/mL). Later, confirmatory urine testing of both patients using
HPLC was negative for tricyclic antidepressants. It was concluded that this
interference has significant clinical implications and can be avoided on urine
testing using a specific chromatographic assay such as HPLC [75].
In this article, the authors discuss 9-hydroxymethyl-10-carbamoyl
acridan (9-OH-CBZ), another metabolite of CBZ found in serum. The
retention time of unconjugated 9-OH-CBZ should be known when using
a chromatographic method, because it appears in concentrations varying
from one-eighth to one-third of the CBZ-E concentration and may
Carbamazepine 175

therefore cause analytical interactions. Liquid chromatography/electrospray

mass spectrometry (LC/ES-MS) in a serum extract identified and con-
firmed 9-OH-CBZ. The amount of 9-OH-CBZ present as conjugate in
serum was between 42% and 65%. The correlation factor values (r) between
serum concentrations of 9-OH-CBZ and 10,11-dihydro-10,11-trans-
dihydroxy-CBZ (trans-CBZ-diol), CBZ-E, and CBZ in 100 serum samples
were 0.77, 0.80, and 0.53, respectively. The origin of 9-OH-CBZ is dis-
cussed [76].
Carbamazepine and diphenhydramine interfered with the assays of hal-
operidol and its metabolite, reduced haloperidol, by reversed-phase HPLC.
Retention times of haloperidol, reduced haloperidol, and the interfering
drugs were very sensitive to the percentage of potassium phosphate buffer
in the mobile phase as well as to the final pH of the eluant. Retention times
were not very dependent upon ionic strength of the eluting solvent mixture.
Haloperidol and reduced haloperidol in the range of 0.510 ng/mL were
analyzed in the presence of 0.2 g/mL diphenhydramine and 5 g/mL of
carbamazepine. The concentrations of all drugs used were in their expected
therapeutic ranges. The isocratic chromatographic conditions were as fol-
lows: 25 cm  4.6 mm C18 column, mobile phase, 75% phosphate buffer
(final concentration, 0.06 M), and 25% acetonitrile; final pH 3.5; flow rate,
2.5 mL/min; and detection by UV absorption at 220 nm. Additional
changes in the percent buffer in the mobile phase may be useful in achieving
separation of other interfering compounds [77].
A fully automated HPLC procedure for the simultaneous determination
of carbamazepine and its main metabolites, epoxy carbamazepine, and
dihydroxycarbamazepine in plasma is described. Liquidsolid extraction
on disposable C18 columns and reversed-phase chromatography on a 3-
m particle size C18 column were combined and automated by using the
Automatic Sample Preparation with Extraction Columns system. UV detec-
tion was performed at 210 nm. 5,6-Dihydro-11-oxo-11H-dibenz[b,e]
azepine-5-carboxamide was used as internal standard. A small plasma vol-
ume (100 L) was required. The total run time for the assay of one sample
was about 10 min. The assay demonstrated good reproducibility. The limit
of quantitation was 0.1 mol/L (about 25 ng/mL) [78].
Felbamate is an investigational AED in clinical trials. An HPLC method
for the simultaneous analysis of felbamate, PHT, 5-(p-hydroxyphenyl)-5-
phenylhydantoin, CBZ, CBZ-E, and carbamazepine-10,11-diol in serum
was developed by a mobile phase optimization technique. Capacity factors
for the compounds of interest and 12 other AEDs and metabolites were
176 S.T. Alrashood

determined with mixtures of methanol, acetonitrile, or tetrahydrofuran and

a 0.01 M ammonium phosphate buffer, pH 6.5, on a reversed-phase C8 col-
umn. An optimized mobile phase composition was determined that could
separate the compounds of interest and three internal standards in less than
15 min. Serum was extracted with CH2Cl2/ethyl acetate (2:1) after addition
of three internal standards. The method was validated for within-day and
between-day precision and accuracy for the six compounds. Coefficients
of variation were generally less than 10% at all concentrations and less than
5% in the typical therapeutic range for each compound. The lower LOD
was estimated at 0.2 g/mL for CBZ and its metabolites and 0.5 g/mL
for felbamate and PHT. For felbamate, the lowest point on the standard
curve was 1.88 g/mL with a between-day variability of 10.3%. The assay
was used to determine the serum concentrations of PHT and CBZ and its
metabolites in a subject before, during, and after felbamate therapy [79].
Simultaneous monitoring of carbamazepine and its metabolities is helpful
in providing information on the induction process via the epoxide-diol
route. The present method involves the LC analysis of 1.0 mL of plasma
for carbamazepine at concentrations of 0.0254.0 mg/L and for both the
10,11-epoxide and 10,11-trans-diol metabolites at concentrations of 0.01
1.0 mg/L. Weighted regression equations, expressing peak area ratios as a
function of concentrations of carbamazepine and its epoxide and trans-diol
metabolites in the standards, were used to determine concentrations in
plasma samples [80].
A simple and rapid LC method is described for the qualitative and quan-
titative determination of carbamazepine in tablet composites and individual
tablets, using the internal standard technique. Analyses were performed on a
C18 reversed-phase column with tetrahydrofuranmethanolwater
(8:37:55) as the mobile phase. A linear relationship was obtained between
detector responses at 254 nm and amounts of carbamazepine injected rang-
ing from 0.2 to 1.7 g. The coefficient of variation for 10 consecutive injec-
tions of a standard preparation was 0.4%. Recoveries of carbamazepine from
100 and 200 mg tablets averaged 101.4% and 99.7%, respectively. Assay
results for commercial tablets analyzed by the proposed method agreed
favorably with those obtained by the method of USP XXI. The assay results
for individual tablets indicated that deviations from the average value and the
range of individual values are much wider with the compendial method than
with the proposed method [81].
LC methods have been developed for the determination of carbamaze-
pine, the impurity 10,11-dihydrocarbamazepine, and related compounds in
Carbamazepine 177

carbamazepine drug substance and tablets. The LC methods specify a 5-m

diol column and a mobile phase of acetonitrilemethanol0.05% aqueous
acetic acid (5:5:90). Iminodibenzyl and iminostilbene, starting materials
for some routes of synthesis, elute late in the LC system; therefore,
a thin-layer chromatographic method for their detection at the 0.05%
level has been developed. A total of 8 tablets and 13 raw material
samples from several sources were examined. The impurities most
frequently found were 10,11-dihydrocarbamazepine and a compound
identified as 10-bromocarbamazepine at levels up to 1.3% and 0.5%, res-
pectively; minimum detectable amounts were about 0.01% and 0.03%,
respectively [82].
An optimized automated LC method for simultaneous measurement of
primidone, phenobarbitone, phenytoin, carbamazepine, and clonazepam
was discussed. A Waters Tri-Module automation system is used and it pro-
vides direct read-out of results after chromatography. A one-step extraction
with ethyl acetate is used to extract the drugs from 100 L serum samples. An
isocratic mobile phase and monitor the column effluent at 210 nm was used.
Drug levels as low as 5 mol/L can be detected. The within-run CVs range
from 1.4% to 2.7%, and the between-run CVs range from 5.2% to 6.1%.
Analytical recovery is in the range from 94% to 108%. The method com-
pares favorably with the enzyme multiple immunoassay technique for rou-
tine AED monitoring, in accuracy, efficiency, and cost effectiveness [83].
The use of a column-switching system for direct injection of samples and
of a sample cleanup on reversed-phase precolumns is described. The
precolumns were filled with spherical C18 silica gel of particle size
30 m. Two applications are reported on (1) the direct injection of serum
samples for the simultaneous analysis of nine AEDs and metabolites and
(2) the determination of phenytoin and carbamazepine in serum ultra-
filtrates. The purge liquid for the sample cleanup was diluted phosphoric
acid, and the eluent mixture for the chromatographic separation was
water/acetonitrile. The analytical column (length 12.5 cm) was filled with
C18 silica gel of particle size 5 m. A gradient elution was chosen for the first
application, while the second application was carried out using isocratic
chromatographic [84].
The chromatographic behavior of nonionic micelles used as the mobile
phase in LC is similar to that of anionic and cationic micellar mobile phases,
with reversal of retention order as the micelle concentration is varied. This
behavior follows the prediction of the model developed for anionic surfac-
tants. Nonionic micelle chromatographic interactions are simplified by
178 S.T. Alrashood

reducing the extent of electrostatic interactions with the solute and, as a

result, their retention dependencies more closely follow the mathematical
predictions. Drugs in blood serum samples are quantitatively determined
by direct injection of the serum onto the chromatographic column, with
no column clogging or pressure buildup. The results are similar to those
found with anionic micellar mobile phases, but are in contrast to the protein
precipitation observed with cationic micelles. Solutemicelle equilibrium
constants and the critical micelle concentration of Brij-35, determined
chromatographically, are reported. The potential usefulness of nonionic
micelles for the determination of theophylline, paracetamol, phenobarbital,
carbamazepine, quinine, quinidine, morphine, codeine, and cocaine is dem-
onstrated [85].
A simple, isocratic LC method was developed for simultaneously mea-
suring ethosuximide, primidone, phenobarbital, phenytoin, carbamazepine,
and their bioactive metabolites within 10 min. The chromatographic system
involves a Waters Radial-NOVA PAK C18 reversed-phase column and
acetonemethanolacetonitrile10 mmol/L phosphate buffer (10:21:8:61
by vol., pH adjusted to 7.95 with NaOH) as mobile phase. The AEDs
are extracted from 50 L of serum by mixing with 50 L of acetonitrile con-
taining 10 mg of tolybarb per liter as internal standard. After centrifugation,
20 L of the supernatant is injected onto the column and eluted with mobile
phase at the rate of 2.8 mL/min at ambient temperature. The column efflu-
ent is monitored at 200 nm. The method can detect the five AEDs in
concentrations as low as 0.5 mg/L. Analytical recovery ranges from 98%
to 102%. Within-run CV ranged from 2.9% to 5.8% and between-run
CV from 4.7% to 7.1%. The method can also be used to measure N-
desmethyl-methsuximide, chloramphenicol, and pentobarbital [86].
Analysis of variance both factorial and nested was used to validate an
HPLC method intended for routine clinical assay of ethosuximide, pheno-
barbital, phenytoin, and carbamazepine. Drugs were salted out, together
with the solvent, from 0.5 mL acetonitrile-deproteinized plasma samples
with 8090% recovery. The acetonitrile extraction solution contained a
known amount of all four drugs. This added amount of any drug was used
when absent from plasma as an internal standard for those present and when
present as a calibrator. Results showed that assay precision was acceptable
(CV 6%) over and above the therapeutic range when additions did not
exceed the lower therapeutic plasma level and if as many replications were
made as there were drugs to assay. In return for some loss of sensitivity,
reciprocal internal standardization provides increased assay reliability owing
Carbamazepine 179

to the usual availability of more than one internal standard and to easier iden-
tification of interfering chromatographic peaks [87].
A sensitive, specific, and very fast LC assay for simultaneously determin-
ing five anticonvulsants (ethosuximide, primidone, phenobarbital, phenyt-
oin, and carbamazepine) by using commercially available 5- or 3-m particle
size reversed-phase columns and a microflow-cell-equipped UV detector
was described. The anticonvulsant drugs are extracted from 200 L of serum
containing 50 mg of cyclopal per liter as an internal standard, by elution from
a Bond-Elut (Analytichem International, Harbor City, CA) column with
300 L of methanol. A 5-L aliquot of the eluate is applied to an analytical
column and eluted with a mobile phase of acetonitrilemethanolphosphate
buffer, 20 mmol/L, pH 3.7 (13.5:35:51.5 by vol.), at a flow rate of 3.0 mL/
min and at 50C. Detection is at 210 or 195 nm. The chromatography is
complete in less than 2.5 min with the 5-m-particle column, and in less
than 1.4 min with the 3-m-particle column. The sensitivity of the method
for all drugs is less than 1 mg/L. Analytical recovery of drugs added to serum
ranged from 92% to 109% for concentrations up to 200 mg/L. Between-run
precision (CV) ranged from 1.3% to 4.1% [88].
An LC method for the simultaneous quantification of carbamazepine and
its 10,11-epoxide metabolite in plasma was described. The method is used
routinely in the analysis of carbamazepine and its epoxide in 0.5 mL of
plasma at concentrations of 120 and 0.25 mg/L, respectively. The use
of peakheight ratios as a measure of detector response appeared to provide
better precision and accuracy than peak area ratios [89].
The object of an LC analysis is to separate, identify, and quantitate the
constituents of interest in a sample mixture within an acceptable analysis
time. This will be achieved by a systematic analysis development rather than
by a trial-and-error approach. Such a systematic procedure requires a knowl-
edge of the chromatographic parameters governing resolution and analysis
time and their relative influences on resolution and its experimental impli-
cation. Furthermore, basic information about the mechanisms of the modes
of LC (adsorption, partition, ion exchange, and steric exclusion) and about
the types of sample which can be preferentially analyzed by them is neces-
sary. This information leads to a rational selection of the separation system
that promises the best chance of success. In the subsequent experimental
work, the analyst systematically measures and calculates resolution and
analysis time as functions of selectivity, capacity, and efficiency of the phase
system under selected chromatographic conditions. The essential chroma-
tograms, tables, and graphs resulting from this systematic method of
180 S.T. Alrashood

development are documented so that it is possible to replicate the analysis

procedure in the laboratories involved. The result is a set of chromato-
graphic conditions capable of achieving an optimum compromise between
resolution and analysis time. The procedure is applied to routine TDM of
AEDs in patient serum [90].
Specific AED concentrations in serum are believed to cause therapeutic
effects in individual patients. Communication between the clinician and the
laboratory performing the drug-level determinations is therefore essential
for safe and effective medication. The now common practice of multiple
drug therapy preferably requires simultaneous separation and quantitation
of the various drugs and their metabolites. The advantages and disadvantages
of current AED analysis methods are discussed. LC was found to be the most
versatile technique for routine and research analysis and can be applied to the
determination of common, less common, and new AEDs and metabolites.
Carbamazepine and its metabolite, CBZ-E, were determined with high
accuracy. The sample preparation procedures, the internal standards, and
mode of LC separation used for the analysis are given. The chromatographic
parameters for optimized resolution and analysis are given. The chromato-
graphic parameters for optimized resolution and analysis time of eight AEDs
and metabolites were devised with special consideration for the influence of
the oven temperature on resolution. About 300 patient serum samples were
analyzed by automatic unattended operation. By this method, some 50 sam-
ples per day can be extracted and analyzed. Quantitative results achieved by
LC and gas liquid chromatography (GLC) on the same patient samples are
reported and chromatograms discussed. Peak scanning was used to demon-
strate the presence of compounds which could eventually interfere with the
detection of phenylethylmalonamide. The overall accuracy of the employed
LC method, the repeatability of retention times on three different columns,
and the measured ranges of AED concentrations are reported [91].
A method for the simultaneous LC determination of ethosuximide, eth-
ylphenacemide, primidone, phenobarbital, carbamazepine, and phenytoin
in serum. The drugs, together with an internal standard, are extracted into
ethyl acetate at pH 7.0. The extract is analyzed isocratically at ambient tem-
perature on a reversed-phase column of SAS Hypersil with a mobile phase
of acetonitrile/tetrabutyl ammonium phosphate solution (2/8 by volume).
The eluted drugs are detected by their absorption at 200 nm, and quantitated
from their peak heights as compared with those of extracted standards. The
day-to-day CV of the method varied between 5.1% and 9.6% for concen-
trations ranging from less than therapeutic to toxic. The results, when
Carbamazepine 181

compared with those by gas chromatography, gave correlation coefficients

of 0.936 for phenytoin, 0.977 for phenobarbital, and 0.939 for primidone.
No drug interference was found except that amobarbital and eth-
ylphenacemide co-chromatographed [92].
A rapid, sensitive, and selective method for the determination of carba-
mazepine and its major metabolite in plasma has been developed. Other
commonly used anticonvulsants can be determined in the same procedure
without interference. After extraction with dichloromethane, the compo-
nents are separated by high-pressure liquid chromatography without further
cleanup or concentration on a column packed with small-particle silica gel.
The mean recovery from plasma is 98.6% with an RSD of 1.6%. The detec-
tion limit for carbamazepine is approximately 2 ng/mL, requiring 1 mL of
plasma [93].

4.2.5.2 Liquid Chromatography

LC methods have been developed for the determination of carbamazepine,
the impurity 10,11-dihydrocarbamazepine, and related compounds in car-
bamazepine drug substance and tablets. The LC methods specify a 5-m diol
column and a mobile phase of acetonitrilemethanol0.05% aqueous
acetic acid (5:5:90). Iminodibenzyl and iminostilbene, starting materials
for some routes of synthesis, elute late in the LC system; therefore, a
thin-layer chromatographic method for their detection at the 0.05%
level has been developed. A total of 8 tablets and 13 raw material
samples from several sources were examined. The impurities most fre-
quently found were 10,11-dihydrocarbamazepine and a compound
identified as 10-bromocarbamazepine at levels up to 1.3% and 0.5%, respec-
tively; minimum detectable amounts were about 0.01% and 0.03%, respec-
tively [82].
Specific AED concentrations in serum are believed to cause therapeutic
effects in individual patients. Communication between the clinician and the
laboratory performing the drug-level determinations is therefore essential
for safe and effective medication. The now common practice of multiple
drug therapy preferably requires simultaneous separation and quantitation
of the various drugs and their metabolites. The advantages and disadvantages
of current AED analysis methods are discussed. LC was found to be the most
versatile technique for routine and research analysis and can be applied to the
determination of common, less common, and new AEDs and metabolites.
Carbamazepine and its metabolite, CBZ-E, were determined with high
accuracy. The sample preparation procedures, the internal standards, and
182 S.T. Alrashood

mode of LC separation used for the analysis are given. The chromatographic
parameters for optimized resolution and analysis are given. The chromato-
graphic parameters for optimized resolution and analysis time of eight AEDs
and metabolites were devised with special consideration for the influence of
the oven temperature on resolution. About 300 patient serum samples were
analyzed by automatic unattended operation. By this method, some 50 sam-
ples per day can be extracted and analyzed. Quantitative results achieved by
LC and GLC on the same patient samples are reported and chromatograms
discussed. Peak scanning was used to demonstrate the presence of com-
pounds which could eventually interfere with the detection of
phenylethylmalonamide. The overall accuracy of the employed LC method,
the repeatability of retention times on three different columns, and the mea-
sured ranges of AED concentrations are reported [91].

4.2.5.3 High-Performance Liquid Chromatography

An increasing number of publications on the dried blood spot (DBS) sam-
pling approach for the quantification of drugs and metabolites have been
spurred on by the inherent advantages of this sampling technique. In the pre-
sent research, a selective and sensitive HPLC method for the concurrent
determination of multiple AEDs (levetiracetam (LVT), LTG, phenobarbital
(PHB), CBZ, and its active metabolite CBZ-E) in a single DBS has been
developed and validated. Whole blood was spotted onto Guthrie cards
and dried. Using a standard punch (6 mm diameter), a circular disc was pun-
ched from the card and extracted with methanol:acetonitrile (3:1, v/v) con-
taining hexobarbital (internal standard) and sonicated prior to evaporation.
The extract was then dissolved in water and vortex mixed before undergo-
ing SPE using HLB cartridges. Chromatographic separation of the AEDs
was achieved using Waters XBridge C18 column with a gradient system.
The developed method was linear over the concentration ranges studied
with r  0.995 for all compounds. The lower limits of quantification
(LLOQs) were 2, 1, 2, 0.5, and 1 g/mL for LVT, LTG, PHB, CBZ-E,
and CBZ, respectively. Accuracy (%RE) and precision (%CV) values for
within and between day were <20% at the LLOQs and <15% at all other
concentrations tested. This method was successfully applied to the analysis of
the AEDs in DBS samples taken from children with epilepsy for the assess-
ment of their adherence to prescribed treatments [94].
In this paper, the online coupling of SPE, based on a restricted-access
support with high-performance reversed-phase chromatography for the
analysis of CBZ and CBZ-E in human plasma samples, is described. A
Carbamazepine 183

precolumn packed with 25 m C(18) alkyl-diol support is used for direct

plasma injection. Using column-switching techniques, the analytes were
enriched on the precolumn by a 5 mM phosphate buffer (pH 7) with 2%
of methanol solution at a flow rate of 0.8 mL/min, while proteins and
endogenous hydrophilic substances in plasma were washed off to waste.
The enriched analytes were then back-flushed onto the analytical C(18) col-
umn, separated by a mixture of 10 mM phosphate buffer (pH 7):acetonitrile
(70:30, v/v) solution at a flow rate of 1.0 mL/min and detected by the UV
absorbance set at 212 and 285 nm and without transfer loss. Linear calibra-
tion graphs were obtained for sample injection volumes of 50 (0.24.0 g of
CBZ mL1 and 0.15.0 g of CBZ-E mL1) and 20 L (5.020.0 g of
CBZ mL1); in either case the r-value was >0.9963. Recoveries from
spiked plasma samples were quantitative for both analytes and the coeffi-
cients of variation were below 3.83%. The lowest sample concentrations that
can be quantified with acceptable accuracy and precision were 0.2 g CBZ
mL1 and 0.1 g CBZ-E mL1 when a sample volume of 50 L was
injected. Concentrations of 0.08 and 0.05 g/mL of CBZ and CBZ-E,
respectively, were considered the LOD for a signal-to-noise ratio of 3. Fur-
thermore, the developed column-switching method was successfully applied
to the determination of CBZ and CBZ-E in plasma samples of patients sub-
mitted to CBZ therapy [95].
A rapid, simple, and robust method is presented for the simultaneous
determination of seven AEDs, including primidone, phenobarbital, phenyt-
oin, carbamazepine with its two major metabolites CBZ-E and carbamaz-
epine-10,11-(trans)-dihydrodiol, and the new AEDs lamotrigine,
hydroxycarbazepine (active metabolite of oxcarbazepine), and zonisamide
in serum by HPLC-DAD. After SPE, separation is achieved on an Alltima
3C18 analytical column using isocratic elution with a mixture of acetoni-
trile, methanol, and phosphate buffer at 45C. The method is exhaustively
validated, including experimental design in combination with statistical
evaluation (ANOVA) to study the robustness of chromatography and sam-
ple preparation. Commonly coadministered AEDs do not interfere with the
method. Intraday precision (RSD < 1.9%), linearity, lower limit of quanti-
tation (LOQ < 0.065 mg/L), and robustness make the method suitable for
daily TDM and pharmacokinetic studies [96].
Monitoring of plasma AEDs is useful for better clinical management in
epileptic patients, particularly in children. CBZ is one of the commonly
prescribed anticonvulsants. The active metabolite of carbamazepine
carbamazepine-10,11-epoxide (CBZ-Epo) also exhibits anticonvulsant
184 S.T. Alrashood

effect. The pineal hormone, melatonin, exerts an anticonvulsant effect in

experimental seizure models and recently has also been used in cases of child-
hood epilepsy. To facilitate the simultaneous plasma estimation of carbamaz-
epine, carbamazepine epoxide, and melatonin, a new HPLC method was
developed. Waters millennium 2010 chromatography manager with a
515 HPLC pump and Waters 24879 dual absorbance UV detector was used.
A 25-L of sample and standards was injected, and chromatographic sepa-
ration was achieved by Merck C18 reversed-phase column particle size
5 m, 250 mm  4 mm. It was quantitated at UV light 210 nm. The reten-
tion times of melatonin, CBZ-Epo, and CBZ were 6.3, 7.5, and 13.9 min,
respectively. The mobile phase used was water:acetonitrile (70:30), pH 3.0
adjusted with orthophosphoric acid at a flow rate of 1 mL/min. The LODs
of melatonin, carbamazepine epoxide, and carbamazepine were 800, 500,
and 1300 pg, respectively [97].
An HPLC method for the simultaneous determination of phenytoin
(CAS 57-41-0), phenobarbital (CAS 50-06-6, phenobarbitone), and carba-
mazepine (CAS 298-46-4) is described. The serum was extracted with ethyl
acetate, the dried extract was reconstituted in mobile phase, and the aliquot
was injected. The eluent drugs were detected at 230 nm. The mobile phase
consisting of methanolwaterglacial acetic acid mixture (67:33:1, v/v/v)
was used at a flow rate of 1 mL/min on C18 column. The absolute recovery
was above 96% of all the three drugs over a concentration range of 0.5
50.0 g/mL. The inter- and intraday precision RSD ranged between
0.795.13% and 0.116.81%, respectively. The method is simple, rapid,
accurate, and sensitive and is presently used for TDM in epileptic patients.
The results obtained with this method showed very good clinical correlation
[98].
To compare restricted-access media high-performance liquid chromato-
graphic (RAM-HPLC) method with FPIA for analysis of CBZ in human
blood, a RAM-HPLC method was established for the determination of
CBZ in plasma. The two methods do not need sample cleanup prior to anal-
ysis and they have almost 100% recovery and good reproducibility. There is
a good correlation between the CBZ concentration in venous plasma sam-
ples determined by FPIA and that in both venous and fingertip plasma sam-
ples obtained by RAM-HPLC, the correlation coefficients being 0.989 and
0.995, respectively. It is shown by t-test that the data sets of venous and fin-
gertip plasma samples given by RAM-HPLC are consistent with each other
but significantly different from the results obtained by FPIA. It was con-
cluded that both direct injection RAM-HPLC and FPIA may be applied
Carbamazepine 185

in determining CBZ in TDM. FPIA is well suited to the routine TDM.

RAM-HPLC is more useful in TDM-related research and especial
cases [99].
An HPLC method has been developed for the simultaneous determina-
tion of oxcarbazepine, 10-hydroxycarbamazepine, epoxy carbamazepine,
carbamazepine, phenobarbital, and phenytoin. After protein precipitation
by acetonitrile, the supernatant was analyzed on a C18 reversed-phase
HPLC column. AEDs and oxazepam (internal standard) were detected by
UV absorbance at 240 nm. Linearity was established for the whole con-
centration range for each compound. Quantitation limits of oxcarbazepine,
10-hydroxycarbamazepine, epoxy carbamazepine, carbamazepine, pheno-
barbital, and phenytoin were 0.58, 3.5, 2.35, 0.66, 1.02, and 3.13 g/mL,
respectively, and mean recoveries added to serum were 105.15%, 84.76%,
94.45%, 96.52%, 98.62%, and 95.08%, respectively. This method has
been used for the simultaneous determination of steady-state serum con-
centration of AEDs in patients treated by one or more anticonvulsive
treatment [100].
An HPLC method with UV detection for the simultaneous analysis
of the AED carbamazepine and five of its metabolites in human plasma
has been developed. The analysis was carried out on a reversed-phase
column (C8, 150 mm  4.6 mm I.D., 5 m) using acetonitrile, methanol,
and a pH 1.9 phosphate buffer as the mobile phase. Under these chro-
matographic conditions, carbamazepine and its metabolites 10,11-dihydro-
10,11-epoxy carbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine,
2-hydroxycarbamazepine, 3-hydroxycarbamazepine, and 10,11-dihydro-10-
hydroxycarbamazepine are baseline separated in less than 18 min. The extrac-
tion of the analytes from plasma samples was performed by means of an original
SPE procedure using Oasis HLB cartridges. The method requires only 250 L
of plasma for one complete analysis. The repeatability (RSD% <2.4), interme-
diate precision (RSD% < 3.5), and extraction yield (84.8103.0%) were very
good for all analytes. The method is suitable for reliable TDM of patients
undergoing chronic treatment with carbamazepine and for kineticmetabolic
studies of this drug [101].
An HPLC method was developed for the measurement of CBZ and
(CBZ-E in human breast milk and plasma. The method involves rapid
C18 SPE of CBZ and CBZ-E. Chromatographic separation was achieved
with a reversed-phase C8 column using a mobile phase of potassium
dihydrogen phosphate (pH 2.5) and acetonitrile (67:33, v/v), with UV
detection at 254 nm. 2-Methyl CBZ was used as the internal standard.
186 S.T. Alrashood

Determination of both CBZ and CBZ-E was possible in the range of 0.01
6.0 and 0.026.0 mg/L in milk and plasma, respectively. The recoveries of
CBZ and CBZ-E added to the milk and plasma were 90.698.0% and 88.9
104.0%, respectively, with coefficients of variation less than 8.3% and 10.5%,
respectively. The method has been used for drug-level monitoring in milk
and plasma samples obtained from CBZ-treated patients. The mean (SD)
levels for CBZ in milk and plasma samples were 3.50 (0.4) and 6.18 (2.9)
mg/L, and for CBZ-E were 1.28 (0.3) and 1.85 (1.0) mg/L, respectively.
The mean (SD) milk/plasma ratios of CBZ and CBZ-E were 0.64 (0.2)
and 0.79 (0.3), respectively. The milk/plasma ratio of CBZ-E was slightly
higher than that of CBZ [102].
An enantioselective HPLC method for the simultaneous determination
of the concentration of the enantiomers of the oxcarbazepine meta-
bolites 10-hydroxycarbazepine (MHD) and carbamazepine-10,11-trans-
dihydrodiol (DHD) in human urine is described. The method is based on
extraction with tert-butylmethyl etherdichloromethane (2:1, v/v) under
alkaline conditions, separation and evaporation of the organic phase, and
dissolution of the residue in the mobile phase. Enantiomers are resolved
on a Diacel Chiralcel OD column (250 mm  4.6 mm I.D.) under isocratic
conditions using as mobile phase n-hexaneethanol2-propanol (18:2:1,
v/v/v) with addition of glacial acetic acid (0.1%). The enantiomers are
detected by UV at 215 nm. The method allows reliable determination of
the MHD and DHD enantiomers in human urine with LOQs of 0.2 and
0.4 mg/L, respectively [103].
CBZ is widely used in the treatment of epilepsy, frequently in combina-
tion with other anticonvulsants. Its metabolite, CBZ-E, is pharmacologi-
cally active and is increased with concurrent use of valproate and other
anticonvulsants. This pharmacokinetic interaction may be particularly
important because CBZ, its epoxide, phenytoin, and lamotrigine all act
on fast voltage-dependent sodium channels. Over a 2-month period, rou-
tine serum requests for CBZ (n 47) (excluding known cases of overdose)
were also analyzed for CBZ epoxide, phenytoin, and lamotrigine using a
simultaneous HPLC method. Valproate was measured using FPIA. With
concurrent phenytoin and lamotrigine administration, there was a relative
increase in CBZ epoxide and a significant decrease in the ratio of CBZ
to epoxide (from more than 5 to 3). If valproate was also present, the con-
centration of parent and metabolite increased significantly, causing potential
toxicity. Two patients in this latter group had significant clinical toxicity,
with parent CBZ concentrations in the reference range; a third patient
Carbamazepine 187

suffered from poor control of seizures. This study illustrates the impor-
tance of awareness of the contribution of active metabolites in TDM and
raises questions about the role of the routine monitoring of such metabolites
[104].
Extrashot-ODS (EXS-ODS) is a syringe-type minicolumn developed
for sample injection into reversed-phase HPLC columns. EXS-ODS con-
sists of (a) a stainless-steel needle fitted to an ordinary syringe-loading sample
injector for HPLC, (b) a 45-L minicolumn tube made of poly-
tetrafluoroethylene (PTFE) and packed with ODS-silica, and (c) a
minicolumn holder made of polystyrene, which is connected to the needle
on one side and the other side is shaped so as to be fitted with a solvent
syringe. Using the device, three antiepileptics in 20 L of human sera were
simultaneously analyzed. First, a 20-L serum specimen diluted with 100 L
of buffer solution into the device was introduced and, second, 100 L of dis-
tilled water. Then the device was attached to the HPLC injector and 130 L
of methanol was introduced into the HPLC column through the device.
Then, reversed-phase HPLC was conducted in the usual manner, with
the chromatogram reading at a wavelength of 210 nm for the assays of
5,5-diphenylhydantoin, phenobarbital, and carbamazepine. The results
obtained by direct peakheight calibration were comparable to those given
by the immunological method [105].
Lamotrigine (LG), phenytoin (PY), carbamazepine (CM), and carba-
mazepine epoxide (CE) are measured with an optimized procedure that uses
thin sorbent extraction disks and a highly selective, sterically protected
bonded silica HPLC column. Routinely, serum (200 L at pH 6.8 with
cyheptamide as internal standard) is applied to an Empore octyl (C8) SPE
disk to isolate the drugs. A water wash removes interferences, and the
retained drugs are eluted with a small volume of solvent. The eluate is
directly injected onto a Zorbax Stable Bond cyanopropyl HPLC column
with quantification at 214 nm. Evaporationconcentration steps are unnec-
essary. Overall, for all drugs, between-run precision CV (n 16 each)
ranged from 2.1% to 4.9% at concentrations from 0.75 to 20.5 mg/L; extrac-
tion recoveries fell within a range of 96110% at concentrations of 2, 10, and
30 mg/L tested for each drug; the lowest LOD was 0.150.35 mg/L. The
analytical response was linear for each drug >80 mg/L (LG) and
>50 mg/L (PY, CM, and CE). Optimization graphs are presented to illus-
trate the rationale for selection of test parameters for a robust method. In
addition, a comparison study between two commercial laboratories demon-
strates accuracy problems associated with LG testing [106].
188 S.T. Alrashood

To study the use of hair analysis in monitoring drug compliance and

historical changes in pharmacokinetics, a method for the quantitative deter-
mination of the antiepileptic drug CBZ and trans-10,11-dihydro-10,11-
dihydroxycarbamazepine (CBZ-diol) in hair from carbamazepine users
was developed. Digestion by 1 M NaOH was found to be the best method
for isolating CBZ and CBZ-diol from hair, followed by SPE and reversed-
phase HPLC with UV detection. Recoveries from spiked hair samples were
7686%. Within-day precision (CV; n 10) for CBZ and CBZ-diol in hair
of a CBZ user containing 10.9 g/g CBZ and 3.2 g/g CBZ-diol was 1.7%
and 5.0%, respectively. Sectional hair analysis of a patient on a constant dos-
age of CBZ demonstrates an exponential decrease in hair concentrations of
CBZ and CBZ-diol with increasing distance from the root, probably caused
by shampooing. No CBZ-E could be detected. However, one component
in the chromatogram is probably CBZ-beta-hydroxythioether, an adduct of
CBZ-epox with cysteine, or acridinethioacetal, its rearrangement product.
The concentration of this component does not decrease with increasing dis-
tance from the root [107].
Theophylline, phenobarbital, amobarbital, and carbamazepine are the
chief therapeutic drugs in clinical practice. Because of the differences among
interindividuals in the metabolic clearance of these drugs and their toxicity at
certain levels of concentration in serum, the dosage should be regulated to
maintain a therapeutic blood drugs level. To achieve this a rapid and accurate
assay method for drugs in blood is necessary. This paper reports that a
reversed-phase HPLC method had been established for the simultaneous
separation and quantitative determination of the four drugs in serum. The
separation was performed on RP-C18 column using methanolwater
(1:1) as mobile phase and 4-aminoantipyrime as internal standard. The
eluted substances were detected at 210 nm [108].
An HPLC method has been developed for the simultaneous analysis of
CBZ and its two metabolites, CBZ-E and carbamazepine-10,11-
dihydroxide (CBZ-diOH), using a recently developed reversed-phase col-
umn with 2-m particles and a 2-L microflow cell equipped with a UV
detector. The separation was achieved using two different C18 reversed-
phase columns (column 1: 100 mm  4.6 mm I.D., particle size 2 m,
TSK gel Super-ODS; column 2: 100 mm  4.6 mm I.D., particle size
5 m, Hypersil ODS-C18) for comparison. The mobile phase was com-
posed of methanolwater (30:70, v/v), and the flow rate was 0.4 mL/min
for both columns. The absorbance of the eluent was monitored at
210 nm. Retention times with column 1 were shorter than with column 2.
Carbamazepine 189

When the three compounds were determined, the sensitivity and LOQ
were about 10 times better with column 1 than with column 2. The relative
recovery and linearity with column 1 were approximately the same as those
with column 2. These results show that the new ODS column packing with
a particle size of 2 m gives higher sensitivity and shorter analysis time than
the conventional ODS column packing [109].
An isocratic LC method employing one extraction step has been devel-
oped for the quantitation of five drugs and three metabolites in human
plasma. The method uses 0.100-mL aliquots of human plasma and two
internal standards. Chromatographic conditions include a
4.6 mm  150 mm Spherisorb ODS2, a 3-m HPLC column, a phosphate
bufferacetonitrilemethanol (700:160:140) mobile phase, and UV absor-
bance detection at 210 nm. Analytes and linear quantitation ranges (g/
mL) were felbamate (FBM), 0.391200; primidone (PRIM), 0.098100;
phenobarbital (PHENO), 0.195100; CBZ, 0.195100; and PHT,
0.195200. For CBZ-trans-diol (CBZ-TR), CBZ epoxide (CBZ-EP),
and the PHT metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin
(HPPH), the range was 0.04925.0 g/mL. Ethosuximide, methsuximide,
2-methyl-2-phenyl-succinimide (methsuximide metabolite), 2-ethyl-2-
phenyl malonamide (PRIM metabolite), 5-ethyl-5-(4-hydroxyphenyl)-
barbituric acid (PHENO metabolite), and mephenytoin do not interfere
with quantitation of the above compounds [110].
A reversed-phase HPLC method for the measurement of LTG simulta-
neously with phenobarbitone (PB), PHT, and CBZ is described using hexo-
barbital as the internal standard. The method uses the chromophore at
307 nm to detect LTG in the presence of interfering CBZ-E detected at
220 nm, the wavelength used to measure the other drugs. This method
requires <10 min/sample for completion. Simultaneous monitoring of
the chromatographs at 220 and 310 nm with a simple calculation allows
LTG to be measured virtually identically to a routine method for monitoring
of the other anticonvulsants. Between-batch precisions for LTG at 2 and
6 mg/L were <5%. Accuracy of LTG estimation was assessed by comparison
with known values of samples supplied by an external quality assessment
scheme [111].
An HPLC method for simultaneous determination of three antiepilep-
tics, phenytoin, phenobarbital, and carbamazepine, in serum for TDM is
described. The drugs were extracted and injected onto a silica-gel column
using a syringe-type minicolumn, Extrashot-Silica, packed with diatoma-
ceous earth granules. We used dichloromethane for extractioninjection
190 S.T. Alrashood

and n-hexane containing 0.2% acetic acid, 2% ethanol, and 15% dic-
hloromethane for the mobile phase of a silica-gel HPLC. The eluent was
monitored with a UV detector set at 240 nm. Linear relationships between
the amount of drug and peak height were confirmed at 120 g/mL in
serum for carbamazepine and 540 g/mL in serum for phenytoin and phe-
nobarbital. When a 5-L aliquot of serum was subjected to this method, the
observed detection limits of the drugs were far less than therapeutic concen-
trations. Thus, our method was simple and accurate enough to be used in
routine TDM and basic pharmacokinetic studies [112].
A precise and accurate HPLC method for the simultaneous assay of car-
bamazepine, phenytoin, phenobarbital, primidone, and their principal
metabolites was established. This method has been used for the analysis of
these drugs and the metabolites in serum, saliva, and urine samples. Aceto-
nitrile is used for the deproteinization of serum and saliva samples, while SPE
is utilized for urine sample pretreatment. Samples of 2 L are injected onto a
3-m ODS-Hypersil column (250 mm  2 mm I.D.) with a column tem-
perature of 40C. The drugs and metabolites are eluted with a mobile phase
containing potassium phosphate bufferacetonitrilemethanol (110:50:30,
v/v/v) at a flow rate of 0.2 mL/min. Signals are monitored by a photodi-
ode-array detector at a sample wavelength of 200 nm with a bandwidth
of 10 nm. These four commonly used AEDs and their six metabolites are
well separated from one another within 15 min. Within-day coefficients
of variation (CV) are within 5% in most cases and between-day CV are from
2.32% to 4.75%. The recovery rates range from 95.12% to 104.42%. This
method has the necessary sensitivity and linearity for routine therapeutic
monitoring of both total and free drug levels and may be employed for phar-
macokinetics studies of drug interactions and metabolism as well [113].
A method is described for the simultaneous measurement of serum levels
of three AEDs, phenobarbital, phenytoin, and carbamazepine, by direct
injection HPLC on a 25-cm Pinkerton internal surface reversed-phase col-
umn. Several commonly available compounds were tested and found not to
co-chromatograph with the three drugs of interest or the internal standard,
5-(p-methylphenyl)-5-phenylhydantoin. Results obtained on patients sam-
ples with this method compared well with those from enzyme-multiplied
immunoassay technique (EMIT) [114].
A rapid, sensitive, and accurate HPLC method for the simultaneous
quantitation of phenobarbitone, phenytoin, CBZ, and CBZ-E in saliva is
described. Only small volumes of saliva (100 L) are required. Separation
of the drugs is achieved by reversed-phase chromatography on a Nova-Pak
Carbamazepine 191

C18 column, with a mobile phase of acetonitrilephosphate buffer at a flow

rate of 2.0 mL/min. Detection is effected by ultraviolet absorption at
215 nm. The total run time is under 12.5 min per assay. A precipitation
but no extraction step is involved, simplifying the assay method. Salivary
concentrations in the range 0.2525 g/mL for carbamazepine, 0.5
20 g/mL for phenytoin and phenobarbitone, and 0.420 g/mL for
CBZ-E can be measured. Recovery varies from 94% to 108%. The method
has been used for routine measurements of anticonvulsants in saliva collected
daily from patients with intractable epilepsy [115].
Another rapid, sensitive, and simple to operate HPLC method for the
simultaneous determination of CBZ and its metabolite (10,11-epoxide car-
bamazepine, ECBZ) in serum has been developed. The sample was
extracted with ethyl acetate. The extract was evaporated to dryness and
taken up with the mobile phase. Separation of CBZ and ECBZ was achieved
by reversed-phase chromatography using a mobile phase consisting of
methanolwater (1:1) at a flow rate of 0.8 mL/min on a 5-m YWG C18
column. Eluent was monitored at 214 nm. The method has a good linearity.
The recoveries of CBZ and ECBZ were found to be 99.7  2.45% and
97.3 4.20%, respectively. Precision studies for both within day and day to
day at different concentrations provided CV values of less than 6%. Some
commonly used anticonvulsants can be determined in the same procedure
without interference. This method is well adapted to the therapeutic moni-
toring of CBZ-treated patients, as well as for pharmacokinetic studies [116].
An HPLC procedure is reported for measuring the plasma concentration
of five anticonvulsants: phenobarbital, phenytoin, carbamazepine, eth-
osuximide, valproic acid, and two bronchodilators: theophylline and caf-
feine. LODs of ethosuximide and valproic acid are 28 mol/L, and
5 mol/L for the other drugs. Linearity limits are always very higher than
the higher therapeutic dose concentration, the interassay RSD are less than
8% for the seven drugs at the three levels of concentration, and the analytical
recoveries ranged from 93% to 104%. The technique is simple and rapid:
four anticonvulsants are simultaneously extracted and dosed, and valproic
acid only has to be dosed lonely. Finally, compared to the other immuno-
assay technique, this method is cheaper [117].
A rapid HPLC method is described for the simultaneous determination
of carbamazepine and the 10,11-epoxide, 10,11-dihydroxy, and 2-hydroxy
metabolites of carbamazepine. The chromatographic system involves the use
of an 18C-microsorb, reversed-phase column with acetonitrile/water
(28:72) as the mobile phase. Detection and quantitation are monitored by
192 S.T. Alrashood

UV absorption at 212 nm. The compounds are extracted from 250 L of

plasma or from 100 L urine with methyl-t-butyl ether and 0.1 M sodium
hydroxide; 2-methylcarbamazepine is added as internal standard. If phenyt-
oin and/or phenobarbital are present in plasma or urine samples, it is nec-
essary to use 1.0 M sodium hydroxide. The limits of quantitation for
carbamazepine and its metabolites are 10 ng/mL [118].
Another rapid, sensitive, and simple to operate HPLC method for the
simultaneous determination of CBZ and carbamazepine 10,11-epoxide
(CBZ-EP) in plasma and saliva is described. The drug and its metabolite
are extracted from both plasma and saliva using commercially available
reversed-phase octadecylsilane bonded silica columns (Bond-Elut C18,
2.8 mL capacity). Separation of CBZ and CBZ-EP was achieved by
reversed-phase chromatography, using a mobile phase consisting of
acetonitrilemethanolwater (19:37:44) at a flow rate of 1.8 mL/min in
conjunction with a Nova-Pak C18 column. The analytical column, in
Radial-Pak cartridge form, was used in combination with a Z-module
RCSS and protected by a Guard-Pak precolumn module containing a
Guard-Pak mu Bondapak C18 insert. Using UV detection at 214 nm, levels
in the region of 50100 ng/mL for CBZ and CBZ-EP can be measured
with only 250 and 500 L of plasma and saliva, respectively. The method,
which has been used to determine steady-state concentrations of the drug
and its metabolite in pediatric patients receiving CBZ monotherapy, is also
suitable for pharmacokinetic studies [119].
An HPLC method is described for the simultaneous determination in
plasma of carbamazepine, ethosuximide, phenobarbitone, phenytoin, and
primidone. The procedure involved the preliminary extraction of the
drugs and the internal standard (hexobarbitone) into a mixture of organic
solvents at pH 2. The dried extract was dissolved in methanol and con-
centration of 25 L was injected into a liquid chromatograph linked to
a reversed-phase column. The drugs and internal standard were eluted
from the column by a mixture of methanol and water, as the mobile phase,
and detected with a UV spectrophotometer at 204 nm. This HPLC assay,
which required 0.5 mL of plasma, was used to determine antiepileptic
drugs levels in 136 mentally handicapped children suffering from epilepsy.
Comparison between different batches of assays showed that recovery of
the drugs from plasma varied from 60% to 98% and with a coefficient of
variance between 3.8% and 9.8%. Detection limit of the method ranged
from 2 g/mL for primidone to 1 g/mL for the remainder of the
antiepileptic drugs [120].
Carbamazepine 193

An HPLC method for the simultaneous determination of carbamaze-

pine, CGP 10000 (a common metabolite of carbamazepine and
oxcarbazepine), desmethylmephenytoin, 10,11-epoxy carbamazepine, eth-
osuximide, GP 47779 (the active metabolite from oxcarbazepine, a new
drug made by Ciba-Geigy), mephenytoin, phenylethylmalonamide,
primidone, pheneturide, phenobarbital, and phenytoin is described. The
serum is extracted with ethyl acetate at pH 3.9 and the dried extract is dis-
solved in 70% ethanol in water and an aliquot is injected into a Hewlett-
Packard 1084 B liquid chromatograph. A reversed-phase (RP-8) column
is used with acetonitrile and water as the mobile phase. The eluted drugs
are detected at 207 nm. The recovery of the compounds varies from 76%
to 95% with the day-to-day precision (CV) between 3.8% and 9.8%, the
within-day precision between 1.8% and 5.8%, and run-to-run precision
between 1.0% and 2.6% [121].
The authors describe a method of assaying antiepileptic and sedative
drugs by HPLC. This method allows for simultaneous separation and quan-
tification of these drugs (ethosuximide, primidone, phenobarbital,
beclamide, prominal, diphenylhydantoin, glutethimide, carbamazepine) in
a relatively short analysis time, by means of a system of radial compression
of the column. The serum is acidified and extracted by ethyl acetate. After
evaporation, the residue is taken up in the mobile phase. The various drugs
are eluted in a 10-m Rad-Pak C 18 column with a mobile phase consisting
of a mixture of methanol and water with a flow rate of 4 mL/min. The chro-
matograph is read at 200 nm and the separation is effective in about 16 min.
The yield is between 75% and 100% and the repeatability of the method is
good (CV less than 9%). The originality of the method resides in the assay of
drugs such as beclamide, methylphenobarbital, and glutethimide at the same
time as the major antiepileptics [122].
A common methodology is reported for the determination of five
major anticonvulsants (carbamazepine, ethosuximide, phenobarbital,
phenytoin, primidone) and their active metabolites (CBZ-E, phenyle-
thylmalonamide) in 30 L of plasma. After a single step of deproteini-
zation and extraction with acetonitrile, leading to an almost complete
recovery of all the analytes, 5 L is injected on a reversed-phase column
(Lichrosorb RP-18, 5 m). The anticonvulsants are eluted isocratically
at a column temperature of 50C with a mobile phase consisting of
acetonitrilephosphate buffer pH 6.9 (40:60 by vol.), and monitored at
208 nm. Quantitation, using peak height or peak area, is based on the
ratio of analyte to internal standard (allylisobutylbarbital) referenced to a
194 S.T. Alrashood

serum-based multiple drug standard. The composition and pH of the

mobile phase, temperature of the column, choice of wavelength of detec-
tion, and size of the column material are crucial for the optimal separation
of these five drugs and their two active metabolites in a chromatographic
time of only 12 min, without sacrificing high sensitivity and column life
[123].
A method for the simultaneous analysis of chloramphenicol and four
AEDs (phenobarbital, phenytoin, carbamazepine, and primidone) in plasma
by HPLC is described. The method involves a preliminary extraction of
0.1 mL of plasma with diethyl ether containing phenacetin as an internal
standard, chromatography with a reversed-phase column with a methanol
water mobile phase, and detection by measuring UV absorbance at 210 nm.
The method demonstrated sufficient precision, sensitivity, and specificity:
the recoveries of the drugs were greater than 95% with the exclusion of
primidone (80.3%); the maximum within-day and day-to-day coefficients
of variation for all drugs were less than 5%; the lower detection limits were
0.5 g/mL or less for all drugs analyzed; and six other antibiotics,
phenylethylmalondiamide, CBZ-E, and chloramphenicol esters did not
interfere with the analysis. The HPLC method was tested for clinical appli-
cability by analyzing plasma samples from a volunteer who received concur-
rent single doses of chloramphenicol, phenobarbital, and phenytoin. This
method can be used for studying drug interactions between chloramphen-
icol and AEDs and for monitoring the concentrations of these drugs in
plasma when administered concurrently, to prevent concentration-related
side effect(s) of each drug [124].
In the last 20 years, there has been considerable improvement in the
determination of anticonvulsive drugs in body fluids. GLC, HPLC, and
immunological methods (radio-, enzyme-, fluoro-, and nephelo-immuno-
assays) have progressively supplanted spectrophotometry and thin-layer
chromatography. As the number of publications shows, these methods have
included a great variety of procedures. At present, GLC and enzyme immu-
noassays are routinely performed, awaiting a wider spread of LC and other
immunological techniques. This paper is a comprehensive review of the
analytical literature on the determination of phenobarbitone, primidone,
phenytoin, carbamazepine, valproic acid, ethosuximide, clonazepam, and
some of their metabolites in physiological fluids. GLC methods are more
particularly reviewed. In order to facilitate the choice between these
procedures, a critical selection of the techniques is given and recom-
mendations are made. Emphasis is laid on technical problems encountered
Carbamazepine 195

with the assays, as well as the need for a rigorous analytical assessment and
internal and external quality controls. This review is completed by consid-
erations on the determination of the free drug fraction and on sample col-
lection and storage [125].
A new instrument for use in assay of therapeutic drugs in serum by
HPLC, the FAST-LC system (Technicon), was discussed. Serum samples
are aspirated directly into the unit, extracted with solvent, and the evapo-
rated and redissolved extract is injected onto a chromatographic column.
The performance of the system by assays in serum for theophylline and four
anticonvulsants (primidone, phenobarbital, phenytoin, and carbamazepine)
plus two of their active metabolites (phenylethylmalonamide and carbamaz-
epine epoxide) was illustrated. For theophylline, final chromatograms are
monitored at 270 nm, at analysis rates of 10 h1. Concentration and absor-
bance are linearly related from 0 to 130 mg of theophylline per liter. For the
anticonvulsants, chromatograms are monitored at 200 nm, at analysis rates of
7.5 h1. The six individual determinations are each linear beyond the ther-
apeutic range. For both drug panels, day-to-day CVs were 46%. Results
correlate well with those by enzyme immunoassay. A total sample volume
of 150 L is required [126].
HPLC was used for simultaneous quantitation of CBZ and carbamaze-
pine 10,11-epoxide (CBZ-EP) in plasma and saliva. Because concentrations
of CBZ can greatly exceed those of CBZ-EP after single doses, two internal
standards, lorazepam and N-desmethyldiazepam, were added to all samples.
Following extraction with chloroform, the components are separated on a
muBondapak CN column with a mobile phase composed of 30% acetoni-
trile in water. Total chromatography time is 10 min. Concentrations of
CBZ and CBZ-EP as low as 18 and 56 ng/mL, respectively, can be detected
using 0.5 L of plasma or saliva. The maximum within-day and day-to-day
coefficients of variation for both compounds are 6.3% and 7.0%, respec-
tively. Specificity of the method was supported by a significant correlation
(r 0.99) between assay results of the present method and those of a previ-
ously published HPLC assay. Application of the method to protein binding
and salivary measurements in a single-dose CBZ disposition study is dem-
onstrated [127].
A procedure for the separation and isolation of the urinary metabolites
of carbamazepine by reversed-phase HPLC is described. After extraction
from urine, the metabolites were separated on either an analytical or
semipreparative C18 mu Bondapak column by gradient elution with
methanolwateracetic acid. Following derivatization, the metabolites
196 S.T. Alrashood

isolated by the use of the semipreparative column were analyzed by gas chro-
matography and gas chromatographymass spectrometry (GCMS) [128].
An HPLC method is described for monitoring plasma concentrations of
cinromide (3-bromo-N-ethylcinnamamide) and its deethylated metabolite.
Carbamazepine levels can be easily measured by the same technique. The N-
isopropyl analog of cinromide is used as internal standard, and all compounds
are easily separated on a reversed-phase column operated at 55C with a
small-diameter precolumn maintained at the same temperature. The extrac-
tion is rapid and generally applicable to plasma and urine samples that are to
be analyzed by reversed-phase chromatography. Short- and long-term
reproducibility studies show less than 4% RSD for replicate determinations
for all drugs. Limits of quantitation are 1020 ng/mL with an internal stan-
dard concentration of 3 g/mL. Another metabolite of cinromide, 3-
bromocinnamic acid, which may have some anticonvulsant effect, can be
analyzed simultaneously by buffering the mobile phase and adding an
ion-pairing reagent [129].
A modified HPLC method for the simultaneous analysis of carbamaze-
pine and its biologically active metabolite, carbamazepine-10,11-epoxide,
was described. Concentrations of both these compounds in the plasma of
35 epileptic patients receiving chronic carbamazepine therapy are presented.
Concentrations of carbamazepine in plasma were related to those of carba-
mazepine-10, 11-epoxide (r 0.495, P < 0.05). Total daily doses
of carbamazepine were better correlated with plasma concentrations of car-
bamazepine-10,11-epoxide (r 0.714, P < 0.001) than of carbamazepine
(r 0.269, P > 0.05). Close correlations were found between results of
the three assay procedures we used to measure plasma carbamazepine con-
centrations: HPLC, GLC, and enzyme immunoassay. Correlation coeffi-
cients exceeded 0.97 and regression slopes were near unity, indicating
that all three procedures were individually specific for the quantification
of plasma carbamazepine [130].

4.2.5.4 Gas Chromatography

An analytical method based on GCMS has been developed to simulta-
neously determine selected acidic and neutral pharmaceuticals and endo-
crine-disrupting substances in surface and tap water. SPE with Oasis HLB
cartridges is followed by derivatization of the target analytes in the eluted
extract. Derivatization was systematically optimized by employing a factorial
experimental design. More specifically a central composite design was
applied to search for the optimal conditions of the derivatization process
Carbamazepine 197

and it was demonstrated that N-methyl-N-tert-butyl-dimethysilyl-tri-

fluoroacetamide (MTBSTFA) had a better overall performance compared
to N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA). The influence of
solvent ratios and elution volumes while using SPE was also studied using
a factorial design. The method was developed successfully for most of the
selected compounds [ie, ibuprofen, salicylic acid, gemfibrozil, naproxen, tri-
closan, propranolol, diclofenac, carbamazepine, 4-octylphenol (OP), 4-
nonylphenol (NP), nonylphenol-monoethoxylate (NP1EO), non-
ylphenoxyacetic acid (NP1EC), estrone (E1), and 17alpha-ethinyloestradiol
(EE2)]. Relative recoveries for spiked river and tap water ranged from 47%
to 130% and 60% to 109%, respectively. Typical LODs were less than 5 ng/
L in tap water and less than 10 ng/L in river water. Twelve target com-
pounds were detected in river and tap water samples using the developed
method. This method is currently used in bench-scale drinking water treat-
ment studies [131].
Carbamazepine, one of the most often used AEDs, undergoes enzyme
biotransformation through epoxidation with the formation of its metabolite,
carbamazepine-10,11-epoxide (carbamazepine epoxide). The determina-
tion of carbamazepine epoxide is clinically significant in TDM as it decreases
the risk of toxic reactions and increases the possibility of reaching the
expected therapeutic result. The aim of this study was to introduce a gas
chromatographic method with mass spectrometric detection to simulta-
neously determine the serum levels of carbamazepine and carbamazepine
epoxide. Blood samples from 80 epileptic patients aged between 1 and 63
years were analyzed. All patients were taking carbamazepine as monotherapy
and had achieved the steady-state serum drug concentration. A micro-
column extraction of carbamazepine and carbamazepine epoxide was
obtained by elution. A gas chromatograph with a mass spectrometer and
a 25 m  0.2 mm ID, 0.33 m film thickness, cross-linked 5% phenyl-meth-
ylsilicone capillary column HP-5 was used. The coefficients of correlation
for the calibration plots obtained for carbamazepine epoxide and carbamaz-
epine were 0.988 and 0.995, respectively. The precision, tested for n 20,
showed coefficients of variation within 1 day as follows: carbamazepine
epoxide: 11.0% and 6.55%; carbamazepine: 11.8% and 5.4%. The coeffi-
cients of variation from day to day were: carbamazepine epoxide: 9.4%
and 6.83%; carbamazepine: 8.5% and 5.7%. The detection limit was
10 g/L and the recovery 82.5% for carbamazepine epoxide and 92.5%
for carbamazepine. A fast and simple gas chromatographic method for the
routine therapeutic monitoring of carbamazepine and carbamazepine
198 S.T. Alrashood

epoxide was developed. The simultaneous determination of the serum levels

of carbamazepine epoxide and carbamazepine offers the possibility of mea-
suring the total drug concentration, as well as that of its metabolite, while
considering the results of clinical response and the special features of car-
bamazepines enzymatic biotransformation through epoxidation with the
formation of its metabolite [132].
A method for the identification and quantification of the antiepileptics
lamotrigine, carbamazepine, and carbamazepine epoxide (active metabolite)
in human serum is described. In refractory epilepsy, the combination of car-
bamazepine and lamotrigine was recently developed to a modern therapeu-
tic concept. The goal of this paper is TDM of these substances. Serum was
extracted with a quick precipitation method using a modified commercial
extraction kit and analyzed by GCMS. A GC temperaturepressure pro-
gram was developed that allowed the determination of lamotrigine by
GCMS. A reference spectrum of pure lamotrigine is herewith published
for the first time. A new mass spectra library was created to support the iden-
tification of the antiepileptics in human serum. In the specified ions mon-
itoring mode a detection limit below the therapeutic range and recoveries
above 90% were obtained. Comparison of results obtained by GCMS or
a commercially available HPLC method (for lamotrigine) and a FPIA (for
carbamazepine) from spiked serum samples showed disagreement of no
more than 10% between the methods and demonstrated the accuracy of
the new method. In addition, quantitative determinations of these antiepi-
leptics in samples from patients under anticonvulsive therapy showed a
strong linear correlation with r2 0.961 for carbamazepine and r2 0.964
for lamotrigine. Only 2 from a total of 46 results differed by more than
10%. Our method for quantifying lamotrigine in serum seems to be highly
specific and capable of measuring lamotrigine in patients on single therapy, as
well as on add-on therapy with carbamazepine or carbamazepine epoxide.
No interference from other coadministered drugs was detected [133].
CBZ levels in the scalp hair of 17 patients (10 males and 7 females), aged
from 5 to 40 years old, receiving the drug systematically were determined
after hair dissolution and SPE procedures using both immunoassay (Abbott
TDx) and GC techniques. Carbamazepine levels in hair ranged from 13.9 to
66.3 g/g (mean 26.6 g/g, median 20.9 g/g) according to GC measure-
ments. The immunoassay technique gave slightly higher results (mean
28.0 g/g, median 22.1 g/g). The blood concentrations of carbamazepine,
using immunoassay (Abbott TDx) technique, ranged from 2.9 to
10.7 g/mL (mean 6.2 g/mL, median 5.7 g/mL). Our data indicate
Carbamazepine 199

the possible use of hair testing as a marker of the dosage history of patients
under long-term treatment with CBZ [134].
The GC quantification of underivatized AEDs such as carbamazepine,
phenytoin, phenobarbital, and primidone in fused-silica capillary columns
was compared with that in packed columns. Excellent correlation was dem-
onstrated between the two column methods by slopes of 0.991.05, by Y-
intercepts of 0.47 to 0.28, and by r values of 0.996 and 0.997. In addition,
some of the specimens from GC analysis by both column methods were
compared with data obtained for these drugs as analyzed with the Abbott
TDx system. Data obtained by both GC column methods showed excellent
correlation [135].
Detection of the anticonvulsants carbamazepine, clonazepam, diazepam,
ethosuximide, mephenytoin, mesuximide, methylphenobarbital, phenobar-
bital, phenytoin, primidone, propylhexedrine, sultiame, trimethadion, and
their metabolites in urine is described. The method presented is integrated in
a general screening procedure (general unknown analysis) for several groups
of drugs, detecting several hundred drugs and over 1000 metabolites. It
includes cleavage of conjugates by acid hydrolysis, isolation by liquidliquid
extraction, derivatization by acetylation, separation by capillary GC, and
identification by computerized MS. Using mass chromatography with the
selective ions m/z 58, 104, 113, 117, 165, 193, 204, and 246, the possible
presence of anticonvulsants and/or their metabolites was indicated. The
identity of positive signals in the reconstructed mass chromatograms was
confirmed by a visual or computerized comparison of the stored full mass
spectra with the reference spectra. The sample preparation, mass chromato-
grams, reference mass spectra, and gas chromatographic retention indices are
documented [136].
This paper reviews GLC methods for the determination of the most
commonly monitored AEDs: phenobarbital, phenytoin, carbamazepine,
primidone, ethosuximide, valproic acid, and clonazepam along with a
new compound, progabide. The individual classes of drugs are first treated
separately to highlight specific aspects of their quantification, and this is
followed by an overview of those methods permitting the concomitant anal-
ysis of two or more antiepileptic compounds. Sample preparation techniques
as well as comparisons between chromatographic and other techniques are
treated more fully in separate sections [137].
Sera of epileptic patients which were routinely examined by GC were
also analyzed using HPLC. Three basic methods for the pretreatment of
samples for HPLC analysis were compared: protein precipitation by adding
200 S.T. Alrashood

acetonitrile to the serum, direct serum extraction using ethylacetate,

and partitioning of serum and buffer solution over a stationary phase and
extraction with dichloromethane/2-propanol. The analytical perfor-
mance and practicability of the three methods were tested under routine
conditions. The following antiepileptic drugs and metabolites were used
in the comparison of HPLC with GC: ethosuximide, primidone, pheno-
barbital, phenytoin, carbamazepine, N-desmethylmethsuximide, and
phenylethylmalonediamide [138].
Free carbamazepine and valproic acid monitoring using the EMIT Free-
Level filtration system was evaluated and compared with reference equilib-
rium dialysis and GC techniques. For carbamazepine, free levels after
filtration or dialysis were essentially identical (mean 1.74 vs 1.77 mg/L,
r 0.940, n 28, EMIT assay). Free levels were 16% higher by EMIT than
by GC, possibly due to cross-reaction with CBZ-E. Free fractions were not
significantly different using any combination of filtration or dialysis with
EMIT or GC (means 0.240.26). There was a significant correlation
between epoxide and parent drug-free fractions (r 0.642). Free fraction
varied from 0.20 to 0.41 among 61 patient samples and was independent
of total drug concentration. For valproic acid, there was a strong correlation
between filtration and dialysis results for free level (r 0.974) and free frac-
tion (r 0.892), but filtration values were 67% higher. Free fraction was
concentration dependent (r 0.597), and lower free fractions by dialysis
were attributed to dilution of total drug concentration. Free fraction varied
from 0.01 to 0.14 among 50 patient samples. For carbamazepine and
valproic acid, the EMIT FreeLevel filtration system compared favorably
with equilibrium dialysis and had the advantage of being rapid [139].
A GC procedure for the simultaneous determination of carbamazepine,
phenobarbital, and phenytoin using SP 2510 DA as the stationary phase is
presented. The AEDs are determined simultaneously without derivatization
under isothermal conditions by a flame ionization detector. The GC proce-
dure can be easily mechanized. The coefficient of variation for the precision
from day to day is 8.9% for carbamazepine, 7.0% for phenobarbital, and 4.3%
for phenytoin as calculated from single determinations. The deviations from
the target value of spiked pool sera range from 4.3% to 9.5%. The GC results
of this method and of determinations by Dexsil 300 for primidone and SP
1000 for ethosuximide are compared with the corresponding enzyme
immunoassays (EMIT). The precision of the GC methods is somewhat bet-
ter than EMIT. When patients sera are analyzed by both procedures, the
results show no clinically relevant differences. It can be concluded that
Carbamazepine 201

the different methods are interchangeable and may be selected according to

practical necessities [140].
Another GC method for the determination of the AEDs carbamazepine,
phenobarbital, phenytoin, and primidone in the same extract of serum is
presented. Saturated ammonium sulfate solution is added to 1 mL serum,
followed by extraction with chloroform. The organic phase is separated
and evaporated. The residue is dissolved in 100 L ethylacetate/acetic acid
(100 mL + 1 mL) for GC. The GC determination is carried out under iso-
thermal conditions without derivatization, using SP 2250 DA as stationary
phase for the determination of phenobarbital and phenytoin, and Dexsil 300
for the determination of carbamazepine and primidone. The coefficient of
variation for the precision from day to day ranges from 4.3% to 7.5% and the
recovery from 93.5% to 111%. The specificity was proven by comparison
with the relative retention times of about 100 drugs. The method is com-
pared with the corresponding EMIT tests [141].
The principles of three independent extraction methods were utilized to
develop an integrated extraction scheme for use in routine therapeutic
monitoring of seven antiepileptic agents. The final method, in which the
three extraction methods were interfaced, permitted routine monitoring
in a single 1 mL volume of human plasma of any one or combination
of the following drugs: PHT, phenobarbital (PB), primidone (PD), 5-
ethyl-5-phenylhydantoin (EPH), ethosuximide (ES), CBZ, and valproic
acid (VPA). An on-column methylation technique was used for simulta-
neous determination of PHT, PB, PD, EPH, and ES. CBZ and VPA were
determined by independent methods as the underivatized compounds. Six
appropriate internal standards were employed in the integrated method for
quantitation of the drugs [142].
An integrated method that overcomes the two main procedural difficul-
ties of GLC, namely, solventsolvent extraction and chemical derivatiza-
tion, was developed. Drugs are extracted from serum by column
chromatography on granular diatomaceous earth (kieselguhr). Subsequent
GLC of underivatized samples can be performed on either of two liquid
phases. A mixed liquid phase, used for quantitative GC assay on patients with
a known therapeutic regimen, has enabled quantitation of 12 drugs in serum.
Alternatively, a single liquid phase, used with the mixed liquid phase, per-
mits the GC identification of unknown drugs on the basis of the character-
istic pattern of the two relative retention times; by this approach more than
40 drugs have been identified in cases of suspected intoxication, both in
serum and in gastric aspirate. Besides providing ease of performance and
202 S.T. Alrashood

wide applicability, the proposed procedure offers a degree of precision and

accuracy that compares favorably with established methods [143].
A quantitative GLC procedure is described for the consecutive determi-
nation of phenytoin, phenobarbital, primidone, phenylethylmalondiamide,
carbamazepine, trimethadione, dimethadione, ethosuximide, and valproate
from a single serum specimen of 1.2 mL. After extraction from serum by two
different procedures, the anticonvulsants are chromatographed without fur-
ther purification on a 3% OV 17 column either with or without derivative
formation by means of "on-column" methylation. Multiple internal stan-
dards are employed in order to enhance the reproducibility of drug-concen-
tration measurement [144].
Serum carbamazepine concentration was determined by GLC method in
14 epileptic patients (32 samples) treated initially with CBZ alone, and the
following results were obtained: 1. The relationship between serum CBZ
concentration and CBZ dose was expressed by a regression line that sloped
sharply upwards at first: high serum concentration was obtained from small
dose up to the end of 3 weeks after initiation of CBZ administration. After
the third to fourth treatment week, regression coefficient was reduced by
half, and the regression line had a gentler slope. 2. As the treatment was con-
tinued longer, serum CBZ concentration and serum concentration/dose
ratio became lower. The relationship between duration of CBZ treatment
and serum concentration/dose ratio was expressed by a regression curve rep-
resenting a quadratic function with negative coefficient, the ratio being high
in early stage of the treatment. This regression curve shows that the ratio
reached its maximum value at the 10.5 days after initiation of the treatment.
The ratio was stabilized at a low level about 34 weeks after that. 3. From the
results, serum CBZ concentration was confirmed to be more dependent on
duration of the treatment than on dose. This was inferred, on the basis of
documentary research, to be attributable to the autoinduction of CBZ
metabolism, the inference of which seemed to be supported by the results
of measurement of time-course change in serum concentration in five cases
of the present series. The results in one of the said five cases suggest that
serum CBZ concentration varies greatly within 12 h. 4. On the basis of these
results, it is proposed that spontaneous decrease in serum CBZ concentra-
tion must be countered not merely by increase in CBZ dose, but by increase
in the number of dosages/day for suppression of excessive diurnal change in
serum concentration as well [145].
A previously published procedure for the GC analysis of carbamazepine
has been modified and expanded to allow simultaneous determination of
Carbamazepine 203

phenylethylmalonamide, a metabolite of primidone. Internal standards that

closely resemble each compound are used, and derivatives are made by
reaction with dimethylformamide dimethylacetal. This change of internal
standard for carbamazepine and the use of a commercial, pretested column-
packing material eliminate the major pitfalls of the original method [146].
A simple, sensitive, and precise GC method for simultaneous extraction,
derivatization, and determination of methsuximide, ethosuximide, diphe-
nylhydantoin, carbamazepine, phenobarbital, and primidone in the presence
of other drugs has been described. The method is especially useful for drug
monitoring in patients on multiple anticonvulsant therapy while also on
combination therapy with psychotropic drugs. It overcomes the analytical
interferences between mephenytoin and phenobarbital; methsuximide
and primidone; kemadrin and primidone; cholesterol and primidone; pro-
lixin, haldol, and other drugs; encountered in other methods using under-
ivatized, trimethylsilylated, or methylated drugs. As little as 0.5 g/mL of a
drug can be determined and if needed the method can be scaled down to
0.3 mL plasma. The method yielded recoveries of 97103% with standard
deviations of 0.71.8. For a constant check of the precision, an internal qual-
ity control using daily analysis of a sample from a frozen plasma pool sup-
plemented with known concentrations of the anticonvulsants was used.
The method is suitable for use in routine clinical laboratory [147].
A comparison of six GC procedures for the simultaneous analysis of
AEDs was made. Methylation with 0.2 M trimethylanilinium hydroxide
was superior to other derivatizing procedures evaluated. The methylated
derivatives of phenobarbital, carbamazepine, primidone, and diphenylhy-
dantoin were separated on the chromatogram, and the peaks were sharp
and symmetrical. The four drugs gave linear curves to twice their toxic levels
[148].
A simple and rapid GC method for the simultaneous estimation of the
anticonvulsant drugs ethosuximide, carbamazepine, phenobarbital, primi-
done, diphenylhydantoin, and the metabolite PEMA in serum is presented.
The method is based on a simple ether extraction of 1 mL serum before and
after precipitation of the proteins by ammonium sulfate and injection of the
extract dissolved in methanol without derivative formation. GC separation
is performed on a highly polar acidic phase (SP 1000, a terephthalic acid-
modified Carbowax 20 M), for detection the instrument is equipped with
a nitrogen-selective detector, and quantitation is performed by automatic
electronic integration of peak areas in relation to the internal standard
Mesantoin. The optimal approach to the GC analysis of problem drugs
204 S.T. Alrashood

like carbamazepine and phenobarbital is discussed, and various stationary

phases and support materials are compared for effectiveness with this
method. In the analysis of over 800 routine serum samples as well as internal
and external quality control samples, this method was found to be reliable
and the results are reproducible [149].
A rapid, simple isothermal GC method for the quantitation of carbamaz-
epine (Tegretol) in serum as its trimethylsilyl derivative has been devel-
oped. A single chloroform extraction using 1.0 mL of serum, a 10-min
derivatization step, and analysis on a 3% OV-1 (methyl silicone) GC column
gives a linear response to a carbamazepine concentration up to 25 mg/L. A
nondrug internal standard (tetracosane) compensates for variables in extrac-
tion, injection, and instrumental changes occurring during analysis. This
method was found to be free of interferences from endogenous substances
in the serum, as well as from toxic concentrations of commonly used drugs.
Day-to-day precision at concentration levels of 2, 5, and 10 mg/L ranged
from 2.9% to 4.4% (CV). Standard addition studies of carbamazepine added
to serum resulted in a mean recovery of 95%. To ensure accurate results, all
standards were prepared in serum [150].
A simple, sensitive method for the determination of phenobarbital, diphe-
nylhydantoin, carbamazepine, and primidone in serum, by use of GLC with
temperature programming, was described. The methylated derivatives of
these anticonvulsants were well resolved, as was 5-(p-methylphenyl)-5-
phenylhydantoin, the internal standard. In this procedure an ion-exchange
resin for separation of the drug from the serum was used. The proposed
procedure requires only 1.0 mL of serum and can be done in less than 1 h.
The lower LOD for each of the drugs is 0.5 mg/L. Analytical recoveries of
drug from serum were excellent and peak height and concentration were
linearly related up to twice the toxic concentration for serum [151].
A GLC method for the simultaneous determination of phenobarbital,
primidone, carbamazepine, and diphenylhydantoin in human serum follow-
ing therapeutic doses has been developed. After extraction with chloroform,
the anticonvulsant drugs were methylated with phenyltrimethylammonium
hydroxide in dimethylformamide at 85C for GLC. Linear temperature
programming of a 1% OV-17 column was used to achieve separation and
quantitation. The procedure described in the present paper is relatively sim-
ple, highly specific, and sufficiently sensitive for use in routine clinical assays
[152].
A method is described to prepare routinely high-resolution support-
coated open tubular (SCOT) columns. A siliceous support material
Carbamazepine 205

(Cab-O-Sil) is deactivated with benzyltriphenylphosphonium chloride and

deposited on the inside wall of a glass capillary column. After additional coat-
ing with a polar stationary phase (OV-225) a number of anticonvulsant drugs
can be analyzed without prior derivatization. The columns described show
high plate numbers and do not deteriorate in use. The repeatability of the
GC analysis is better than 1.2% (Orel). The minimum detectable quantity
is of the order of 1010 g. An improved procedure for isolating the drugs
from serum is given, which results in an extraction recovery of better than
90% for the drugs of interest [153].
A simple, rapid, and specific GC method for the measurement of plasma
carbamazepine concentration is presented. The extraction is a single-step
method, and the drug is measured by derivatization using on-column meth-
ylation and standard instrumentation. Other anticonvulsant agents do not
coextract. Other drugs do not interfere in the chromatogram. Stability,
recovery, and reproducibility make this procedure suitable for routine car-
bamazepine measurements [154].
Another simple, sensitive determination of phenobarbital, diphenylhy-
dantoin, carbamazepine, and primidone in serum, by use of GLC with
temperature programming, was developed. The methylated derivatives of
these anticonvulsants are well resolved, as was 5-(p-methyl-phenyl)-5-
phenylhydantoin, the internal standard. The proposed procedure requires
only 0.20 mL of serum and can be done in less than 30 min. The lower
LOD for each of the drugs is 0.5 mg/L. Analytical recoveries of drug from
serum were excellent and peak height and concentration were linearly
related up to twice the toxic concentration for serum [155].

4.2.5.5 Capillary Chromatography

An overview of the electrokinetic chromatographic methods for the analysis
of AED levels in biological samples is presented. In particular, micellar elec-
trokinetic capillary chromatography (MEKC) is a very suitable method for
the determination of these drugs, because it allows a rapid, selective, and
accurate analysis. In addition to the electrokinetic chromatographic studies
on the determination of AEDs, some information regarding sample pre-
treatment will also be reported: this is a critical step when the analysis of bio-
logical fluids is concerned. The electrokinetic chromatographic methods for
the determination of recent AEDs (eg, lamotrigine, levetiracetam) and clas-
sical anticonvulsants (eg, carbamazepine, phenytoin, ethosuximide, valproic
acid) will be discussed in depth, and their pharmacological profiles will be
briefly described as well [156].
206 S.T. Alrashood

A very rapid and simple MEKC method was developed for the simulta-
neous determination of four AEDs, ethosuximide (Etho), primidone (Pri),
phenytoin PHT, and CBZ in human serum. Sample analysis required only
100 L of human serum which only needed to be centrifuged, decanted, and
combined with the running buffer [5.3 mM Na2HPO4/3.2 mM borax
buffer (pH 9.5) containing 55 mM SDS and 3.5% (v/v) acetone]. The anal-
ysis was performed in only 10 min into fused-silica capillaries (57 cm total
length with 50 m I.D. and 50 cm to the detector) using the MEKC meth-
odology with diode-array detection at 220 nm. The calibration graphs were
established for ethoximide, primidone, phenytoin, and carbamazepine
between 0 and 20 mg/L. Recoveries were between 85% and 87%. The sim-
plicity of the proposed methodology makes it suitable for routine clinical
use, especially for epileptic patients on polytherapy [157].
A zero-dead volume column end and a conical shaped slurry reservoir for
wide-bore stainless-steel packed capillary LC columns were designed and
evaluated. A detailed procedure for the preparation of reversed-phase stain-
less-steel packed capillary columns with 0.51.0 mm I.D is described. The
influences of the column length and the packing material on the column
performance have been studied. Columns were evaluated by the reduced
plate height vs reduced velocity curve and the peak asymmetric factor.
Experimental results showed that the column efficiency and the reproduc-
ibility were better than 75% of theoretical value and 6% RSD, respectively.
Separations of AEDs and chlorinated benzenes are demonstrated [158].
In CBZ therapy the concomitant monitoring of concentrations of CBZ
and its metabolites is strictly recommended, primarily to avoid toxic side
effects. Currently, clinical routine monitoring of CBZ is accomplished by
HPLC or immunological methods. In this study a micellar electrokinetic
capillary chromatographic (MECC) method was developed for routine drug
monitoring of CBZ and its main metabolites, carbamazepine 10,11-diol and
CBZ-E, in human serum or plasma samples. The MECC method enabled
baseline separation of all analytes within 2.5 min. The assay revealed suffi-
cient precision and sensitivity and the results of either an automated HPLC
or the MECC chromatography assay were in good agreement (r 0.97).
The maximum deviation for CBZ was 0.26 M [159].

4.2.6 Capillary Electrophoresis Methods

This study establishes a method, using different buffer conductivities and
large-volume sample stacking-sweeping CE, for analysis of CBZ and its five
metabolites in serum. The capillary (50/60 cm) was filled with a high
Carbamazepine 207

concentration of background electrolyte (150 mM phosphate, pH 3.5, con-

taining 15% methanol), followed by a large volume of samples (10 psi, 20 s)
with low-concentration buffers (5 mM phosphate, pH 3.5, with 5% meth-
anol). When high voltage was applied (20 kV), the sodium dodecyl sulfate
(SDS) started to sweep the analytes to an outlet. Meanwhile, the analytes
decelerated at the boundary between low- and high-conductivity buffers.
Finally, a narrow sample zone was formed. The procedure of sweeping
and separation was simultaneously carried out by a sweeping buffer
(150 mM phosphate, pH 3.5) with 15% methanol and 50 mM SDS added,
and the detection was performed by UV at 214 nm. The method was val-
idated for linearity (r  0.997), precision, and accuracy. The calibration cur-
ves were established for CBZ and its five metabolites between 0.0325 and
0.033 g/mL. The LODs (S/N 3) were 0.01 g/mL for each analyte.
Compared with simple MEKC (0.5 psi, 5 s), this system can improve the
sensitivity about 300-fold. Finally, this method was successfully applied to
five patients, who had taken 200 mg CBZ daily, and CBZ levels were found
to be from 3.72 to 5.82 g/mL [160].
In order to adopt a general workflow for complex biological matrices
with respect to a new bloodbrain barrier (BBB) model, a micellar electro-
kinetic chromatography method has been developed. The cells forming the
BBB have been cultivated in a special cell growth medium in which six
drugs (acetaminophen, caffeine, carbamazepine, cimetidine, indometacin,
and propranolol) have been dissolved and tested for their penetration prop-
erties. The results showed good to very good accordance to the reference
values. Samples were directly injected onto the capillary without any pre-
treatment (fused-silica capillary, I.D.: 50 m, L: 48 cm, l: 40 cm). After
method development, separations were carried out using a 60 mM borate
buffer containing 200 mM of SDS at 30 kV, leading to an analysis time of
less than 10 min. Between two runs, the capillary was rinsed with a mixture
of equal parts of running buffer and isopropanol (70%, v/v), which proved to
be very effective to remove matrix compounds. An appropriate choice of the
detection wavelength (220 or 254 nm) could avoid major interferences
between analytes and matrix. The typical RSD% for migration times was
approximately 2%; for peak areas, it ranged from 2% to 6%, which was very
well acceptable for the generic method used in this study; and the low con-
centrations investigated. The LODs ranged from 10 to 30 ng/mL [161].
A CD-modified microemulsion electrokinetic chromatography method
has been developed and validated for dexamphetamine sulfate which allows
the simultaneous determination of charged and uncharged impurities of the
208 S.T. Alrashood

drug including the levorotary (R)-enantiomer. The optimized background

electrolyte consisted of 1.5% (w/w) SDS, 0.5% (w/w) ethyl acetate, 3.5%
(w/w) 1-butanol, 2.5% (w/w) 2-propanol, and 92% (w/w) 50 mM sodium
phosphate buffer, pH 3.0, containing 5.5% (w/w) sulfated -CD. Separa-
tions were performed in a 50.2/40 cm, 50 m I.D. fused-silica capillary at
a temperature of 20C and an applied voltage of 14 kV. Carbamazepine
was used as internal standard. The assay was validated in the range of 0.1
1.0% for the related substances and 0.15.0% for levoamphetamine based
on a concentration of 3 mg/mL of dexamphetamine sulfate. The LOD of
all analytes ranged between 0.05% and 0.2%. In commercial samples of
dexamphetamine sulfate, levoamphetamine was found at concentrations
between 3.2% and 3.8%, whereas none of the other impurities could be
detected [162].
Drug monitoring in serum samples was performed using second-order
data generated by CE-DAD, processed with a suitable chemometric strat-
egy. Carbamazepine could be accurately quantitated in the presence of its
main metabolite (carbamazepine epoxide), other therapeutic drugs
(lamotrigine, phenobarbital, phenytoin, phenylephrine, ibuprofen, acet-
aminophen, theophylline, caffeine, acetyl salicylic acid), and additional
serum endogenous components. The analytical strategy consisted of the fol-
lowing steps: (i) serum sample cleanup to remove matrix interferences; (ii)
data preprocessing, in order to reduce the background and to correct for
electrophoretic time shifts; and (iii) resolution of fully overlapped CE peaks
(corresponding to carbamazepine, its metabolite, lamotrigine, and unex-
pected serum components) by the well-known multivariate curve resolu-
tion-alternating least-squares (MCR-ALS) algorithm, which extracts
quantitative information that can be uniquely ascribed to the analyte of
interest. The analyte concentration in serum samples ranged from 2.00 to
8.00 mg/L. Mean recoveries were 102.6% (s 7.7) for binary samples,
and 94.8% (s 13.5) for spiked serum samples, while CV (%) 4.0 was
computed for five replicates, indicative of the acceptable accuracy and pre-
cision of the proposed method [163].
In this paper, the determination of pharmaceuticals employing micro-
emulsion electrokinetic chromatography (MEEKC) with atmospheric
pressure photoionization-mass spectrometric (APPI-MS) detection was
presented. This recent hyphenated technique allows overcoming some dis-
advantages of MEEKC, namely its inherent incompatibility with MS
detection. Important parameters like microemulsion (ME) composition,
the composition of the sheath liquid, and APPI-MS detection parameters
Carbamazepine 209

have been investigated. Using the optimized set of parameters, the eight
selected substances could be detected down to concentrations between 3
(phenacetin) and 41 mg/L (diltiazem). Switching to the MS2 mode, the
use of specific transitions for the detection of each analyte provided
improved detection limits in the range of 0.6 (carbamazepine) to 6 mg/L
(metoprolol). Calibration curves were linear over one to two orders of
magnitude with correlation coefficients better than 0.98 [164].
Microemulsion electrokinetic chromatography was applied for the
separation of levetiracetam from other AEDs (primidone, phenobarbital,
phenytoin, lamotrigine, and carbamazepine) that are potentially coad-
ministered in therapy of patients. The influence of the composition of the
microemulsion system (with SDS as charged surfactant) was investigated,
modifying the kind of cosurfactant (lower alcohols from C3 to C5), the
pH (and salinity) of the aqueous background electrolyte, and the ratio of
aqueous phase to organic constituents forming the microdroplets of the
oil-in-water emulsion. Separation selectivity was depending on all these
parameters, resulting even in changes of the migration sequence of the
analytes. Only moderate correlation was observed for the microemulsion
system compared with a micellar system, both consisting of the aqueous
borate buffer (pH 9.2) and SDS as micelle former (linear correlation coef-
ficient for analyte mobilities is 0.974). The sample solvent plays an important
role on the shape of the resulting chromatograms: methanol at concentra-
tions higher than 35% impairs peak shape and separation efficiency. The
microemulsion method (with 93.76% aqueous borate buffer (pH 9.2,
10 mM), 0.48% n-octane, 1.80% SDS, 3.96% 1-butanol, all w/w) is suitable
for the determination of levetiracetam in human plasma (combined with a
sample pretreatment based on SPE) [165].
Single isomer octakis-(2,3-dihydroxy-)6-sulfato-gamma-cyclodextrin
used as pseudostationary phase of the background electrolyte interacts with
dibenzo[b, f]azepines (consisting of a condensed three-ring system) and
forms negatively charged complexes. Hydroxygroups in positions 2 and
3 at carbamazepine increase the extent of interaction, whereas substitu-
tion by oxygen at position 10 and/or 11 reduces it. The complex
constants for the analytes are ranging from few tens of L/mol (10-
hydroxycarbamazepine, 10,11-dihydroxycarbamazepine, 10,11-epoxy car-
bamazepine, oxcarbazepine) to several hundreds of L/mol (carbamazepine,
2-hydroxycarbamazepine, 3-hydroxycarbamazepine) and are much larger
than those of the analytes with octakis-(2,3-dimethyl-)-6-sulfato-gamma-
cyclodextrin. Full enantiomeric separation of the chiral metabolites of
210 S.T. Alrashood

carbamazepine and oxcarbazepine is obtained at octakis-(2,3-dihydroxy-)6-

sulfato-gamma-cyclodextrin concentrations of about 10 mM (3 mM borate
buffer, pH 8.5). Compared to heptakis-6-sulfato-beta-cyclodextrin, selec-
tivity differs and stereoselectivity is more pronounced [166].
Quality assurance is an important aspect in TDM. CE assays for deter-
mination of (i) ethosuximide via direct injection of serum or plasma, (ii)
lamotrigine after protein precipitation by acetonitrile and analysis of an ali-
quot of the acidified supernatant, and (iii) CBZ and CBZ-E after
solute extraction followed by analysis of the reconstituted extract are
characterized via analysis of a large number of commercial quality control
sera containing up to 14 analytes (9 of them are anticonvulsants) in
subtherapeutic, therapeutic, and toxicologic concentration levels. CE
data obtained in single determinations are shown to compare well with
the spike values and the mean of data determined in other laboratories
using immunoassays and/or HPLC, values that are reported by the
external quality control scheme. Carbamazepine and ethosuximide drug
levels are also shown to agree well with those determined in our depart-
mental drug assay laboratory using automated immunoassays. The presented
data reveal the effectiveness of assay assessment via analysis of quality control
sera and confirm the robustness of the assays for TDM in a routine setting
[167].
TDM of CBZ, a widely used AED, is required for optimization of phar-
macotherapy with this drug and for assessment of the patients compliance to
therapy. The suitability of employing MEKC in the absence of electroosmo-
sis for the determination of CBZ and its main metabolite CBZ-E in extracts
of human serum and plasma is reported. Using micelles formed by dodecyl
sulfate, analyses performed in untreated fused-silica capillaries at acidic pH
and in commercially available coated capillaries under application of
reversed polarity are compared. Uncoated and polyvinyl alcohol-coated
capillaries proved to be unsuitable for this purpose, whereas capillaries
coated with linear polyacrylamide and N-acryloylaminoethoxyethanol
and operated at pH 7.6 are shown to provide high-quality and reliable data
on a short timescale. Assay performance is discussed via statistical analysis of
the data produced from a set of quality control sera that contain up to 14
different drugs and via analysis of patient samples. Intra- and interday
imprecision data for concentrations between 4.0 and 84 M are demon-
strated to be <10%. Run times are shown to be <50% compared to
those observed in conventional MEKC at alkaline pH (ie, in the presence
of electroosmosis) [168].
Carbamazepine 211

Methods for the determination of drug residues in water have been

developed based on the combination of LC or CE with MS. For HPLC-
MS, two types of interfaces (pneumatically assisted ESI interface or an atmo-
spheric pressure chemical ionization interface, respectively) were employed
and compared in terms of detection limits. 2 mM ammonium acetate at pH
5.5 and a methanol gradient were used for the HPLC-MS allowing the sep-
aration of a number of drugs such as paracetamol, clofibric acid, penicillin V,
naproxen, bezafibrate, carbamazepine, diclofenac, ibuprofen, and mef-
enamic acid. A 20 mM ammonium acetate solution, pH 5.1, was employed
for the separation of clofibric acid, naproxen, bezafibrate, diclofenac, ibu-
profen, and mefenamic acid by CE-MS. Sample pretreatment was per-
formed by SPE for HPLC-MS or by a combination of liquidliquid
extraction and SPE for CE-MS. The applicability of both the HPLC-MS
and CE-MS method was demonstrated for several river water samples [169].
Conditions were worked out for the separation of carbamazepine,
olanzapine, and their main metabolites CBZ-E, 10-hydroxycarbamazepine,
and desmethylolanzapine. The separation was based on electrokinetically
driven methods in the capillary format. The main difficulty in separating these
compounds is related to their different chemical classes. Whereas the carba-
mazepine members are amides and are electrically neutral, the olanzapine
members have aliphatic amino groups and are thus cationic under most
experimental conditions. Different additives were applied as pseudosta-
tionary phases to implement selectivity. Poly(diallyldimethylammonium),
PDADMA, is a polycationic replaceable and soluble polymer that interacts
mainly according to the polarizability of the analyte molecules. The MEKC
principle was applied with the common SDS as micelle former. In both sys-
tems, only partial resolution of the analytes was obtained. The most favorable
system consisted of a charged, oligomeric additive: full separation of all
analytes within 4 min was achieved with heptakis-6-sulfato-beta-cyclodextrin
(7 mM) in 30 mM borate buffer, pH 8.5 [170].
The use of CE for TDM of AEDs was examined. MEKC with a DAD
simultaneously determined concentrations of ZNS, a new type of AED, and
phenobarbital, phenytoin, and carbamazepine, typical AEDs, in human
serum. ZNS levels in human serum obtained by MEKC correlated well with
levels obtained by HPLC. The serum levels of phenobarbital, phenytoin,
and carbamazepine determined by MEKC were almost equal to those
obtained by FPIA. The reproducibility of separation and quantification with
MEKC for intra- and interday assays was appropriate. This MEKC method
could provide a simple and efficient TDM method for AEDs, especially in
212 S.T. Alrashood

patients treated with a combination of ZNS and other AEDs. MEKC may be
an attractive method for TDM, because of its specificity of separation, auto-
mation of procedure, ease of method development, low cost, small aqueous
buffer amounts, speed of analysis, small injection volume, and high environ-
ment-directed performance [171].
A systematic screening method has been developed for the detection
of 29 CNS drugs in human plasma, urine, and gastric juice by high-
performance capillary electrophoresis. The first step is sample preparation.
The patients or normal human plasma (0.5 mL) spiked with CNS drugs
was extracted with 2  4 mL dichloromethane, while 2 mL of patients
or spiked urine was extracted with 2  6 mL chloroform. The combined
extract from plasma or urine was evaporated to dryness in a rotation evap-
orator at 35C. The residue was dissolved in 100 L methanol and
subsequently 400 L of redistilled water was added. The patient gastric
juice (3 mL) was centrifuged at 2000 r/min for 5 min. The supernatant
was filtered through 0.45 m microporous membrane for injection onto
capillary columns. The second step was to perform CZE separation in
acidic buffer composed of 30 mmol/L (NH4)3PO4 (pH 2.50) and 10%
acetonitrile (condition A). Most of the benzodiazepines (diazepam, nitra-
zepam, chlordiazepoxide, flurazepam, estazolam, alprazolam) and meth-
aqualone were baseline separated and detected at 513 min, while
thiodiphenylamines showed group peaks at 35 min and barbiturates
migrate with electroosmotic fluid (EOF) together. The third step is to
separate the drugs in basic buffer constituted of 70 mmol/L Na2HPO4
(pH 8.60) and 30% acetonitrile (condition B). The thiodiphenylamines
and some other basic drugs could be well separated, which include
trihexyphenidyl, imipramine, amitriptyline, diphenhydramine, chlor-
promazine, doxepin, chlorprothixene, promethazine, and flurazepam,
while the rest of the CNS drugs did not interfere with the separation.
The last step was to separate the drugs by MEKC in such a buffer as
70 mmol/L SDS plus 15 mmol/L Na2HPO4 (pH 7.55) and 5% methanol
(condition C). Barbiturates (barbital, phenobarbital, methylphenobarbital,
amobarbital, thiopental, pentobarbital, secobarbital) and some hydrophobic
drugs (glutethimide, alprazolam, clonazepam, carbamazepine, trifluopera-
zine, oxazepam) could be well separated. These drugs might be identified
by both the relative migration time (rtm tdrug/tEOF) and the ratios of
peak heights (rh) monitored at different wavelength, since the ratios are
characteristic of the spectrum of a drug. This method has been used in several
real clinical samples of intoxication. For example, perphenazine and doxepin
Carbamazepine 213

were detected in the gastric juice and phenobarbital in blood and gastric
juice of an intoxicated patient [172].
Charged carboxymethyl-beta-cyclodextrin was successful in the CE sep-
aration of a series of tricyclic antidepressants. The cyclodextrin alone was
successful in the separation of carbamazepine, protriptyline, desipramine,
clomipramine, and opipramol using a 3-(trimethoxysilyl)propyl methacry-
late capillary coating to reduce the electroosmotic flow. The ideal buffer pH
was found to be in the range of 67 and the ideal cyclodextrin concentration
to be 10 mM. All nine antidepressants were resolved using the charged
cyclodextrin in the MEKC mode with SDS as the surfactant. Neither
the cyclodextrin nor the surfactant alone was successful in resolving the
whole series of compounds under investigation but a combination of
both produced the separation. Separations were performed on a linear poly-
acrylamide-coated capillary. The ideal pH of the buffer was in the range of
57 [173].

5. BIOLOGICAL ASSAY
P450 metabolic enzymes are expressed in the human and rodent brain.
Recent data support their involvement in the pathophysiology of epilepsy.
However, the determinants of metabolic enzyme expression in the epileptic
brain are unclear. We tested the hypothesis that status epilepticus (SE) or
exposure to phenytoin or phenobarbital affects brain expression of the met-
abolic enzyme CYP2E1. SE was induced in C57BL/6J mice by systemic
kainic acid (KA). Brain CYP2E1 expression was evaluated 1824 h after
SE by immunohistochemistry. Colocalization with neuronal nuclei, glial
fibrillary acidic protein, and CD31 was determined by confocal microscopy.
The effect of phenytoin, carbamazepine, and phenobarbital on CYP2E1
expression was evaluated in vivo or by using organotypic hippocampal cul-
tures in vitro. CYP2E1 expression was investigated in brain resections from a
cohort of drug-resistant epileptic brain resections and human endothelial
cultures (EPI-EC). Immunohistochemistry showed an increase of CYP2E1
expression limited to hippocampal CA2/3 and hilar neurons after severe SE
in mice. CYP2E1 expression was also observed at the astrocytevascular
interface. Analysis of human brain specimens revealed CYP2E1 expression
in neurons and vascular endothelial cells (EC). CYP2E1 was expressed in
cultured human EC and overexpressed by EPI-EC. When analyzing the
effect of drug exposure on CYP2E1 expression, we found that, in vivo
or in vitro, ethanol increased CYP2E1 levels in the brain and liver.
214 S.T. Alrashood

Treatment with phenytoin induced localized CYP2E1 expression in the

brain, whereas no significant effects were exerted by carbamazepine or phe-
nobarbital. Our data indicate that the effect of acute SE on brain CYP2E1
expression is localized and cell specific. Exposure to selected antiepileptic
drugs could play a role in determining CYP2E1 brain expression. Additional
investigation is required to fully reproduce the culprits of P450 enzyme
expression as observed in the human epileptic brain [174].
This study aimed to investigate the prevalence and association of HLA-
B*15:02 with carbamazepine-induced StevensJohnson syndrome and
toxic epidermal necrolysis (CBZ-SJS/TEN) in the Indian population in
Malaysia, which mostly originated from Southern India. HLA-B alleles in
5 Indian case patients with CBZ-SJS/TEN and 52 CBZ-tolerant controls,
and followed by a pooled sample of 7 cases from 2 centers in Malaysia, were
analyzed. Positive association for HLA-B*15:02 with CBZ-SJS/TEN was
detected in Indians (40% [2/5] vs 3.8% [2/52], odds ratio [OR] 16.7,
P 0.0349), of which 80% (4/5) of the Indian patients originated from
Southern India. A pooled sample of seven cases showed stronger association
between HLA-B*15:02 and CBZ-SJS/TEN (57.1% [4/7] vs 3.8% [2/52],
OR 33.3, 95% CI 4.25162.21, P 1.05  103). Subsequent meta-analysis
on Indians from Malaysia and India further demonstrated a significant and
strong association between HLA-B*15:02 and CBZ-SJS/TEN (OR
38.54; 95% CI 6.83217.34, P < 1.0  104). Our study is the first on
Indians predominantly from Southern India that demonstrated HLA-
B*15:02 as a strong risk factor for CBZ-SJS/TEN despite a low population
allele frequency. This stressed the importance of testing for HLA-B*15:02,
irrespective of the ancestral background, including populations with low
allele frequency [175].
Approximately 60% of morphine is glucuronidated to morphine-3-glu-
curonide (M3G) which may aggravate preexisting pain conditions. Accu-
mulating evidence indicates that M3G signaling through neuronal Toll-
like receptor 4 (TLR4) may be central to this proalgesic signaling event.
These events are known to include elevated neuronal excitability, increased
voltage-gated sodium (NaV) current, tactile allodynia, and decreased opioid
analgesic efficacy. Using an in vitro ratiometric-based calcium influx analysis
of acutely dissociated small and medium-diameter neurons derived from
lumbar dorsal root ganglion (DRG), we observed that M3G-sensitive neu-
rons responded to lipopolysaccharide (LPS) and over 35% of these M3G/
LPS-responsive cells exhibited sensitivity to capsaicin. In addition, M3G-
exposed sensory neurons significantly increased excitatory activity and
Carbamazepine 215

potentiated NaV current as measured by current and voltage clamp, when

compared to baseline-level measurements. The M3G-dependent excitabil-
ity and potentiation of NaV current in these sensory neurons could be
reversed by the addition of CBZ, a known inhibitor of several NaV currents.
We then compared the efficacy between CBZ and morphine as independent
agents, to the combined treatment of both drugs simultaneously, in the
tibial nerve injury (TNI) model of neuropathic pain. The potent anti-
nociceptive effects of morphine (5 mg/kg, i.p.) were observed in TNI
rodents at postinjury day (PID) 714 and absent at PID 2128, while admin-
istration of CBZ (10 mg/kg, i.p.) alone failed to produce antinociceptive
effects at any time following TNI (PID 728). In contrast to either drug
alone at PID28, the combination of morphine and CBZ completely atten-
uated tactile hyperalgesia in the rodent TNI model. The basis for the
potentiation of morphine in combination with CBZ may be due to the
effects of a latent upregulation of NaV1.7 in the DRG following TNI.
Taken together, our observations demonstrate a potential therapeutic
use of morphine and CBZ as a combinational treatment for neuropathic
pain [176].
In the present study, a sensitive and accurate isotope dilution method
was developed for the simultaneous determination of 17 polar pharmaceu-
tical and personal care product (PPCP) residues (log Kow 1.405.74),
including 14 pharmaceuticals and 3 personal care products, in biological
organs and tissues. The proposed method involved enzymatic hydrolysis,
followed by sequential cleanup using silica-gel chromatography and gel
permeation chromatography, and analysis via ultra-HPLC with tandem
MS. This method yielded acceptable absolute recoveries (4888%) and
internal standard-corrected recoveries (90130%) for 17 PPCPs. MDLs
were between 0.0092 and 3.2 ng/g wet weight, and the LOQs were
between 0.020 and 8.7 ng/g wet weight. The method can be used to read-
ily detect the target compounds at trace levels while minimizing the
required sample volume. The developed method was applied to the deter-
mination of 17 PPCPs in the liver and kidney of 17 birds collected from
Japan and also in the plasma, liver, and brain of 7 cyprinoid fish from an
effluent-dominated stream in Japan. Triclosan was detected in 5 of 11 fish-
eating birds but not in nonfish-eating birds, suggesting the contamination
of prey fish by the chemical. Nonsteroidal antiinflammatory drugs,
antibacterial agents, and psychotropic agents were frequently detected in
the fish tissues. In addition, seven of the target compounds were found
in fish brain. The median brain/plasma ratios of the psychotropic agents
216 S.T. Alrashood

ranged from 1.6 (carbamazepine) to 12 (diphenhydramine), indicating high

transportability to fish brain [177].
Many people with schizophrenia do not achieve a satisfactory treatment
response with just antipsychotic drug treatment, and various adjunct med-
ications are used to promote additional response. The antiepileptic carba-
mazepine is one such drug. So in order to examine whether
carbamazepine or oxcarbazepine alone is an effective treatment for schizo-
phrenia and schizoaffective psychoses and whether carbamazepine or
oxcarbazepine augmentation of neuroleptic medication is an effective treat-
ment for the same illnesses, The Cochrane Schizophrenia Groups Register
of Trials (Dec. 2001), The Cochrane Library (Issue 3, 2001), MEDLINE
(19662001), EMBASE (19802001), Biological Abstracts (19802001),
PsycLIT (18862001), and PSYNDEX (19742001) were searched. For
the most recent update, the Cochrane Schizophrenia Groups Register of
Trials in Jul. 2012 was investigated. In addition, references of all identified
studies for further trials and contacted relevant pharmaceutical companies
and authors for additional data were inspected. All randomized controlled
trials (RCTs) comparing carbamazepine or compounds of the carbamaze-
pine family with placebo or no intervention, whether as sole treatment or
as an adjunct to antipsychotic medication for the treatment of schizophrenia
and/or schizoaffective psychoses, were included. Data were extracted inde-
pendently. For homogeneous dichotomous data, fixed-effect, risk ratio
(RR), with 95% CIs on an intention-to-treat basis were calculated. For con-
tinuous data, MDs were calculated. The risk of bias for the present studies
was assessed. It was found that the updated search did not reveal any further
studies that met the inclusion criteria. The number of included studies there-
fore remains at 10 with the number of participants randomized still 283.One
study comparing carbamazepine with placebo as the sole treatment for
schizophrenia was abandoned early due to high relapse rate with 26 of 31
participants relapsing by 3 months. No effect of carbamazepine was evident
with no difference in relapse between the two groups (1 RCT n 31, RR
1.07 CI 0.781.45). Another study compared carbamazepine with antipsy-
chotics as the sole treatment for schizophrenia. No differences in terms of
mental state were found when comparing 50% reduction in Brief Psychiatric
Rating Scale (BPRS) scores (1 RCT n 38, RR 1.23 CI 0.781.92). A
favorable effect for carbamazepine was found when more people who
received the antipsychotic (perphenazine) had parkinsonism (1 RCT
n 38, RR 0.03 CI 0.000.043). Eight studies compared adjunctive carba-
mazepine vs adjunctive placebo. Adding carbamazepine to antipsychotic
Carbamazepine 217

treatment was as acceptable as adding placebo with no difference between

the numbers leaving the study early from each group (8 RCTs n 182,
RR 0.47 CI 0.161.35, very low quality evidence). Carbamazepine
augmentation was superior compared with antipsychotics alone in
terms of overall global improvement, but participant numbers were low
(2 RCTs n 38, RR 0.57 CI 0.370.88). There were no differences for
the mental state outcome of 50% reduction in BPRS scores (6 RCTs
n 147, RR 0.86 CI 0.671.12, low-quality evidence). Less people in
the carbamazepine augmentation group had movement disorders than
those taking haloperidol alone (1 RCT n 20, RR 0.38 CI 0.141.02).
No data were available for the effects of carbamazepine on subgroups of
people with schizophrenia and aggressive behavior, negative symptoms,
or EEG abnormalities or with schizoaffective disorder. Based on the
currently available randomized trial-derived evidence, it was concluded
that carbamazepine cannot be recommended for routine clinical use for
treatment or augmentation of antipsychotic treatment of schizophrenia.
At present, large, simple well-designed and reported trials are justified
especially if focusing on people with violent episodes and people with
schizoaffective disorders or those with both schizophrenia and EEG
abnormalities [178].
Previously, a gene expression signature in rat liver for detecting a specific
type of oxidative stress (OS) related to reactive metabolites (RM) was
reported. High doses of the drugs disulfiram, ethinyl estradiol, and
nimesulide were used with another dozen paradigm OS/RM compounds,
and three other drugs flutamide, phenacetin, and sulindac were identified by
this signature. In a second study, AEDs were compared for covalent binding
and their effects on OS/RM; felbamate, carbamazepine, and phenobarbital
produced robust OS/RM gene expression. In the present study, liver RNA
samples from drug-treated rats from more recent experiments were exam-
ined for statistical fit to the OS/RM signature. Of all 97 drugs examined,
in addition to the nine drugs noted earlier, 19 more were identified as
OS/RM-producing compoundschlorpromazine, clozapine, cyproterone
acetate, dantrolene, dipyridamole, glibenclamide, isoniazid, ketoconazole,
methapyrilene, naltrexone, nifedipine, sulfamethoxazole, tamoxifen, cou-
marin, ritonavir, amitriptyline, valproic acid, enalapril, and chlorampheni-
col. Importantly, all of the OS/RM drugs listed earlier have been linked to
idiosyncratic hepatotoxicity, except chloramphenicol, which does not have
a package label for hepatotoxicity, but does have a black box warning for
idiosyncratic bone marrow suppression. Most of these drugs are not acutely
218 S.T. Alrashood

toxic in the rat. The OS/RM signature should be useful to avoid idiosyn-
cratic hepatotoxicity of drug candidates [179].
Concerns are being expressed recently over possible environmental
effects of human pharmaceuticals. Although the likelihood of acute toxicity
is low, the continuous discharge of pharmaceuticals into the aquatic envi-
ronment means that sublethal effects on nontarget organisms need to be seri-
ously considered. One-year-old Atlantic salmon parr were exposed to
7.85  0.13 g/L of the antidepressant drug CBZ for 5 days to investigate
the changes of mRNA expression in the brain by means of a custom 17k
Atlantic salmon cDNA microarray. The selected concentration is similar
to upper levels that can be found in hospital and STP effluents. After treat-
ment, 373 features were differently expressed with 26 showing up- or
downregulation of 2-fold (P  0.05). Among the mRNAs showing the
highest change were the pituitary hormones encoding features somatolactin,
prolactin, and somatotropin, or growth hormone. Functional enrichment
and network analyses of up- and downregulated genes showed that CBZ
induced a highly different gene expression profile in comparison to
untreated organisms. CBZ induced expression of essential genes of the focal
adhesion and extracellular matrixreceptor interaction pathways most likely
through integrin alpha-6 (itga6) activation. Negative regulation of apoptotic
process, extracellular matrix organization, and heme biosynthesis were the
most enriched biological process-related GO terms, with the simultaneous
enrichment of collagen and extracellular region-related cellular component
GO terms, and extracellular matrix structural constituent, hormone activity,
and chromatin binding molecular function-related GO terms. These results
show that relatively low doses of CBZ may affect brain physiology in
exposed salmon parr, targeting similar processes as in human, indicating a
high degree of conservation of targets of CBZ action. However, and since
the mRNAs showing most changes in expression are critical for adaptation
to different stressors and life history transitions in Atlantic salmon, more
research should be undertaken to assess CBZ effects to avoid impairment
of normal development and maintenance of natural populations [180].
SCN1A encodes the alpha subunit of the voltage-gated sodium channel
and plays a crucial role in several epilepsy syndromes. The common SCN1A
splice-site polymorphism rs3812718 (IVS5N + 5 G > A) might contribute to
the pathophysiology underlying genetic generalized epilepsies and is associ-
ated with electrophysiological properties of the channel and the effect of
sodium channel-blocking AEDs. The effects of the rs3812718 genotype
on cortical excitability at baseline and after administration of carbamazepine
Carbamazepine 219

in order to investigate the mechanism of this association were assessed.

Paired-pulse transcranial magnetic stimulation (TMS) was applied in 92
healthy volunteers with the homozygous genotypes AA or GG of
rs3812718 at baseline and after application of 400 mg of carbamazepine
or placebo in a double-blind, randomized, crossover design. Resting motor
threshold (RMT), short interval intracortical inhibition (SICI), intracortical
facilitation (ICF), and cortical silent period (CSP) were determined. At base-
line, there was no significant difference in any TMS parameter. Genotype
GG was associated with a higher carbamazepine-induced increase in CSP
duration as compared to AA (multivariate analysis of covariance [MAN-
COVA], P 0.013). An expected significant increase in RMT was geno-
type independent. It was found that the rs3812718 genotype modifies the
effect of carbamazepine on CSP duration (mainly reflecting modulation
of -aminobutyric acid (GABA)ergic inhibition), but not on RMT (mainly
reflecting modulation of voltage-gated sodium channels). This provides evi-
dence that rs3812718 affects the pharmacoresponse to carbamazepine via an
effect on GABAergic cortical interneurons. The results also confirm that
TMS is useful to investigate the effect of genetic variants on cortical excit-
ability and pharmacoresponse [181].
A considerable heterogeneity exists in the literature on the role of differ-
ent HLA alleles in CBZ-induced cutaneous adverse drug reactions (cADRs)
of varying severity among diverse ethnic groups. The aim of the present
study was to understand and summarize this heterogeneity and evaluate
the contribution of common HLA alleles to susceptibility to cADRs in
patients treated with CBZ through a meta-analysis. A literature search of
Embase, Medline, Web of Knowledge, and Cochrane Database of System-
atic Reviews was performed up to 28 Sep. 2013. A total of 20 reports were
identified as eligible studies, which included 720 CBZ-intolerant [SJS/TEN
(bullous lesions): n 277; hypersensitivity syndrome/maculopapular
exanthema (nonbullous lesions): n 359; others: n 84], 1512 CBZ-
tolerant, and 1113 normal controls. It was observed HLA-A*3101 and
HLA-B*1502 as risk markers and HLA-B*4001 as a protective marker
for susceptibility to cADRs when comparing intolerant with tolerant
patients. Stratification by clinical outcome showed HLA-B*1502 and
HLA-B*1511 as risk and HLA-A*2402 as protective markers for
bullous lesions in the Asians [HLA-B*1502: odds ratio (OR) 80.70;
95% CI 45.62142.77; P 1.8  1051; I(2) 33%, HLA-B*1511:
OR 17.43; 95% CI 3.1297.40; P 1.1  103; I(2) 0%, HLA-
A*2402: OR 0.27; 95% CI 0.110.64; P 2.7  103; I(2) 0%].
220 S.T. Alrashood

Furthermore, HLA-A*3101 was observed to be a universal risk marker,

irrespective of cADR type [OR (bullous lesions) 5.65; 95% CI 2.70
11.78; P 4.03  106; I(2) 49%, OR (nonbullous lesions) 8.58; 95%
CI 5.5513.28; P 4.46  1022; I(2) 0%]. Sensitivity analysis showed
HLA-B*4001 as a protective marker in Chinese population for showing
bullous lesions (OR 0.14; 95% CI 0.060.32; P 3.2  106; I(2)
0%). In summary, the meta-analysis showed the presence of HLA alleles
contributing toward risk of as well as protection against various CBZ-
induced cADRs [182].
Biomarkers are useful tools as indicators/predictors of disease severity
and drug responsiveness and, thus, are expected to make drug development
more efficient and to accelerate proper use of approved drugs. Many aca-
demic achievements on biomarkers have been reported, but only several
biomarkers are used in drug development and clinical settings. The results
were first shown on the pharmacogenomic analysis of the anticancer drug
irinotecan and of SJS/TEN. UGT1A1*6 and *28 were significantly associ-
ated with altered pharmacokinetics of an irinotecan metabolite, SN-38, and
with increased frequency of severe neutropenia. HLA*58:01 and HLA-
B*15:11/HLA-A*31:01 were associated with SJS/TEN by allopurinol
and carbamazepine, respectively. In addition to these genomic biomarkers,
metabolomic biomarkers, which can reflect the disease phenotype and drug
responsiveness, have been exploring for 12 major diseases in Japan, as a part
of a multiomics team with multinational centers. In animal models of dilated
cardiomyopathy and Alzheimers disease, it was found several changes in
lipid metabolite levels in the diseased tissues. Moreover, two oxidized fatty
acids were correlatively changed in the brain and plasma from Alzheimers
model mice before its onset, and thus, could be candidates for predictive bio-
markers. Finally, several key issues for academic researches on biomarker dis-
covery and development, especially for newly coming researchers in the
field of pharmaceutical sciences, were proposed/discussed. We hope that
this review would help novel biomarker identification and qualification
in Japan [183].
Increased studies that reported genetic susceptibility to drug hypersensi-
tivity reactions, as exemplified by the HLA-A*31:01 and HLA-B*15:02
association with CBZ-induced hypersensitivity reactions, such as mac-
ulopapular exanthema (MPE), drug rash with eosinophilia and systemic
symptoms (DRESS), and SJS/TEN were observed. This studys objective
is to carry out a comprehensive analysis on the clinical spectrum and
HLA genotypephenotype correlations in CBZ-induced hypersensitivity
Carbamazepine 221

reactions. The clinical information of 194 patients with CBZ hypersensitiv-

ity (51 MPE, 23 DRESS, 112 SJS/TEN, and 8 cases with other phenotypes),
and 152 CBZ-tolerant controls were analyzed. All are Han Chinese. The
HLA-A/HLA-B genotypes, gene dosage, and drug dosage effects were
examined. CBZ-SJS/TEN showed the strongest association with the
HLA-B*15:02 allele (P(c) 5.8  1043; odds ratio (OR) (95% CI)
97.6 (42.0226.8)), in which HLA-B*15:02 was identified in all patients
(25/25) with SJS/TEN with >5% body surface area (BSA) skin detachment,
but lost its 100% association (85.1%, 74/87) in SJS with <5% BSA detach-
ment. In contrast, HLA-B*40:01 showed negative association with CBZ-
induced SJS/TEN (P(c) 8.3  105; OR (95% CI) 0.22 (0.10.4)). By
comparison, CBZ-induced MPE/DRESS had no association with HLA-
B*15:02, but linked to HLA-A*31:01 (P(c) 2.7  103; OR (95%
CI) 6.86 (2.419.9)), and HLA-B*51:01 (P(c) 0.01; OR (95% CI)
4.56 (2.010.5)). No gene dosage or CBZ dosage effects were observed.
Finally, this study reported the different strengths of HLA association with
CBZ hypersensitivity in Han Chinese. With the increasing application of
pharmacogenetic markers, the HLA genotypephenotype correlations
and the results of the test need to be carefully interpreted for CBZ-induced
hypersensitivity reactions [184].
A simple, rapid, and efficient method, based on surfactant assisted dis-
persive liquidliquid microextraction (SA-DLLME), followed by HPLC
has been developed for simultaneous preconcentration and trace detection
of ZNS and carbamazepine in biological samples. A conventional cationic
surfactant called cetyltrimethyl ammonium bromide (CTAB) was used as a
disperser agent in the proposed approach. 1.5 mL of CTAB (0.45 mmol/L)
(disperser solvent) containing 50.0 L of 1-octanol (extraction solvent) was
injected rapidly into 7.0 mL of water or diluted plasma or urine. A cloudy
solution (water, 1-octanol, and CTAB) was formed in the test tube. After
the formation of cloudy solution, the mixture was centrifuged and 20 L of
the collected phase was injected into HPLC for subsequent analysis. Some
parameters such as the type and volume of the extraction solvent, the type
and concentration of surfactant, pH, ionic strength, and centrifugation time
were evaluated and optimized. Under optimum extraction conditions, the
LODs were 2.1 and 1.5 g/L (based on 3Sb/m) for urine samples, and 2.3
and 1.6 g/L for plasma samples. Linear dynamic range of 5300 and 5
200 g/L was obtained for ZNS and carbamazepine in all samples. Finally,
the applicability of the proposed method was evaluated by extraction and
determination of the drugs in urine and plasma samples [185].
222 S.T. Alrashood

Interactions of the drug carbamazepine with the serum protein alpha(1)-

acid glycoprotein (AGP) were examined by high-performance affinity
chromatography. Frontal analysis studies with an immobilized AGP column
and control column indicated that carbamazepine had both low-affinity
interactions with the support and high-affinity interactions with AGP.
When a correction was made for binding to the support, the association
equilibrium constant measured at pH 7.4 and 37C for carbamazepine
with AGP was 1.0 (0.1)  105 M1, with values that ranged from 5.1 to
0.58  105 M1 in going from 5 to 45C. It was found in competition
studies that these interactions were occurring at the same site that binds
propranolol on AGP. Temperature studies indicated that the change in
enthalpy was the main driving force for the binding of carbamazepine to
AGP. These results provide a more complete picture of how carbamazepine
binds to AGP in serum. This report also illustrates how high-performance
affinity chromatography can be used to examine biological interactions
and drugprotein binding in situations in which significant interactions
for an analyte are present with both the chromatographic support and an
immobilized ligand [186].
Carbamazepine is metabolized to an active metabolite known as CBZ-E,
or simply the epoxide metabolite. The presence of this metabolite can
have clinically significant implications in TDM of carbamazepine, but
accurate quantification of the epoxide metabolite is currently limited to
chromatographic techniques. In this study, mathematical equations are
proposed for the estimation of carbamazepine and epoxide metabolite con-
centrations based on values generated by common carbamazepine immuno-
assays. Three immunoassays were studied: particle-enhanced turbidimetric
inhibition immunoassay (PETINIA, Siemens Healthcare Diagnostics,
Deerfield, IL), ADVIA Centaur (Siemens Healthcare Diagnostics), and a
cloned enzyme donor immunoassay (CEDIA; Roche, Indianapolis, IN).
Equations were based on observed cross-reactivity of epoxide with the
PETINIA (average, 96.2%; range, 86.6105.7%) and epoxide cross-reactiv-
ity with the ADVIA Centaur assay (average, 6.5%; range, 5.37.7%). In
addition, equations were developed using average cross-reactivity of
epoxide with the PETINIA and with the CEDIA. Values determined
by calculation correlated well with carbamazepine and epoxide concentra-
tions in supplemented and patient samples, for which values of
carbamazepine (2.212.0 g/mL [951 mol/L]) and the epoxide metabo-
lite (0.62.4 g/mL) were also verified by liquid chromatography-tandem
MS [187].
Carbamazepine 223

Carbamazepine is a potent inducer of drug-metabolizing enzymes,

which results in a number of clinically significant drugdrug interactions.
Deinduction occurs when long-term carbamazepine therapy is discon-
tinued. The goal of this study was to develop a population pharmacokinetic
(PPK) model to describe the time course of carbamazepine deinduction. Sta-
ble-labeled carbamazepine was administered intravenously on three occa-
sions during the deinduction period to 15 patients with epilepsy for
whom carbamazepine therapy was being discontinued. Data were analyzed
using a nonlinear mixed-effects model (NONMEM). An enzyme turnover
model consisting of a one-compartment model linked with a hypothetical
enzyme compartment was applied to characterize the time course of carba-
mazepine deinduction. Model evaluation was performed using the bootstrap
approach and a visual predictive check. In the final model, the deinduction
process was accomplished by decreasing the rate of enzyme synthesis,
resulting in a decrease in the relative amount of enzymes. The estimated rate
constant for enzyme degradation was 0.00805 h1, corresponding to a half-
life of the combined enzymes of 86.1 h (3.6 days). It was included an enzyme
turnover model adequately characterized the experimental data. Based on
the predicted enzyme half-life from the final model, the deinduction process
should be completed within 2 weeks after carbamazepine therapy is termi-
nated [188].
A newly developed high-throughput desorption electrospray ionization
(DESI) source was characterized in terms of its performance in quantitative
analysis. A 96-sample array, containing pharmaceuticals in various matrices,
was analyzed in a single run with a total analysis time of 3 min. These solu-
tion-phase samples were examined from a hydrophobic PTFE ink printed
on glass. The quantitative accuracy, precision, and LOD were
characterized. Chemical background-free samples of propranolol (PRN)
with PRN-d7 as internal standard (IS) and CBZ with CBZ-d10 as IS were
examined. So two other sample sets consisting of PRN/PRN-d7 at varying
concentration in a biological milieu of 10% urine or porcine brain total lipid
extract, were measured with total lipid concentration 250 ng/L. The back-
ground-free samples, examined in a total analysis time of 1.5 s/sample,
showed good quantitative accuracy and precision, with a relative error
(RE) and RSD generally less than 3% and 5%, respectively. The samples
in urine and the lipid extract required a longer analysis time (2.5 s/sample)
and showed RSD values of around 10% for the samples in urine and 4% for
the lipid extract samples and RE values of less than 3% for both sets. The
LOD for PRN and CBZ when analyzed without chemical background
224 S.T. Alrashood

was 10 and 30 fmol, respectively. The LOD of PRN increased to 400 fmol
when analyzed in 10% urine, and 200 fmol when analyzed in the brain
lipid extract [189].
The objective of the study was to prepare and evaluate carbamazepine-
loaded enteric microparticles produced by a novel coacervation method. An
aqueous polymeric stabilizer solution was added to an organic carbamaze-
pine/Eudragit L100-55 solution. Water, which is a nonsolvent for the drug
and the enteric polymer, caused phase separation and the formation of coac-
ervate droplets. These droplets hardened into microparticles upon further
addition of the aqueous phase. The microparticles were characterized with
respect to particle size distribution, morphology, encapsulation efficiency,
yield, physical state and physical stability of the drug, wettability, in vitro
release, and in vivo bioavailability. Microparticles with a smooth surface
and dense structure were obtained with high encapsulation efficiency
(>85%) and yield (>90%). The drug was in a noncrystalline state in the
matrix and physically stable for 5 months at room temperature. Under sink
conditions, the drug dissolution rate from the microparticles was signifi-
cantly enhanced compared to the physical mixture and to the pure drug;
the release profile of the microparticles was stable after 5 months. Under
nonsink conditions, an unstable supersaturated solution of carbamazepine
was obtained from microparticles with the subsequent formation of nee-
dle-shaped crystals. The high surface area and good wettability of the micro-
particles, the noncrystalline state of the drug in the matrix, and the fast
dissolution rate contributed to a significantly enhanced oral bioavailability
from the microparticles when compared to the physical mixture [190].
The genotoxic effect of CBZ has been investigated in few studies. There
is little evidence linking CBZ with any genotoxic effects, particularly in
vitro micronucleus test using cytogenesis-block technique. In this study,
the genotoxicity of the AED, carbamazepine, was tested using cytokine-
sis-block (CB) micronucleus assay. In vitro analysis was performed in human
blood lymphocytes from four healthy persons at five different concentrations
of carbamazepine (6, 8, 10, 12, and 14 g/mL). Genotoxic potential and
cytotoxic effects of carbamazepine were evaluated by using micronucleus
assay and CB proliferation index, called the parameter of cytotoxicity in
human peripheral blood lymphocyte cultures, respectively. The results of
this study indicate that CBZ caused the genotoxic effect under in vitro con-
ditions, except at the dose of 6 g/mL, and cytotoxic effects of carbamaz-
epine were revealed by a decrease in the CB proliferation index at all the
concentrations [191].
Carbamazepine 225

The in vitro dissolution of CBZ was investigated using an automated

artificial stomachduodenum (ASD) model. Successful simulation of the
dog physiology in the fasted state showed that the rank order of the ASD
estimated bioavailabilities is as follows: Form III > Form I > dihydrate. This
result is in excellent agreement with those found in the literature. Additional
simulations comparing different gastric transit times during fasted and fed
states are also discussed [192].
The occurrence of pharmaceutically active compounds in the aquatic
environment has been recognized as one of the emerging issues in environ-
mental chemistry. However, the ecotoxicological effects of pharmaceuticals
have still not been researched adequately. Carbamazepine, which is com-
monly present in surface and groundwater, was studied using 6 ecotoxico-
logical model systems with 18 endpoints evaluated at different exposure time
periods. The battery included the immobilization of D. magna, biolumines-
cence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga
Chlorella vulgaris, and micronuclei induction and root growth inhibition in
the plant Allium cepa. Cell morphology, neutral red uptake, total protein
content, MTS metabolization, lactate dehydrogenase leakage, and activity
and glucose-6-phosphate dehydrogenase activity were studied in the salmo-
nid fish cell line RTG-2. The total protein content, LDH activity, neutral
red uptake, and MTT metabolization in Vero monkey kidney cells were also
investigated. The most sensitive system to carbamazepine was the Vero cell
line, followed by C. vulgaris, V. fischeri, D. magna, A. cepa, and RTG-2 cells.
EC50 values from 19 M in Vero cells at 72 h to more than 1200 M in
other systems were obtained. Comparing the concentrations in water and
the toxicity quantified in our assay systems, carbamazepine is not expected
to produce acute toxic effects in the aquatic biota under these circumstances,
but chronic and synergistic effects with other chemicals cannot be excluded
[193].
The present work extended previous physicochemical investigations on
the effects of solid dispersion on the solubility, the dissolution rate, and the
pharmacokinetic profile of carbamazepine. Solubility studies showed a linear
increase in carbamazepine solubility with the increase of PEG 6000 concen-
tration. There is no marked difference between physical mixtures and solid
dispersions for the enhancement of carbamazepine solubility by PEG 6000.
Less than 60% of pure carbamazepine was dissolved in 90 min. Physical mix-
tures (carbamazepine phase III), solid dispersions (carbamazepine phase II),
and dissolution rates were higher compared to the parent drug. The disso-
lution of carbamazepine phase III was more pronounced than that evoked by
226 S.T. Alrashood

the phase II. The dissolution profiles indicated that the percentage of the
drug dissolved was dependent on the proportion of PEG 6000. In solid dis-
persions, there was a remarkable enhancement in the dissolution rates of the
drug in the vicinity of the eutectic composition as compared with those of
corresponding physical mixtures. Hence, the optimum value for the solid
dispersion was 80.5  1.7% of carbamazepine having dissolved within the
first 10 min compared to 40  1% for the corresponding physical mixtures
of the same composition. Statistical analysis of pharmacokinetic parameters
confirmed that the carbamazepine:PEG 6000 binary systems displayed
higher bioavailability of the drug than the pure carbamazepine. The area
under the curve (AUC) values highlighted the evidence that only slight dif-
ferences in the bioavailability of the drug occur between physical mixtures
and solid dispersions prepared at the 80:20 and 50:50 drug:carrier compo-
sitions. However, the mean normalized plasma concentrations showed that
standard error deviations are rather wide intervals for pure drug and physical
mixtures in comparison to solid dispersions. One additional interesting point
to consider is the disappearance of the multiple peaks on the individual
kinetic curves of the 50:50 solid dispersion composition. Furthermore,
the investigations have highlighted the interest of solid dispersions prepared
at <<near>>-eutectic composition as our preliminary data show that the
plasma concentration (C(5 h)) of the drug for the 15:85 dispersed sample
containing 150 mg of carbamazepine is not significantly different from that
obtained for the 50:50 dispersed sample containing 300 mg of the drug
[194].
Although opposite mood and psychomotor disturbances usually occur in
mania and melancholia, clinical features may also be shared in common or
may be present at the same time in both phases of manic-depressive psycho-
sis. In a parallel fashion, most pharmacological agents are selectively effective
in one mood phase (tricyclics and monoamine oxidase inhibitors for depres-
sion, and neuroleptics for mania) and frequently precipitate or exacerbate the
opposite phase. These agents, therefore, may be affecting biological sub-
strates mediating the opposing phase of affective illness. With the exception
of electroconvulsive therapy and lithium chemotherapy, few treatments are
effective in both mania and melancholia. It is noteworthy, therefore, that
carbamazepine may be useful in the acute and prophylactic treatment of
mania and melancholia, including some lithium nonresponders and patients
vulnerable to tricyclic-induced mood switches. The clinical and biolog-
ical effects of carbamazepine will be discussed with special emphasis on its
biochemical action and the possible mechanisms by which it might influence
Carbamazepine 227

biological substrates mediating both phases of manic-depressive illness. In

addition, the theoretical implications of the presence of both shared and
opposing clinical, pharmacological, and biochemical characteristics of the
illness will be discussed [195].
Reagents are now available for the measurement of a range of AEDs by
fluorescence polarization immunoassay using the Roche Cobas Fara II Ana-
lyzer. Evaluation data are presented that demonstrate that these assays rep-
resent a convenient, cost-effective, and analytically reliable alternative to
other commercially available systems for the measurement of drugs during
TDM. The use of a general-purpose analyzer, the opportunity to perform
different drug assays simultaneously, and long calibration stability confer sig-
nificant benefits on these methods. These are especially applicable for the
laboratory that undertakes only small or moderate numbers of such investi-
gations during TDM [196].
Five therapeutic drug assays, carbamazepine, phenobarbital, phenytoin,
theophylline, and valproic acid, were evaluated using an automated random
access system for performing thin dry film multilayer competitive immuno-
assays, the OPUS analyzer. All reagents for the therapeutic drug assays are
contained in a coated multilayer film chip encased within a plastic bar-coded
test module and require no external or supplementary reagents. A serum or
plasma sample is applied to the test module by the instrument and the fluo-
rescence intensity from the module is measured after 6 min. It was found the
OPUS assays acceptable for clinical use. Within-run coefficient of variations
was 2.36.7%, between-run, 2.97.6%. These methods correlated well with
the Abbott TDx, having correlation coefficients of 0.920.97. Because of
the instrument design and the stability of the reagents, weekly calibration
is not needed and samples can be run immediately upon receipt in a random
access fashion or can be batched together [197].
Performance characteristics of substrate-labeled fluorescent immunoas-
says for the drugs phenytoin, phenobarbital, primidone, carbamazepine, the-
ophylline, gentamicin, tobramycin, amikacin, and quinidine run on an
Optimate automated fluorometric analyzer were compared with those of
automated EMITs for the same drugs performed on a Cobas centrifugal ana-
lyzer for patient samples and controls. For 100 patient samples assayed by
both systems for each drug, excellent correlations were obtained, with cor-
relation coefficients ranging from 0.96 to 0.99. Likewise, very good within-
run (n 10) and between-run (n 30) precision was obtained by both
methods. Values for controls and clinical specimens by the Optimate
methods calculated using the same-day calibration curves were not
228 S.T. Alrashood

significantly different from those calculated from calibration curves stored 14

days, indicating at least 14-day curve stability for all assays [198].
The analytical performance of a new automated substrate-labeled fluo-
rescence immunoassay system (Ames TDA/Optimate) was evaluated by
comparative TDM of carbamazepine, phenobarbital, phenytoin, theophyl-
line, and quinidine. Using Optimate, the coefficients of variation of the
within- and between-run variability in the lower therapeutic range were
4.8% and 4.7%, respectively, for carbamazepine (by HPLC: 4.3% and
8.2%), 4.5% and 7.2%, respectively, for phenytoin (by GLC: 1.9% and
6.2%), 2.4% and 2.9%, respectively, for phenobarbital (by GLC: 11.4%
and 14.7%), 3.2% and 4.6%, respectively, for theophylline (by HPLC: 7.2
and 12.5%), and 3.0% and 22.8%, respectively, for quinidine (by fluorom-
etry: 3.0% and 4.2%). Stored calibration curves in connection with a nor-
malization factor could be used for at least 4 weeks for the measurement
of human specimens. Linear regression analysis of parallel therapeutic
drug-level monitoring demonstrated good agreement between the
Optimate (y) and comparative technique (x) for each drug. The corres-
ponding expressions for carbamazepine are y 0.85x + 0.49 (r 0.09,
n 44), for phenobarbital y 0.99x + 0.75 (r 0.98, n 45), for phenytoin
y 0.97x  0.35 (r 0.99, n 50), for theophylline y 1.0x + 0.43
(r 0.99, n 44), and for quinidine y 0.94x + 0.11 (r 0.96, n 32) [199].
The Abbott TDx is a fully automated method for drug-level monitoring.
The system consists of a competitive FPIA performed by a microprocessor-
controlled fluorometer with integral pipetting and data reduction systems.
The method for the stat measurement of phenobarbital (PHENO), phenyt-
oin (PTN), carbamazepine (CARB), and theophylline (THEO) was evalu-
ated. The threshold of detection is PHENO, 0.8 mg/L; PTN, 0.8 mg/L;
CARB, 0.1 mg/L; and THEO, 0.4 mg/L. Between-run precision in the
therapeutic range expressed as CV is between 3.3% and 9.1% for all four
drugs. Recovery of each drug from a variety of matrices was essentially
100%, with no significant interference by elevated levels of bilirubin, triglyc-
erides, or hemoglobin. The TDx methods were correlated to established
methods for 100 patient specimens: PHENO: Y(TDx) 0.95 (GLC)
+ 1.4 mg/L R 0.98, PTN: Y(TDx) 0.91 (GLC) + 1.1 mg/L R 0.99,
CARB: Y(TDx) 0.91 (EMIT) + 0.4 mg/L R 0.98, THEO: Y(TDx)
0.93 (HPLC) + 0.5 mg/L R 0.99 The calibration stability is no shorter
than 1 week (PTN) and as long as 5 weeks (CARB). The analytic system
is an accurate, precise, and highly stable method for the stat measurement
of these four drugs [200].
Carbamazepine 229

6. STABILITY
The aims of this study were to characterize the alterations in total and
free CBZ and in total and free carbamazepine epoxide (CBZ-EPO) clear-
ances during pregnancy, to calculate the change in free fractions of CBZ and
CBZ-EPO during pregnancy, and to determine whether seizure worsening
is associated with a low ratio to nonpregnant baseline concentration of total
or free CBZ or CBZ-EPO. Women on CBZ were enrolled before concep-
tion or during pregnancy in this prospective, observational study. Concom-
itant medications and seizure frequency were recorded. Serum total and free
CBZ and CBZ-EPO were collected at each visit. Changes in the clearance
of all four compounds and free fractions of CBZ and CBZ-EPO were com-
pared with nonpregnant baseline. During pregnancy, the ratios to baseline
concentrations of total and free CBZ and CBZ-EPO were compared for
months with and without increased seizure frequency. Total and free
CBZ and CBZ-EPO clearances were calculated in 15 pregnancies in 12
women. Clearances did not change for any of these compounds during
pregnancy. The free fraction of CBZ increased from 0.23 at baseline to a
maximum of 0.32 in the third trimester (P 0.008). In the six women on
CBZ monotherapy with adequate seizure diaries and blood sampling, sei-
zure worsening did not correspond to a ratio to baseline concentration of
less than 0.65 for total or free CBZ or CBZ-EPO. In conclusion, total
and free CBZ and CBZ-EPO clearances did not change substantially during
pregnancy, and seizure frequency worsening was not associated with
decreased concentrations of total or free CBZ; therefore, TDM may not
be necessary for all women on CBZ during pregnancy. Further studies
with larger sample sizes are needed before definitive recommendations
can be made. Carbamazepine monotherapy may be a relatively safe and
cost-effective treatment option for women with focal epilepsy syndromes
during pregnancy [201].
This study investigates the application of hot-melt extrusion for the for-
mulation of (CBZ solid dispersions, using polyethylene glycolpolyvinyl
caprolactampolyvinyl acetate graft copolymer (Soluplus, BASF, Germany)
and polyoxyethylenepolyoxypropylene block copolymer (Poloxamer
407). In agreement with the current Quality by Design principle, formula-
tions of solid dispersions were prepared according to a D-optimal mixture
experimental design, and the influence of formulation composition on
the properties of the dispersions (CBZ heat of fusion and release rate) was
230 S.T. Alrashood

estimated. Prepared solid dispersions were characterized using differential

scanning calorimetry, attenuated total reflectance infrared spectroscopy,
and hot stage microscopy, as well as by determination of the dissolution rate
of CBZ from the hot-melt extrudates. Solid dispersions of CBZ can be suc-
cessfully prepared using the novel copolymer Soluplus. Inclusion of Pol-
oxamer 407 as a plasticizer facilitated the processing and decreased the
hardness of hot-melt extrudates. Regardless of their composition, all hot-
melt extrudates displayed an improvement in the release rate compared to
the pure CBZ, with formulations having the ratio of CBZ:Poloxamer
407 1:1 showing the highest increase in CBZ release rate. It was concluded
that interactions between the mixture components (CBZ and polymers), or
quadratic effects of the components, play a significant role in overall influ-
ence on the CBZ release rate [202].
Although pharmaceuticals have been detected in the environment only in
the range from ng/L to g/L, it has been demonstrated that they can adversely
affect the health status of aquatic organisms. Lysosomal membrane stability
(LMS) has previously been applied as an indicator of cellular well-being to
determine health status in bivalve mussels. The objective of this study is to
evaluate LMS in Ruditapes philippinarum hemolymph using the neutral red
retention assay. Clams were exposed in laboratory conditions to caffeine
(0.1, 5, 15, 50 g/L), ibuprofen (0.1, 5, 10, 50 g/L), and carbamazepine
and novobiocin (both at 0.1, 1, 10, 50 g/L) for 35 days. Results show a
dose-dependent effect of the pharmaceuticals. The neutral red retention time
measured at the end of the bioassay was significantly reduced by 50% after
exposure to environmental concentrations (P < 0.05) (caffeine 15 g/L;
ibuprofen 10 g/L; carbamazepine 1 g/L; and novobiocin 1 g/L),
compared to controls. Clams exposed to these pharmaceuticals were con-
sidered to present a diminished health status (retention time < 45 min),
significantly worse than controls (96 min) (P < 0.05). The predicted no envi-
ronmental effect concentration results showed that these pharmaceuticals are
very toxic at the environmental concentrations tested. Measurement of
the alteration of LMS has been found to be a sensitive technique that
enables evaluation of the health status of clams after exposure to pharmaceu-
ticals under laboratory conditions, thus representing a robust Tier-1 screening
biomarker [203].
To assess the physical, chemical, and microbiological stability of two
oral suspensions of carbamazepine at concentrations of 2.5% and 5%,
both oral suspensions were compounded from powdered carbamazepine
and Ora-Sweet SF and Ora-Plus commercial compounding excipients.
Carbamazepine 231

At the 2-, 4-, and 6-month marks, different quality assays were performed,
comprising physical (pH, state of the suspension, organoleptic properties),
chemical (HPLC), and microbiological assays. Results: The final concentra-
tion at 6 months for both the 2.5% and 5% carbamazepine suspensions was
22.9 and 45.9 mg/mL, respectively, with calculated richness values between
90% and 110% fulfilling USP23 NF18 requirements. No changes in physical
properties and no culture growth were observed during the study period.
Both oral suspensions are physically, chemically, and microbiologically
stable for at least 6 months when preserved at room temperature in amber
glass flasks [204].
The focus of this investigation was to prepare the cocrystal of CBZ using
nicotinamide as a coformer and to compare its preformulation properties and
stability profile with CBZ. The cocrystal was prepared by solution cooling
crystallization, solvent evaporation, and melting and cryomilling methods.
They were characterized for solubility, intrinsic dissolution rate, chemical
identification by Fourier transform infrared spectroscopy, crystallinity by
differential scanning calorimetry, powder X-ray diffraction, and morphol-
ogy by scanning electron microscopy. Additionally, mechanical properties
were evaluated by tensile strength and Heckel analysis of compacts. The
cocrystal and CBZ were stored at 40C/94% RH, 40C/75% RH, 25C/
60% RH, and 60C to determine their stability behavior. The cocrystals
were fluffy, with a needle-shaped crystal, and were less dense than CBZ.
The solubility profiles of the cocrystals were similar to CBZ, but its intrinsic
dissolution rate was lower due to the high tensile strength of its compacts.
Unlike CBZ, the cocrystals were resistant to hydrate transformation, as rev-
ealed by the stability studies. Plastic deformation started at a higher compres-
sion pressure in the cocrystals than CBZ, as indicated by the high yield
pressure. In conclusion, the preformulation profile of the cocrystals was sim-
ilar to CBZ, except that it had an advantageous resistance to hydrate trans-
formation [205].
In this paper, the thermodynamics of the anhydrate/dihydrate carbamaz-
epine (CBZA/CBZH) in ethanolwater mixtures was studied by measuring
the solubility of anhydrate and dihydrate carbamazepine at 060C. Both
stable form solubility and metastable form solubility were measured, the lat-
ter with the assistance of Raman immersion probe. The thermodynamic
properties of the anhydrate/dihydrate system, such as the relative stability,
and enthalpy and entropy of dissolution, were estimated by plotting the
measured solubility data according to the vant Hoff equation. The
anhydrate/dihydrate carbamazepine showed an enantiotropic relationship
232 S.T. Alrashood

in the studied mixtures and temperature ranges. It was shown that at a certain
temperature, there was an equilibrium water activity value at which the
anhydrate and dihydrate carbamazepine were in equilibrium. This equilib-
rium water activity value depends significantly on the temperature. The
lower the temperature, the smaller is the water activity value needed to attain
equilibrium between anhydrate and dihydrate. The obtained results are use-
ful in determining crystallization parameters to achieve a desired anhydrate
or hydrate phase. The approach can be applied to other anhydrate and
hydrate systems [206].
There are few studies in the literature that deal with the effect of excip-
ients on the kinetics of vapor phase induced hydrateanhydrate phase trans-
formations. The main purpose of this study was to probe the phase stability
of hydrateanhydrate systems in the presence of hygroscopic and non-
hygroscopic excipients following exposure to either dehydrating or hydrat-
ing conditions. Physical mixtures and compacts of model hydrate formers
(theophylline and carbamazepine) and excipients (mannitol, microcrystal-
line cellulose (MCC), polyvinylpyrrolidone (PVP) K12 and K90) were
stored at 22C and varying relative humidities. Raman spectroscopy was
used to monitor the kinetics of transformation between hydrate and
anhydrate. In general, excipients were found either to have no effect or
to promote dehydration. For hydrate formation, excipients could accelerate,
retard, or have no influence on hydration kinetics. MCC was found to have
only minimal effects on either the dehydration or hydration kinetics of
model compounds, whereas mannitol enhanced dehydration but had little
effect on hydration. Different PVP grades showed a variety effects: PVPK12
greatly enhanced the dehydration of both theophylline monohydrate (MT)
and carbamazepine dihydrate (DC). PVPK90 also enhanced the dehydration
of DC, but had a negligible effect on MT. For hydrate formation, PVPK12
was found to have a retarding effect on theophylline anhydrous (AT) trans-
formation, but enhanced the conversion of carbamazepine anhydrous
(AC) to DC, PVPK90 also retarded the hydration of AT, but had no
effect on AC. Optical microscopy and X-ray powder diffraction studies
suggested that PVP (in particular K12), when stored at high RH, was
able to result in the partial dissolution of the active pharmaceutical ingre-
dient and hence changed the hydration process from a solid state to a solu-
tion-mediated transformation. In summary, the effect of excipients on the
kinetics of dehydration and hydration is complex and needs to be rational-
ized in terms of several excipient properties including physical state,
chemical composition, and the possibility of specific APIexcipient
Carbamazepine 233

interactions. It is concluded that a multitude of factors will dictate, and often

complicate, the final effect of excipients on the phase transformation kinetics
of hydrate formers [207].
The transition temperature, T(t), of polymorphs is estimated from both
their heats of solution and solubilities (or intrinsic dissolution rates) deter-
mined at any one temperature (eg, ambient). At a given temperature, T,
the enthalpy difference, H, between polymorphs, I and II, is equal to
the difference between their heats of solution, whereas the free energy dif-
ference, G, can be estimated by the equation, G  RT ln (c(I)/c(II)) or
G RT ln (J(I)/J(II)), where c is the solubility and J is the intrinsic dis-
solution rate. The entropy difference, S, is evaluated as (H  G)/T.
Because the heat capacity difference, C(p), between polymorphs is
small enough to be neglected, the transition temperature may be estimated
by the equation, T(t) H/S. The thermodynamic stability relationships
of the polymorphs (ie, whether they are enantiotropes or monotropes)
are predicted from the value of T(t) and the melting temperature. The T
(t) values for auranofin, carbamazepine, chloramphenicol palmitate, cyclo-
penthiazide, gepirone hydrochloride, lamivudine, MK571, premafloxacin,
sulfamerazine, sulfamethoxazole, sulfathiazole, and urapidil were calculated
from reported values of the heats of solution and solubilities (or dissolution
rates). The stability relationships deduced from the calculated values of T(t)
are in good agreement with those reported using other methods, such as
differential scanning calorimetry and interpretation of melting data [208].
The stability of therapeutic drugs in sera collected in Becton-Dickinson
Vacutainer serum separator SST tubes has been well studied. Although most
therapeutic drugs are stable, certain drugs such as phenytoin, carbamazepine,
and phenobarbital decrease in concentrations over a long storage time. To
circumvent this problem, Becton-Dickinson devised a new gel formulation.
The authors studied the stability of 14 commonly monitored drugs in sera
when stored on the new gel of the SST II tubes and compared the concen-
trations of drugs in sera stored in plain tubes (no gel), those stored in the old
SST tubes, and those stored in the SST II tubes containing a new serum sep-
arator gel. The concentrations of most drugs studied did not decline even
after 24 h of storage in SST II tubes. After storage for 7 days in SST II tubes,
the concentration of carbamazepine declined by 10% and that of phenytoin
decreased by 4%. This is a significant improvement over the existing tube,
where concentrations of several drugs declined with prolonged storage. The
authors conclude that new SST II tubes are effective in collecting blood for
TDM [209].
234 S.T. Alrashood

The effect of packaging and storage on CBZ tablets was examined using
Tegretol and Tegral, dispensed in strip seals, and Finlepsin, dispensed in bot-
tles. Tegretol and Tegral tablets were stored in their original strips at 40, 50,
and 60C for 6, 3, and 1 month, respectively, at 75% relative humidity
(RH). Also, tablets were removed from their strips, placed in bottles, and
exposed daily to 97% RH at 40C for 5 min for 30 days. Finlepsin tablets
were exposed to 97% RH at 25 or 40C for 1 month by removing bottle
caps daily for 5 min. Dissolution was used to assess in vitro tablet perfor-
mance, and HPLC was used to evaluate the chemical stability of CBZ.
Results show that Tegretol tablets were not affected by the tested stress con-
ditions. Tegral tablets, stored in their strips at 50 or 60C and 75% RH,
showed increased disintegration and dissolution. The effect of 40C/75%
RH for 6 months was similar to 1-month storage at 40C/97% RH; the tab-
lets hardened and dissolved less than fresh Tegral tablets. Removal of Tegral
tablets from their original strips resulted in only 7% dissolved in 60 min. For
Finlepsin, the effect of 97% RH at 40C was more profound than 97% RH
at 25C, but both conditions caused a decrease in dissolution, the extent of
which was dependent on tablet position in the bottle. Stressed CBZ tablets,
however, showed no change in the chemical stability of CBZ under all
tested conditions [210].
The stability of four commonly used anticonvulsant drugs, viz., valproic
acid, carbamazepine, phenytoin, and phenobarbital in whole blood was
investigated after storage at conditions simulating storage and transport from
outlying rural clinics. Storage conditions included 24 h at 2325C, 24 h at
37C, 48 h at 37C, and 48 h at 2325C. Valproic acid, carbamazepine,
and phenobarbital were stable for 48 h at both storage temperatures. Phenyt-
oin was stable at 2325C for 48 h. However, small but statistically signif-
icant decreases in phenytoin concentrations were observed in samples that
were stored for 24 h or longer at 37C. These changes may not be clinically
significant [211].
The tablet surface was evaluated without physical damage by means of
Fourier transform infrared reflection-absorption spectroscopy (FT-IR-
RAS) and colorimetric measurement (color difference, E) of the carba-
mazepine polymorphs I, II, and III, after photodegradation at two irradiation
intensities (3.0 and 12.0 J/cm2/s) under a near-UV fluorescent lamp. The
surface of sample pellets of all crystalline forms turned gradually from white
to yellow-orange upon exposure to light, and the discoloration rate of form
II was faster than that of forms I and III, indicating that form II was the most
Carbamazepine 235

unstable of the three. The major photoproducts were identified by HPLC,

NMR, and MS analyses. The carbamazepine content on the surface of the
tablet was determined based on the absorption at 1685 cm1 attributable to
C]O stretch vibration in the FT-IR-RAS spectra before and after irradi-
ation by a near-UV fluorescent lamp. The semilogarithmic plots of the
photodegradation profiles of the various polymorphs were straight lines,
including the induction period, indicating that degradation of the drug
on the surface followed first-order kinetics. The induction periods of all
forms were not significantly different. However, the degradation rate con-
stant of form II at 12.0 J/cm2/s was 5.1 and 1.5 times larger than those of
forms I and III, respectively [212].
The stability of carbamazepine in commercially available suspension
that had been repackaged in various single-dose containers was studied.
Carbamazepine suspension was repackaged in 2- and 8-mL aliquots in
amber glass vials, polypropylene vials, and amber polypropylene syringes,
and in 2-mL aliquots in amber glass oral syringes. Containers were stored
at room temperature and continuously exposed to fluorescent light for up
to 12 weeks. Samples from each container type and volume were assayed
for carbamazepine content by HPLC at various intervals during storage.
Carbamazepine concentrations in the samples were compared with the car-
bamazepine concentration in the original manufacturers container. The
pH of the samples was also determined, and the suspensions were inspected
for color, odor, and large particles. There was no significant decrease in
carbamazepine concentration of more than 10% in samples stored for up
to 8 weeks. After 12 weeks, significant decreases in concentration were
observed in all but one container type. No changes in color, odor, or
consistency were observed during the 12 weeks, and there were no sig-
nificant changes in pH. In commercially available suspension repackaged
in volumes corresponding to common pediatric doses, carbamazepine
(20 mg/mL) is stable for at least 8 weeks when stored at room temperature
in the containers tested [213].
The stability of therapeutic concentrations of 11 drugs (amikacin, carba-
mazepine, digoxin, gentamicin, lithium, methotrexate, phenobarbital,
phenytoin, quinidine, theophylline, tobramycin) and 2 trace elements (cop-
per and zinc) in plasma stored in serum separator (Corvac brand) blood
collection tubes was investigated over a 1-week period of storage in the
refrigerator. No significant change in concentration was noted for any ana-
lyte during the study period. Concentrations were also not significantly
236 S.T. Alrashood

different from those observed during concurrent storage of the same plasma
samples in nonserum separator (Vacutainer brand) blood collection tubes
[214].
Derivation of standard curves for the EMIT TDM system involves sev-
eral mathematical algorithms, all of which can be rewritten in the form of a
linear equation y mx + b. The stability of the standard curve in terms of
slope and y-intercept for three drug assays (procainamide, gentamicin,
and carbamazepine) by generating calibration curves intermittently for
periods as long as 90 days was examined. Controls at three concentrations
were assayed after each calibration to validate the standard curves. On the
basis of 98% CIs, the slopes of standard curves for procainamide, gentamicin,
and carbamazepine were stable for 89, 80, and 57 days, respectively. Control
values generated from standard-curve manipulations (adjustments to the y-
intercept) indicated consistent accuracy and precision throughout the entire
study, as compared with control values determined after each calibration.
The increased utility of the standard curve and reagents suggests that full rec-
alibration on a regular basis is not always necessary [215].
The stability of carbamazepine in four suspending vehicles is reported.
Suspensions of carbamazepine 200 mg/5 mL in sorbitol 70%, simple syrup,
modified Hospital of the University of Pennsylvania Suspending Vehicle
(HUP), and diluted HUP (HUP-A) were prepared. The first three suspen-
sions were stored in amber glass bottles and oral syringes at 4, 25, and 37C,
and the HUP-A suspension was stored at 4C. Physical stability was assessed
by visual inspection of sedimentation, ease of pouring, and foaming upon
shaking. Carbamazepine concentrations were determined periodically over
90 days by an EMIT. The assay was validated by acid-heat degradation of the
drug, separation of breakdown products by thin-layer chromatography, and
confirmation of nonreactivity of the breakdown products with the assay.
The concentration of carbamazepine in sorbitol 70%, simple syrup, and
HUP-A was at least 90% of the prepared concentration at all sampling times.
Although separation occurred, the simple syrup suspensions could be
redispersed. The suspension in HUP-A remained homogeneous, was easy
to pour, and produced less foam than the HUP suspension. Extemporane-
ously compounded suspensions of carbamazepine in HUP-A or in simple
syrup can be used for patients who require a liquid dosage form. Even
though sorbitol 70% produced a pharmaceutically acceptable product, its
use is not recommended because it has been reported to cause intractable
diarrhea [216].
Carbamazepine 237

7. PHARMACOKINETICS, METABOLISM, AND EXCRETION

7.1 Pharmacokinetics
CBZ is a leading molecule in the management of epilepsy. Surveys have rev-
ealed that a sufficient lack of therapeutically efficient CBZ transbuccal for-
mulation exists. Therefore, this investigation was directed toward designing
multiparticulate composite construct (MCC) for the transbuccal delivery of
CBZ. The MCC was formulated using interphase, coparticulatecosolvent
homogenization technique, and lyophilization. In vitro, ex vivo, and in vivo
investigations were performed. The mesoporous (pore width 80.1233 A)
MCC was mechanically stable ((D) 0.0290 J, M(F) 8.5490 N/mm)
and resilient (M(R) 5.5040%). It demonstrated distinctive controlled-
release (9.9800%/h), permeation enhancing (10.8730%/h), drug loading
(90.0541%), and bioadhesive ((adh) 0.0034 J, F(det) 1.0751 N) capac-
ities. In vivo studies on pigs showed the ability of the MCC to effectively
initiate and regulate transbuccal permeation of CBZ as visualized by out-
comes of the quantitative and qualitative assessments of isolated plasma sam-
ples. Furthermore, comparisons of in vitro and in vivo data of MCC with a
conventional product highlighted its capability to attain higher bioavailabil-
ity and more controlled release trends. Histological and cytological investi-
gations confirmed that the MCC is biocompatible. The mathematical model
produced relevant pharmacokinetics and in vitro/in vivo correlation infor-
mation [217].
The authors describe a case of a 37-year-old Malay lady with an unusu-
ally slow carbamazepine clearance, which may be related to genetic poly-
morphisms of drug-metabolizing enzymes and transporters. When given a
small daily dose of 200 mg immediate-release carbamazepine, this patient
experienced drowsiness. Subsequently, she reduced her carbamazepine dose
to 200 mg twice a week (on Mondays and Fridays), resulting in poor seizure
control. At the same time, the patient was diagnosed with hyperthyroidism
and was given carbimazole and propranolol. Hyperthyroidism and the con-
current use of these antihyperthyroid agents may have further slowed down
the metabolism of carbamazepine. TDM of carbamazepine was carried out,
and a slow carbamazepine clearance of 1.45 L/h per 70 kg was observed.
Genotyping of selected genetic variants in CYP3A4, CYP3A5, EPHX1,
ABCB1, and ABCC2 revealed that she has CYP3A5*3/*3 and ABCB1
3435-CC genotypes. Both genotypes have been shown to be associated with
238 S.T. Alrashood

higher adjusted mean serum carbamazepine concentration in Chinese and

Korean patients with epilepsy. Physicians should be vigilant about the risk
of adverse effects among patients with a slow carbamazepine clearance, espe-
cially in Malays. Simulations of carbamazepine dosing regimen based on the
pharmacokinetic parameters of this patient were performed to allow individ-
ualization of drug therapy [218].
The effects of CYP3A5 polymorphisms on CBZ pharmacokinetic
parameters when CBZ is used as either monotherapy or coadministered
with PHT, phenobarbital (PB), or VPA were determined. So, retrospective
data were collected from an electronic database and medical records. Blood
samples were obtained and drug concentrations analyzed as a part of routine
TDM. Screening for wild-type CYP3A5*1 and CYP3A5*3 single nucleo-
tide polymorphism (rs776746) by allelic discrimination assay using real-time
polymerase chain reaction technique (real-time PCR) was performed. Phar-
macokinetic parameters of CBZ, and clearance and dose-adjusted CBZ
levels in patients with different genotypes were calculated and compared.
The obtained results of the 70 patients assessed, 8 (11%) patients were
homozygous CYP3A5*1/*1, 28 (40%) patients were heterozygous
CYP3A5*1/*3, and 34 (49%) patients were homozygous CYP3A5*3/*3.
The CBZ clearance and dose-adjusted CBZ levels did not significantly differ
between patients with CYP3A5*1 and CYP3A5*3 alleles when CBZ was
used as monotherapy. For patients who used CBZ in combination with an
enzyme-inducing AED (PHT or PB), individuals carrying the CYP3A5*1
allele (CYP3A5 expressers) showed a trend of having higher CBZ clearance
and lower dose-adjusted CBZ level as compared to individuals carrying the
CYP3A5*3 allele, even though no statistical significance was recorded.
Nevertheless, it was observed that AEDs significantly increased CBZ
clearance only in patients carrying the active CYP3A5*1 allele. Finally, it
was concluded that when CBZ was used in combination with enzyme-
inducing AED, CYP3A5 expressers yielded a trend toward greater sus-
ceptibility to change in CBZ clearance and showed lower dose-adjusted
CBZ levels compared to CYP3A5 nonexpressers. The dosage regimen
should be adjusted accordingly to gain a better clinical outcome. This article
is open to POST-PUBLICATION REVIEW. Registered readers (see For
Readers) may comment by clicking on ABSTRACT on the issues con-
tents page [219].
In silico approaches to predict absorption, distribution, metabolism, and
excretion of new drug candidates are gaining a relevant importance in drug
discovery programs. When considering particularly the pharmacokinetics
Carbamazepine 239

during the development of oral AEDs, one of the most prominent goals is
designing compounds with good bioavailability and brain penetration.
Thus, it is expected that in silico models able to predict these features
may be applied during the early stages of AED discovery. The present inves-
tigation was mainly carried out in order to generate in vivo pharmacokinetic
data that can be utilized for development and validation of in silico models.
For this purpose, a single dose of each compound (1.4 mmol/kg) was orally
administered to male CD-1 mice. After quantifying the parent compound
and main metabolites in plasma and brain up to 12 h postdosing, a noncom-
partmental pharmacokinetic analysis was performed and the corresponding
brain/plasma ratios were calculated. Moreover, the plasma protein binding
was estimated in vitro applying the ultrafiltration procedure. The present in
vivo pharmacokinetic characterization of the test compounds and corres-
ponding metabolites demonstrated that the metabolism extensively com-
promised the in vivo activity of CBZ derivatives and their toxicity.
Furthermore, it was clearly evidenced that the time to reach maximum peak
concentration, bioavailability (given by the AUC), and metabolic stability
(given by the AUC012 h ratio of the parent compound and total systemic
drug) influenced the in vivo pharmacological activities and must be consid-
ered as primary parameters to be investigated. All the test compounds pres-
ented brain/plasma ratios lower than 1.0, suggesting that the BBB restricts
drug entry into the brain. In agreement with in vitro studies already per-
formed within our research group, CBZ, CBZ-E, and oxcarbazepine
exhibited the highest brain/plasma ratios (>0.50), followed by
eslicarbazepine, R-licarbazepine, trans-diol, and BIA 2-024 (ratios within
0.050.50). BIA 2-265 was not found in the biophase, probably due to
its high plasma protein-bound fraction (>90%) herein revealed for the first
time. The comparative in vivo pharmacokinetic data obtained in the present
work might be usefully applied in the context of discovery of new AEDs that
are derivatives of CBZ [220].
To establish using DBS as a surrogate to plasma for TDM of CBZ, the
PPK estimates from concurrent DBS and plasma levels were compared. The
doseconcentration relationship, estimated parameter, and variability were
determined. A total of 98 observations from 97 people with epilepsy (PWE)
were included in this study. Data were split into 3:1 ratio for the respective
index group and validation group. Nonlinear mixed-effects regression
with one-compartment, first-order absorption, and elimination model
was utilized. Covariates were screened for inclusion into final model via
forward stepwise addition and backward elimination method. Predictive
240 S.T. Alrashood

performances of the final models were assessed for bias and precision. The
typical clearance for CBZ was estimated to be 5.85 and 5.68 L/h from
plasma and DBS concentrations, respectively. The final models for clearance
estimates obtained from plasma concentrations (Cplasma) included total daily
CBZ dose per unit weight (DD) and gender while from DBS concentrations
(Cdbs) included only DD. The final models were both precise and nonbias.
The developed PPK models had comparable estimates, errors, and predic-
tive performances. Our findings suggest that Cplasma and Cdbs could be used
interchangeably for pharmacokinetic studies of CBZ [221].
The objective of the study was to investigate the pharmacokinetics (PK)
of unbound and total plasma CBZ concentrations following simultaneous
administration of intravenous and oral formulations. The hypothesis that
age-related alterations in physiology and patient characteristics influence
CBZ disposition and protein binding was tested. Patients (n 113) on main-
tenance therapy received a 100 mg dose of a novel, intravenous, stable-
labeled (SL) CBZ formulation as partial replacement of their morning
CBZ dose. A two-compartment model described unbound and total SL-
CBZ data. The stable-labeled intravenous dosing methodology enabled
the estimation of the CBZ clearance (CL) and volumes of distribution.
The CL of CBZ was dependent on race through the model equation
unbound CL (L/h) 11.2  (1.30) (Race), where Race 1 for Caucasian
and 0 for African American. Total body weight (TBW) explained 57%
and 70% of the interindividual variability in the central and peripheral vol-
umes of distribution, respectively. Age, sex, smoking, plasma albumin, and
AGP concentrations had no effect on CL, binding, or volumes of distribu-
tion. The model was evaluated via bootstrap and predictive check. Results
may support race-specific dosing for CBZ where an average African-Amer-
ican individual would receive 70% of the standard dose prescribed for the
Caucasian person [222].
The aim of this study was to evaluate the association of genetic variants
in the major genes involved in CBZ metabolism and transport with its PK
in epilepsy patients. Twenty-five SNPs within 7 CBZ pathway genes,
namely CYP3A4, CYP3A5, EPHX1, NR1I2, UGT2B7, ABCB1, and
ABCC2, were analyzed for association with CBZ PK in 90 epilepsy pa-
tients. The CYP3A4*1B SNP was significantly associated with CBZ
clearance. Significant association of EPHX1 SNPs was observed with
greater carbamazepine-10,11-trans-dihydrodiol:carbamazepine-10,11 epo-
xide ratios. Among drug transporters, ABCB1 and ABCC2 SNPs were
significantly associated with altered CBZ clearance. SNPs within CBZ
Carbamazepine 241

pathway genes contribute to interpatient variation in CBZ PK and might

contribute to pharmacoresistant epilepsy. Although the results need further
clinical validation in a larger patient cohort, they indicate that genetic var-
iation in CBZ pathway genes could influence its PK and hence would have
clinical significance [223].
Carbamazepine is commonly used as AED in elderly patients. This study
analyzed prospective data collected as part of a randomized, double-blinded
trial of newly diagnosed epilepsy patients. The aims of this study were to
determine the PK parameters and their variability of carbamazepine in
elderly patients and to quantify the effect of covariates on these parameters.
Prospectively collected carbamazepine concentrations from 121 patients
aged 60 years or older were used to develop a PPK model. Data were ana-
lyzed by a NONMEM. A 1-compartment model with first-order absorption
and elimination was used to characterize the time course of carbamazepine
concentration. Model evaluation and the predictive performance of the final
model were assessed using the nonparametric bootstrap approach. The
apparent clearance (CL/F) of carbamazepine in this community-dwelling
elderly population was estimated to be 3.59 L/h with an interindividual var-
iability of 18.1%. The CL/F increases 23% in patients comedicated with
phenytoin. The volume of distribution (V/F) was estimated to be 102 L
with an interindividual variability of 74.7%. Carbamazepine clearance was
not associated with body weight or any parameterization of body size nor
was age or race or any marker of hepatic or renal function in commu-
nity-dwelling elderly patients. Elderly patients on concurrent phenytoin
therapy may require a smaller 23% higher dose on average, about half that
reported for younger patients [224].
Owing to the lack of an intravenous formulation, the absolute bioavail-
ability, absolute clearance, and half-life in patients at steady state of carba-
mazepine have not been determined. An intravenous, stable-labeled (SL)
formulation in order to characterize carbamazepine pharmacokinetics in
patients was developed. Ninety-two patients received a 100-mg infusion
of SL-carbamazepine as part of their morning dose. Blood samples were col-
lected up to 96 h after drug administration. Plasma drug concentrations were
measured with LCMS, and concentrationtime data were analyzed using a
noncompartmental approach. Absolute clearance (L/h/kg) was significantly
lower in men (0.039  0.017) than in women (0.049  0.018; P 0.007)
and in African Americans (0.039  0.017) when compared with Caucasians
(0.048  0.018; P 0.019). Half-life was significantly longer in men than in
women as well as in African Americans as compared with Caucasians. The
242 S.T. Alrashood

absolute bioavailability was 0.78. Sex and racial differences in clearance may
contribute to variable dosing requirements and clinical response [225].
The effect of dosing time on the bioavailability of carbamazepine imme-
diate-release (IR) tablets was investigated when administered to beagle dogs
who were fasting, with coadministration of food (Co-food), and 0.5 h
before food and 2 h after food. The study was conducted using a single dose
of 200 mg (tablets/solution) with a 2-week washout period in a crossover
design. Food intake significantly increased the rate and extent of tablet
absorption. The Cmax (g/mL, 8.13/3.65) and tmax (h, 1.83/0.92) were
increased more than twofold and the AUC024 (g h/mL, 20.09/8.19)
was 2.5 times that of the values obtained under fasting conditions. The bio-
availability of the tablets under fasting conditions was 91.2%, but increased
to 223.5%, 182.8%, and 148.4% in the Co-food, 0.5 h before food, and 2 h
after food groups, respectively (P < 0.05). Although there was no significant
difference in the C(max) or AUC024 between the treatments with food,
the absorption appeared to be reduced to some extent when the tablets were
given 2 h after food. The oral bioavailability of CBZ IR tablets was signif-
icantly affected by the timing of the food intake. This is maybe favored by
the fluctuations in the level of bile salts with the timing of food intake. To
obtain acute therapy for a drug with narrow therapeutic window, attention
should be given to the dosing time and food intake interactions [226].
Species differences in the oral PK and absolute bioavailability (F(abs)) of
carbamazepine polymorphs (form I and form III) and dihydrate were stud-
ied. The PK of each form was investigated in rats following a single oral/
intravenous administration of 10 mg/kg and an oral dose of 80 mg/kg,
which were compared with the published data obtained from dogs and
humans. No significant differences were found in their C(max), T(max),
AUC(01), and F(abs) among the forms at the low dose. However, signif-
icant differences were observed at the high dose. The F(abs) of each form
was markedly reduced with increasing of doses in species (eg, F(abs) in rats
ranged from >82% to 38.456.0%). At a comparable dose, the C(max), and
AUC(01) of rats and humans were about 310 times higher than in dogs.
The absorption rate of form III in rats exhibited a similar trend to that in
humans and was far higher in dogs. A multipeak phenomenon in plasma cur-
ves was observed in rats and humans, but not in dogs. In conclusion, rats
appear to be a better predictor of carbamazepine polymorphs absorbed in
humans, and form III may be more suitable as a pharmaceutical crystal [227].
The PK of CBZ and its active 10,11-epoxide metabolite (CBZ-E) were
evaluated after intravenous and oral administration of 5 mg/kg CBZ to rats
Carbamazepine 243

with hyperlipidemia induced by Poloxamer 407 (HL rats) and controls. The
total area under the plasma concentrationtime curve (AUC) of CBZ in HL
rats after intravenous administration was significantly greater than that in
controls due to their slower nonrenal clearance (CL(NR)). This was due
to slower hepatic CL(int) for metabolism of CBZ to CBZ-E in HL rats
via CYP3A1/2. This result was consistent with a previous study, indicating
reduced hepatic CYP3A1/2 expression in HL rats. Interestingly, the AUC
of CBZ-E was also increased in HL rats, while AUC(CBZ-E)/AUC(CBZ)
ratios remained unchanged. These results suggested that further
metabolism of CBZ-E to the inactive metabolite trans-10,11-dihydoxyl-
10, 11-dihydro-CBZ (CBZ-D) via microsomal epoxide hydrolase (mEH)
was also slowed in HL rats. The significantly reduced hepatic mRNA level
and expression of mEH protein in HL rats compared to controls confirmed
the earlier hypothesis. Similar PK changes were observed in HL rats after oral
administration of CBZ. These findings have potential therapeutic implica-
tions assuming that the HL rat model qualitatively reflects similar changes in
patients with hyperlipidemia. Caution is required regarding pharmacother-
apy in the hyperlipidemic state in cases where drugs that are metabolized
principally by CYP3A1/2 or mEH and have a narrow therapeutic range
are in use [228].
The aim of the present study was to build PPK models for the clearance
of CBZ in two separate populations of Serbian patients with epilepsy, chil-
dren, and adults. Analysis was performed using 114 and 53 steady-state con-
centrations of CBZ collected from 98 children and 53 adult epileptic
patients, respectively. Mean values of TBW and age were 31  13 kg and
8  3 years in the population of children, and 67  13 kg and 32  15 years
in the population of adults. The one-compartment model with first-order
elimination and without absorption was used from the PREDPP (Prediction
for Observation Population Pharmacokinetics) library of NONMEM soft-
ware. The derived final models of CBZ clearance were similar in the target
populations. The most important factors which affected typical mean value
of CBZ clearance in both populations studied were age of the patients and
total daily dose; the CBZ clearance linearly followed increase of these fac-
tors. However, the influence of the patients age was almost 3.4 times higher
in the pediatric population than that in adults, while the influence of total
daily dose of CBZ is similar. On the other hand, final model in the adult
population revealed also influence of concomitant therapy with phenobar-
bital (PB). The magnitude of this effect was +1.61 L/h. The PK models
obtained were validated in groups of 18 children and 13 adults with epilepsy.
244 S.T. Alrashood

So, the derived models describe well CBZ clearance in terms of Serbian
pediatric and adult epileptic patient characteristics, offering a basis for ratio-
nal individualization of CBZ dosage regimens [229].
Carbamazepine belongs to the class II biopharmaceutical classification
system which is characterized by a high per-oral dose, a low aqueous solu-
bility, and a high membrane permeability. The bioavailability of such a drug
is limited by the dissolution rate. The present study deals with the formu-
lations of immediate-release tablets of poorly soluble carbamazepine. As
model tablets for this investigation, two formulations (named A and
B formulations) of carbamazepine tablets labeled to contain 200 mg were
evaluated. The aim of this study was to establish possible differences in dis-
solution profile of these two formulations purchased from the local market.
The increased crystallinity together with enlarged particle size, enhanced
aggregation and decreased wettability of the drug, resulted in insufficient
dissolution rate for formulation B. From the dissolution point of view, this
formulation was inferior to the formulation A, due to the solubilization
effect [230].
CBZ is metabolized mainly by the CYP3A family of enzymes, which
includes CYP3A4 and CYP3A5. Several studies have suggested that the
CYP3A5*3 genotype influences the PK of CYP3A substrates. The present
study aimed to assess the effect of the CYP3A5*3 genotype on serum con-
centration of CBZ at the steady state in Korean epileptic patients. The serum
concentrations of CBZ in 35 Korean epileptic patients were measured and
their CYP3A5 genotype was determined. Fourteen patients were CYP3A5
expressors (2 for CYP3A5*1/*1 and 12 for CYP3A5*1/*3) and 21 patients
were CYP3A5 nonexpressors (CYP3A5*3/*3). Dose-normalized concen-
trations (mean  SD) of CBZ were 9.9  3.4 ng/mL/mg for CYP3A5
expressors and 13.1  4.5 ng/mL/mg for CYP3A5 nonexpressors
(P 0.032). The oral clearance of CBZ was significantly higher in CYP3A5
nonexpressors than that of CYP3A5 expressors (0.056  0.017 L/h/kg vs
0.040  0.014 L/h/kg, P 0.004). The CYP3A5 genotype affected the
CBZ concentrations in Korean epileptic patients and is a factor that may
contribute to interindividual variability in CBZ disposition in epileptic
patients [231].
Influence of soybean administration on the bioavailability of carba-
mazepine and omeprazole was studied after single-dose administration of
soybean (10 g/kg p.o.) or after chronic administration of soybean (50%
(w/w) mixed with normal feed) for 15 days in rats. Carbamazepine was
administered orally at a dose of 10 mg/kg and omeprazole at a dose of
Carbamazepine 245

20 mg/kg. Soybean decreased the bioavailability of carbamazepine after

both single dose and chronic administration. It produced a significant
decrease in C(max), T(max), and AUC(0t) of carbamazepine after
single-dose administration and increased the plasma clearance and V(d)
along with decrease in C(max), T(max), AUC(0t), and AUC(0infinity)
after chronic administration. On the contrary, soybean administration
increased the bioavailability of omeprazole by producing an increase in
C(max), AUC(0t), and AUC(0infinity) and a decrease in V(d) after
single-dose administration and a decrease in plasma clearance along
with increase in C(max), AUC(0t), and AUC(0infinity) after chronic
administration. The half-life of omeprazole was also increased after
both acute and chronic administration of soybean. It was concluded
that soybean decreases the bioavailability of carbamazepine and increases
the bioavailability of omeprazole after both single-dose and chronic
administration [232].
The dose of carbamazepine required to achieve optimal seizure control
varies widely from patient to patient. Polymorphic variants in various
genes involved in the pharmacokinetics and pharmacodynamics of carba-
mazepine in an effort to identify predictors of maintenance dose were inves-
tigated. A total of 70 patients with epilepsy (49% were males; median age,
34 years; range, 1472 years) who had benefited (>50% reduction in
seizure frequency for at least 12 months) from treatment with carbamaze-
pine monotherapy were included in the analysis. Known variants in
drug-metabolizing enzyme genes, including those encoding cytochrome
P450s, uridine 50 -diphosphate-glycosyltransferase, and mEH, together
with a sodium channel polymorphism in SCN2A, were screened using poly-
merase chain reaction-restriction fragment length polymorphism or direct
sequencing. Associations between demographic and genetic variables and
carbamazepine dose were identified by univariate and multivariate regression
analyses. All genotype frequencies were consistent with HardyWeinberg
equilibrium (P > 0.05). No single demographic or genetic variable was
of sufficient strength to independently influence carbamazepine dosing
requirements. However, a multivariate model, incorporating patient age
and specific genotypes (c.337T>C, c.416A>G) of the EPHX1 gene
encoding mEH, revealed a significant association with the maintenance
dose of carbamazepine (r2 0.362, P 0.002). This proof-of-principle
study suggests that genetic variants in EPHX1 can be used to predict main-
tenance doses of carbamazepine. A large-scale prospective investigation of
genetic influences on drug dosing strategies in epilepsy, with specific focus
246 S.T. Alrashood

on whole gene variability for those proteins involved in the pharmacoki-

netics and pharmacodynamics of antiepileptic agents, is warranted [233].
This work describes the PK of a novel carbamazepine nanoemulsion.
The plasma concentration profiles were determined in beagle dogs after
i.v. bolus administration of a 5 mg/kg carbamazepine nanoemulsion
and compared to the corresponding carbamazepine/hydroxypropyl-beta-
cyclodextrin complex solution. Both formulations showed similar PK pro-
files and could represent valuable formulations in case of emergencies, when
a rapid action in the CNS is desirable [234].
Previous evidence has shown that chronic 3-mercaptopropionic acid
(MP) administration induced brain P-gp overexpression altering target site
accumulation of phenytoin. The aim of the present work was to assess the
involvement of P-gp in carbamazepine and phenobarbital hippocampal PK
in an experimental model of epilepsy, induced by repetitive MP administra-
tion. Seizures were induced in Wistar rats by injection of MP (45 mg/kg, i.p.)
during 10 days. Control rats (C) were injected with saline solution. In order
to monitor extracellular brain antiepileptic levels, a concentric probe was
inserted into the hippocampus. Animals were administered with carba-
mazepine (10 mg/kg, i.v.) or phenobarbital (20 mg/kg, i.v.) 30 min after
intraperitoneal administration of vehicle or nimodipine (2 mg/kg), a
well-known P-gp inhibitor. No differences were found in hippocampal
concentrations of carbamazepine comparing all groups. In vehicle pretreated
rats, hippocampal phenobarbital concentrations were lower in MP (maximal
concentration, C(max): 6.0  0.6 g/mL, P < 0.05) than in animals (C
(max): 9.4  0.9 g/mL). Control rats pretreated with nimodipine showed
similar results (C(max): 10.7  0.6 g/mL) than those pretreated with vehi-
cle. Nimodipine pretreatment in MP rats enhanced hippocampal phenobar-
bital concentrations (C(max): 10.2  1.0 g/mL, P < 0.05) as compared
with vehicle pretreatment. Results of our work suggest that P-gp over-
expression by repetitive seizures induced by MP administration does not
modify brain bioavailability of carbamazepine. Conversely, hippocampal
levels of phenobarbital are reduced in MP rats with regard to nonepi-
leptic rats, suggesting a potential role of P-gp overexpression in
pharmacoresistance to Phenobarbital [235].
The purpose of this study was to perform PPK analysis on carbamazepine
and to determine the population model of clearance of this drug in terms of
individual patient characteristics. A total of 107 steady-state serum concen-
trations from 97 adult and pediatric epileptic patients, collected during rou-
tine clinical care, were used for the analysis. To determine the influence of
Carbamazepine 247

different covariates on the estimate of carbamazepine clearance, the NON-

MEM software package with ADVAN1 subroutine were used. This is a
one-compartment model with first-order elimination and without absorp-
tion. The typical mean value for carbamazepine clearance, estimated by the
base model (without covariates), in our population was 3.43 L/h. The final
results of the analysis show that carbamazepine clearance increased
nonlinearly with TBW and age, and linearly with concomitant administra-
tion of valproate. The magnitude of the effect of valproate was +0.874 L/h.
The interindividual variability (coefficient of variation) for clearance and the
residual variability (including intraindividual variability), described by an
exponential error model, were 16.76% and 31.14%, respectively. The results
of this PPK analysis were validated in a group of 16 epileptic patients and
suggested good predictive performance of the final model. The derived
model describes carbamazepine clearance in terms of characteristics of Ser-
bian patients, using minimal data obtained from routine clinical care of epi-
leptic patients. This is the basis for future PK studies on a specific epileptic
population, which will lead to better overall management of epilepsy in Ser-
bia [236].
The purpose of the present study was to evaluate the effect of KA-
induced acute seizures on the PK profiles of AED, CBZ in mice.
Experimental acute seizure in mice was induced by intraperitoneal injec-
tion of KA (30 mg/kg), and mice were provided for experiments after
48 h of KA treatment. The portal plasma concentrations of CBZ and its
metabolite carbamazepine-10,11-epoxide (CBZ-epo) had trends to
decrease as compared to the control mice, whereas the brain CBZ and
CBZ-epo concentrations were actually lower in KA-treated mice. On
the other hand, the exsorption of CBZ from blood to the intestinal lumen
via P-gp in KA-treated mice was significantly increased in parallel with
that of Rhodamine-123 (Rho123), a P-gp substrate. Western blotting
analysis for intestinal and cerebral P-gp showed that the P-gp expression
was induced in the KA-treated mice. The apparent brain-to-plasma concen-
tration ratio (Kp) of CBZ in the KA-treated mice showed significant
decrease but that of CBZ-epo did not. Moreover, in the KA-treated mice,
the percentage of protein binding was significantly increased and found to
be an inverse proportion in the relationship between the Kp and protein
binding of CBZ. In conclusion, the mechanism responsible for a decreased
brain CBZ concentration in the KA-induced seizure mice is based on the
upregulation of P-gp function in tissues and plasma protein binding of
CBZ [237].
248 S.T. Alrashood

Individualization of CBZ dosage regimen in patients with epilepsy based

on TDM followed by estimation of PK parameters can help in better control
of epilepsy. The objective was to establish a population (POP) PK model of
CBZ for Egyptian adult and pediatric patients with epilepsy. Single steady-
state (SS) trough plasma concentrations of CBZ were available for 302
patients with epilepsy (55.6% men and 44.4% women) who were catego-
rized as children (n 118) and adults (n 184) with mean age (years) 
SD of 10.6  4.8 and 29.4  9.9, respectively. Carbamazepine was given
as an oral suspension (n 19) or controlled release tablet (n 283) with aver-
age dose of 15.0  7.8 mg/kg per day. A one-compartment model with first-
order absorption and elimination for SS conditions (ADVAN2, SS2,
TRANS2) was applied using NONMEM 6.2. Separate absorption rate con-
stants were modeled for the two formulations. The mean POP CL, its inter-
subject variability (ISV), as well as residual error of CBZ concentration were
estimated. The POP estimate for CL was 3.5 L/h with coefficient of vari-
ation value of 2.6%, which was consistent with literature data. The ISV on
CL was 44.5%. The POP PK model was validated by bootstrap resampling,
and the individual estimates were within the 95% CI of the bootstrap results.
Different covariates that might affect CBZ CL have been evaluated, but
the limited number of samples per individual prevented precise covariate
analysis. Finally, it was concluded that the POP PK model we have devel-
oped for CBZ shows good predictive performance in Egyptian adult and
pediatric patients with epilepsy. Another PK study to better define the
effect of different covariates would improve on the model for dosage indi-
vidualization [238].
CBZ clearance decreases from childhood to adulthood and the factors
determining this change could include age, size, autoinduction, or matura-
tional changes. This study aims to describe the PPK of CBZ in children and
young adults and test the hypothesis that CBZ clearance correlates with
weight, surface area, and age. CBZ TDM data (sparse data) were collected
from child and adult epileptics, and rich data were obtained from a bioequiv-
alence study of CBZ in young adults. PPK analysis was performed using
NONMEM V. Forward stepwise, multiple regression was performed on
the covariates. Bootstrap validation was performed. A total of 946 observa-
tions from 91 subjects, ages 0.737 years, were collected and analyzed. A
one-compartment, first-order absorption, and elimination model,
with exponential interindividual error and additive residual error models,
was developed. The population model was: clearance (L/h)
(2.24  surface area (m2)) + (0.047  dose (mg/kg)); volume of distribution
Carbamazepine 249

(L) 0.37  weight (kg); absorption rate constant 0.013 h1. CBZ clear-
ance increased with surface area and dose [239].
Proper use of AEDs in the elderly involves knowledge of their PK to
ensure a patient-specific balance between efficacy and toxicity. However,
populations of epileptic patients on chronic CBZ therapy which have been
studied have included data of relatively few elderly patients. The aim of the
present study was to evaluate the PPK of CBZ in elderly patients on chronic
monotherapy. The nonparametric expectation maximization program in
the USC*PACK collection of PC programs to estimate individual and pop-
ulation postinduction PK of CBZ in epileptic elderly patients who received
chronic CBZ monotherapy was used. Age-related changes of CBZ PPK
were evaluated from routine TDM data of 37 elderly and 35 younger
patients with epilepsy. As a historical control previously published popu-
lation modeling results from 99 young epileptic patients on chronic CBZ
monotherapy were used. In that control group, TDM was performed in
the same PK laboratory, using the same sampling strategy as in the present
study, and the same PK population modeling software was used for data
analysis. A poor correlation was found between daily CBZ dose and serum
concentrations in the elderly patients (r 0.2, P 0.25). Probably statisti-
cally significant difference in the median values of the CBZ metabolic rate
constant (P < 0.001) between elderly and relatively young epileptic patients
was found. The results showed that age-related influences in CBZ PK in
elderly patients should be considered in the optimal planning of CBZ dosage
regimens. Most elderly patients with epilepsy will usually need CBZ dosages
lower than those based on the median population PK parameter values
obtained from younger patients. The present population model is also
uniquely well suited for the new multiple model design of dosage regi-
mens to hit target therapeutic goals with maximum precision [240].
To investigate the characteristics of CBZ transport and drug interactions
at the BBB, cultured rat brain microvascular endothelial cells (rBMEC) were
used as an in vitro model of the BBB. When cells became confluent, CBZ
uptake over time was recorded by incubation of the cells in a medium con-
taining 10 mg/L CBZ at 37C. The steady-state uptake of CBZ by rBMEC
was tested for different CBZ concentrations at 37C. The effects of various
agents on the steady-state uptake of CBZ and efflux of CBZ from rBMEC
were also studied. The uptake of CBZ by rBMEC was time- and concen-
tration-dependent. The steady-state uptake occurred at 30 min for incuba-
tion. The steady-state uptake was significantly increased (P < 0.01) by
treatment with dinitrophenol. The coadministration of cyclosporine
250 S.T. Alrashood

A significantly increased the steady-state uptake of CBZ by the rBMEC,

whereas coadministration of olanzapine significantly decreased the uptake
in a concentration- and temperature-dependent manner. The efflux of
CBZ from rBMEC was inhibited by CsA. As a conclusion, the transport
of CBZ at the BBB is mediated by many transporters. Some specific
ABC (ATP-binding cassette) efflux transporters may be involved in the
transport of CBZ. Drugs influence the transport of CBZ at the BBB in dif-
ferent ways [241].
Population models can be important extensions of TDM, as they allow
estimation of individual PK parameters based on a small number of measured
drug concentrations. This study used a Bayesian approach to explore the
utility of routinely collected and sparse TDM data (1 sample per patient)
for CBZ monotherapy in developing a PPK model for CBZ in pediatric
patients that would allow prediction of CBZ concentrations for both imme-
diate- and controlled-release formulations. Patient and TDM data were
obtained from a pediatric neurology outpatient database. Data were analyzed
using an iterative two-stage Bayesian algorithm and a nonparametric adap-
tive grid algorithm. Models were compared by final log likelihood, mean
error (ME) as a measure of bias, and root mean squared error (RMSE) as
a measure of precision. Fifty-seven entries with data on CBZ monotherapy
were identified from the database and used in the analysis (36 from males, 21
from females; mean [SD] age, 9.1 [4.4] years [range, 221 years]). Prelimi-
nary models estimating clearance (CL) or the elimination rate constant (K
(el)) gave good prediction of serum concentrations compared with measured
serum concentrations, but estimates of CL and K(el) were highly correlated
with estimates of volume of distribution (V(d)). Different covariate models
were then tested. The selected model had zero-order input and had age and
body weight as covariates. CL (L/h) was calculated as K(el)  V(d), where K
(el) [K(i)  (K(s)  age)] and V(d) [V(i) + (V(s)  body weight)]. Median
parameter estimates were V(i) (intercept) 11.5 L (fixed); V(s) (slope)
0.3957 L/kg (range, 0.012001.5730); K(i) (intercept) 0.173 h1 (fixed);
and K(s) (slope) 0.004487 h1  y1 (range, 0.00018000.02969). The
fit was good for estimates of steady-state serum concentrations based on
prior values (population median estimates) (R 0.468; R2 0.219) but
was even better for predictions based on individual Bayesian posterior
values (R2 0.991), with little bias (ME 0.079) and good precision
(RMSE 0.055). Based on the findings of this study, sparse TDM data
can be used for PPK modeling of CBZ clearance in children with epilepsy,
and these models can be used to predict CL at steady state in pediatric
Carbamazepine 251

patients. However, to estimate additional PK model parameters (eg, the

absorption rate constant and V(d)), it would be necessary to combine sparse
TDM data with additional well-timed samples. This would allow develop-
ment of more informative PPK models that could be used as part of Bayesian
dose-individualization strategies [242].
The objective of this study was to investigate the effects of SLS upon the
saturation solubility of carbamazepine, its dissolution kinetics, and T50%
defined as the time required for dissolving 50% of carbamazepine. Water,
0.1 N-HCl, and phosphate buffers at pH 4.0 and 6.8 containing 0.1%,
0.5%, 1%, and 2% SLS were used as dissolution media. The dissolution study
was conducted by using the USP dissolution apparatus II with an agitation
rate of 75 rpm. Samples of the dissolution media were taken in 7, 15, 30, 45,
60, 75, and 90 min, and the amounts of carbamazepine were determined
spectrophotometrically at 285 nm. All dissolution data were fitted well into
a four-parameter exponential equation: Q a(1  e(bt)) + c(1 e(dt)). In
this equation, Q represented % carbamazepine dissolved at a time t, and a, b,
i, and d were constants. This equation led to the calculation of dissolution
rates at various time points and T50%. It was found that the dissolution rate
of carbamazepine was directly proportional to the aqueous concentration of
SLS. In addition, under our experimental conditions T50% values ranged
from 37.8 to 4.9 min. It was interesting to note that T50% declined rapidly
as the surfactant concentration increased from 0.1% to 0.5%, whereas it
declined more slowly at concentrations greater than 1%. These results clearly
demonstrated that the dissolution rate of carbamazepine and duration of its
dissolution test could be tailored by optimizing the amount of SLS in a
dissolution medium [243].
Various extended-release CBZ formulations have been developed pre-
viously, in order to reduce the frequency of dosing in chronic therapy and to
decrease the variability in drug plasma concentration. In the present study,
the suitability of different grades of Gelucires (G, glyceride-based excipients)
to formulate CBZ extended-release capsules by the application of semisolid
matrix (SSM) filling capsule technology was investigated. The possible mod-
ification of CBZ release kinetics by using Gelucire blends or inclusion of
hydrophilic additives in the SSM was studied. The effect of aging on some
selected formulations was also evaluated, using scanning electron micros-
copy and differential thermal analysis. Twenty-one capsule formulations
were prepared and assessed for their release characteristics. The mechanism
of drug release from the test formulations was studied. The following results
were obtained: (a) release data could not be correlated to the melting point
252 S.T. Alrashood

(mp) of Gelucires used, pointing to relative lipophilicity of the base as a more

important determinant of drug release. Among Gelucire grades having melt-
ing points higher than 37C, the release rate proved to be highly dependent
on matrix composition. (b) CBZ release occurred by different mechanisms,
including matrix disintegration, diffusion, and/or erosion depending on
the vehicle employed. (c) Zero-order release profiles of CBZ were
obtained from SSM-based on G50/13, G53/10, and their blends in ratios
higher than 1:1 and G53/10 containing croscarmellose sodium. (d) The
aging study revealed that these latter formulations, except those based on
G50/13, also showed high dissolution stability during 1 year of shelf
aging. (e) PVP, as a polymorphic transformation inhibitor, can be used to
reduce the storage-induced changes of some grades of Gelucires. From
the earlier data, it can be concluded that different grades of Gelucires and
their blends as well as hydrophilic additives could be successfully used to
formulate CBZ extended-release SSM filled capsules with various release
kinetics [244].
Although epileptic crises are equally frequent in women and men, several
factors cause female epileptics to present a series of gender-specific problems.
To date, few studies have been published on the kinetics of CBZ and CBZ-
E active metabolite in a Mexican population, and no information for epilep-
tic women of reproductive age is available. The aim of the present work was
to study the PK behavior of this group of women during steady state. Four-
teen epileptic women under chronic treatment receiving only the anticon-
vulsant CBZ to control their crises were studied. A blood sample was taken
before breakfast, before the morning dose of 200 mg, and after the dose at 1,
2, 3, 4, 5, and 8 h. Serum was separated by centrifugation at 1350  g. Serum
concentrations of CBZ and of the metabolite CBZ-E were measured by
HPLC. Pharmacokinetic parameters were calculated by statistical moment
method after obtaining serum concentrations. Maximum time (T(max))
for CBZ was reached at 2.72  0.71 h and for CBZ-E, it was
3.60  0.79 h. C(max) for CBZ was 7.30  2.30 g/mL, while C(min)
for CBZ was 6.30  2.49. Maximum serum values for CBZ-E were
1.01  0.57, equivalent to 13.80% of CBZ; t(12) value for CBZ and CBZ-E
was 18.20 and 16.10 h, respectively. AUC values for CBZ and metab-
olite were 70.33  17.10 and 9.20  2.50 g/L/h, respectively. CBZ
and CBZ-E clearance did not show differences and were 0.37 and
0.40 mL/kg/min, respectively. Extraction index for serum concentrations
of CBZ and CBZ-E AUC(CBZ)/AUC(CBZ-E) was 0.13; positive corre-
lation was observed between serum concentrations of CBZ and E-CBZ, with
Carbamazepine 253

r 0.94. The suggested schedule for therapeutic monitoring of

serum concentrations of CBZ in chronic treatments is 3 h for maximum peak
concentration of C(max) after dose administration and for minimum
peak concentration, C(min) prior to subsequent administration of the dose
was obtained [245].
To assess the influence of aging on the steady-state PK of CBZ in a large
population of patients evaluated in a TDM setting, the database of a large
TDM service was used to identify retrospectively steady-state serum CBZ
concentrations in 157 elderly patients with epilepsy (65 years and older)
treated with CBZ alone or in combination with Phenobarbital (PB).
CBZ apparent oral clearance (CL/F) values were calculated and compared
with those determined in an equal number of controls aged 2050 years, and
matched for gender, body weight, and comedication. Compared with
corresponding controls, mean CBZ CL/F values were 23% and 24% lower,
respectively, in the groups of elderly patients receiving monotherapy
(57.1  20.6 vs 74.6  28.3 mL/h/kg; P < 0.0001) and PB comedication
(74.7  25.5 vs 98.7  34.9 mL/h/kg; P < 0.01). Within each age group,
patients comedicated with PB showed significantly higher CBZ CL/F
values than those on monotherapy. A negative correlation between CL/F
and age was found both within the monotherapy and the PB comedicated
groups. In addition, CL/F values showed a positive relation with the admin-
istered daily dosage, which persisted within subgroups homogeneous for age
and comedication. The independent influence of age, CBZ dosage, and
comedication on CBZ CL/F was confirmed by multiple regression analysis.
So, it was found that carbamazepine CL/F is decreased in an age-dependent
manner in elderly patients compared with younger subjects, presumably
because a reduction in the rate of CYP3A4-mediated drug metabolism.
Elderly patients retain their sensitivity to dose-dependent autoinduction
and to heteroinduction by enzyme-inducing AEDs, but their metabolic
rates remain considerably below those observed in matched controls. As
a result of this, patients in old age will require lower CBZ dosages to
achieve serum concentrations comparable with those found in nonelderly
adults [246].
To model and reevaluate the PK of CBZ and CBZ-E after 5 day b.i.d.
dosing with either Carbatrol (extended-release beads) or Tegretol-XR (an
osmotic pump tablet, an Oros tablet) using compartmental method. Plasma
concentration time profile data from 15 normal healthy adults received, in a
randomized crossover fashion, Carbatrol (2  200 mg capsules), b.i.d. for 5
days and Tegretol-XR (400 mg), b.i.d. for 5 days were available for
254 S.T. Alrashood

analysis from the previous study. The compartmental kinetic parameters of

CBZ and CBZ-E were simultaneously fitted by assuming: (i) one-compart-
ment open model with zero-order absorption with lag time, and first-order
elimination for CBZ and (ii) one-compartment open model with
MichaelisMenten formation with a sigmoidity factor, and first-order elim-
ination for CBZ-E. Time to 50% of CBZ plateau concentrations (TC50)
was estimated and statistically compared between the two products. There
was a good agreement between simulated and observed plasma concentra-
tions. For CBZ, the fitted parameters were: the first-order elimination rate
constant (K(10)) 0.024 and 0.022 h1, t1/2 27.3 and 30.3 h, and volume of
central compartment (V(1)) 1.119 and 1.160 L/kg, for Carbatrol and
Tegretol-XR, respectively. For CBZ-E, the fitted parameters were: the
first-order elimination rate constant (K(30)) 0.128 and 0.157 h1, t1/2 6.1
and 5.1 h, volume of central compartment (V(3)) 0.728 and 0.644 L/kg,
V(max) 0.085 and 0.076 mg/h/kg, and K(m) 28.639 and 33.138 mg/mL,
for Carbatrol and Tegretol-XR, respectively. The fitted PK parameters of
CBZ and CBZ-E were generally consistent with published values from
the previous studies. A minimal rise in CBZ-E concentrations was observed
during the first 12 h, the finding of which has not been reported before.
Consequently, the CBZ-E plasma profiles appear as sigmoid curves and
have a different shape compared to those of the CBZ profiles. The inclusion
of the sigmoidity factor allowed flexibility in the fitting and optimized
the simulation results. When compared to published literature of single-
dose data, the investigation of CBZ and CBZ-E PK from this study
suggested that autoinduction might occur by the fifth day of dosing and
might partly contribute to the sigmoidal shape of CBZ-E profiles. The fitted
model well described the plasma profiles of both CBZ and CBZ-E.
Carbatrol and Tegretol-XR were similar in their PK based on compartmen-
tal analysis [247].
A sizeable number of epilepsy patients remain uncontrolled with CBZ
monotherapy. While the therapeutic plasma concentration range of CBZ
is only vaguely defined, PK differences in the disposition of CBZ among
subjects could be responsible for the inadequate control of seizures in some.
This study was aimed at associating serum CBZ levels with seizure control
and elucidating any PK differences between patients with controlled and
uncontrolled epilepsy. The study was conducted in 16 controlled and 15
uncontrolled adult epileptic patients receiving CBZ monotherapy for the
previous 3 or more months, without any dosage change. Blood samples
were drawn from the patients before and 0.5, 1, 2, 3, 4, 8, 12, and 24 h after
Carbamazepine 255

ingestion of their total daily dose of CBZ. Serum CBZ levels were measured
by HPLC and the PK parameters were calculated. The uncontrolled epilep-
tic patients were receiving a higher daily dose of CBZ (difference not sig-
nificant). The trough and peak serum CBZ levels were relatively higher in
the uncontrolled group, and at no time point were the drug levels lower in
these patients compared to the controlled group. The absorption kinetics,
volume of distribution, and plasma half-life of CBZ were similar in the
two groups. Thus, nonattainment or nonmaintenance of therapeutic
CBZ level or other PK difference was not responsible for occurrence of
seizures in the uncontrolled patients. A high percentage of patients with
generalized tonicclonic seizures (73%) and simple partial seizures (SPS)
with generalization (66%) were controlled by CBZ, while SPS and complex
partial seizures (CPS) were largely uncontrolled. It appears that phar-
macodynamic resistance of the seizure to CBZ rather than PK factors is
responsible for lack of efficacy of CBZ in nonresponding epileptic
patients [248].
The dissolution behaviors of CZP polymorphs and pseudopolymorphs
(form I, form III, and dihydrate) and the bioavailabilities (BA) of each form
in dogs after oral administration were investigated. Bioavailability tests were
carried out at a dose of either 40 or 200 mg/body. The results of dissolution
tests in JP13 first fluid (pH 1.2) at 37C indicated that the initial dissolution
rate was in the order of form III > form I > dihydrate, while form III was
transformed to dihydrate more rapidly than form I, resulting in decrease
of the dissolution rate. The solubilities of both anhydrates (form I and form
III), calculated from the initial dissolution rate of each anhydrate, were 1.5
1.6 times that of the dihydrate. At the dose of 40 mg/body, there were no
significant differences in the AUC between forms; their AUCs were nearly
equal to that of CZP solution using polyethylene glycol 400. These findings
suggested that most crystalline powder of each form administered at the low
dose was rapidly dissolved in gastrointestinal (GI) fluid. On the other hand,
for the dose of 200 mg/body, significant differences in plasma concentra-
tiontime curves of CZP among polymorphic forms and dihydrate were
observed. The order of AUC values was form I > form III > dihydrate.
The inconsistency between the order of initial dissolution rates and that
of AUC values at the high dose may have been due to rapid transformation
from form III to dihydrate in GI fluids [249].
Carbamazepine produces dose-related anticonvulsant effects in epilepsy
models including the genetically epilepsy-prone rat (GEPR) model and the
rat maximal electroshock (MES) model. Doseresponse relationships are
256 S.T. Alrashood

quantitatively different among the models. Against electroshock seizures in

Sprague-Dawley rats the ED50 dose is 7.5 mg/kg, whereas the ED50 against
audiogenic seizures in severe seizure GEPRs (GEPR-9s) is 3 mg/kg. In con-
trast, the ED50 in moderate seizure GEPRs (GEPR-3s) is 25 mg/kg. The
present study was designed to ascribe doseresponse differences among
the three rat strains to pharmacokinetic or pharmacodynamic factors. After
systemic carbamazepine, PK studies revealed differences in area under the
concentration-vs-time curve. In other experiments, carbamazepine-
induced serotonin release from hippocampus was used as a pharmacody-
namic marker. In a concentration-controlled design using intracerebral
microdialysis, hippocampal carbamazepine infusions produced similar
concentrationresponse relations for the three rat strains. These data support
the hypothesis that doseresponse differences among the three rat strains are
primarily pharmacokinetic in nature [250].
In this study, the aim was to assess PK effects and adverse cognitive effects
of switches between generic and branded formulations of CBZ. Twelve
patients were included in a randomized open-label, observer-blind, cross-
over design with a double-baseline period, comparing three different for-
mulations of carbamazepine in monotherapythe innovatory branded
form Tegretol and two generic forms, CBZ Pharmachemie and CBZ
Pharbita. Cognitive assessment was carried out at baseline and 3 days after
a crossover. AUC and a number of PK properties (serum concentration
day curves, change in serum concentration (delta scores), peak/trough
concentrations, and peak time) did not differ among the three CBZ
formulations. Therefore, the basic assumption for this study, ie, to test
PK-related differences in cognitive profile, was not met. In line with
these findings, none of the cognitive variables showed statistically signi-
ficant differences with respect to the cognitive profile during the day.
Switches between the investigated generic CBZ formulations and the
branded product did not result in any difference in cognitive profiles. These
results are not necessarily valid, though, for other generic forms of CBZ,
for other types of AEDs, or for CBZ treatment in higher doses or in
polytherapy [251].
The PK and adverse effects of an oral loading dose of carbamazepine
administered in tablet or suspension form were studied. Patients on a hospital
epilepsy unit who were to receive carbamazepine as a discharge medication
were randomly assigned to receive either an oral 8-mg/kg loading dose of
the tablet formulation or the same dose of the suspension on an empty stom-
ach. Blood samples were drawn before and at intervals up to 12 h after the
Carbamazepine 257

loading dose. Adverse effects were evaluated subjectively and objectively.

Total and free serum CBZ and CBZ-E concentrations were determined
by HPLC. Six adult patients were enrolled in and completed the study.
All the patients achieved therapeutic total carbamazepine levels; the suspen-
sion group did so within 2 h and the tablet group within 5 h. Maximum
serum carbamazepine concentrations ranged from 7.10 to 9.92 mg/L, area
under the concentration-vs-time curve from 54.85 to 82.23 g h/L, and
terminal elimination half-life from 14.05 to 15.71 h. Adverse effects were
mild, few, and short-lived; none of the patients developed GI toxicity.
Adverse effects were not associated with total or free CBZ and CBZ-E con-
centrations or with total or free CBZ-E:CBZ ratios. An oral loading dose of
carbamazepine 8 mg/kg achieved therapeutic levels within 2 h when given
as a suspension and within 5 h when given as tablets and was well tolerated in
all patients [252].
A new capsule dosage form of CBZ has been developed, consisting of
three different types of beads (immediate-release, extended-release, and
enteric-release) that may be taken sprinkled on food or swallowed for easy
administration. We compared the PK of the extended-release dosage form
of CBZ (Carbatrol capsules) twice daily with the conventional immediate-
release formulation of CBZ four times daily. The randomized, double-
blind, two-way, crossover study was conducted at two sites, with a planned
sample size of 24 adult patients with epilepsy. Each treatment was adminis-
tered for 2 weeks. At the end of the 2-week period, blood samples were
obtained hourly for a 24-h period. The 90% CIs of the ratio of the means
of the extended-release formulation twice daily to the immediate-release
formulation four times daily were within the range of 0.801.25 for each
of the PK parameters for CBZ and for the summation of CBZ and CBZ-E.
There was no difference in the frequency of seizures between treatment
(P 0.103). The results demonstrate that extended-release CBZ twice daily
was bioequivalent to immediate-release CBZ four times daily, with regard
to CBZ levels and summation of CBZ and CBZ-E levels, based on the PK
parameters evaluated. Substituting one formulation for the other did not
cause patients to have a significant change in seizure frequency [253].
The aim of the authors study was to investigate the factors affecting CBZ
clearance (CL) in children with epilepsy. The factors evaluated were TBW,
age, dose, sex, and phenobarbital (PB) and valproic acid (VA) comedication.
A total of 387 steady-state serum concentration samples were analyzed.
These were collected during CBZ therapy from 201 children, aged 114
years and weighing 978 kg. Population CL was calculated by using
258 S.T. Alrashood

NONMEM, with a one-compartment model with first-order absorption

and elimination. The absorption rate, bioavailability, and volume of distri-
bution were set at values found in the literature. The model found best to
describe the data was CL (0.0122 TBW + 0.0467 dose) age 0.331 (1.289
PB). The interindividual variability in CL had a CV of 11.8%, and the resid-
ual error, described by using an additive model, was 1.5 mg/L. The results
show that CL increases linearly with TBW and nonlinearly with age; thus
older children have a lower CL with respect to TBW than do younger ones.
Likewise, CL was seen to increase with the increase in the CBZ dose,
suggesting a dose-dependent autoinduction of CBZ metabolism. Concom-
itant PB administration affected CL; however, sex and VA comedication did
not affect it significantly. The final regression model for CL was validated in
a different group of 74 children. The standardized prediction error (SPE) was
not significantly different from zero (SPE 0.028), indicating that the model
proposed for CL can be used to make accurate dosage recommendations.
With these population estimates, CBZ doses that would be suitable for pedi-
atric patients of different ages are proposed [254].
To study the transfer and metabolism of oxcarbazepine (OCBZ) and 10-
hydroxy-10,11-dihydrocarbamazepine (10-OH-CBZ) and CBZ metabo-
lism and its possible induction in human placenta,A dual recirculating
human placental perfusion system, blood sampling, HPLC, reverse tran-
scriptase-polymerase chain reaction (RT-PCR), and enzyme assays. OCBZ
was metabolized into 10-OH-CBZ in five human placental cotyledons per-
fused for 2 h in a dual recirculating perfusion system. The same metabolite
was found by HPLC in three sample pairs of maternal and cord blood taken
during delivery from patients on OCBZ therapy. In all of the clinical sam-
ples, 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine (10,11-D) was
also found, but not in the perfusions. In addition, 10-OH-CBZ was not
metabolized in the placental perfusions. The transfer of OCBZ through
the perfused placentas was quicker than the transfer of antipyrine, while
the transfer of 10-OH-CBZ was slower. Both OCBZ and 10-OH-CBZ
also accumulated in placental tissue. CBZ metabolism was studied in three
perfusions using placentas from mothers on CBZ therapy. No metabolism
could be detected in the perfused placentas, while metabolites were found in
both maternal and cord blood of the same mothers. Another series of pla-
centas of mothers on CBZ therapy did not differ significantly from the pla-
centa of a healthy mother as to CYP activities or the level of CYP3A4
mRNA. OCBZ is metabolized into 10-OH-CBZ to some extent in human
placenta in vitro, suggesting that the placenta also participates in the
Carbamazepine 259

metabolism of OCBZ in vivo. On the contrary, the placenta does not par-
ticipate in the metabolism of CBZ. No induction of placental CBZ metab-
olism in vitro can be detected after maternal CBZ treatment during
pregnancy [255].
The aim of the study is to compare carbamazepine PK parameters
between obese and lean subjects following the administration of a single
200 mg tablet. A single-dose intervention, open study was conducted.
The experiments subjects were 18 obese (group A) otherwise healthy subjects,
referred to the metabolic outpatient clinic, and 13 healthy lean (group B)
volunteers. Inclusion criterion for the obese subjects was a body mass
index (BMI weight/height2) of more than 30 kg/m2. In the obese group,
mean  SD TBW, BMI, and percent of ideal body weight (IBW) were
111.4  19.9 kg, 38.8  6.0 kg/m2, and 182.7  30.7%, respectively. These
values were significantly greater than the respective values of 63.2  8.3 kg,
22.4  1.6 kg/m2, and 105.8  5.8% obtained in the lean group (P < 0.001).
Single-dose oral administration of carbamazepine 200-mg tablet was used.
Carbamazepine elimination half-life (t1/2), apparent volume of distribution
(Varea/F), and its oral clearance (Clpo/F) were derived from the drug con-
centrationtime curves. Carbamazepine Varea/F and t1/2 were significantly
greater in group A than in group B (98.4  26.9 vs 60.7  8.5 L, respectively,
P < 0.001; and 59.4  14.7 vs 31.0  5.0 h, respectively, P < 0.001), but its
Clpo/F was reduced only slightly in obese as compared with lean subjects
(19.8  5.2 vs 23.0  4.6 mL/min, respectively, P 0.07). Correction for
IBW yielded similar results for Varea/F and t1/2, but Clpo/F per kg of
IBW was significantly smaller in the obese than in the lean subjects
(0.32  0.07 vs 0.39  0.06 mL/min/kg of IBW, respectively, P < 0.02).
Linear correlations were observed between Varea/F and TBW for both
group A (r 0.92, P < 0.001) and group B (r 0.77, P < 0.002). In
comparison with lean subjects, carbamazepine Varea/F is significantly greater
in obese subjects and its t1/2 is markedly prolonged. The minor nonsignif-
icant effect of obesity on carbamazepine Clpo/F suggests that in obese
subjects, the carbamazepine daily dose should be based on IBW, not on
TBW [256].
The relative bioavailability of three carbamazepine generics available in
Turkey was investigated in five healthy male volunteers. When issuing a
license to any drug, FDA stipulates at most a difference of 20% from the ref-
erence drug only in peak concentration and AUC. This condition may cause
some problems, as two generics of the same drug can yield the same total
amount (AUC) and can be accepted as bioequivalent despite different curves
260 S.T. Alrashood

of the two drugs. In this study, to compare drugs from the point of view of
bioequivalency, a new calculation method was suggested that takes into
account ka (absorption rate constant), ke (elimination rate constant), tmax
(time to peak), MRT (mean residence time), and AUC. Should this formula
be used in comparison of bioequivalency, all the parameters related to the
kinetics of drugs will have been taken into account. However, among three
carbamazepine genericsTegretol, Temporol, and Karazepinthe most
desirable curve is that of Tegretol, while bioavailability values are, respec-
tively, F 0.86, 0.93, 0.85 and AUC 145, 161, 127. The A parameter
values are, respectively, 49.3, 47.2, 42.9 [257].
A major metabolite of CBZ, CBZ-10,11-epoxide (EPO), has been
reported to possess anticonvulsant properties. Therefore, the present study
was undertaken in order to develop a PK model to predict the behavior
of EPO in the body after administration of CBZ. The serum concentration
time curves after oral administration of solution of CBZ (200 mg) or EPO
(150 mg) in six healthy subjects showed the characteristic nose, suggesting
that disposition of CBZ or EPO could be described by the two-compart-
ment model. The kinetic parameters of disposition for CBZ and EPO were
calculated by the method of Wagner, assuming the absolute bioavailabilities
of CBZ and EPO to be 1.0 and 0.81, respectively. Total body clearance and
elimination rate constant of EPO were very much larger than those of the
parent drug, but there was no statistically significant difference in the distri-
bution volume between CBZ and EPO. The formation rate of EPO was
calculated by a deconvolution method and obeyed MichaelisMenten
kinetics. Based on these findings, a PK model of the fate of CBZ and
EPO in humans was developed and the time courses of CBZ and EPO
in serum after oral administration of three tablet preparations and a solution
containing 200 mg of CBZ were simultaneously fitted to this model by solv-
ing the differential equations by the RungeKuttaGill method. There was
good agreement between calculated and observed serum values, suggesting
that the present model is appropriate to describe the formation and dispo-
sition of EPO from CBZ. The formation rate constant of EPO (Vmax/
Km/V1) was approximately 1/15th of the elimination rate constant of
EPO. This suggested a flip-flop model in which the formation of EPO
was rate-limiting in humans. The observation that the serum concentrations
of EPO after administration of CBZ were 1/10th to 1/20th of those of the
parent drug was well explained by the flip-flop kinetics of EPO, together
with the large differences in total body clearance and elimination rate con-
stant between CBZ and EPO [258].
Carbamazepine 261

Carbamazepine is a first-line drug in the treatment of most forms of epi-

lepsy and also the drug of first choice in trigeminal neuralgia. Furthermore, it
is now frequently used in bipolar depression. Most oral formulations of car-
bamazepine are well absorbed with high bioavailability. The drug is 75%
bound to plasma proteins. The degree of protein binding shows little vari-
ation between different subjects, and there is no need to monitor free rather
than total plasma concentrations. Carbamazepine is metabolized in the liver
by oxidation before excretion in the urine. A major metabolite is CBZ-E
which is further metabolized by hydration before excretion. This epoxide
diol pathway is induced during long-term treatment with carbamazepine.
Comedication with phenytoin or phenobarbitone further induces this met-
abolic pathway. Some but not all studies indicate an increased metabolism of
carbamazepine during pregnancy. The drug crosses the placenta, and the
newborns who are exposed to the drug during fetal life eliminate the drug
readily after birth. There seems to be no problem to nurse children during
treatment with carbamazepine. Metabolism of carbamazepine is comparable
in children and adults. Several studies have tried to establish a relationship
between plasma carbamazepine and clinical effect in epilepsy, but very
few of these are controlled. The best anticonvulsant effect seems to be
obtained at plasma concentrations of 1540 mol/L, and a similar optimal
plasma concentration range was found in a controlled study in trigeminal
neuralgia. Side effects are more frequent at higher plasma concentrations
but are also seen within that range. In some patients, with pronounced fluc-
tuation of plasma concentrations during the dosage interval, side effects may
be avoided by more frequent dosing. CBZ-E is a potent anticonvulsant in
animal models. During treatment with carbamazepine, the plasma concen-
trations of this metabolite are usually 1050% of those of the parent drug. It
has not been possible to establish the relative contribution of the two com-
pounds to the pharmacological effects. The epoxide has therefore been
given to humans with the aim of determining the relative potency of the
parent drug and its metabolite. After single oral doses of CBZ-E to healthy
subjects, the compound was rapidly absorbed. As a mean of 90% of the
given dose was recovered in urine as trans-10,11-dihydroxy-10,11-
dihydro-carbamazepine, a complete absorption of unchanged epoxide
was shown [259].
Carbamazepine seems to as effect as phenytoin in the treatment of grand
mal and psychomotor epilepsy. It is the drug of first choice in trigeminal
neuralgia. After single oral doses of carbamazepine, the absorption is fairly
complete and the elimination half-life is about 35 h (range, 1865 h).
262 S.T. Alrashood

During multiple dosing, the half-life is decreased to 1020 h, probably

due to autoinduction of the oxidative metabolism of the drug. Phenytoin
and barbiturates also induce the metabolism of carbamazepine. After
single doses of carbamazepine, elimination follows dose-dependent
first-order kinetics. Carbamazepine is metabolized by oxidation before
excretion in the urine. In experimental animals, the metabolite CBZ-E
has anticonvulsant activity comparable with that of the parent drug. The
plasma concentration of the metabolite during long-term treatment of epi-
leptic patients varies between 5% and 81% of that of the parent drug. The
plasma protein binding of the metabolite is about 50% compared with about
75% for the parent drug. Less than 50% of a given carbamazepine doses has
been identified as metabolites in the urine. The quantitatively most impor-
tant metabolites is the trans-10,11-dihydro-10,11-diol. The kinetics of car-
bamazepine have been explored to some extent in pregnant women,
newborns, and children. Plasma levels of carbamazepine seem to decrease
during pregnancy, possibly as a result of increased metabolism. The drug
readily crosses the placenta and the levels measured in newborns are com-
parable with maternal plasma concentrations. In newborns exposed to the
drug during fetal life, the plasma half-lives were relatively short (8.2
28.1 h) indicating an induction of carbamazepine metabolism during gesta-
tion. The PK of carbamazepine in children aged 0.315 years are compara-
ble with that in adults. A single daily dose of carbamazepine is insufficient;
two doses per day are appropriate in most cases, but some patients may ben-
efit from more frequent dosing to avoid side effects. Compared with phe-
nytoin, for example, very few controlled studies have been performed to
establish the plasma level range of carbamazepine associated with the best
therapeutic outcome. However, the best anticonvulsant effect of carbamaz-
epine seems to be obtained at plasma levels of about 510 g/mL (20
40 mol/L). Side effects are most frequent at higher levels but may also
be seen at lower levels [260].
The time courses of plasma carbamazepine concentrations were followed
in six apparently healthy adult subjects who, at different times, took single
oral drug doses of 200, 400, 500, 600, 700, 800, and 900 mg. There were
some suggestions of impaired bioavailability of the drug when given in tablet
form. The following values were obtained for various PK parameters:
kabs 0.176  0.209 h1; k 0.0203  0.0055 h1; t1/2 37.5  13.1 h;
VD 0.825  0.1041 kg1; clearance 0.0163  0.00611 kg1. The elimi-
nation rate constant showed a statistically significant increase with increasing
drug dose. This may help explain the clinical observation that the rate of rise
Carbamazepine 263

of steady-state plasma carbamazepine concentrations tends to decrease with

dose increase in patients taking carbamazepine alone [261].
The excretion of carbamazepine in the saliva of six normal adults after
receiving a single oral dose of 400 mg carbamazepine is described. There
was a good correlation between carbamazepine concentrations in the plasma
and saliva (r 0.94, P < 0.001). This indicates that the concentrations of car-
bamazepine in the saliva can be used to monitor carbamazepine therapy. The
half-life of carbamazepine in the plasma was not significantly different from
the half-life in the saliva. Thus areas under concentrationtime curves,
apparent volumes of distribution (Vd), and the total body clearances were
significantly dependent (P < 0.001) upon the distribution of carbamazepine
between plasma and saliva. Calculated from saliva concentrations, 75% of the
total carbamazepine plasma concentration is bound to protein, while 25% is
unbound in diffusional equilibrium with saliva. These figures are consistent
with data in the literature [262].

7.2 Metabolism
Hepatic intrinsic clearance (CLint) of drugs is often predicted based on in vitro
data that are obtained from the MichaelisMenten analysis. While most of the
metabolic ratesubstrate concentration kinetic curves fit to the Michaelis
Menten equation, cytochrome P450 (CYP) and uridine 50 -diphosphate
(UDP)-glucuronosyltransferases exhibit sigmoidal kinetics for certain drugs.
In our study, the kinetics of CYP3A4-catalyzed carbamazepine 10,11-epox-
idation in human liver microsomes (HLMs) was sigmoidal and fitted to the
Hill equation, revealing the S50 value of 358 M, n of 2.0, and the Vmax value
of 463 pmol/min/mg. While the intrinsic clearance calculated from
MichaelisMenten parameters (CLint) overestimated the observed in vivo
intrinsic clearance (CLint, in vivo), the maximum intrinsic clearance calculated
based on the Hill equation (CLmax) exhibited better predictions of CLint, in
vivo. Such better prediction using the CLmax was also observed for other four
drugs, all of which also exhibited sigmoidal metabolic rateconcentration cur-
ves, according to the literature data. However, even if we assume such Hill
equation, intrinsic clearances predicted at their therapeutic concentrations
from in vitro data were still much lower than their CLint, in vivo, suggesting
the existence of unknown factors causing discrepancy between in vitro intrin-
sic clearance in HLMs and in vivo data. Thus, even if we assume sigmoidal
kinetics, that would not be enough for accurate prediction of CLint, in vivo,
and it would be preferable to use CLmax to quantitatively extrapolate the in
vitro data to in vivo clearance [263].
264 S.T. Alrashood

Carbamazepine and diclofenac were frequently detected in water bodies.

In this study, crude lignin peroxidase, produced from a white rot fungus
Phanerochaete chrysosporium, was studied on its in vitro degradation of both
drugs. The influencing parameters were studied, including pH, the hydro-
gen peroxide concentration, veratryl alcohol, and the temperature. It was
found that LiP completely degraded diclofenac at pH 3.04.5 and 3
24 ppm H2O2, while the degradation efficiency of carbamazepine was
mostly below 10%. The addition of veratryl alcohol and the increased tem-
perature did not enhance the degradation of carbamazepine [264].
To develop a PPK model to evaluate the effects of variety of covariates
on clearance of CBZ and its main metabolite CBZ-E in Chinese population.
Serum samples at steady trough state (n 459) were collected prospectively
from 408 compliant outpatients during their routine clinical care. CBZ and
CBZ-E concentrations were simultaneously determined by HPLC. Popu-
lation clearance (CL) of CBZ and CBZ-E was estimated by nonlinear mixed
effect modeling and NONMEM program with a one-compartment model
of first-order absorption and elimination. TBW, dose, and concomitant
medication were all important determinants of CL of CBZ and CBZ-E.
The final regression model for CBZ was: VPA 1 for patients comedicated
with valproic acid and its dose greater than 18 mg/kg, otherwise VPA 0;
PHT 1 for patients comedicated with phenytoin, otherwise PHT 0;
PB 1 for patients comedicated with phenytoin, otherwise PB 0. The
final regression CL model for the CBZ-E was: where VPA 1 for patients
comedicated with valproic acid, otherwise VPA 0. CONCLUSION: The
current models, which describe CL of CBZ and CBZ-E in terms of patient-
specific details, can be used as a reference to optimize CBZ therapy in
Chinese epilepsy patients [265].
In vitro studies were conducted to identify the cytochromes P450 (P450s)
involved in the formation of 2- and 3-hydroxycarbamazepine, metabolites
that may serve as precursors in the formation of protein-reactive metabolites.
HLMs converted carbamazepine (30300 M) to 3-hydroxycarbamazepine
at rates >25 times those of 2-hydroxycarbamazepine. Both the 2- and 3-
hydroxylation of carbamazepine appeared to conform to monophasic
MichaelisMenten kinetics in HLMs (apparent Km values, approximately
1640, and approximately 217 M; apparent Vmax values, approximately 5.71,
and approximately 46.9 pmol/mg of protein/min, respectively). Rates
of carbamazepine 2- and 3-hydroxylation correlated strongly with
CYP2B6 activity (r  0.757) in a panel of HLMs (n 8). Carbamazepine
3-hydroxylation also correlated significantly with CYP2C8 activity at a
Carbamazepine 265

carbamazepine concentration of 30 M. Formation of 2- and 3-

hydroxycarbamazepine did not correlate significantly with any other
P450 activities. The chemical inhibitors ketoconazole (CYP3A) and 7-
EFC (CYP2B6) inhibited both 2- and 3-hydroxycarbamazepine forma-
tion, whereas 4-methylpyrazole (CYP2E1) markedly decreased 2-
hydroxycarbamazepine formation. Several recombinant P450s catalyzed
carbamazepine 2- and 3-hydroxylation, but after adjustment for relative
hepatic abundance, CYP3A4 and CYP2B6 appeared to be the major cata-
lysts of carbamazepine 3-hydroxylase activity, and at least five P450s were
significant contributors to 2-hydroxycarbamazepine formation; CYP2E1
made the greatest contribution to the CLint of carbamazepine 2-hydroxyl-
ation (30%), but P450s CYP1A2, 2A6, 2B6, and 3A4 also made significant
contributions (1318%). These results suggest that CYP2B6 and CYP3A4
are largely responsible for the formation of 3-hyrdoxycarbamazepine,
whereas multiple P450s (CYP1A2, 2A6, 2B6, 2E1, and 3A4) contributed
to 2-hydroxycarbamazepine formation [266].
CBZ-E is a major metabolite of CBZ. It has anticonvulsive properties
and may be responsible for side effects of CBZ treatment. Fifty-two children
between the age of 2 weeks and 15 years were treated with CBZ (mean dos-
age 17 mg/kg body weight) either as mono- (n 36) or in polytherapy
(n 16). The drug was delivered as an oral solution, as a nonretarded tablet,
or, most frequently, as a retarded tablet. The duration of treatment ranged
from 1 to 94 months with 23 patients being on treatment for less than 3
months. Blood samples were taken with random timing after the last inges-
tion of the drug. The relative concentration of CBZ epoxide (expressed in %
of CBZ) was higher in infants (median 48.9%) than in older children
(median 14.9% in the 12- to 15-year-old group). A significant linear corre-
lation with age was found (P < 0.001). In addition to young age, polytherapy
(P < 0.01) and administration as a nonretarded formulation rather than as a
retarded tablet (P < 0.05) induced a higher relative concentration of the
epoxide. The relative concentration of the epoxide did not correlate with
the serum CBZ concentration and the duration of treatment. Although
in our study high epoxide levels were not related to clinical side effects,
we recommend that in very young children polytherapy treatment with
carbamazepine should be performed with caution and in difficult cases a
determination of the epoxide level should be considered [267].
The disposition of CBZ was investigated in the SWV mouse. A
14
C-CBZ dose was administered to CBZ-pretreated mice, and the distribu-
tion of radiolabeled material was determined. Twenty-four hours after
266 S.T. Alrashood

the 14C-CBZ dose, 92.5% of the dose was accounted for in urine (56%), in
the viscera and carcass (22%), in feces (11%), and expired as 14CO2 (2%).
CBZ metabolites present in hydrolyzed urine were also identified using a
combination of spectroscopic techniques. CBZ, CBZ-E, 2- and 3-
hydroxy-CBZ, methylsulfonyl-CBZ, and glucuronides of CBZ and
CBZ-E accounted for 64% of total urinary radioactivity (024 h) in
CBZ-pretreated mice. Minor metabolites of CBZ included novel cysteine
and N-acetylcysteine conjugates of CBZ, as well as a methylsulfonyl conju-
gate of CBZ-E not previously reported. The urinary excretion of these
thioether conjugates was increased in CBZ/phenobarbital-pretreated
mice and decreased in CBZ/stiripentol-pretreated mice in comparison
with CBZ-only treated mice. Preliminary studies of the effects of pheno-
barbital and stiripentol on the urinary abundance of these metabolites are
consistent with the modulation of teratogenicity in the SWV mouse by
the same pretreatments. These data suggest that the formation of thioether
metabolites of CBZ may be related to CBZ teratogenicity in the SWV
mouse [268].
CBZ-E was found to decompose in gastric juice in vitro. An antacid did
not affect the bioavailability of single CBZ doses given to three subjects and
was therefore used to neutralize gastric juice when administering CBZ-E.
CBZ-E was given orally as a suspension in two single doses ranging from 10
to 200 mg to each of four healthy subjects. Plasma concentrations of CBZ
and CBZ-E were determined with HPLC. Plasma concentrations and uri-
nary excretion of the end metabolite trans-10,11-dihydroxy-10,11-dihydro-
CBZ (trans-CBZ-diol) were measured by mass fragmentography. After dos-
ing with CBZ-E, peak plasma concentrations of the parent compound were
reached within 2 h. Urinary recovery of trans-CBZ-diol was 90  11%
(mean  SD) of the dose, indicating almost complete absorption. Plasma
kinetics of the epoxide fitted an open one-compartment model with elim-
ination half-lives (t1/2) of 6.1  0.9 h. Clearance was 89  25 mL/kg/h. The
urinary excretion t1/2 of the trans-CBZ-diol was 12.4  0.9 h, which
is longer (P < 0.001) than the epoxide plasma t1/2. There was no indication
of dose-dependent kinetics of the epoxide. After 200 mg CBZ to the
same subjects, plasma CBZ t1/2 was 26.0  4.6 h and clearance was
23.4  4.6 mL/kg/h. Of the CBZ dose, 20.5  2.9% was excreted as the
trans-CBZ-diol, which gives an estimate of the percentage of CBZ that is
metabolized by the epoxidediol pathway in healthy subjects. These obser-
vations provide a basis for the administration of CBZ-E in patients to assess
its clinical effects [269].
Carbamazepine 267

7.3 Excretion
The kidneys have the capability to both excrete and metabolize drugs.
An understanding of mechanisms that determine these processes is required
for the prediction of PK, exposures, doses, and interactions of candidate
drugs. This is particularly important for compounds predicted to have
low or negligible nonrenal clearance (CL). Clinically significant interac-
tions in drug transport occur mostly in the kidneys. The main objective
was to evaluate methods for prediction of excretion and metabolic renal
CL (CL(R)) in humans. CL(R) is difficult to predict because of the
involvement of bidirectional passive and active tubular transport, differ-
ences in uptake capacity, pH and residence time on luminal and blood
sides of tubular cells, and limited knowledge about regional tubular resi-
dence time, permeability (P(e)), and metabolic capacity. Allometry pro-
vides poor predictions of excretion CL(R) because of species differences
in unbound fraction, urine pH, and active transport. The correlation
between fraction excreted unchanged in urine (f(e)) in humans and animals
is also poor, except for compounds with high passive P(e) (extensive/
complete tubular reabsorption; zero/negligible f(e)) and/or high nonrenal
CL. Physiologically based in vitro/in vivo methods could potentially be
useful for predicting CL(R). Filtration could easily be predicted. Prediction
of tubular secretion CL requires an in vitro transport model and
establishment of an in vitro/in vivo relationship and does not appear to
have been attempted. The relationship between passive P(e) and tubular
fraction reabsorbed (f(reabs)) for compounds with and without apparent
secretion has recently been established and useful equations and limits
for prediction were developed. The suggestion that reabsorption has a
lipophilicity cutoff does not seem to hold. Instead, compounds with passive
P(e) that is less than or equal to that of atenolol are expected to have neg-
ligible passive f(reabs). Compounds with passive P(e) that is equal to or
higher than that of carbamazepine are expected to have complete f(reabs).
For compounds with intermediate P(e), the relationship is irregular and f
(reabs) is difficult to predict. Tubular cells are comparably impermeable (for
passive diffusion) and show regional differences in enzymatic and trans-
porter activities. This limits the usefulness of microsome data and makes
microsome-based predictions of metabolic CL(R) questionable. Renal
concentrations and activities of CYP450s are comparably low, suggesting
that CYP450 substrates have negligible metabolic CL(R). The metabolic
CL(R) of high-P(e) UDP-glucuronosyltransferase substrates could contrib-
ute to the total CL [270].
268 S.T. Alrashood

The mood stabilizers, lithium, CBZ, and valproate (VPA), have differing
PK, structures, mechanisms of action, efficacy spectra, and adverse effects. 1.
Lithium has a low therapeutic index and is renally excreted and hence
has renally mediated but not hepatically mediated drugdrug interac-
tions. 2. CBZ has multiple problematic drugdrug interactions due to
its low therapeutic index, metabolism primarily by a single isoform
(CYP3A3/4), active epoxide metabolite, susceptibility to CYP3A3/4 or
epoxide hydrolase inhibitors, and ability to induce drug metabolism (via
both cytochrome P450 oxidation and conjugation). In contrast, VPA has
less prominent neurotoxicity and three principal metabolic pathways, ren-
dering it less susceptible to toxicity due to inhibition of its metabolism.
However, VPA can increase plasma concentrations of some drugs by
inhibiting metabolism and increase free fractions of certain medications
by displacing them from plasma proteins. 3. Older anticonvulsants such as
phenobarbital and phenytoin induce hepatic metabolism, may produce tox-
icity due to inhibition of their metabolism, and have not gained general
acceptance in the treatment of primary psychiatric disorders. 4. The newer
anticonvulsants felbamate, lamotrigine, topiramate, and tiagabine have dif-
ferent hepatically mediated drugdrug interactions, while the renally
excreted gabapentin lacks hepatic drugdrug interactions but may have
reduced bioavailability at higher doses. 5. Investigational anticonvulsants
such as oxcarbazepine, vigabatrin, and ZNS appear to have improved PK
profiles compared to older agents. 6. Thus, several of the newer anticonvul-
sants lack the problematic drugdrug interactions seen with older agents,
and some may even (based on their mechanisms of action and preliminary
preclinical and clinical data) ultimately prove to have novel psychotropic
effects [271].
Urinary excretions of CBZ, CBZ-E, carbamazepine-10,11-trans-diol,
9-hydroxyacridan, and 2- and 3-hydroxycarbamazepine were measured at
various stages of pregnancy, and in the postnatal period, in 10 epileptic
women, 6 of whom took no other enzyme-inducing anticonvulsant and
4 of whom took such comedication. Mean plasma carbamazepine apparent
clearance was increased in pregnancy, but only by virtue of the increased
clearance in the anticonvulsant comedicated women. Alterations in the pro-
portions of the carbamazepine dose cleared via the various excretion path-
ways studied were quantitatively minor, but there was evidence consistent
with impaired conversion of CBZ-E to carbamazepine-10,11-trans-diol
during all pregnancies studied. Clearances of carbamazepine to the various
excretory products studied were consistent with there being (i) increased
Carbamazepine 269

urinary excretion of unmetabolized drug in pregnancy, possibly related to

the increased glomerular filtration rate (GFR), (ii) increased formation of
oxidative metabolites of the drug, particularly in women comedicated
with enzyme-inducing anticonvulsants, this effect being offset, in full (in
non-comedicated women) or in part (in comedicated women) by (iii) inhi-
bition of the epoxidediol pathway in pregnancy, an inhibition to which
folate intake may have contributed [272].
In eight epileptic patients receiving chronic antiepileptic treatment, a
study of the excretion in sweat of phenytoin, phenobarbitone, and carba-
mazepine was performed. All three drugs were found to be present in sweat.
Phenytoin sweat concentration was found to correspond to the free fraction
in plasma and to be independent of sweat flow. Phenobarbitone sweat con-
centration was found to increase with increasing sweat flow. Regarding
drug-level monitoring, it is proposed that under changing climatic condi-
tions the phenomenon may be of clinical significance [273].
The PK of carbamazepine were evaluated in four male rhesus monkeys.
A 20-mg/kg dose was administered by intravenous (5-min) infusion and
orally (nasal-gastric intubation) in a propylene glycolethanolwater sol-
vent. Plasma and urine determinations were performed by GLC. All semi-
logarithmic intravenous curves exhibited an irregular decay behavior in the
first 3-h period, followed by a linear disappearance phase (t1/2 equals 1.0
2.4 h). Urinary excretion measurements confirmed the short elimination
half-life and showed that less than 1% of the dose was excreted unchanged.
Oral studies also yielded a short elimination half-life (1.01.60 h), which was
confirmed by urinary excretion measurements. The oral curves were ana-
lyzed pharmacokinetically. The fraction of the dose reaching the systemic
circulation ranged between 58% and 87%. Measurable (but insignificant)
amounts of drug were found in the feces after intravenous and oral admin-
istrations [274].
Carbamazepine (amizepine) is a widely used psychotropic agent. A much
easier accessibility of this drug, observed during the recent years, may
account for an increasing number of acute intoxications with carbamaze-
pine. The aim of this study was to determine the elimination kinetics of car-
bamazepine and its metabolite CBZ-E, and to identify the quantitative
relationship between concentrations of these compounds, in serum. The
subjects were 41 patients with acute carbamazepine intoxication. Serum
CBZ and CBZ-E concentrations were determined every 6 h during the first
24 h of hospitalization, and then every 12 h. At the same time, urinalyses
were performed for each patient to confirm or exclude homogeneity of
270 S.T. Alrashood

poisoning. Depending on the type of intoxication (homogeneous or com-

bined), three groups of patients, and on the method of treatment (symp-
tomatic, charcoal administration), two groups of patients were
distinguished. The statistical analysis of the results revealed that among
the investigated parameters (time-integrated concentrations of CBZ and
CBZ-E in serum, the presence of drugs, and/or ethanol, charcoal treat-
ment) only carbamazepine concentrations had statistically significant effect
on the duration of coma regarded as a critical effect. The kinetics of car-
bamazepine elimination was determined on the basis of the mean carba-
mazepine concentrations at the same timing of sampling for each patient
in all the three groups; the mean carbamazepine elimination in serum
followed zero-order kinetics. In individual groups, the decrease in serum
carbamazepine concentrations ranged from 0.5 to 0.8 mg/L/h. Contrary
to the suggestions found in the literature, CBZ-E determination does
not seem to enhance the possibility of anticipating the course of intoxica-
tion or the time of recovery [275].

8. PHARMACOLOGY
To study strength-duration properties of motor and sensory axons to
evaluate whether there is a change in current through the persistent sodium
(Na+) channels of sensory and motor axons in peripheral nerves of epileptic
patients before and after VPA and CBZ treatment due to the presence of
similar channels in the CNS and peripheral nervous system (PNS). This
study, conducted in Baskent University Faculty of Medicine, Adana,
Turkey from Jan. 2011 to Feb. 2012, involved 10 patients with partial
epilepsy, 10 patients with primary generalized epilepsy who were not cur-
rently prescribed anticonvulsant therapy, and 10 control subjects. Using
an electromyography machine, stimulus intensity was performed to pro-
duce the target (40% of maximum) compound muscle action potentials
and compound sensory action potentials. The currents required for different
stimulus durations, 0.05, 0.1, 0.2, 0.3, 0.5, and 1 ms, were produced.
Stimulusresponse curves were then constructed, and the strengthduration
time constants were estimated using Weiss formula. The rheobase of motor
and sensory fibers was lower in the control group than the values of patients
before and after CBZ and VPA therapy. In the PNS of epileptic patients,
CBZ and VPA therapy results in decreased axonal excitability. This method
may be used in investigating the underlying pathology of peripheral nerve
diseases in vivo [276].
Carbamazepine 271

Voltage-gated sodium channels are inhibited by many local anesthetics,

antiarrhythmics, and AEDs. The local anesthetic lidocaine appears to be able
to access its binding site in the sodium channel only from the membrane
phase or from the internal face of the channel. In contrast, the AED carba-
mazepine was found to inhibit voltage-gated sodium channels only with
external, but not internal, application, implying a major difference. This
point was investigated using both whole-cell and inside-out patch record-
ings from human Na(v)1.7 channels in a stable cell line. In the whole-cell
configuration, carbamazepine inhibited sodium current within seconds
when applied externally, but had little or no effect when applied internally
for up to 15 min, confirming previous results. However, carbamazepine
inhibited sodium channels effectively and rapidly when applied to the inter-
nal face of the membrane using inside-out patch recording. It was found that
lidocaine also has little or no effect when applied intracellularly in whole-cell
recording, but blocks effectively and rapidly when applied to the internal
surface using inside-out patches. In contrast, the cationic lidocaine deriva-
tive QX-314 (N-ethyl-lidocaine) blocks effectively when applied internally
with whole-cell dialysis, as well as when applied to inside-out patches. It was
concluded that carbamazepine and lidocaine access the sodium channel in
similar ways and hypothesize that their lack of effect with internal dialysis
in whole-cell recording reflects rapid exit through membrane near the
pipette recording site. This effect likely limits the ability of any compound
with significant membrane permeability to be applied intracellularly by
whole-cell dialysis [277].
Onset of the mitochondrial permeability transition (MPT) plays a caus-
ative role in ischemia/reperfusion (I/R) injury. Current therapeutic strate-
gies for reducing reperfusion injury remain disappointing. Autophagy is a
lysosome-mediated, catabolic process that timely eliminates abnormal or
damaged cellular constituents and organelles such as dysfunctional mito-
chondria. I/R induces calcium overloading and calpain activation, leading
to degradation of key autophagy-related proteins (Atg). CBZ, an FDA-
approved anticonvulsant drug, has recently been reported to increase
autophagy. The effects of CBZ on hepatic I/R injury were investigated.
Hepatocytes and livers from male C57BL/6 mice were subjected to simulate
in vitro as well as in vivo I/R, respectively. Cell death, intracellular calcium,
calpain activity, changes in Atg, autophagic flux, MPT, and mitochondrial
membrane potential after I/R were analyzed in the presence and absence
of 20 M CBZ. CBZ significantly increased hepatocyte viability after
reperfusion. Confocal microscopy revealed that CBZ prevented calcium
272 S.T. Alrashood

overloading, the onset of the MPT, and mitochondrial depolarization.

Immunoblotting and fluorometric analysis showed that CBZ blocked cal-
pain activation, depletion of Atg7 and Beclin-1, and loss of autophagic flux
after reperfusion. Intravital multiphoton imaging of anesthetized mice dem-
onstrated that CBZ substantially reversed autophagic defects and mitochon-
drial dysfunction after I/R in vivo. In conclusion, CBZ prevents calcium
overloading and calpain activation, which, in turn, suppresses Atg7 and
Beclin-1 depletion, defective autophagy, onset of the MPT, and cell death
after I/R [278].
Limited and conflicting data exist for the influence of AEDs on thyroid
function in children. The aim of this study was to investigate the effects of
phenobarbital, valproate, carbamazepine, oxcarbazepine, and levetiracetam
monotherapy on thyroid function in daily clinical practice during a 12-
month treatment period. A total of 223 children (103 females and 120 males)
with new onset and controlled epilepsy treated with valproate (n 129),
phenobarbital (n 33), carbamazepine (n 36), oxcarbazepine (n 14),
levetiracetam (n 11) were enrolled in the study. Serum-free thyroxine
(fT4) and thyroid-stimulating hormone (TSH) levels were measured before
and at 1st, 6th, and 12th month of therapy. At baseline, average fT4 and TSH
concentrations were not different between the drug groups. Valproate-
treated patients had decreased fT4 and increased TSH levels at months 1,
6, and 12. Carbamazepine-treated patients had decreased fT4 levels at
months 1, 6, and 12 and increased TSH levels at months 1 and 6. Phenobar-
bital-treated patients had decreased fT4 levels at months 1 and 6, and
increased TSH levels at months 6 and 12. Oxcarbazepine-treated patients
had decreased fT4 levels at month 1. Levetiracetam-treated patients showed
no significant change of fT4 and TSH at any times. The frequency of sub-
clinical hypothyroidism at month 12 was 28% in valproate, 21.4% in
oxcarbazepine, 18.2% in phenobarbital, 13.9% in carbamazepine, and 0%
in levetiracetam groups. The data suggest that all AEDs studied except
levetiracetam had varying degrees of deleterious effects on thyroid function
[279].
The hyperplasia of synovial fibroblasts is considered to be essential for the
evolution of joint destruction in rheumatoid arthritis (RA). Previously, it
was reported that antirheumatic drugs, both COX inhibitors, and disease-
modifying antirheumatic drugs inhibit proliferation of synoviocytes in vitro.
The presented study investigates the effect of antiepileptic drugs on the via-
bility and proliferation of synovial fibroblasts in vitro. Experiments were
conducted on human synoviocytes derived from an RA patient and rabbit
Carbamazepine 273

synoviocytes cell line HIG-82. Cell proliferation and viability were assessed
by means of BrdU assay and MTT assay, respectively. The IC50 value (the
concentration of drug necessary to induce 50% inhibition) together with
confidence limits was calculated. Carbamazepine inhibited proliferation of
human fibroblasts and viability of HIG-82 with IC50 values of 86 and
82 M, respectively. Diphenylhydantoin, valproate, and phenobarbital
inhibited viability of HIG-82 cells with the IC50 values of 110, 500, and
1031 M, respectively. Based on these findings, it can be suggested that anti-
epileptic drugs may have a disease-modifying effect on rheumatoid synovial
proliferation [280].
Anticonvulsants have been used to manage psychiatric conditions for
over 50 years. It is recognized that some, particularly valproate, carbamaz-
epine, and lamotrigine, are human teratogens, while others including
topiramate require further investigation. It was aimed to appraise the doc-
umentation of this risk by psychiatrists and review discussion around con-
traceptive issues. A retrospective review of prescribing patterns of four
anticonvulsants (valproate, carbamazepine, lamotrigine, and topiramate)
in women of child-bearing age was undertaken. Documented evidence
of discussion surrounding teratogenicity and contraceptive issues was
sought. Valproate was most commonly prescribed (n 67). Evidence of ter-
atogenic risk counseling at medication initiation was suboptimal40% of
individuals prescribed carbamazepine and 22% of valproate. Documentation
surrounding contraceptive issues was also low17% of individuals pre-
scribed carbamazepine and 13% of valproate. It was found both low rates
of teratogenic risk counseling and low rates of contraception advice in
the cohort. Given the high rates of unplanned pregnancies combined with
the relatively high risk of major congenital malformations (MCMs), it is
essential that a detailed appraisal of the risks and benefits associated with anti-
convulsant medication occurs and is documented within patients psychiat-
ric notes [281].
Although it is well documented that long-term therapy with older AEDs
leads to an increase in risk for atherosclerosis, there has been only limited
information regarding the vascular risk in patients who are treated with
new AEDs. It was therefore conducted a prospective longitudinal study
to assess the potential effects of new AEDs on the circulatory markers for
vascular risk in patients with newly diagnosed epilepsy. Adult patients with
epilepsy who began to receive monotherapy with one of the new AEDs,
including levetiracetam (LEV), OXC, and topiramate (TPM), were rec-
ruited. Circulatory markers of vascular risk were measured twice before
274 S.T. Alrashood

and after 6 months of AED monotherapy. A total of 109 patients completed

the study (LEV, n 40; OXC, n 40; TPM, n 29). Six months of mon-
otherapy resulted in significant increases in low-density lipoprotein choles-
terol (LEV, from 90.2 to 98.5 mg/dL, 9.2% increase, P 0.025; OXC, from
96.5 to 103.2 mg/dL, 7.0% increase, P 0.049), homocysteine (LEV, from
7.9 to 10.4 m, 31.6% increase, P 0.001; OXC, from 8.7 to 11.5 m,
32.2% increase, P < 0.001; TPM, from 8.3 to 12.3 m, 48.2% increase,
P < 0.001), apolipoprotein B (LEV, from 63.6 to 77.4 mg/dL, 21.7%
increase; OXC, from 67.0 to 83.2 mg/dL, 24.2% increase; TPM, from
66.7 to 84.4 mg/dL, 26.5% increase; all P < 0.001), and apolipoprotein
B/apolipoprotein A1 ratio (LEV, from 0.51 to 0.61, 19.6% increase;
OXC, from 0.52 to 0.67, 28.8% increase; TPM, from 0.50 to 0.67,
34.0% increase; all P < 0.001). Serum apolipoprotein A1 and folate were sig-
nificantly decreased in TPM (from 139.1 to 132.1 mg/dL, 5.0% decrease,
P 0.014) and OXC (from 8.1 to 6.4 ng/mL, 21.0% decrease, P 0.046)
groups, respectively. There were no significant changes in total cholesterol,
triglyceride, high-density lipoprotein cholesterol, lipoprotein(a), and vita-
min B12 in all three groups. The findings suggest that treatment with some
new AEDs might be associated with alterations in circulatory markers of vas-
cular risk, which could contribute to the acceleration of atherosclerosis and
increased risk of vascular diseases [282].
Changes within the immune system have been reported to contribute to
the pathophysiology of bipolar disorder and epilepsy. Interestingly, over-
lapping results regarding the cytokine system have been found for both dis-
eases, namely alterations of interleukins IL-1, IL-2, IL-4, IL-6, and tumor
necrosis factor- (TNF-). However, the effect of mood stabilizers and
AEDs on these cytokines has not been systematically evaluated, and their
effect on IL-17 and IL-22, other immunologically important cytokines,
has not been reported. Therefore, we systematically measured levels of
IL-1, IL-2, IL-4, IL-6, IL-17, IL-22, and TNF- in stimulated blood of
14 healthy female subjects in a whole blood assay using the toxic shock
syndrome toxin TSST-1 as stimulant. Blood was supplemented with the
mood stabilizers or antiepileptic drugs primidone (PRM), CBZ,
levetiracetam (LEV), LTG, VPA, OXC, topiramate (TPM), phenobarbital
(PB), lithium, or no drug. IL-1 production was significantly decreased by
PRM, CBZ, LEV, LTG, OXC, PB, and lithium. IL-2 significantly
decreased by PRM, CBZ, LEV, LTG, VPA, OXC, TPM, and PB. IL-
22 significantly increased by PRM, CBZ, LEV, OXC, TPM, and lithium
and decreased by VPA. TNF- production significantly decreased under
Carbamazepine 275

all applied drugs. The mechanism of action and side effects of mood stabi-
lizers and AEDs may involve modulation of IL-1, IL-2, IL-22, and TNF-
signaling pathways. IL-22 may be a research target for specific therapeutic
effects of mood stabilizers and AEDs. These drugs might influence cytokine
production by modulating ion channels and GABA receptors of immune
cells [283].
The effects of AEDs on bone metabolism and the endocrine system are
not fully known, and publications on the subject are inconsistent. The study
aimed to examine the mutual effects of VPA, CBZ, and PBAEDs fre-
quently used in childhoodon bone mineral metabolism and thyroid func-
tion tests. Children monitored with a diagnosis of idiopathic epilepsy by the
pediatric neurology clinic, using AEDs for at least 6 months and with epi-
sodes under control, were included in the study. Patients were divided into
groups on the basis of the drugs used. Thyroid function tests and 25-hydro-
xyvitamin D or 25(OH)D levels were measured from blood specimens. The
data obtained were then compared with those of the control group. A sig-
nificantly high level of subclinical hypothyroidism was seen in patients using
VPA (P < 0.001). There was no significant difference between any of the
three study groups and the control group in terms of 25(OH)D
(P > 0.05). Pediatric patients using AEDs, particularly VPA, should be mon-
itored for subclinical hypothyroidism. VPA, CBZ, and PB have no effect on
vitamin D levels [284].
CBZ, a widely used anticonvulsant and mood stabilizer, activates mul-
tiple proliferative and prosurvival pathways. Here, it was hypothesized that
CBZ may promote hepatocellular proliferation and ameliorate liver regen-
eration. C57BL6/J mice were orally administered CBZ or vehicle and
underwent a 70% partial hepatectomy (PHx) and 85% PHx or treatment
with carbon tetrachloride (CCl4). Liver regeneration was determined by
liver to body weight ratio, hepatocyte proliferation markers, and activation
of intracellular signaling pathways. Two to 5 days after the 70% PHx, the
liver to body weight ratio was significantly higher in the CBZ-treated mice
than in the vehicle-treated mice. CBZ treatment upregulated the number of
proliferative hepatocytes following PHx or CCl4 treatment, as assessed by
intrahepatic Ki-67 staining, BrdU uptake, and PCNA protein expression.
PHx surgery induced the expression of several cyclins and activated Akt/
mTOR signaling pathways, all of which were enhanced by CBZ treatment.
The administration of the mTOR inhibitor temsirolimus abrogated the
hepatoproliferative effect of CBZ. CBZ treatment significantly improved
the survival rate of the mice that underwent lethal 85% massive
276 S.T. Alrashood

hepatectomy. CBZ demonstrated a novel hepatoproliferative effect through

the activation of the mTOR signaling pathway in hepatectomized mice.
CBZ has the potential to be a therapeutic option for facilitating efficient liver
regeneration in patients subjected to liver surgery [285].
AEDs are currently used in both neurology and psychiatry. It is generally
accepted that GABAergic compounds have sedative and anxiolytic proper-
ties, whereas channel blockers are mood stabilizers. However, this paradigm
is often challenged. This is related to the variety of mechanisms of action of
individual AEDs on biological systems, only some of which are related to the
desired CNS effect. At present, just a few AEDs are licensed for psychiatric
indications, namely carbamazepine, valproate, lamotrigine, and pregabalin.
Data on other AEDs show potential benefits, but are still inconclusive in
some cases. This article discusses molecular targets of AEDs relevant for their
psychotropic properties with special attention to newest compounds. Cur-
rent knowledge gaps are also highlighted [286].
To compare the levels of homocysteine, vitamin B12, and folic acid
before and after 6 months of carbamazepine therapy and to correlate them
with carbamazepine level at 6 months, a prospective comparative study was
held in a Tertiary care center in North India using 51 children (212 years of
age) presenting with motor partial seizures taking CBZ (1020 mg/kg/day)
for 6 months. Change in serum homocysteine, B12, and folic acid level was
observed. Fasting venous samples were collected before CBZ therapy and
after 6 months. Homocysteine was analyzed using homocysteine enzyme
immunoassay. Vitamin B12 and folic acid were estimated using electro-
chemiluminesence technique. Carbamazepine levels were measured at 6
months. Of the 51 children, 36 (males21) were followed up and their data
were analyzed. Mean homocysteine level was 11.51  3.95 mol/L at
recruitment and 11.77  6.65 mol/L at 6 months (P 0.785). At recruit-
ment 6 (16%) children had homocysteine level above 15 mol/L which
increased to 10 (27%) at 6 months. Mean vitamin B12 at recruitment was
292.1  111.2 and 297.8  82.9 pg/mL at 6 months (P 0.764). Mean folic
acid at recruitment was 9.98  3.45 and 10.66  3.97 ng/mL at 6 months
(P 0.358). There was no correlation between carbamazepine levels with
homocysteine, vitamin B12, and folic acid (P > 0.05). There was no effect
of age, sex, or dietary pattern on homocysteine levels. Hence 6 months of
carbamazepine therapy did not cause a significant change in serum levels of
homocysteine, vitamin B12, and folic acid [287].
In clinical trials, the total incidence of reported adverse reaction to car-
bamazepine is 4.5 per million at defined daily doses, corresponding to 2.7 per
Carbamazepine 277

million at prescribed daily doses. Among the adverse reactions of carbamaz-

epine, most often reported are skin reactions (48%), hematological (14%),
and hepatic disorder (10%). Herein, we present a case with erythematous
skin rashes and hepatosplenomegaly [288].
Carbamazepine is used to control seizures. Its common side effects are
sleep disorders, anorexia, nausea, vomiting, polydipsia, irritability, ataxia,
and diplopia. Involvement of the immune system is rare, and few cases of
decreased immunoglobulin levels have been reported. A patient with low
immunoglobulin levels due to carbamazepine use who presented with
recurrent urinary tract infection was described. Intravenous immunoglobu-
lin was administered, and immunoglobulin levels increased to safer levels
after discontinuation of carbamazepine. Previous reports describe severe
infection after carbamazepine-induced hypogammaglobulinemia. There-
fore, in patients using antiepileptics, particularly carbamazepine, serum
immunoglobulin levels should be checked in those with recurrent infections
[289].
It was sought to elucidate the influence of centrally active drugs on inter-
hemispheric inhibition (IHI) between primary motor cortices in healthy
humans. Studies of IHI before and 2 h after intake of a single oral dose of
carbamazepine, dextromethorphan, lorazepam, or placebo were conducted
and compared it with the well-known results for SICI and ICF. Drugs were
tested in separate sessions and in random order. While SICI and ICF were
not altered by carbamazepine, IHI was reduced at the interstimulus interval
of 8 ms. Dextromethorphan tended to enhance SICI and to reduce ICF and
had no effect on IHI. Lorazepam reduced ICF as expected and enhanced IHI
at the long intervals of 50 and 80 ms. A moderate trend for interhemispheric
facilitation was inconsistently observed at the interval 2 ms and blocked by
carbamazepine. In addition, carbamazepine increased the motor threshold.
It was concluded that circuits mediating short interstimulus intervals of IHI
are susceptible to sodium channel blockade. The results increase the knowl-
edge of interhemispheric transmission [290].
Data on the effects of AEDs on the immune system are frequently incon-
sistent and sometimes conflicting because the effects of drugs cannot be sep-
arated from those of seizures, first-generation drugs have been most
intensively investigated, the patients genetic background, the mechanism
of action and the PK profile of AEDs and the concurrent use of immuno-
suppressant drugs may act as confounders. Valproate, carbamazepine, phe-
nytoin, vigabatrin, levetiracetam, and diazepam have been found to
modulate the immune system activity by affecting humoral and cellular
278 S.T. Alrashood

immunity. AEDs are associated with PK interactions (most frequently

occurring with carbamazepine, phenytoin, phenobarbital, and valproate).
Hepatic metabolism is the primary site of interaction for both AEDs and
immunotherapies (ACTH, dexamethasone, hydrocortisone, methylpred-
nisolone, cyclophosphamide, methotrexate, rituximab), which entail induc-
tion or inhibition of drug effects. However, the clinical importance of these
drug interactions is still far from defined. An important adverse effect of the
action of AEDs on the immune system is antiepileptic hypersensitivity syn-
drome (AHS), a life-threatening, idiosyncratic cutaneous reaction to aro-
matic AEDs resulting in end organ damage. Phenytoin, carbamazepine,
phenobarbital, lamotrigine, oxcarbazepine, felbamate, and ZNS have been
implicated. The pathogenic mechanisms of AHS are incompletely under-
stood [291].
In epilepsy, allegedly, a neurotransmitter imbalance between the inhib-
itory GABA and the excitatory glutamate prevails. Therefore, some AEDs
are thought to increase GABA release. Because little is known about
corresponding presynaptic effects of AEDs in the human brain, this study
investigated the effects of carbamazepine, lamotrigine, phenytoin,
gabapentin, pregabalin, levetiracetam, and valproate on 3H-GABA release
from human neocortical synaptosomes preincubated with 3H-GABA. To
obtain information on possible species differences, rat neocortical synapto-
somes were investigated concomitantly. Release was evoked by either verat-
ridine (1, 3.2, or 10 M), which prevents activated voltage-dependent Na+
channels from closing, or elevation of extracellular [K+] from 3 to 15 mM.
The exocytosis inhibitor tetanus toxin (TeT) or withdrawal of buffer Ca2+
(Ca (e) (2+)) reduced K+-evoked release in both species, while the blockade
of Na+ channels with tetrodotoxin had no effect. K+-evoked release was
characterized as predominant, Ca2+-dependent and Na+-independent, exo-
cytosis. Carbamazepine and phenytoin in the rat and carbamazepine, phe-
nytoin, lamotrigine, and valproate in human tissue reduced K+-evoked
3
H-GABA release. With respect to veratridine-evoked release, Ca (e) (2+)
withdrawal did not reduce release in the rat; it even increased the release
in human tissue. TeT was slightly inhibitory in the rat. Blockade of GABA
transport diminished veratridine-evoked 3H-GABA release in either species.
This release was characterized as mediated mainly by transporter reversal.
Carbamazepine, lamotrigine, and phenytoin in rat tissue and carbamazepine
and phenytoin in human decreased veratridine-induced 3H-GABA release.
Interestingly, no AED increased 3H-GABA release. The reduction by AEDs
of veratridine-evoked release was more intense than that of K+-evoked
Carbamazepine 279

release. In conclusion, reduction of GABA release by AEDs may be the

actual objective in a pathologically altered neuronal network where GABA
acts in a depolarizing fashion [292].
Aim of this study was to learn whether the AEDs carbamazepine,
lamotrigine, phenytoin, gabapentin, pregabalin, levetiracetam, and val-
proate (all at 100 M) presynaptically influence the neurotransmission of
the excitatory transmitter glutamate. The effects of these frequently used
AEDs were examined on 3H-glutamate release from superfused synapto-
somes of both rat and human neocortex. Release was evoked by elevation
of buffer [K+] from 3 to 15 mM or by the Na+ channel activator veratridine
(1, 3.2, and 10 M). Buffer [K+] elevation induced 3H-glutamate exocyto-
sis, which was Ca2+-, but not Na+-, dependent and which was accompanied
only in human tissue by release through transporter reversal. In rat tissue,
release was diminished by the Na+ channel inhibitors carbamazepine,
lamotrigine, and phenytoin, which therefore may also affect presynaptic
Ca2+ channels. Interestingly, levetiracetam increased 3H-glutamate release.
In contrast, the tested AEDs did not affect K+-evoked 3H-glutamate release
in human tissue, neither when the transporters were operative nor when
exocytosis was isolated by transporter blockade. Veratridine-evoked 3H-
glutamate release was a Na+-dependent transmitter efflux through reversed
transporters in both species which in human synaptosomes was accompanied
by exocytosis. The latter depended on external Ca2+. Carbamazepine,
lamotrigine, and phenytoin reduced this release from both rat and human
tissue. There is an obvious species difference in the effects of carbamazepine,
lamotrigine, and phenytoin on K+-evoked 3H-glutamate release, while their
inhibitory effects on veratridine-evoked release were similar. Thus, the
depression of 3H-glutamate release by carbamazepine, lamotrigine, and phe-
nytoin may be due to inhibited synaptosomal Na+ or Ca2+ influx [293].
Drug-induced liver injury is a major safety concern in drug development
and clinical pharmacotherapy; however, advances in the understanding of
the mechanisms of drug-induced liver injury are hampered by the lack of
animal models. CBZ is a widely used antiepileptic agent. Although the drug
is generally well tolerated, only a small number of patients prescribed CBZ
develop severe hepatitis. In the present study, a mouse model of CBZ-
induced liver injury and elucidated the mechanisms accounting for the
hepatotoxicity of CBZ was developed. Male BALB/c mice were orally
administered CBZ for 5 days. The plasma levels of alanine aminotransferase
and aspartate aminotransferase were prominently increased, and severe liver
damage was observed via histological evaluation. The analysis of the plasma
280 S.T. Alrashood

concentration of CBZ and its metabolites demonstrated that 3-hydroxy-

CBZ may be relevant in CBZ-induced liver injury. The hepatic glutathione
levels were significantly decreased, and OS markers were significantly
altered. Mechanistic investigations found that hepatic mRNA levels of
TLR4, receptor for advanced glycation end products, and their ligands were
significantly increased. Moreover, the plasma concentrations of
proinflammatory cytokines were also increased. Prostaglandin E(1) admin-
istration ameliorated the hepatic injury caused by CBZ. In conclusion, met-
abolic activation followed by the stimulation of immune responses was
demonstrated to be involved in CBZ-induced liver injury in mice [294].
CBZ is a first-line AED, although it is also used for the treatments of psy-
chiatric disorders and neuropathic pain. The CBZ utilization has been asso-
ciated with male reproductive damage, including hormonal alterations,
sexual dysfunction, and reduction of sperm quality. The wide and long-term
use of the CBZ is a common schedule in children and adolescents and alters
the testosterone level in adult rats and humans. The objective of this work
was to evaluate the CBZ side effects on the ventral prostate of rats from pre-
puberty to sexual maturation, since the prostate is an androgen-dependent
organ. Twenty-three-day-old male albino Wistar rats received CBZ diluted
in propylene glycol (20 mg/kg/i.p. via). The treatment lasted 20, 40, and 70
days, according to the different stages of the rat sexual maturation. At the end
of each treatment period, ventral prostates were removed and histologically
processed. The prostate sections were submitted to the histopathological,
morphological, and stereological analyses using image analysis system.
Reductions of the glandular epithelium, glandular lumen, and fibromuscular
stroma volume of the ventral prostate were observed in adult rats treated
with CBZ since the weaning. Triggering and degranulation of mast cells
were observed in the fibromuscular stroma of prepubertal and pubertal
CBZ-treated rats. The results suggest a direct effect of the CBZ on rat ven-
tral prostate, evidenced by increase of mast cell and macrophage populations
during prepuberty and puberty causing a ventral prostate accentuated dam-
age in the adult phase [295].
AED had an effect on bone metabolism in children. This study was con-
ducted in order to determine the relationships between serum leptin levels,
bone mineral density (BMD), and bone turnover markers in epileptic chil-
dren. Fifty-three patients were treated with VPA and 23 with CBZ mon-
otherapy; 50 healthy children were included in the study as controls.
Serum alkaline phosphatase (ALP) and cross-linked C-telopeptide (CTx)
levels were statistically significantly higher in the CBZ group than in the
Carbamazepine 281

VPA group and the control group (P < 0.0001, P < 0.010, respectively).
Serum osteocalcin and ALP levels were significantly lower in the VPA group
than in the control group (P < 0.012, P < 0.030, respectively). Although we
found slightly higher serum leptin levels in both the CBZ and VPA groups,
they were not significantly different from the control group (P > 0.05). We
demonstrated that the markers of bone formation and resorption increased
with CBZ and decreased with VPA treatment without affecting BMD and
vitamin D levels in prepubertal epileptic children [296].
Although the US FDA recommends screening for HLA-B*1502 allele in
most of Asian ancestry before initiating carbamazepine therapy, the HLA
associations with carbamazepine hypersensitivity in non-Chinese Asian
populations remain unclear. This study investigated the association between
the HLA class I genotype and carbamazepine-induced severe cutaneous
adverse reaction (SCAR) in Koreans. Twenty-four patients who had devel-
oped carbamazepine-induced SCAR (7 SJS, 17 drug hypersensitivity syn-
drome (HSS)), 50 carbamazepine-tolerant controls from the Korean
Pharmacogenetic Adverse Drug Reaction Research Network, and data of
485 Korean general population from a previously published study were rec-
ruited. HLA-A, -B, and -C genotyping was performed by direct DNA
sequence analysis. Only one of the seven SJS patients was positive for the
B*1502 allele, but the frequency of B*1511 was much higher in the patients
with CBZ-SJS than in the CBZ-tolerant control patients (P 0.011, P(c)
not significant; OR 18.0 (2.3141.2)). The frequencies of A*3101 in car-
bamazepine-induced HSS and SCAR were significantly higher than those in
carbamazepine-tolerant controls (P(c) 0.011, OR 8.8 (2.530.7) and P
(c) 0.013, OR 7.3 (2.322.5), respectively). The frequencies of B*1511
in carbamazepine-SJS and A*3101 in carbamazepine-HSS/SCAR were
significantly higher than those in the general population. HLA-B*1502
does not seem to be an effective predictive marker for carbamazepine-
induced SCAR, while HLA-B*1511 and A*3101 were associated with car-
bamazepine-induced SJS and HSS/SCAR, respectively, in the Korean pop-
ulation [297].
The aromatic anticonvulsants CBZ and phenytoin (PHN) are associated
with a relatively high incidence of idiosyncratic drug reactions (IDRs). If
biomarkers could be found that would predict the risk that a drug candidate
would cause IDRs, it would significantly decrease the risks associated with
drug development. The IDRs associated with CBZ and PHN appear to be
immune-mediated. The danger hypothesis posits that for something to
induce an immune response, it must cause some type of cell damage that
282 S.T. Alrashood

ultimately causes upregulation of costimulatory molecules on anti-

gen-presenting cells; without this, the response will be immune tolerance.
If the danger hypothesis is correct, the ability of a drug or its RM to induce
cell damage or stress may be related to its risk of causing IDRs. In a parallel
study reported elsewhere, we found that major metabolites of these two
drugs, 3-OH-CBZ and 4-OH-PHN, can be oxidized by peroxidases to
phenoxyl free radicals, which could cause OS by redox cycling. In this study
using mRNA microarrays, we found that CBZ and PHN treatment induced
changes in mRNA expression in mice. Many of the changes were in genes
related to Keap1Nrf2ARE signaling pathways and enzymes involved in
responding to oxidant stressors and RMs such as glutathione transferase
and heat-shock proteins. The similar patterns of genes induced by these
two drugs are consistent with the clinical observation that those two drugs
exhibit cross-sensitivity. These findings are consistent with the induction of
cell stress by CBZ and PHN, most likely due to RMs. Such changes may
represent a danger signal and represent a biomarker of the potential that a
drug will cause IDRs; however, different drugs likely cause cell stress by dif-
ferent mechanisms and, therefore, the biomarkers for other drugs would
likely be different [298].
Maternal use of AEDs during pregnancy has been associated with an
increased risk of major congenital abnormalities in the fetus. CBZ is an
AED that was developed and marketed mainly for the treatment of epileptic
seizures. Some investigators described an increased rate of major congenital
anomalies following treatment with CBZ during pregnancy, while others
found no such increase. In order to quantify better the risks of exposure
to CBZ during pregnancy, data from prospective studies were pooled. It
was found in prospective studies involving 1255 cases of exposure that
CBZ therapy increased the rate of congenital anomalies, mainly neural tube
defects, cardiovascular and urinary tract anomalies, and cleft palate. CBZ
may also induce a pattern of minor congenital anomalies and developmental
retardation, but the study did not address these endpoints. CBZ also appears
to reduce gestational age at delivery. A combination of CBZ with other
AEDs is more teratogenic than CBZ monotherapy. Children born to
untreated epileptic women do not appear to have an increased rate of major
birth defects. In light of these results, it was recommended performing a level
2 ultrasound and fetal echocardiography in women treated with CBZ during
pregnancy [299].
Some epileptic drugs may change antioxidant enzyme activities in
humans and experimental animals. Recent studies suggest that membrane
Carbamazepine 283

lipid peroxidation may be causally involved in some forms of epilepsy, and

the differences are reported in free radical scavenging enzyme levels.
GSHpX, SOD, and GSH are important parameters of antioxidant defense
mechanisms. This study was undertaken to evaluate the effects of VPA
and CBZ therapy on erythrocyte glutathione (GSH), glutathione peroxidase
(GSHpX), superoxide dismutase (SOD), and lipid peroxidation. During the
treatment with VPA or CBZ, the erythrocyte GSHpX and GSH levels of
epileptic children were significantly changed as compared to those of health
control subjects. The mean levels of serum lipid peroxidation and erythro-
cyte SOD were not statistically different from controls. The methods used
for investigation of GSHpX, SOD, GSH, and serum lipid peroxidation were
all based on spectrophotometric measurement [300].
The effect of lithium and carbamazepine in the treatment of bipolar
affective disorder is well established. Although a number of biochemical
effects have been found, the exact molecular mechanisms underlying their
therapeutic actions have not been elucidated nor are the target regions in the
brain identified. Taken into account the important role of the cyclic AMP
second messenger system in the regulation of neuronal exitability and the
indications of its involvement in the pathophysiology of bipolar affective dis-
order, it was focused on the drug effects on cyclic AMP levels. The objec-
tives of this investigation were to measure the effects on basal cyclic AMP
levels and to locate target regions within the rat brain after long-term admin-
istration of lithium and carbamazepine. Drug treatments were carried out for
a period of 28 days. After either drug treatment the cyclic AMP level was
increased three to four times in frontal cortex but unchanged in hippocam-
pus, hypothalamus, thalamus, amygdala, and cerebellum. In neostriatum the
cyclic AMP level was decreased to about 30% after treatment with lithium.
The common region-selective effect was suggested, observed for both drugs
in frontal cortex, to be essential for the therapeutic actions of lithium and
carbamazepine [301].
The major AEDs used for the control of seizures can induce develop-
mental toxicity when administered during pregnancy. Vitamin A and reti-
noids are thought to control many processes of embryonic development
including growth, differentiation, and morphogenesis. It was therefore stud-
ied if the teratogenic action of antiepileptic agents could be mediated via
alteration of the endogenous vitamin Aretinoid metabolism. Retinol and
its oxidative metabolites all-trans-, 13-cis-, and 13-cis-4-oxo-retinoic acid
were measured in the plasma of 75 infants and children treated with various
AEDs for the control of seizures, and in 29 untreated controls of comparable
284 S.T. Alrashood

age. Retinol levels increased with age, while the concentrations of retinoic
acid compounds did not exhibit age dependency. Valproic acid mon-
otherapy increased retinol levels in the young age group and a trend toward
increased retinol concentrations was also observed in all other patient
groups. The plasma levels of the oxidative metabolites 13-cis- and 13-cis-
4-oxo-retinoic acids were strongly decreased in all patient groups treated
with phenytoin, phenobarbital, carbamazepine, and ethosuximide, in com-
bination with valproic acid, to levels which were below 1/3rd and 1/10th of
corresponding control values, respectively. Little changes were observed
with all-trans-retinoic acid except in one patient group treated with valproic
acid/ethosuximide cotherapy where increased levels of this retinoid were
found. The study indicates that therapy with antiepileptic agents can have
a profound effect on the endogenous retinoid metabolism. Because of the
importance of retinoids for the signaling of crucial biological events during
embryonic development, such altered retinoid metabolism may be highly
significant in regard to AED teratogenesis [302].
Thyrotropin-releasing hormone is an endogenous tripeptide with endo-
crine-independent neurophysiologic properties that may be relevant to
affective or seizure disorders. The effect of carbamazepine was studied,
which has both mood-stabilizing and anticonvulsant properties, on cerebro-
spinal fluid thyrotropin-releasing hormone levels in affectively ill patients.
Paired cerebrospinal fluid samples were collected from nine inpatients with
mood disorders, both while medication free and while taking carbamazepine
for an average of longer than 1 month at 950 mg/d, achieving blood levels of
8.8 mg/L. Carbamazepine treatment was consistently and significantly asso-
ciated with increased cerebrospinal fluid thyrotropin-releasing hormone
levels (P < 0.0001). As carbamazepine-induced increases in thyrotropin-
releasing hormone levels could be relevant to its either psychotropic or anti-
convulsant properties, further clinical and preclinical investigation of this
finding appears as indicated [303].

9. TOXICITY
Hemoperfusion (HP) or dialysis is occasionally used following
CBZ toxicity but it remains unclear which is the most efficient modality.
A case of severe CBZ intoxication treated was described with different
extracorporeal modalities during which CBZ toxicokinetics were com-
pared. Case details: A 58-year-old man was transferred to our facility 24 h
after ingesting over 14 g of sustained-release CBZ. Because of worsening
Carbamazepine 285

neurological condition requiring mechanical ventilation and CBZ levels

reaching 47.6 g/mL, he underwent three intermittent hemodialysis
(IHD), two continuous venovenous hemofiltration (CVVH), and one IHD
with HP (IHD-HP). IHD and CVVH removed 1.73 g of carbamazepine
over 43 h. Mean apparent half-life was 8.8 h during IHD 49.1 h during
CVVH, and 5.1 h during IHD-HP, while measured endogenous half-life
after extracorporeal therapies was 81.4 h. Mean CBZ clearances were
106.2 mL/min during IHD and 21.2 mL/ min during CVVH. His neuro-
logical status improved during extracorporeal elimination, and he was dis-
charged without sequel after 16 days. Treatments were well tolerated
aside from thrombocytopenia during IHD-HP. All extracorporeal treat-
ments facilitated CBZ elimination, although CVVH was significantly less
efficient than IHD and IHD-HP. IHD-HP may be better than IHD alone
but must be weighed against its risks. IHD appears sufficient to eliminate
CBZ and may need to be repeated or prolonged according to the clinical
context if CBZ absorption is delayed [304].
The aim of this study was to investigate the effects of valproate and car-
bamazepine on renal glomerular and tubular functions. The patient group
comprised 54 children with new-onset epilepsy treated with valproate
(n 30) and carbamazepine (n 24). Twenty-six healthy children were in
the control group. The serum creatinine and cystatin C levels and urinary
excretion of N-acetyl--D-glucosaminidase (NAG) levels were measured
and the GFR was estimated. Serum creatinine and cystatin C concentrations
were not different between patients and controls. The GFR of the patient
groups was higher than those of the control group. Thus, both drugs
probably lead to glomerular hyperfiltration and toxicity for glomerular
functions. However, urinary NAG/creatinine levels were significantly
higher in patients receiving only valproate (6.1  5). The difference between
carbamazepine and control groups was not significant for urinary NAG/
creatinine levels. Our data suggest that valproate has adverse effects on renal
tubular functions [305].
Nonsteroidal human pharmaceuticals are prevalent in domestic waste-
water and may find their way into the environment at low concentrations.
Since most pharmaceuticals are designed to be biologically active at low
concentrations, there is a risk that these compounds may affect aquatic wild-
life. Of particular concern is the occurrence of pharmaceutical mixtures,
which may lead to increased adverse effects compared to individual com-
pounds. Interactive effects were previously demonstrated for amphibians
exposed to pesticide mixtures, but no such studies investigating responses
286 S.T. Alrashood

of amphibians to pharmaceutical mixtures are apparently available. Results

demonstrated increased toxicity (loss of tactile response) of striped marsh
frog (Limnodynastes peronii) tadpoles exposed to a mixture of naproxen, car-
bamazepine, and sulfamethoxazole, compared to exposures to the individual
compounds. Significant time  treatment interactions were observed for
tadpole development following chronic exposures to 10 or 100 g/L of each
compound and the mixture; however, responses were weak and main treat-
ment effects were not significant. Despite minor effects at low exposure con-
centrations, results demonstrated a potential for mixtures of nonsteroidal
pharmaceuticals commonly occurring in wastewater to influence amphibian
development. With the vast numbers of pharmaceuticals that exist and are
found in the environment, this work highlights a need for further research
into mixtures of pharmaceutically active wastewater contaminants. Further,
since pharmaceuticals exert extremely varied biological actions, it is
suggested that future investigations would benefit from inclusion of end-
points that are indicative of physiological or metabolic performance, as well
as assessment of sensitive behavioral responses [306].
Carbamazepine overdose is a common, toxic ingestion, manifesting as
CNS and respiratory depression. Carbamazepine is highly protein bound
with a large volume of distribution and, therefore, inefficiently removed
by conventional hemodialysis. We describe the successful use of continuous
venovenous hemodialysis (CVVHD) with 5% albumin-enhanced dialysate
in a 31-year-old female who developed CNS depression, hypotension, and
respiratory failure, requiring mechanical ventilation, after an intentional
ingestion of approximately 10 g of extended-release carbamazepine,
Tegretol CR(). The peak drug level was 26 g/mL, therapeutic range
812 g/mL, with toxicity often developing a level above 15 g/mL. Nor-
mal half-life of drug elimination is 3560 h in carbamazepine nave patients.
In contrast, with albumin-enhanced dialysis, we observed a drug half-life of
18 h. She was extubated on day 2 and was transferred to inpatient psychiatry
by day 3 without significant neurologic sequelae. In vitro studies have been
done with bovine blood demonstrating significant carbamazepine removal
using CVVHD with albumin-enhanced dialysate. There has been very lim-
ited experience using albumin-enhanced CVVHD in an adult patient with
carbamazepine toxicity [307].
CBZ intoxication can be associated with severe toxicity, including neu-
rological and cardiorespiratory abnormalities. Highly protein-bound CBZ is
not removed efficiently through conventional hemodialysis. Charcoal
hemoperfusion is the most effective extracorporeal elimination therapy
Carbamazepine 287

for CBZ intoxication. Recent reports have indicated that continuous

venovenous hemodiafiltration (CVVHDF), albumin-enhanced continuous
venovenous hemodialysis, high-flux hemodialysis, and plasma exchange can
be as effective as charcoal hemoperfusion. In contrast to recent reports,
which demonstrated the effectiveness of CVVHDF with high dialysate flow
in CBZ intoxication, we observed that serum CBZ level was decreased min-
imally by albumin-enhanced CVVHDF with low dialysate flow. Therefore,
albumin-enhanced CVVHDF with high dialysate flow should be considered
in severe CBZ intoxication, if HP is unavailable because of the lack of facil-
ities or if it cannot be performed [308].
Carbamazepine, an antiepileptic pharmaceutical agent commonly found
in wastewater, is highly recalcitrant to standard wastewater treatment prac-
tices. This study investigated the mixture toxicity of carbamazepine transfor-
mation products formed during UV photolysis using three standard
ecotoxicity assays (representing bacteria, algae, and crustaceans). UV treat-
ment of 6 mg/L CBZ solution was carried out over a 120-min period and
samples were removed periodically over the course of the experiment.
Quantification results confirmed the degradation of carbamazepine
throughout the treatment period, together with concurrent increases in acri-
dine and acridone concentrations. Ecotoxicity was shown to increase in par-
allel with carbamazepine degradation, indicating that the mixture of
degradation products formed was more toxic than the parent compound,
and all three ecotoxicity endpoints were still inhibited >60% relative to con-
trol populations upon dosing with 90+ min UV-treated carbamazepine
solution. Single compound toxicity testing also confirmed the higher tox-
icity of measured degradation products relative to the parent compound.
These results show that transformation products considerably more toxic
than carbamazepine itself may be produced during UV treatment of waste-
water effluents and/or photoinduced degradation of carbamazepine in nat-
ural waters. This study highlights the need to consider mixture toxicity and
the formation and persistence of toxicologically relevant transformation
products when assessing the environmental risks posed by pharmaceutical
compounds [309].
Use of AEDs in pregnancy is associated with congenital malformations
and developmental delay. Previous studies have suggested that women who
have had one child with a congenital malformation are at increased risk
of having other children with malformations. We sought to confirm the
magnitude of risk in a large cohort drawn from the UK Epilepsy and Preg-
nancy Register. The UK Epilepsy and Pregnancy Register is a prospective,
288 S.T. Alrashood

observational registration, and follow-up study setup to determine the rel-

ative safety of AEDs in pregnancy. Data were extracted for those women
who prospectively registered more than one pregnancy and calculated the
recurrence risks for fetal malformations. Outcome data were available for
1534 pregnancies born to 719 mothers. For women whose first child had
a congenital malformation, there was a 16.8% risk of having another child
with a congenital malformation, compared with 9.8% for women whose first
child did not have a malformation (relative risk 1.73, 95% CI 1.012.96).
The risk for recurrence was 50% for women who had had two previous chil-
dren with a congenital malformation. There was a trend toward a higher risk
for recurrent malformations in pregnancies exposed to valproate (21.9%, rel-
ative risk 1.47, 95% CI 0.683.20) and topiramate (50%, relative risk 4.50,
95% CI 0.9720.82), but not for other drugs such as carbamazepine and
lamotrigine. Recurrence risks were also higher for pregnancies exposed
to polytherapy regimens and for those where the dose of AED treatment
had been increased after the first pregnancy. As a conclusion, women
who have had a child with a malformation are at increased risk of having
other children with malformations. This is in keeping with previous reports
that have suggested that genetic influences may be one of the factors deter-
mining the teratogenic risk of AEDs [310].
Awareness of residual pharmaceutically active compounds (PhACs) in
the aquatic environment is growing as investigations into these pollutants
are increasing and analytical detection techniques are improving. However,
the toxicological effects of PhACs have not been adequately researched. In
this study, the toxic effects of CBZ, an anticonvulsant drug commonly pre-
sent in surface and groundwater, were studied in juvenile rainbow trout,
Oncorhynchus mykiss, by acute semistatic bioassay. Blood parameters, liver
xenobiotic-metabolizing response, and tissue antioxidant status were evalu-
ated. Compared to the control group, fish exposed to CBZ (96 h LC50)
showed significantly higher Er, Hb, MCHC, monocytes, neutrophil
granulocytes, and plasma enzymes activity, and significantly lower MCV
and lymphocytes. CF and HSI were not significantly different among groups
such as hepatic EROD. SOD, CAT, GPx, and GR activities were signifi-
cantly higher in liver of experimental groups, but decreased significantly in
brain and gill. In general, antioxidant enzyme activity in intestine and muscle
was less evident than in liver. OS indices (levels of LPO and CP) were sig-
nificantly higher in gill and brain, despite a trend to increased values were
manifested in the remaining tissues. In short, CBZ-induced stress responses
in different tissues were reflected in the oxidant stress indices and
Carbamazepine 289

hematological parameters. However, before those parameters are used as

special biomarkers for monitoring residual pharmaceuticals in aquatic envi-
ronment, more detailed experiments in laboratory need to be performed in
the future [311].
AED exposure in utero has been associated with MCMs and adverse
cognitive outcomes in the offspring of women with epilepsy (WWE). How-
ever, determining the exact risk and the relative risks of AEDs for these out-
comes has been challenging and, only in recent years, has improved study
designs enabled us to get a clearer picture of the risks. Still, there is a startling
lack of information for many of the newer and widely used AEDs. At this
point of time, studies clearly show that (VPA as a part of polytherapy or
when used as a monotherapy is associated with an increased risk of MCMs,
and that it poses about threefold the risk of CBZ. It is unclear if any other
AEDs studied pose an increased risk of MCM occurrence; in the best avail-
able large study the absolute rates of MCMs with other several other AEDs
were not different from untreated WWE. The absolute risks have been
reported as CBZ 2.2%, LTG 3.2%, PHT 3.7%, and untreated WWE
3.5%, with VPA as the outlier at 6.2%. In utero VPA exposure is also asso-
ciated with a risk of lower verbal intelligence quotient (IQ) in children, at
approximately 10 points lower than controls. CBZ appears to pose no risk to
cognitive outcome, and there is some evidence that PHT and phenobarbital
(PB) may be associated with risk of reduced cognitive outcome. Polytherapy
is associated with greater risk than monotherapy for both MCMs and cog-
nitive outcome. Although more information is needed and hopefully will be
obtained from ongoing prospective studies, it is clear that WWE taking VPA
and planning pregnancy should have a discussion with their physician about
considering changing to another AED before pregnancy, if possible [312].
A case of coma due to carbamazepine ingestion with the intention of
committing suicide at 33 weeks gestation is presented. Management
included activated charcoal administration and exchange plasmapheresis.
A fetal nonstress test was nonreassuring but the Apgar score, cord blood
gases, and early neonatal outcome were normal. Differential diagnosis of
coma in pregnancy should include investigation for drug intoxication [313].
Newly designed AEDs are being evaluated for their efficacy in
preventing seizures and for their toxic profiles. The toxic effects of two
dibenz[b, f]azepine derivatives with anticonvulsant activity, 10,11-dihydro-
10-hydroxyimino-5H-dibenz[b, f]azepine-5-carboxamide (BIA 2-024) and
(S)-()-10-acetoxy-10,11-dihydro-5H-dibenz[b, f]azepine-5-carboxamide
(BIA 2-093), with the structurally related compounds CBZ and OXC, both
290 S.T. Alrashood

in current use for the treatment of epilepsy were investigated and compared.
Primary rat hippocampal neurons were used to evaluate neuronal morphol-
ogy and biochemical changes induced by the AEDs used in this study.
Immunocytochemical staining against MAP-2 was used to evaluate neuro-
nal morphology. Reactive oxygen species (ROS) and changes in mitochon-
drial membrane potential (Psim) were measured by fluorescence techniques.
Intracellular adenosine triphosphate (ATP) levels were quantified by HPLC.
Hippocampal neurons treated for 24 h with CBZ or OXC (300 M)
showed degeneration and swelling of neurites, but this effect was not
observed in neurons treated with BIA 2-024 or BIA 2-093 (300 M).
ROS production also was increased in neurons treated with OXC, but
not in neurons treated with the other AEDs. ATP levels were significantly
decreased only in neurons treated with OXC, although the energy charge
was not altered. Furthermore, OXC led to a decrease of Psim. In all param-
eters assayed, OXC was more toxic than the other AEDs used. Because the
new putative AEDs have previously been shown to have an efficacy in
preventing seizures similar to that of CBZ and OXC, and are less toxic
to neuronal cells, they may be considered as alternatives to the current avail-
able therapies for the treatment of epilepsy [314].
Carbamazepine toxicity on cardiovascular system in the course of acute
poisonings and long-term therapy are observed rarely. Its toxic influence on
action potential in Purkinje fibers and four depolarization phase expresses
clinically as the His bundle and atrioventricular blocks especially in patients
with cardiologic disturbances. A case of 64-year-old woman with ischemic
heart disease poisoned with carbamazepine who died because of severe
arrhythmias in the course of myocardial infarction during first 24 h of intox-
ication was presented. Heightened awareness of high-risk lethal cardiovas-
cular complications in patients intoxicated with carbamazepine with history
of heart diseases is needed [315].
To examine common signs and symptoms of mild to moderate CBZ
overdose in young children. The medical records of previously healthy chil-
dren admitted to the pediatric departments for acute accidental CBZ poison-
ing during the years 199398 were evaluated retrospectively. Information
was retrieved on serum CBZ levels, signs and symptoms on admission
and during hospitalization, ECG findings, and chemical laboratory test.
There were 14 exposed children all under the age of 5 years. These children
accidentally took CBZ prescribed for a family member. The diagnosis of
CBZ poisoning in seven children was unknown on admission because of
inadequate history and was revealed only on toxicology screen. Nystagmus
Carbamazepine 291

and drowsiness occurred in 8 of the 14 children, nystagmus and ataxia in 4

children, and drowsiness and tachycardia in another 2 children. The peak
CBZ serum levels in these children ranged from 18 to 32 g/mL, mean
+ SD; 25 + 4.64 g/mL (therapeutic range: 510 g/mL). Based on a certain
group of young pediatric patients with mild to moderate CBZ poisoning, it
is concluded that nystagmus is the most common sign of this overdose.
Other common signs are drowsiness and ataxia. The presence of nystagmus
and CNS depression of unknown etiology in a young child should suggest
the possibility of CBZ toxicity [316].
Postimplantation rat embryo culture is used widely for studies of
embryotoxic effects on the isolated embryo after in vitro exposure to xeno-
biotic compounds. In this study, the relevance of three routes of exposure of
the embryo in vitro was evaluated using the embryotoxic anticonvulsant
carbamazepine. Embryotoxic effects were assessed, and analyses in concep-
tus tissues were done to reveal uptake and metabolism of the compound.
Exposure via the culture medium resulted in neural tube defects and general
retardation of growth and development. After injections into the amniotic
or exocoelomic space, local membrane adhesions were found. Intra-amni-
otic exposure caused adhesions of the amniotic membrane with the embry-
onic neural plate, resulting in trapping of the membrane in the closing neural
tube as well as in open neural tube defects occurring in various areas of the
neural tube. Only after exposure via the culture medium were amounts of
carbamazepine detectable in the embryonic tissue, correlating with the sys-
temic effects found. It is concluded that uptake from the culture medium via
the yolk sac circulation is the relevant exposure route to be used for
embryotoxicity effect assessment [317].
Seven psychiatric inpatients receiving carbamazepine 600 mg/day were
coadministered clarithromycin 400 mg/day for 5 days to treat atypical pneu-
monia. Blood samples were taken after clarithromycin coadministration
and at 1 and 4 weeks after its discontinuation. Plasma concentrations of
CBZ and CBZ-E were measured using HPLC. During clarithromycin
coadministration, four of the seven patients developed moderate-to-severe
toxic symptoms of carbamazepine, such as drowsiness, dizziness, and ataxia,
which resolved within 5 days after clarithromycin discontinuation. In these
four patients, plasma carbamazepine concentrations after clarithromycin
coadministration were approximately twice as high as those after its discon-
tinuation. In the seven patients, the mean plasma concentration of carba-
mazepine, but not of CBZ-E, after clarithromycin coadministration was
significantly (P < 0.01) higher than those at 1 and 4 weeks after its
292 S.T. Alrashood

discontinuation. The present report suggests that clarithromycin

coadministration induces increased plasma carbamazepine concentrations,
which may result in carbamazepine toxicity. Therefore, care should be given
to prescribing clarithromycin for patients receiving carbamazepine [318].
The mechanism of CBZ-related teratogenicity was investigated in the
SWV mouse by contrasting the effects of CBZ-E and OXC treatments. Die-
tary CBZ-E administration was initiated 2 weeks before mating and contin-
ued through day 18 of gestation. OXC was administered to pregnant dams
by gavage on day 6 of gestation and continued through day 18 of gestation.
Maternal plasma concentrations of CBZ-E ranged from 1.4 to 17.7 g/mL
and OXC ranged from 6.1 to 15.9 g/mL. In comparison, clinical plasma
concentrations of CBZ-E ranged from 1 to 2 g/mL and OXC plasma con-
centrations were 1 g/mL or less. The incidence of malformation was 14%,
27%, and 26% after daily CBZ-E doses of 300, 600, and 1000 mg/kg,
respectively, compared with a 6% incidence in no-drug control mice,
P < 0.05. The incidence of malformation was 8% after exposure at the
highest tolerable dose of OXC (1100 mg/kg/day), compared with a 5%
incidence in no-drug controls, P > 0.05. Phenobarbital cotreatment
(45 mg/kg/day) with OXC (1100 mg/kg/day) did not lead to changes in
the incidence of malformation when compared with OXC (1100 mg/kg/
day) dosed alone. These data are consistent with a teratogenic CBZ metab-
olite, possibly CBZ-E, or with oxidation of CBZ-E or CBZ at positions on
the aromatic ring leading to the formation of reactive intermediates such as
arene oxides or quinines [319].
Carbamazepine (Tegretol, CBZ) is an anticonvulsant drug that is very
effective in the treatment of tonicclonic seizures and is gaining acceptance
as a treatment for various psychiatric disorders. The drug is embryotoxic in
rodents and has been reported to produce neural tube defects in approxi-
mated 1% of prenatally exposed human offspring. It is metabolized by the
cytochrome P-450 system to a stable, pharmacologically active epoxide
intermediate, carbamazepine-10,11-epoxide. It is currently unknown
whether the parent compound, the epoxide intermediate, or some other
metabolite is the embryotoxic agent. The present study was designed to
determine the embryotoxicity of CBZ and its epoxide intermediate
(CBZ-E) in a rodent whole embryo culture system. Rat embryos were
cultured beginning on day 9 of gestation (GD 9), and mouse embryos
were cultured beginning in GD 8. All embryos were cultured for 48 h in
medium containing various concentrations of either CBZ or CBZ-E. Mice
were more sensitive to the effects of CBZ than were rats. The parent
Carbamazepine 293

compound was embryotoxic to mouse embryos at concentrations as low as

12 g, but it was only embryotoxic at 60 g/mL to rat embryos. CBZ-E was
not embryotoxic to either species at concentrations as high as 48 g/mL.
These results suggest that the parent compound is the embryotoxic agent
and that the epoxide intermediate plays no role in the drugs embryotoxic
mechanism [320].
To evaluate the cardiovascular effects of carbamazepine in patients pre-
senting to the emergency department, a retrospective case series from Feb. 1,
1985 to Jul. 30, 1993 was conducted. Seventy-two adults and pediatric
patients with serum carbamazepine concentrations greater than 12 g/mL
and concurrent 12-lead ECGs participated. The mean carbamazepine level
was 24 g/mL (range, 12.655 g/mL). Minor ECG abnormalities were
noted but no clinically significant arrhythmias were found. No correlation
was found between carbamazepine concentration and heart rate or PR,
QRS, or corrected QT intervals. Four adult patients had transient hypoten-
sion. Clinically significant cardiovascular toxicity occurs rarely in patients
with toxic carbamazepine concentrations. ECG findings do not correlate
with serum carbamazepine concentration [321].
Data from carbamazepine poisonings reported to the Kentucky Regional
Poison Center from Jan. 1986 through Mar. 1992 to identify information
available at the time of poison center contact which correlates to outcome
were reviewed. The Spearman rank correlation test was used to describe
the relationship between two ordinal variables and interval-level variables.
The KruskalWallis test was used to determine the relationship between cat-
egorical and ordinal variables. Two-way analysis of variance was used to test
the effect of routine carbamazepine use on final severity and carbamazepine
level of 345 reports involving carbamazepine poisoning; 263 (76%) involved
only carbamazepine ingestion and formed the database. One hundred eighty-
four (70%) carbamazepine ingestions occurred in victims 17 years old, 79
(30%) occurred in adults. Severity assigned at the time of initial poison center
contact was significantly correlated with outcome severity for children and
adults (r  0.9, P < 0.00001). The amount reported ingested influenced the
correlation between initial and final severity, whereas time elapsed between
ingestion and poison center contact did not alter the correlation between ini-
tial and final severity. The reason for ingestion was significantly correlated
with outcome (P < 0.00001). A significant correlation between outcome
and peak carbamazepine level for each age group was observed (pediatric
R 0.5, P < 0.00001, and adult r 0.4, P 0.008). Carbamazepine levels
>85 mol/L (>20 g/mL) were associated with more severe toxicity [322].
294 S.T. Alrashood

Pulmonary toxicity caused by carbamazepine seems to be a nondose-

dependent process involving pulmonary infiltrates, eosinofilia, and skin rash.
A case was reported with acute presentation that resolved successfully after
the drug was withdrawn and the patient was treated with corticosteroids.
Bronchoalveolar lavage showed an inversion of the lymphocyte ratio
CD4/CD8 supporting the suggestion by other authors that drug-induced
pulmonary toxicity [323].
Owing to marked fluctuations in plasma concentrations, circadian
CNS toxicity (maximum in the early afternoon) occurred in a 69-year-
old female patient being treated with an instant-release formulation of
CBZ. The neurologic syndrome was reversible after administration of the
same daily dose as sustained-release formulation. This case illustrates the
importance of correct timing of blood sampling to detect drug-induced tox-
icity and of use of sustained-release formulations in antiepileptic therapy
with CBZ [324].
Generic substitution is practiced widely in both hospital and community
settings. There have been several reports of reduced serum concentrations
and seizure exacerbation following generic substitution of Tegretol. The
first two cases of carbamazepine toxicity resulting from the substitution of
Tegretol with Epitol were described. Two 6-year-old children experienced
increases in the maximum serum carbamazepine concentration, one of 22%
and one of 41%. Both became asymptomatic when their serum concentra-
tions were lowered and had no residual effects [325].
Acute liver toxicity caused by carbamazepine is a well-known though
infrequent event. Severe toxicity with hepatocellular insufficiency is even
more rare. A case is presented of a patient who suffered of partial epilepsy
on treatment with valproate and carbamazepine, who was admitted because
of severe acute liver insufficiency attributable to carbamazepine. He had
started treatment with the latter drug 2 weeks earlier, when he developed
fever, jaundice, rash, and signs of encephalopathy in association with eleva-
tion in serum transaminases levels and a decrease in prothrombin index
(24%). Discontinuation of both AEDs, together with the usual supportive
measures, was followed by a complete resolution. Valproate was restarted
without complications. Liver biopsy suggested acute hepatitis of drug-
related origin. Granulomas or steatosis was not found. The histologic picture
together with the relation between carbamazepine administration and the
development of hepatotoxicity allow us to dismiss valproate as the possible
causal agent of this patients disease. Therefore, it was believed that it was an
acute hepatocellular failure secondary to carbamazepine [326].
Carbamazepine 295

Review of the medical records of 2 major adult teaching hospitals for a 4-

year period revealed 33 instances of carbamazepine overdose. These patients
had a mean age of 30 years and 58% were known epileptics. They ingested a
mean of 12 g carbamazepine (range, 1.645 g), with 51% of cases involving
other drugs, particularly alcohol. The clinical manifestations of toxicity
formed a recognizable clinical picture of diminished conscious state
(100% of patients), mydriasis (42%), abnormal muscle tone and tendon
reflexes (55%), and ataxia, nystagmus, or ophthalmoplegia (48%). Twenty
percent of cases were complicated by seizures. The incidence of hypergly-
cemia and hypokalemia was related to higher drug concentrations. Twelve
percent showed hyponatremia and 50% had transient evidence of hepatic
dysfunction. The PK properties of carbamazepine play a role in determining
management strategies. Management is largely supportive through avoid-
ance of drug interactions, large doses of activated charcoal, careful airway
management, and correction of electrolyte disturbances [327].
A 5-year-old mentally retarded child developed laboratory evidence of
pancreatitis during accidental acute CBZ intoxication. He had been seizure-
free with CBZ for 4 years for a seizure disorder with no obvious toxicity.
CBZ had been discontinued 5 months before he was admitted to the hos-
pital. After he accidentally ingested a CBZ overdose, he was found vomiting
and lethargic. Serum amylase and lipase levels were increased for several
days. With supportive treatment and no CBZ, he recovered and serum amy-
lase and lipase levels returned to normal. No other causes of pancreatitis were
identified. Therefore, most likely the chemical pancreatitis was associated
with the acute CBZ intoxication [328].
A retrospective study of consecutive cases of massive carbamazepine poi-
soning treated in an intensive care unit during the period 198191 was per-
formed, mainly to determine whether serum carbamazepine levels were
predictive of toxicity. Out of a total of 51 admissions with a diagnosis of car-
bamazepine self-poisoning, 28 (25 patients) were included. The reasons for
exclusion were coingestion of other drugs (11 cases), incorrect diagnosis or
inadequate information (6 cases), a peak observed serum concentration of
carbamazepine below 76 mol/L (18 mg/L) (4 cases), and lack of any docu-
mented serum carbamazepine assay (2 cases). The peak serum concentra-
tions ranged from 78 to 285 mol/L (18.467.4 mg/L). It was found that
serum levels equal to or above 170 mol/L (40 mg/L) were significantly
associated with an increased risk of serious complications such as coma, sei-
zures, respiratory failure, and cardiac conduction defects. In 60% of the 10
patients with a serum level 170 mol/L at least two of these symptoms
296 S.T. Alrashood

occurred, in 50% at least three, and in 40% all four. There were two fatalities.
Among the 16 patients (18 admissions) with a serum carbamazepine concen-
tration below 170 mol/L, only one was comatose and none had any of the
other severe symptoms. It is concluded that serum carbamazepine levels
accurately predict the severity of toxicity in massive carbamazepine poison-
ing in adults [329].
Murine myeloma cells were exposed to toxic, growth-retarding levels of
two AEDs, PHT, and CBZ. J558L cells were treated for 12 days, washed
free of drug, and, upon recovery of growth, cloned to determine the fre-
quency of lambda light-chain secreting lines. The results indicate that
short-term exposure to high toxic levels (510 times the therapeutic dose)
of PHT and CBZ reduces or eliminates lambda light-chain secretion at a
high frequency. Furthermore, although most cloned lines tested positive
for cytoplasmic lambda light chain, some lines had no detectable cytoplasmic
immunoglobulin (Ig). The data are consistent with the hypothesis that long-
term changes in fully differentiated B cell function may occur after toxic
level AED exposure [330].
The teratogenicity of CBZ was investigated in Sprague-Dawley CD
rats at doses of 0, 200, 400, and 600 mg/kg administered by gavage in corn
oil on days 718 of gestation in a dosage volume of 2 mL/kg. The CBZ-600
dose was maternally toxic in that dams in this group weighed 30.6% less
than controls by E20. This group had significantly increased resorptions,
reduced live fetal weight (51.6% less than controls), and increased skeletal
and visceral abnormalities. The CBZ-400 dose also significantly reduced
maternal weight gain during gestation to 26.6% less than controls by E20.
No significant increase in resorptions occurred in this group; live fetuses
weighted 42.9% less than controls and showed an increase in visceral, but
not skeletal, abnormalities. The CBZ-200 dose did not significantly affect
maternal weight gain or increase resorptions or fetal abnormalities but did
reduce fetal body weight (20.3% less than controls). Maternal serum total
CBZ concentrations 1 h after the final dose were 22.9, 27.9, and
34.4 g/mL for the 200, 400, and 600 mg/kg groups, respectively. These
levels were little changed 6 h posttreatment. CBZ was 6570% serum pro-
tein bound across dose groups. Human therapeutic levels of CBZ are 4
12 g/mL and the drug is typically 80% serum protein bound. This
suggests that abnormalities in rats occur at concentrations well above the
human therapeutic range. However, a no-effect level was not found for
fetal body weight. Further experiments will be required to determine
how much lower doses will need to be in order to find a no-effect level
Carbamazepine 297

for fetal body weight. Nevertheless, the present data suggest that CBZ is not
potent at inducing malformations in rats [331].
A cooperative prospective study of consecutive cases of carbamazepine
overdose was conducted to determine if serum levels were predictive of tox-
icity and if risk factors such as age, chronic exposure, or previous disorder or
cardiovascular disease could be used as prognostic indicators. Seventy-three
consecutive cases were collected from two regional certified poison control
centers from Jan. 1989 to Aug. 1989. There were 25 exposures in children
less than 6 years, 11 exposures in adolescents, and 37 exposures in adults.
Ten adult cases and one adolescent case were excluded from the study
due to the presence of coingestants or inadequate information. Peak mea-
sured serum levels ranged from 0.3 to 56 g/mL. Using the presence of
coma, seizure activity, or respiratory depression requiring mechanical ven-
tilation as measures of toxicity, it was found poor correlation between rising
serum levels of carbamazepine and toxicity. Increased serum levels of carba-
mazepine did appear to correlate with increased hospital stay, but not with
ICU stay. History of a seizure disorder appears to pose increased risk of a
seizure in carbamazepine overdose. In this series chronic exposure to carba-
mazepine did not appear to increase the risk of coma or respiratory depres-
sion for a given toxic serum level and may add some protective effect. Serum
levels below 40 g/mL do not appear to accurately predict the severity of
toxicity. Cardiac conduction defects were rare (one child). Anticholinergic
findings, as evidence by decreased bowel motility and sinus tachycardia,
were common. Previous cardiovascular disease and age did not appear to
be important prognostic indicators [332].
Folate depletion has often been a problem in chronic AED therapy.
CBZ, a commonly used AED, has been implicated in some clinical studies.
A rat model was developed to examine the effects of chronic CBZ treatment
on folate concentrations in the rat. In the course of developing this model, a
common vehicle, propylene glycol, by itself in high doses, was found to
exhibit protective properties against induced seizures and inhibited weight
gain. Seizures induced by hexafluorodiethyl ether (HFDE) were also found
to be a more sensitive measure of protection by CBZ than seizures induced
by MES. Oral administration of CBZ as an aqueous suspension every 8 h at a
dose of 250 mg/kg was continuously protective against HFDE-induced sei-
zures and was minimally toxic as measured by weight gain over 8 weeks of
treatment. The CBZ levels measured in plasma and brain of these animals,
however, were below those normally considered protective. This treatment
with CBZ had no apparent adverse effect on folate concentrations in the rat,
298 S.T. Alrashood

and, indeed, the folate concentration increased in liver after 6 weeks of treat-
ment and in plasma at 8 weeks of treatment [333].
Carbamazepine is the drug of first choice in the treatment of SPS and
CPS and trigeminal and glossopharyngeal neuralgias. It is usually preferred
to phenobarbitone or phenytoin because of its powerful antiepileptic activ-
ity combined with a relative lack of adverse effects. In this article the mech-
anisms of action and pharmacological properties of carbamazepine are
outlined in order to explain the pathogenesis of most side and toxic effects.
Most of these effects, namely those affecting the nervous or cardiovascular
systems, correlate well with an increased concentration of the drug in plasma
and disappear spontaneously upon discontinuation of therapy. Other less
frequent toxic effects, namely aplastic anemia or fatal hepatitis, may be
ascribed to unforeseeable idiosyncratic reactions. Carbamazepine poisoning,
usually accidental and sometimes secondary to the coadministration of other
drugs, yields a clinical picture with neurological and cardiovascular signs.
The outcome is usually favorable, sometimes with spontaneous improve-
ment, and death is a distinct rarity. No specific antidotes are available.
The oral administration of activated charcoal has been shown to be an effec-
tive therapeutic measure significantly reducing the plasma half-life of the
drug [334].
Overdose of CBZ can be fatal. The case of a patient with near-lethal tox-
icity due to delayed absorption of drug was reported. A 36-year-old woman
was admitted with coma, hypotension, and unusual movements. CBZ level
several hours later was 36 mg/L. Gastric lavage revealed no pill fragments,
and activated charcoal was administered. CBZ level initially fell, reaching
28 mg/L 36 h after admission. Blood level then rose sharply, reaching
54 mg/L 64 h after admission. The pattern of rise suggested renewed
absorption of drug. Vigorous cathartics were given, and further doses of
charcoal were administered. Three hours after onset of diarrhea, roving
eye movements occurred. Two hours later she grimaced to pain. Eight hours
after the onset of diarrhea, she was awake. In CBZ overdose, activated char-
coal therapy coupled with aggressive intestinal purging helps prevent con-
tinued absorption of drug, late exacerbation of symptoms, and potentially
fatal outcome [335].
A patient who developed anemia with an isolated erythroid toxicity fol-
lowing chronic carbamazepine administration is reported. The anemia
quickly resolved with discontinuation of this drug. Other hematologic tox-
icities of carbamazepine are well described; however, an isolated erythroid
toxicity is unusual. In addition, the onset of this drug-induced toxicity
Carbamazepine 299

developed later than is expected for carbamazepine-associated hematological

toxicities. This case demonstrates the suddenness with which hematological
toxicities can occur with carbamazepine, and affirms the need for regular
monitoring of patients. Any significant decrease in the patients hemoglobin
or hematocrit level requires close monitoring for the sudden development of
serious anemia [336].
Human peripheral blood mononuclear cells (PBMCs) were isolated
from healthy volunteers and exposed in vitro to phenytoin or carbamaze-
pine, two widely used AEDs. This study investigated the effects of these
drugs on natural killer (NK) cell activity and antibody-dependent cell-medi-
ated cytotoxicity (ADCC), which are both thought to protect against devel-
oping neoplasms. Also, the genotoxicity of phenytoin on human PBMCs
was investigated by gravity-flow alkaline elution. Concentrations of phe-
nytoin considered therapeutic (10 and 20 g/mL) and a dose considered
acutely toxic (40 g/mL) were used, while carbamazepine levels of
8 g/mL (therapeutic) and 10 and 16 g/mL (acutely toxic) were tested.
Phenytoin at all three concentrations significantly suppressed NK cell
activity in a dose-dependent manner. Carbamazepine had no significant
effect on NK cell activity at the dose levels studied. Incubation in propyl-
ene glycol, the diluent for carbamazepine, significantly decreased NK cell
activity compared to saline. Phenytoin also significantly depressed inter-
feron augmentation of NK cell cytotoxicity in a dose-dependent manner.
ADCC activity was significantly depressed with 20 and 40 g/mL phenyt-
oin. Alkaline elution showed a slight but significant increase in DNA
single-strand breaks of PBMCs exposed to 40 g/mL phenytoin for 18
or 72 h. These results show phenytoin may induce pronounced immuno-
suppression of NK cell and ADCC activity in patients receiving anti-
epileptic therapy and that this agent has a potential for genotoxic side
effects. Phenytoin may also increase the potential for neoplasm develop-
ment by a direct interaction with cellular DNA and/or an indirect
mechanism by immunosuppression [337].
The effects of phenytoin (30 g/mL), phenobarbital (64 g/mL), and
carbamazepine (24 g/mL) were assessed in cerebral cortical cell cultures.
After AED exposure for 11 days, cultures were assayed for total protein,
number of neurons, tetanus toxin fixation, high-affinity uptake of GABA
and beta-alanine, activity of choline acetyltransferase, and benzodiazepine
binding. Carbamazepine-exposed cultures demonstrated minimal effects,
whereas highly significant deficits related to generalized toxicity were
observed in cultures exposed to phenytoin or phenobarbital [338].
300 S.T. Alrashood

Anticonvulsant effect and neurological toxicity were investigated in

mice for CBZ and its main metabolites, CBZ-E and 10,11-dihydro-
10,11-trans-dihydroxy-CBZ. The compounds were first tested separately
and the results were expressed in terms of brain concentrations. Brain pen-
etration was very poor for 10,11-dihydro-10,11-trans-dihydroxy-CBZ,
which had neither anticonvulsant nor neurotoxic activity. Against MES,
CBZ was slightly more potent than CBZ-E and both were ineffective
against pentylenetetrazole and bicuculline. CBZ and CBZ-E displayed
similar neurotoxicity. Combined administration of CBZ and CBZ-E rev-
ealed a slightly synergistic interaction with regard to both anticonvulsant
and neurotoxic action, the therapeutic index of the mixture being similar
to that of CBZ. These results suggest that antiepileptic activity and neuro-
logical toxicity of CBZ are proportional to the sum of the concentrations of
CBZ and its metabolite CBZ-E. Furthermore, variable levels of CBZ-E in
relation to CBZ do not affect the overall therapeutic index. Finally, the
results do not indicate that CBZ-E alone has any advantage over CBZ with
regard to neurotoxic side effects and therapeutic index [339].
The objective of this investigation was to study the teratogenic effects of
dosage levels and time of administration of three anticonvulsant drugs (car-
bamazepine [CMZ], sodium valproate [NaV], and diphenylhydantoin
[DPH]) on craniofacial development in the CD-1 mouse fetus. Pregnant
females were intubated on each of days 810, 1113, 1416, and 816 of
gestation with the following dose levels for each drug: 375, 563, 938 mg/
kg CMZ; 225, 338, 563 mg/kg NaV; 50, 75, 125 mg/kg DPH. Appropriate
control groups were maintained for each drug. On gestation day 17, preg-
nant females were killed and implantation sites were recorded as live, dead,
or resorbed. All live fetuses were examined for craniofacial defects. Results
of examination of 1398 fetuses indicated that CMZ, NaV, and DPH were
teratogenic and embryotoxic at all dose levels. This study indicated
that the observed decrease in mean fetal weight was drug-, dose-, and
time-dependent. There was a drug-, dose-, and time-dependent increase
observed in the number of dead fetuses, whereas the number of resorbed
fetuses was observed to be only time-dependent. The observed frequencies
of hydrocephalies, secondary palatal clefts, and submucous palatal clefts were
significant for all three factors (drug, dose, and time), whereas the observed
frequencies of hematomas and exencephalies were significant only for drug
and time. Cleft lips were observed only in the highest dose level of DPH.
Uterine horn distribution of defects indicated that fetuses located at the
proximal end of the horns were less subject to major defects than those
Carbamazepine 301

fetuses located at the distal end of the uterine horns. Fetuses with craniofacial
hematomas were found in the proximal one-third of the uterine horn,
resorbed fetuses, and fetuses with submucous palatal clefts in the middle
one-third of the uterine horns and dead fetuses and fetuses with
exencephalies, cleft lips, and secondary palatal clefts were localized in the
distal one-third of the uterine horns. In comparing the effect of drug, dosage,
and time on the development of craniofacial malformations in the CD-1
mouse fetus, CMZ was the least teratogenic and embryotoxic of the three
anticonvulsant drugs employed in this study [340].
Four patients with an acute overdose of carbamazepine were examined
with serial blood level determinations. The clinical spectrum consisted of
coma, respiratory depression, seizures, myoclonus, nystagmus, hyperre-
flexia, hyporeflexia, delayed gastric emptying with cyclic coma, ataxia, sinus
tachycardia, and atrioventricular conduction delay. Carbamazepine elimina-
tion half-lives varied from 10 to 29 h, and in one case carbamazepine-10,11-
epoxide was measured and had a half-life of 24 h [341].
Saliva is a body fluid which, like serum, can be used for determination of
concentrations of certain drugs, both in pharmacotherapy and in acute poison-
ings. The aim of this study was to determine carbamazepine concentrations in
both saliva and serum in acute poisoning in order to show if there is a corre-
lation between the obtained values, as well as to monitor toxicokinetics of car-
bamazepine in body fluids. Saliva and serum samples were obtained from 26
patients treated with carbamazepine and 20 patients acutely poisoned by the
drug immediately after their admission in the Emergency Toxicology Unit.
Determination of salivary and serum carbamazepine concentrations was per-
formed by the validated high-pressure liquid chromatographyultraviolet
(HPLC-UV) method. A significant correlation of salivary and serum carba-
mazepine concentrations in both therapeutic application and acute poisoning
(r 0.9481 and 0.9117, respectively) was confirmed. In acute poisonings the
mean ratio between salivary and serum concentrations of carbamazepine
(0.43) was similar to the mean ratio after its administration in therapeutic doses
(0.39), but there were high interindividual variations in carbamazepine con-
centrations in the acutely poisoned patients, as a consequence of different
ingested doses of the drug. In acute poisoning the halftime of carbamazepine
in saliva and serum was 12.57 and 6.76 h, respectively. The results suggest a
possible use of saliva as an alternative biological material for determination of
carbamazepine concentrations in therapeutic application and acute poisoning
as well, and a possible extrapolation of the results obtained in saliva to serum
concentrations of carbamazepine [342].
302 S.T. Alrashood

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 CHAPTER FIVE

Pioglitazone
A. Al-Majed, A.H.H. Bakheit, H.A. Abdel Aziz, H. Alharbi,
F.I. Al-Jenoobi
King Saud University, Riyadh, Kingdom of Saudi Arabia

Contents
1. Description 380
1.1 Nomenclature 380
1.2 Formulae 381
1.3 Elemental Analysis 382
1.4 Appearance 382
2. Uses and Applications 382
3. Methods of Preparation 382
4. Physical Characteristics 387
4.1 Color/Form 387
4.2 Melting Point 387
4.3 Dissociation Constant 387
4.4 Octanol/Water Partition Coefficient 387
4.5 Solubility 387
4.6 Vapor Pressure 387
4.7 X-Ray Powder Diffraction Pattern 388
4.8 Thermal Analysis 388
4.9 Spectroscopy 389
4.10 Mass Spectroscopy 390
4.11 NMR Spectrometry 390
5. Method of Analysis 391
5.1 Compendial Methods 391
5.2 Reported Method of Analysis 399
5.3 Electrochemical Method 406
5.4 Chromatography 409
6. Stability 427
7. Clinical Applications 428
7.1 Pharmcodynamics 428
7.2 Mechanism of Action 429
7.3 Pharmacokinetics 429

Profiles of Drug Substances, Excipients, and Related Methodology, Volume 41 # 2016 Elsevier Inc. 379
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/bs.podrm.2015.11.002
380 A. Al-Majed et al.

7.4 Absorption 430

7.5 Distribution 430
7.6 Metabolism 430
7.7 Excretion 431
7.8 Elimination Half-Life 431
References 431

1. DESCRIPTION
1.1 Nomenclature
1.1.1 Systemic Chemical Names
5-[[4-[2-(5-Ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,
3-thiazolidine-2,4-dione hydrochloride [1].
(RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl)thiazolidine-2,
4-dione [2].
(
)-5-{p-[2-(5-Ethyl-2-pyridyl)-ethoxy] benzyl}-2,4-thiazolidinedione
hydrochloride [3].
[(
)-5-[[4-[2-(5-ethyl-2-pyridinyl) ethoxy] phenyl] methyl]-2,4-]
thiazolidine-dione [4].
5-(4-(2-(5-ethylpyridin-2-yl)ethoxy)benzyl)thiazolidine-2,4-dione
hydrochloride [5].
5-[[4-[2-(5-ethylpyridin-2-yl) ethoxy] phenyl] methyl] thiazolidine-
2,4-dione.
5-[4-[2-(5-ethyl-2-pyridyl)ethoxy]benzyl]-2,4-thiazolidinedione.
(
)-5-[[4-[2-(5-Ethyl-2-pyridinyl)-ethoxy] phenyl] methyl]-2,
4-thiazolidinedione.
2,4-Thiazolidinedione, 5-[[4-[2-(5-ethyl-2-pyridinyl)ethoxy]phenyl]
methyl]-(9CI).
2,4-Thiazolidinedione, 5-((4-(2-(5-ethyl-2-pyridinyl)ethoxy)phenyl)
methyl)-.
5-({4-[2-(5-ethylpyridin-2-yl) ethoxy] phenyl}methyl)-1,
3-thiazolidine-2,4-dione.
5-[[4-[2-(5-ethyl-2-pyridinyl)ethoxy]phenyl]methyl]thiazolidine-2,
4-dione.
5-{4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl}-4-hydroxy-1,3-thiazol-2
(5H)-one.
2-Amino-5-{4-[2-(5-ethyl-pyridin-2-yl)-ethoxy]-benzyl}-thiazol-4-
one [6].
Pioglitazone 381

1.1.2 Nonpropriety Name

Generic: Pioglitazone Hydrochloride [1].
Synonyms: Pioglitazonum [INN-Latin]; Pioglitazona [INN-Spanish];
Duetact, AD-4833; Pioglitazone [INN:BAN]; U 72107, pioglitazone
(INN); Pioglitazone [BAN:INN].
1.1.3 Propriety Name
Brand names [3]:
Actos (Takeda Pharmaceuticals); Glustin (Eli Lilly and Company);
Glados (Tabuk Pharmaceuticals). Arg.: Actos; Cereluc; Higlucem; Pioglit;
Piotamax; Austral.: Actos; Austria: Actos; Belg.: Actos; Braz.: Actos;
Canad.: Actos; Chile: Actos; Diabestat; Tiazac; Cz.: Actos; Glustin;
Denm.: Actos; Fin.: Actos; Fr.: Actos; Ger.: Actos; Gr.: Actos; Hong
Kong: Actos; India: Diaglit; G-Tase; Glita; Glizone; Opam; P-Glitz;
Pepar; Piomed; Piosafe; Piozulin; Indon.: Actos; Deculin; Ital.: Actos;
Jpn: Actos; Malaysia: Actos; Mex.: Zactos; Neth.: Actos; Glustin; Norw.:
Actos; NZ: Actos; Philipp.: Actos; Prialta; Zypi; Port.: Actos; Glustin;
Rus.: Actos (); S.Afr.: Actos; Spain: Actos; Swed.: Actos; Switz.:
Actos; Thai.: Actos; UK: Actos; USA: Actos; Venez.: Actos.
Multiingredient [3]:
Oseni (Alogliptin/pioglitazone systemic); Duetact (Glimepiride/
pioglitazone systemic); ActoPlus Met, ActoPlus Met XR (Metformin/
pioglitazone systemic); Cz.: Competact; Glubrava; Tandemact; Fr.:
Competact; Tandemact; India: Exermet P; P-Glitz M; Piomed M;
Piosafe MF; Port.: Competact; Tandemact; UK: Competact; USA:
Actoplus Met; Duetact.
1.2 Formulae
1.2.1 Empirical Formula, Molecular Weight, and CAS Number

Pioglitazone C19H20N2O3S 356.44 g/ [111025-46-8] [2]

mol
Pioglitazone C19H20N2O3SHCl 392.90 g/ [112529-15-4] [1,3]
Hydrochloride mol

1.2.2 Structural Formula

O
O
S
HN
N CH3

O
382 A. Al-Majed et al.

1.3 Elemental Analysis

Pioglitazone: C: 64.02%, H: 5.66%, N: 7.86%, O: 13.47%, S: 9.00%.
Pioglitazone Hydrochloride: C: 58.08%, H: 5.39%, N: 7.13%, O
12.22%, S: 8.16%, Cl: 9.02%.

1.4 Appearance
Colorless prisms from ethanol [1] and needles from dimethylformamide and
water [7].

2. USES AND APPLICATIONS

Pioglitazone is a thiazolidinedione antidiabetic with actions similar to
those of rosiglitazone. Pioglitazone depends on the presence of insulin for its
mechanism of action [8]. It is used to treat diabetes mellitus type 2. It may be
used alone or with other medicines such as insulin, metformin, or sulfonyl-
urea agents. Pioglitazone is used together with a proper diet and exercise to
help control blood sugar levels. It decreases insulin resistance in the periph-
ery and in the liver, resulting in increased insulin-dependent glucose disposal
and decreased hepatic glucose output. In animal models of diabetes,
pioglitazone reduces the hyperglycemia, hyperinsulinemia, and hyper-
triglyceridemia characteristic of insulin-resistant states such as type 2 diabe-
tes. The metabolic changes produced by pioglitazone result in increased
responsiveness of insulin-dependent tissues and are observed in many animal
models of insulin resistance. Pioglitazone is not intended for treating type 1
diabetes [3].

3. METHODS OF PREPARATION
A number of syntheses of pioglitazone have been disclosed. Fischer et al.
[9] and Les et al. [10] described two related syntheses of pioglitazone hydro-
chloride (Scheme 1). The tosylate of 2-(5-ethylpyridin-2-yl) ethanol 2, formed
in situ with tosyl chloride, was displaced by 4-hydroxybenzaldehyde 3 by
means of benzyltributylammonium chloride and NaOH to give 4-[2-(5-eth-
ylpyridin-2-yl)ethoxy]benzaldehyde 6. Alternatively, a nucleophilic aromatic
substitution reaction between alcohol 2 and 4-fluorobenzonitrile 4 using NaH
as the base provided 4-[2-(5-ethylpyridin-2-yl)ethoxy] benzonitrile 5, which
was reduced with Raney nickel and formic acid to aldehyde 6. Then
Pioglitazone 383

CN CN
H3C
H3C
NaH
+ F N O
N OH 4 5
2
RaNi, HCOOH
TsCl, PhCH2NBu3Cl,
+ aq. NaOH
O
CHO S
CHO NH
H3C
HO O N
3 N O
6
, H

S
H3C O S
H3C O
O N
N O H2 or NaBH4
H N O N
O H
7 1

Scheme 1 Fischer and Les method for synthesis of pioglitazone 1.

condensation of 6 with thiazolidine-2,4-dione in basic medium afforded 5-[-

4-[2-(5-ethylpyridin-2-yl)ethoxy]benzylidene] thiazolidine-2,4-dione 7.
Finally, this compound was hydrogenated to provide pioglitazone 1.
Scientists from Takeda chemical industries Ltd. [11] synthesized
pioglitazone 1, by condensed 5-ethyl-2-pyridyl ethanol 2 with 4-fluoro
nitro benzene 3 to obtain nitro compound 4. Hydrogenation of nitro com-
pound 4 in methanol using 10% Pd/C afforded amine compound 5. Com-
pound 5 was treated with sodium nitrate in the presence of HBr to give diazo
compound 6, which when reacted with methyl acrylate in the presence of
cuprous oxide by applying Meerwein arylation conditions afforded -
Bromo ester compound 7. Condensation of compound 7 with thio urea
in the presence of sodium acetate followed by acid catalyzed hydrolysis
resulted pioglitazone 1 (Scheme 2).
Meguro et al. [12] reported an alternative process for the synthesis of 1.
This involves protection of 2 with p-toluene sulfonyl chloride in the pres-
ence of phase transfer catalyst (benzyl tert-butyl ammonium chloride
(BTBAC)) resulted intermediate 3. Subsequently, intermediate 3 was sub-
jected to nucleophilic substitution. In particular, intermediate 3 was reacted
with 4-hydroxy benzaldehyde 4 in the presence of NaOH to give aldehyde
compound 5. The compound 5 was reacted with 2,4-thiazolidinedione 6 in
the presence of piperidine by employing knoevengal reaction conditions to
obtain benzylidene intermediate, which was hydrogenated using Pd/C in
dioxane to obtain (Scheme 3).
384 A. Al-Majed et al.

F NO2
Et NO2
OH Et Et NH2
3 10% Pd/C
N N O
2 NaH, DMF MeOH N O
4 5
O
1. NaNO2, HBr O
Et CH3
O Thio urea Et
Br NH
2. Methylacrylate, S
N O Sodium acetate
Cu2O N O
6 7 NH

O
2N HCl
Et
NH
S
N O
O
1

Scheme 2 Synthesis of pioglitazone 1 from (intermediate methyl 2-bromo-3-(4-(2-(5-

ethylpyridin-2-yl)ethoxy)phenyl)propanoate (6) from 1-fluoro-4-nitrobenzene through
SNAr and Meerwein arylation).

OH

Et Et CHO
OH OHC
TsCL Et 4
OTs
N BTBAC, DCM N O
2 NaOH, water
N 5
O NH O 3 O
O
Et
S 6 Et
NH 5% Pd/C
S NH
Piperidine N O S
O Dioxane N O
ethanol, water O
7 1

Scheme 3 Synthesis of pioglitazone 1 from the Knoevenagel condensation of

thiazolidine-2,4-dione 6 and 4-(2-(5-ethylpyridin-2-yl)ethoxy)benzaldehyde 5.

Huber [13] reported synthesis of pioglitazone 1 that involves a reduction

of 2 using sodiumborohydride in the presence of cobalt chloride/dimethyl-
glyoxime catalyst system (Scheme 4). An improved procedure by using sim-
ilar reagents was described by Andrzej Les [10] and coworkers for the
preparation of 1.
Scientists from Smithkline Beecham pharmaceuticals [14] reported a
reduction of benzylidene intermediate 2 using microbial reductase, derived
from suitable red yeast (Scheme 5).
Bipin Pandey et al. [15] described a process for the synthesis of 1 that
involves the usage of halohydrin compounds as intermediates. Reaction
of 5-ethyl-2-vinyl pyridine 2 with N-bromo succinamide provided bromo-
hydrin compound 3, which was reacted with 4 in the presence of base
(NaOH, K2CO3, or NaH) to afford compound 5. Condensation of 2,4-
thiazolidinedione 6 with compound 5 by employing Knoevenagel reaction
conditions resulted in benzylidene compound 7, which was hydrogenated
with sodium borohydride in the presence of cobalt chloride and
Pioglitazone 385

O
O
Et NaBH4
NH Et
S CoCl2, DMG NH
N O S
O THF, water N O
2 1 O

Scheme 4 Synthesis of pioglitazone from (Z)-5-(4-(2-(5-propylpyridin-2-yl)ethoxy)

benzylidene)thiazolidine-2,4-dione 2 using sodium borohydride in the presence of
cobalt chloride/dimethyl-glyoxime catalyst.

O
O
Et
NH Et
S Yeast NH
N O S
O Dioxane N O
2 1 O

Scheme 5 Synthesis of pioglitazone from (Z)-5-(4-(2-(5-propylpyridin-2-yl)ethoxy)

benzylidene)thiazolidine-2,4-dione 2 using microbial reductase.

OH
Et
Et
OHC Et CHO
CH2
N NBS Br 4
2 N
H NaOH, water N O
3 OH K2CO3 (or) NaH
O N O OH 5
O
S O
Et NaBH4
6 NH Et
S CoCl2, DMG NH
N O S
Piperidine O N O
OH DMF O
acetic acid 7 OH 8
etanol, water
O
O
PCl5 Et
NH Et
S Zn, acetic acid NH
CHCl3 N O S
O N O
Cl O
9 1

Scheme 6 Synthesis of pioglitazone 1 from intermediate halohydrin compounds.

dimethylglyoxime to furnish compound 8. Chlorination of 8 with PCl5,

POCl3, or SOCl2, followed by reaction with zinc and acetic acid, resulted
pioglitazone 1 (Scheme 6). Reactions were also performed by replacing OH
group with other groups (Cl, Br. OMs, OTs, and OSO3H).
Takao et al. [16] reported an alternative synthesis of pioglitazone 1,
where 2,4-thiazolidinedione 3 was condensed with 4-hydroxy benzalde-
hyde 2 in the presence of sodium acetate, acetic anhydride, and
dimethylacetamide to obtain intermediate 4, which was hydrogenated with
Pd/C and H2 in acetic acid to furnish 5. The resultant compound 5 was sub-
jected to N-alkylation with triphenyl methyl chloride in methylene chloride
resulted 6. Hydrolysis of 6 using sodium methoxide in toluene afforded 7.
Condensation of 7 and tosylate intermediate 8 in basic medium (K2CO3)
386 A. Al-Majed et al.

H CH3COONa O O
OH N
O O (CH3CO)2O O 10% Pd/C O
NH NH
OHC + S
CH3CON(CH3)2 H3C O
S
H3C O
S
2
CH3COOH 5
3 O O
4 Et
O OTs
O
PPh3CCl O NaOMe N
NCPh3 NCPh3
S S
DCM, TEA H3C O Toluene HO K2CO3
O O
6 7

O O
Et Et
H+
NCPh3 NH
S S
N O N O
O O
8 1

Scheme 7 Synthesis of pioglitazone 1 from intermediate (Z)-4-((2,4-dioxothiazolidin-5-

ylidene)methyl)phenyl acetate 4 from condensation of 2,4-thiazolidinedione 3 with
4-hydroxybenzaldehyde 2.

O
CHO O O
Cl OtBu OtBu
OtBu
3 10% Pd/C
BnO O O
2 tBuOK, tBuOH BnO Ethyl acetate HO
5
4
Et
O
O
N OMs Et MsCl
6 OMe Et
TEA OCH3
OH
N O OMs
K2CO3, ACN N O
7
8

O
Et O
NH Et
2MHCl
Sodium acetate S NH
N O S
NH N O
Thiourea
9 O
ethanol
1

Scheme 8 Synthesis of pioglitazone 1 from intermediate (Z)-4-((2,4-dioxothiazolidin-5-

ylidene)methyl)phenyl acetate 4 from condensation of 4-benzylloxybenzaldehyde 2
with tert-butyl chloroacetate.

followed by deprotection of trityl group in the presence of hydrochloric acid

furnished compound 1 (Scheme 7).
Thijs and coworkers [17] have synthesized pioglitazone 1, where 4-
benzyloxybenzaldehyde 2 was condensed with tert-butyl chloroacetate 3,
employing Darzens condensation conditions, afforded ,-epoxy ester 4,
which underwent for debenzylation using 10% Pd/C and hydrogen to obtain
intermediate 5. Intermediate 5 was reacted with 5-ethylpyridine-2-ethy
mesylate 6 in the presence of K2CO3 afforded -hydroxy compound 7,
which was mesylated using methanesulfonyl chloride in the presence of
TEA to provide mesylate compound 8. Requisite compound 1 was prepared
by condensation of thio urea with mesylated intermediate 8 in the presence
of sodium acetate, followed by hydrolysis of imine compound 9 using
hydrochloric acid (Scheme 8).
Pioglitazone 387

4. PHYSICAL CHARACTERISTICS
4.1 Color/Form
Pioglitazone: Colorless needles from dimethylformamide and water.
Pioglitazone hydrochloride: Colorless prisms from ethanol [7], Odorless
white crystalline [18].

4.2 Melting Point

Pioglitazone: 183184C [7].
Pioglitazone hydrochloride: 193194C [7].

4.3 Dissociation Constant

pKa 5.19 [7].

4.4 Octanol/Water Partition Coefficient

Log Kow 3.96 [6].

4.5 Solubility
Soluble in DMF, DMSO (79 mg/mL); slightly soluble in ethanol (4 mg/
mL), acetone, or acetonitrile; practically insoluble in water; insoluble in
ether. Soluble in 25 mM of DMSO [18]; in water, 46.85 mg/L at 25C [6].
Pioglitazone hydrochloride very soluble in dimethyl formamide; slightly
soluble in ethanol; very slightly soluble in acetone, acetonitrile. Practically
insoluble in water and ether [7,19].
Tao et al. [20] measured the solubility of pioglitazone hydrochloride
(form I) in methanol, ethanol, 1-propanol, acetic acid, and N,N-
dimethylacetamide between 278.15 and 323.15 K at atmospheric pressure.
The solubility of pioglitazone hydrochloride (form I) increases with increasing
temperature and the order is N,N-dimethylacetamide> methanol > acetic
acid > ethanol > 1-propanol.

4.6 Vapor Pressure

2.88  1014 mm Hg at 25C [6].
388 A. Al-Majed et al.

4.7 X-Ray Powder Diffraction Pattern

The X-ray powder diffraction pattern of Pioglitazone HCl has been mea-
sured using a Scintag XTRA X-ray powder Diffractometer, equipped with
a solid state Si(Li) detector thermoelectrically cooled and single channel ana-
lyzer and using a copper K ( 1.5418 A) radiation (tube operated at
40 kV, 40 mA). The data were collected over an angular range from 2 to
40 degree two theta continuous scan mode using a step size of 3 degree
two theta and a step time of 1 s. The peaks (reflections) of crystalline
pioglitazone hydrochloride are found at values of two theta of about 9.2,
10.4, 15.2, 16.4, 18.6, and 21.4
0.2 [21].

4.8 Thermal Analysis

4.8.1 Melting Behavior
Shirolkar et al. [22] determined the melting temperature of Pioglitazone by
melting point apparatus. It was found to be 194C which is acceptable to the
values of the reported melting temperature [18].

4.8.2 Differential Scanning Calorimetry

The differential scanning calorimetry (DSC) thermogram of Pioglitazone
was recorded on microspheres using DSC. Samples were accurately weighed
and put into aluminum pans and then sealed with aluminum lids. The ther-
mograms of the samples were obtained at a scanning rate of 10C/min. The
peak of pure drug was found at 192193C [22].

4.8.3 Thermogravimetric Analysis

Thermogravimetric analysis, derivative thermogravimetry, and differential
thermal analysis were carried out using simultaneous DTA-TGA thermal
analyzer apparatus (Shimadzu DTG-60H). The samples (47 mg) were
placed in platinum pan and heated up to 900C at a rate of 10C/min
under nitrogen purge (30 mL/min). Pioglitazone decomposed four stages
of decomposition: At the first stage (145225.9C), pioglitazone exhibits
a weight loss of 9.53% due to the loss of HCl molecule. A weight loss
of 57.09% observed between 225.9C and 327.73C which may be
attributed to the loss of C10H8NO3S. Beyond 389.34C, the drug
decomposed in two stages due to the loss of C4H9 at 389.34468C
(weight loss of 14.71%) and the loss of C5H3N at 468551.55C (weight
loss of 19.47%) [23].
Pioglitazone 389

4.9 Spectroscopy
4.9.1 Ultraviolet Spectroscopy
The ultraviolet (UV) absorption spectra of 20 g/mL pioglitazone HCl in
methanol is shown in Fig. 1. The figures were recorded using a Shimadzu
UV spectrophotometer; model no. UV-1800 with 1 cm matched quartz
cells was used for experiments. The absorption spectra of reference and
test solution were carried out in a 1 cm quartz cell over the range of
200400 nm.
The molar absorptivity of pioglitazone HCl at 268 nm is
6561.43 L/mol cm.

4.9.2 Infrared Spectroscopy

Infrared (IR) spectrum of pioglitazone was recorded as KBr disk using
the Shimadzu FT-IR Spectrum BX apparatus. The IR absorption
spectrum of pioglitazone showed two carbonyl functions in the range of
16841743 cm1. The NH absorption band appeared at 3258 cm1 (Fig. 2).

2.000
4

1.000
3
Abs.

1
5

0.000

0.572
200.00 250.00 300.00 350.00 400.00
nm.
Figure 1 Ultraviolet absorption spectrum of pioglitazoe HCl dissolved in methanol.
390 A. Al-Majed et al.

Figure 2 Infrared absorption spectrum of pure pioglitazone HCl.

4.10 Mass Spectroscopy

The mass spectrum of pioglitazone (C19H20N2O3S, 356.44) was obtained
using an Agilent 6320 Ion trap mass spectrometer (Agilent technologies,
USA) equipped with an electrospray ionization interface (ESI). A connector
was used instead of column. Mobile phase composed of a mixture of solvents
A and B (50:50), where A is high-performance liquid chromatography
(HPLC) grade water and B is acetonitrile. Compound was prepared by
weighing the solid substances to 1 mg/mL in DMSO and diluted with
mobile phase. Test solution was prepared by diluting the stock solutions
to 1030 mg mL depending on the ion intensitieswith mobile phase.
Flow rate was 0.4 mL/min and run time was 5 min. MS parameters were
optimized for each compound. The scan was ultra-scan mode. MS2 scans
were performed in the mass range of m/z 501000. The ESI was operated
in positive mode. The source temperature was set to 350C nebulizer gas
pressure of 55.00 psi with dry gas flow rate of 12.00 L/min. Fig. 3 shows
the molecular ion peak of pioglitazone at m/z 357.1 [M]+.

4.11 NMR Spectrometry

4.11.1 1H NMR Spectrometry
1
H NMR spectrum of pioglitazone was scanned in DMSO-d6 on a Brucker
NMR spectrometer operating at 500 MHz. Chemical shifts are expressed in
Pioglitazone 391

102 +Scan(0.297 min) pg00002.d

357.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2 320.1
0.1
0
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
Counts vs mass-to-charge (m/z)
Figure 3 Mass spectrum of pioglitazone.

-values (ppm) relative to TMS as an internal standard. Coupling constants

( J) are expressed in Hz (Table 1 and Fig. 4AC).

4.11.2 13C NMR Spectrometry

13
C NMR spectrum of pioglitazone was scanned in DMSO-d6 on a
Brucker NMR spectrometer operating at 125 MHz. Chemical shifts are
expressed in -values (ppm) relative to TMS as an internal standard
(Table 2 and Fig. 5).

5. METHOD OF ANALYSIS
5.1 Compendial Methods
5.1.1 United States Pharmacopeia Methods [24]
Definition
Pioglitazone Hydrochloride contains NLT 98.0% and NMT 102.0% of
C19H20N2O3SHCl, calculated on the anhydrous basis.
Identification
A. IR absorption <197K >
B. Identification testsGeneral, Chloride <191 >: Dissolve 25 mg of
Pioglitazone Hydrochloride in 0.5 mL of nitric acid, and add 2 mL of
dilute nitric acid. It meets the requirements of the test for chloride.
C. The retention time of the pioglitazone peak of the sample solution cor-
responds to that of the standard solution, as obtained in the Assay.
392 A. Al-Majed et al.

Table 1 1H NMR of Pioglitazone (DMSO-d6)

6 8 11 O
7 9 O 16
5 12 S
10
N H
1 N
3 11 13 15 17
2 4 12 14
O
Pioglitazone

Signal Location () Shape Integration Correspondences

1 1.211.26 m 3H dCH2dCH3 (Hs1)
2 2.762.82 m 2H dCH2dCH3 (Hs2)
3 3.033.09 m 1H dArdCH2dCH < (Hs14)
4 3.273.32 m 1H dArdCH2dCH < (Hs14)
5 3.493.51 m 2H dCH2dCH2dOdArd
(sH8)
6 4.404.41 m 2H dCH2dCH2dOdArd
(Hs9)
7 4.854.89 m 1H ArdCH2dCH< (H15)
8 6.866.89 m 2H ArHs (11 + 11)
9 7.137.17 m 2H ArHs (12 + 12)
10 7.977.99 dd, J 3.5, 1H Pyridine H6
8.0 Hz
11 8.408.42 d, J 3.0, 8.0 Hz 1H Pyridine H5
12 8.728.73 d, J 6.0 Hz 1H Pyridine H4
13 12.05 s 1H NdH

Assay
Procedure:
Mobile phase: Acetonitrile, 0.1 M ammonium acetate, and glacial
acetic acid (25:25:1)
Standard solution: Prepare a 0.5 mg/mL solution of USP
Pioglitazone Hydrochloride RS in methanol and dilute with Mobile
phase to obtain a solution containing 50 g/mL of pioglitazone
hydrochloride.
Pioglitazone 393

Figure 4 (A) 1H NMR spectrum of pioglitazone. (B) 1H NMR spectrum of pioglitazone

(aliphatic region).
(Continued)
394 A. Al-Majed et al.

Figure 4Cont'd (C) 1H NMR spectrum of pioglitazone (aromatic region).

System suitability stock solution: 0.5 mg/mL of USP Pioglitazone

Hydrochloride RS and 0.13 mg/mL of benzophenone in methanol.
System suitability solution: Dilute system suitability stock solution
with mobile phase to obtain a solution containing 50 g/mL of
pioglitazone hydrochloride and 13 g/mL of benzophenone.
Sample solution: Prepare a 0.5 mg/mL solution of pioglitazone
hydrochloride in methanol and dilute with mobile phase to obtain
a solution containing 50 g/mL of pioglitazone hydrochloride.
Mode: LC
Detector: UV 269 nm
Column: 4.6 mm  15 cm; 5 m packing L1
Column temperature: 25
2.5C
Flow rate: 0.7 mL/min
(NOTEAdjust the flow rate so that the retention time of the
pioglitazone peak is about 7 min.)
Injection size: 20 L
System suitability
Pioglitazone 395

13
Table 2 C NMR of Pioglitazone (DMSO-d6)
6 8 11 O
7 9 O 16
5 12 S
10
N H
1
N
3 11 13 15 17
2 4 12 14
O
Pioglitazone

Signal Location () Correspondences

1 14.08 CH3 (C1)
2 25.07 CH2 (C2)
3 32.79 CH2 (C8)
4 36.67 CH2 (C14)
5 53.42 CH (C15)
6 65.91 CH2 (C9)
7 114.90 (2C) CH (C11 + C11)
8 127.53 CH (C6)
9 129.56 C (C13)
10 130.88 (2C) CH (C12 + CC12)
11 140.67 CH (C4)
12 141.71 C (C10)
13 145.56 CH (C5)
14 151.74 C (C3)
15 157.49 C (C7)
16 172.12 CdO (C16)
17 176.15 CdO (C17)

Samples: System suitability solution and standard solution

(NOTEThe approximate relative retention times for pioglitazone
and benzophenone are 1.0 and 2.6, respectively.)

Suitability requirements:
Tailing factor: NMT 1.5 for pioglitazone and benzophenone, system
suitability solution
396 A. Al-Majed et al.

13
Figure 5 C NMR spectrum of pioglitazone.

Resolution: NLT 15 between pioglitazone and benzophenone,

system suitability solution
Relative standard deviation: NMT 2.0% for six replicate injections,
standard solution.

Analysis:
Samples: Standard solution and Sample solution
Calculate the percentage of C19H20N2O3SHCl in the portion of
Pioglitazone Hydrochloride taken:

   
rU CS
Result   100
rS CU

rU peak response from the Sample solution

rS peak response from the Standard solution
CS Concentration of USP PioglitazoneHydrochloride RS in
the Standard solution (g/mL).
Pioglitazone 397

CU Concentration of Pioglitazone Hydrochloride in the Sam-

ple solution (g/mL).
Acceptance criteria: 98.0102.0% on the anhydrous basis

Impurities
Inorganic impurities:
Residue on ignition <281 >: NMT 0.1%
Heavy metals
 Sodium sulfide solution: 5 g of sodium sulfide in 10 mL of water
and 30 mL of glycerin
 Magnesium nitrate solution: 100 mg/mL of magnesium nitrate in
alcohol
 Standard solution: Place 10 mL of magnesium nitrate solution in a
platinum or porcelain crucible. Ignite the alcohol to burn. Cool,
add 1 mL of sulfuric acid, heat carefully, and ignite at 550
50C.
Cool and add 3 mL of hydrobromic acid. Proceed as directed from
this point under Sample solution, adding 1.0 mL of Standard Lead
Solution (see Heavy Metals <231 >, Special Reagents) before
adding water to make 50 mL.
 Sample solution: Place 1.0 g of pioglitazone hydrochloride in a
platinum or porcelain crucible. Mix with 10 mL of magnesium
nitrate solution. Ignite the alcohol to burn and carbonize by gradual
heating. Cool, add 1 mL of sulfuric acid, heat carefully, and incin-
erate by ignition at 550
50C. If carbonized substances remain,
moisten with a small amount of sulfuric acid and incinerate by
ignition. Cool, dissolve the residue in 3 mL of hydrobromic acid,
and evaporate on a water bath to dryness. Wet the residue with three
drops of hydrochloric acid, add 10 mL of water, and dissolve by
warming. Add one drop of phenolphthalein TS and add ammonia
TS dropwise until a pale red color develops. Add 2 mL of 1 N acetic
acid, filter if necessary, wash with 10 mL of water, transfer the filtrate
and washings to a Nessler tube, and add water to make 50 mL.
 Analysis: Add one drop of Sodium sulfide solution to each of the
tubes containing the Standard solution and Sample solution. Mix
thoroughly and allow to stand for 5 min. Compare the colors of
both solutions by viewing the tubes downward or transversely
against a white background. The Sample solution has no more
color than the Standard solution.
Acceptance criteria: NMT 10 ppm
398 A. Al-Majed et al.

Organic impurities
Procedure
 Mobile phase and System suitability stock solution: Proceed as
directed in the Assay.
 System suitability solution: Dilute the System suitability stock
solution with Mobile phase to obtain a solution containing
25 g/mL of pioglitazone hydrochloride and 6.5 g/mL of
benzophenone.
 Sample solution: 0.2 mg/mL of pioglitazone hydrochloride dis-
solved in 20% of the final volume with methanol, then diluted
with Mobile phase to final volume.
 Standard solution: 1 g/mL of pioglitazone hydrochloride pre-
pared by diluting the Sample solution with Mobile phase.
 Mode: LC
 Detector: UV 269 nm
 Column: 4.6 mm  15 cm; 5 m packing L1
 Column temperature: 25
2.5C
 Flow rate: 0.7 mL/min
 (NOTEAdjust the flow rate so that the retention time of the
pioglitazone peak is about 7 min.)
 Injection size: 40 L
 Run time: At least 4  the retention time of pioglitazone.
System suitability
 Samples: System suitability solution and Standard solution
 Suitability requirements
A. Tailing factor: NMT 1.5 for pioglitazone and benzophenone,
System suitability solution
B. Resolution: NLT 15 between pioglitazone and benzophe-
none, System suitability solution
C. Relative standard deviation: NMT 3.0%, Standard solution
Analysis
 Samples: Standard solution and Sample solution
 Calculate the percentage of each impurity in the portion of
Pioglitazone Hydrochloride taken:

 
rU
Result  D  100
rS
Pioglitazone 399

rU peak response of each individual impurity from the

Sample solution
rS peak response of pioglitazone from the Standard solution
D dilution factor used to prepare the Standard solution,
0.005
Acceptance criteria
Individual impurities: See Table 3.
Total impurities: NMT 0.5%.
5.2 Reported Method of Analysis
5.2.1 Spectroscopy Methods
5.2.1.1 UV Spectrometry, Colorimetry, and Thermal Analysis
Ulu et al. [25] developed three rapid, sensitive, and simple spectrophoto-
metric methods for the determination of pioglitazone in pure and pharma-
ceutical preparations. For the first method, UV spectrophotometry,
standard solutions were measured at 270.2 nm. It was linear from 5.0 to
20.0 g/mL. For the second method, the distances between two extre-
mum values (peak-to-peak amplitudes), 272.0 and 287.4 nm, were mea-
sured in the second-order derivative spectra of standard solutions. The
linearity was found to be 2.012.0 g/mL for pioglitazone standards in
acetonitrile. The third method was based on the formation of an ion asso-
ciation complex with bromocresol green (BCG), bromocresol purple
(BCP), bromophenol blue (BPB), and bromothymol blue (BTB). The
assay was linear over the concentration range of 20.0100.0 g/mL for
BCG, 10.0100.0 g/mL for BCP, 20.0120.0 g/mL for BPB, and
10.0100.0 g/mL for BTB. The three proposed methods have been suc-
cessfully applied to the assay of pioglitazone in pure and in pharmaceutical
preparations.

Table 3 Impurity
Relative Retention Acceptance Criteria,
Name Time NMT (%)
Hydroxypioglitazonea 0.7 0.15
Pioglitazone 1.0
Didehydropioglitazoneb 1.4 0.15
N-alkylpioglitazonec 3.0 0.15
Any other individual impurity 0.10
400 A. Al-Majed et al.

Adhikari et al. [26] used a spectroscopic method to quantify three anti-

diabetics in multicomponent formulations. In the present study, three wave-
length spectroscopic method and multiwavelength method was carried out
for determination of metformin hydrochloride, glipizide, pioglitazone
hydrochloride in their bulk and preparations using acetonitrile:methanol:
water in the proportion of 5:4:1. The max was found at 236.5, 226.4,
and 227.3 nm, respectively. The isobestic point was found to be 254 nm.
Method II is based on multiwavelength spectroscopy. All the three drugs
obey the BeerLambert limit within the concentration range of 550 g/
mL. The method can be used for routine quantitative analysis of metformin,
glipizide, and pioglitazone in pure and tablet dosage forms.
Amanlou et al. [27] developed an extractive spectrophotometric method
for the determination of pioglitazone hydrochloride in pure and pharmaceu-
tical formulations. This method is based on the formation of yellow ion-pair
complex between the basic nitrogen of the drug and bromocresol green
(BCG) in phthalate buffer of pH 2.4. The formed complexes were extracted
with chloroform and measured at 419 nm. Beers law was obeyed in the
range 2.514 g/mL. The proposed method has been applied for the deter-
mination of drug in commercial tablets dosage forms.
Deepa et al. [28] developed a method of analysis to determine gimepride
(GLM), pioglitazone hydrochloride, and metformin hydrochloride (MET)
in combined dosage forms using second-derivative spectrophotometry. The
combined preparations were quantified using the second-derivative
responses at 233.4 nm for gimepride, 265.4 nm for pioglitazone hydrochlo-
ride, and 252.6 nm for MET in spectra of their solution in methanol. The
calibration curves were linear in the concentration range 525 g/mL for
glimepiride, 525 g/mL for pioglitazone hydrochloride, and 212 g/
mL for metformin hydrochloride. The method was applied for estimation
of glimepiride, pioglitazone hydrochloride, and metformin hydrochloride
in combined tablet formulation.
Dhole et al. [29] described a UV spectrophotometric method for the
simultaneous determination of pioglitazone HCl, metformin HCl, and
glibenclamide in combined tablet dosage form using ethanol (95%) as sol-
vent. The wavelengths selected for the analysis were 237, 268, and 300 nm
for estimation of metformin HCl, pioglitazone HCl, and glibenclamide,
respectively. Beers law was obeyed in the concentration ranges of 3
30, 10100, and 110 g/mL for pioglitazone HCl, metformin HCl,
and glibenclamide, respectively. The mean percentage drug content for
Pioglitazone 401

pioglitazone HCl, metformin HCl, and glibenclamide were found to be

99.48%, 99.77%, and 99.35%, respectively. The method was found to
be suitable for the routine quality control analysis of pioglitazone HCl,
metformin HCl, and glibenclamide in pure and pharmaceutical dosage
forms.
Game [30] developed a spectrophotometric method for simultaneous
estimation of glimepiride and pioglitazone HCl in capsules by employing
first order derivative zero crossing method. The wavelengths selected for
quantitation were 230.0 nm (zero cross point of glimepiride) for
pioglitazone HCl and 250.0 nm (zero cross point of pioglitazone HCl)
for glimepiride. Linearity was maintained within a wide concentration range
from 4.0 to 30.0 g/mL for glimepiride and 6 to 30 g/mL for pioglitazone
HCl. The limit of detection and limit of quantification for glimepiride were
found to be 2.0 and 4.0 g/mL, respectively, and for pioglitazone HCl 4.0
and 6.0 g/mL, respectively. The method was applied in the analysis of
commercial capsules.
Havele et al. [31] developed a spectrophotometric method for simulta-
neous estimation of atorvastatin and pioglitazone in bulk and tablet. The
method showed maximum absorbance at 210 nm for atorvastatin while
showed maximum absorbance for pioglitazone at 225 nm. The overlain
spectra showed maximum absorbance at 242 nm. The method was applied
for simultaneous determination for both drugs in tablet dosage form.
Kishore et al. [32] developed spectrophotometric methods for the simul-
taneous estimation of pioglitazone and glimepiride. First method used was
the simultaneous equation method, in which two wavelengths (216 and
225 nm) were selected for the measurement of absorbance. Second method
was the absorption ratio method in which measurements are made based on
the absorptivity at the isosbestic point (228 nm) and absorption maxima of
pioglitazone (216 nm). The absorption maximum wavelengths of
pioglitazone and glimepiride were observed at 216 and 225 nm, respec-
tively, and the isosbestic point at 228 nm. Linearity ranges were 525 g/
mL for both drugs. The proposed methods were recommended for routine
analysis of pioglitazone and glimepiride as they are rapid, precise, accurate,
and reproducible.
Ali et al. [33] developed a UV spectrophotometric method for the quan-
titative estimation of pioglitazone in bulk and pharmaceutical dosage forms.
Pioglitazone hydrochloride has absorption maxima at 224.4 nm in ethanol
and obeyed Beers law in a concentration range of 525 g/mL.
402 A. Al-Majed et al.

Mohd et al. [34] developed another UV spectroscopic method for the

estimation of pioglitazone hydrochloride in bulk and tablet dosage form.
Pioglitazone hydrochloride shows maximum absorption at 269 nm with
molar absorptivity of 9.6013  104 L/mol cm. Beers law was obeyed in
the concentration range of 1070 g/mL. The proposed method was found
to be accurate and precise for estimation of pioglitazone hydrochloride in
bulk and tablet dosage form.
Patil Pallavi et al. [35] developed UV derivative spectrophotometric
methods for the simultaneous determination of glimepiride, metformin
HCL, and pioglitazone HCL in tablets. The first derivative UV spectropho-
tometric method was performed at 227, 233, and 265.5 nm for glimepiride,
metformin HCL, and pioglitazone HCL, respectively, in 0.1 N NaOH
solution and distilled water (50:50).
Patil et al. [36] developed two visible spectrophotometric methods (A
and B) for the quantitative estimation of pioglitazone, in bulk drug and phar-
maceutical dosage forms. Methods were based on the formation of pale yel-
low colored and green colored chromo gens, which were measured 267 and
297 nm, respectively. For the first method, UV spectrophotometry, stan-
dard solutions were measured at 267 nm. The first method was linear from
2.5 to 20 mg/mL. The second method was based on the formation of an ion
association complex with Methyl orange (MO) and Bromocresol Green
(BCG). The assay was found to be linear over the concentration range of
2.520 g/mL. The two methods have been successfully applied to the assay
of pioglitazone.
Rathod et al. [37] developed and validated simple spectrophotometric
method for simultaneous quantitation of metformine hydrochloride and
pioglitazone hydrochloride in tablet dosage form without previous sepa-
ration. In simultaneous equation method, metformine hydrochloride
and pioglitazone hydrochloride were quantified using their absorptivity
values at selected wavelengths, 233 and 265.5 nm, respectively. The simul-
taneous equation method permits simple, rapid, and direct determination
of metformine hydrochloride and pioglitazone hydrochloride in commer-
cially available tablet dosage form without previous separations and can
therefore be used for routine analysis of both drugs in quality control
laboratories.
Shakya et al. [38] developed and validated a UV spectrophotometric
method for the analysis of pioglitazone in tablets. The proposed method
was performed in phosphate buffer (pH 7.4). Beers law was valid in a con-
centration range of 1050 g/mL and UV detection was done at 238 nm.
Pioglitazone 403

The proposed method was applied to the determination of pioglitazone in

two pharmaceutical formulations.
Singhvi et al. [39] developed one simple, accurate, economical, and
reproducible UV spectrophotometric method for simultaneous estimation
of pioglitazone and glimepiride in combined tablet dosage form. The devel-
oped method employs multiwavelength spectroscopy using 280 and 238 nm
as two wavelengths for estimation of pioglitazone and glimepiride,
respectively.
Sujana et al. [40] developed difference spectrophotometric methods for
the estimation of pioglitazone and metformin in bulk drug and in pharma-
ceutical formulations. Difference spectrum obtained by keeping
pioglitazone and metformin separately in 0.1 M NaOH in the sample cell
and 0.1 M HCl as blank, showed characteristic peaks (max) at 228.1 nm
pioglitazone and 228.2 nm metformin and the characteristics peaks for phar-
maceutical formulations were also found. The proposed method can be used
for routine estimation of pioglitazone and metformin in pharmaceutical dos-
age form.
Sujana et al. [41] developed two new UV spectrophotometric methods
for simultaneous estimation of pioglitazone hydrochloride and metformin
hydrochloride in tablets. The first method was based on application of
Vierordts method which involves the formation and solving of simulta-
neous equations at 225 and 237 nm, as absorbance maxima of pioglitazone
hydrochloride and metformin hydrochloride, respectively. The second
method employed was absorption correction method which involves direct
estimation of pioglitazone hydrochloride at 267 nm, as at this wavelength
metformin hydrochloride has zero absorbance and shows no interference.
For estimation of metformin hydrochloride, corrected absorbance was cal-
culated at 237.0 nm due to the interference of pioglitazone hydrochloride at
this wavelength. Calibration curves were linear with a correlation coefficient
of 0.999 over the concentration ranges of 614 and 15 g/mL for both the
drugs. The proposed methods can be used in the quality control of pharma-
ceutical formulations and routine laboratory analysis.
Jani et al. [42] developed another analytical method for simultaneous esti-
mation of valsartan and pioglitazone hydrochloride. This method involves
solving of simultaneous equations based on the measurement of absorbance
at two wavelengths, 248 and 268 nm, from 0.1 N HCl and phosphate buffer
solutions. Both drugs obey the Beers law in the concentration ranges
employed for this method. The method can be used to estimate the amount
of valsartan and pioglitazone hydrochloride in pharmaceutical formulations.
404 A. Al-Majed et al.

Abdelmonem et al. [43] represented simple atomic absorption spectro-

scopic and spectrophotometric methods for determination of pioglitazone
hydrochloride and carvedilol based on formation of ion-pair associates
between drugs and inorganic complex, bismuth(III) tetraiodide (Method A)
and between drugs and organic acidic dyes, fast green and orange G (Method
B). Method A is based on formation of ion-pair associate between drugs and
bismuth (III) tetraiodide in acidic medium to form orange-red ion-pair asso-
ciates, which can be quantitatively determined by two different procedures.
The formed ion-pair associate is extracted by methylene chloride, dissolved
in acetone, and quantified spectrophotometrically at 490 nm. Method B is
based on formation of ion-pair associate between drugs and either fast green
dye or orange G dye in acidic medium to form ion-pair associates. The
formed ion-pair associate is extracted by methylene chloride and quantified
spectrophotometrically at 630 nm (for fast green dye method) or 498 nm
(for orange G dye method).
Okdeh et al. [44] developed simple extractive spectrophotometric
method for the rapid determination of pioglitazone in pure form and phar-
maceutical formulations. The method was based on the formation of binary
complex (ion-pair complex) between the Pioglitazone and Chromotrope
2R in an acidic buffer, giving purple color, and the absorbance of dic-
hloromethane extracted complex was measured at 514 nm. The complexa-
tion reaction was extremely rapid at room temperature and the absorption
values remains unchanged up to 24 h. Beers law was obeyed in the concen-
tration range of 1.065.0 g/mL, detection limit was 0.16 g/mL, and the
molar absorptivity coefficients were 9.934  103 L/mol/cm. Recoveries
were between 99.13% and 102.17%. Interferences of the other ingredients
and excipients were not observed.
Dubey et al. [45] developed a UV spectroscopy method for the estima-
tion of Pioglitazone hydrochloride tablet dosage form and validated by
ICH guidelines. The standard (10 g/mL) was scanned between 200 and
400 nm and maximum absorption was recorded at 231.5 nm. The assay
results were found to be 100.52%. The linearity range of 1565 g/mL
proved that it obeyed Beers Law, and the correlation coefficient (r2) was
found to be 0.9983 at 270 nm with an intercept of 0.0008 and a slope
of 0.0018 with RSD less than 2% complied ICH. The pH degradation
study of API was found to be less at pH 712. The force degradation
study of Pioglitazone were done on Stress degradation by hydrolysis
under alkaline condition by using 0.1 N NaOH and was found to be
5.76% for 60 min, 9.61% for 90 min. Stress degradation by hydrolysis under
Pioglitazone 405

acidic condition by using 3 N HCl and product degradation was found to

be 11.53% for 60 min and 21.15% for 90 min for API. Dry heat-induced
degradation was done by using 70C temperature and was found to
be 1.93% for API for 48 h. Oxidative degradation was done by using
hydrogen peroxide and product degradation was found to be 19.23% at
15 min. Photolytic degradation was found to be 9.61% for 3 h and
15.38% for 5 h for API.
Kashyap et al. [46] developed a UV spectroscopic method for the simul-
taneous estimation of Alogliptin and Pioglitazone bulk and pharmaceutical
dosage forms. First order derivative and dual wavelength methods were
developed and validated using solvent methanol. Both methods show line-
arity at 530 g/mL. The first order derivative spectra of each solution were
obtained. ZCP of Alogliptin was found to be 275.60 nm and ZCP of
Pioglitazone was found to be 268.20 nm. Pioglitazone was measured at
the zero crossing point (ZCP) of Alogliptin and Alogliptin. In dual wave-
length method, spectra two wavelengths 270.20 and 265 nm were selected
as 1 and 2 for the estimation of Alogliptin. Pioglitazone shows the same
absorbance at these wavelengths. Similarly, wavelengths 280 and 271 nm
were selected as 3 and 4 for estimation of Pioglitazone. Alogliptin shows
the same absorbance at these wavelengths.
Dubey et al. [47] developed a simple procedure for the estimation of
Pioglitazone by first order derivative spectroscopy. The method is based
upon determination of D1 value of Pioglitazone at 231.5 nm, in 0.1 N
NaOH. Pioglitazone at its max shows linearity in the concentration range
of 1565 g/mL.

5.2.1.2 Atomic Absorption Spectroscopic

Sarat et al. [48] developed a method of determination of Cobalt (Co) in the
Pioglitazone hydrochloride sample by the atomic absorption spectroscopy
(AAS). The linearity with a correlation coefficient value of 0.9993 and accu-
racy recoveries at LOQ are ranging from 93.2% to 105.2%. The limit of
detection (LOD) obtained under the optimum condition was 0.3 g/g
and relative standard deviation for six replicate determinations of 10 g/L
was 2.66%.
Abdelmonem et al. [43] represented simple atomic absorption spectro-
scopic and spectrophotometric methods for determination of pioglitazone
hydrochloride and carvedilol based on the formation of ion-pair associates
between drugs and inorganic complex, bismuth(III) tetraiodide (Method
A). Method A is based on the formation of ion-pair associate between drugs
406 A. Al-Majed et al.

and bismuth (III) tetraiodide in acidic medium to form orange-red ion-pair

associates, which can be quantitatively determined by two different proce-
dures. The formed ion-pair associate is extracted by methylene chloride, dis-
solved in acetone, dried, and then decomposed by hydrochloric acid, and
bismuth content is determined by direct atomic absorption spectrometric
technique.

5.2.1.3 Spectrofluorimetric Method

Alarfaj et al. [49] established a convenient and sensitive spectrofluorimetric
method for the determination of two antidiabetic drugs, ie, pioglitazone
HCl and glimepiride, in pharmaceutical formulations and biological fluids.
The method is based on the native fluorescence of the studied drugs in meth-
anol. The fluorescence intensity was measured in methanol at 512 and at
522 nm for pioglitazone HCl excitation and glimepiride, respectively.
The range of 0.0051.3 g/mL for pioglitazone HCl with lower limit of
detection (LOD) of 1.61  103 g/mL.

5.3 Electrochemical Method

5.3.1 Potentiometric Measurement
Saber et al. [50] developed simple poly(vinyl chloride) membrane sensors for
the determination of pioglitazone in biological samples (urine) and pharma-
ceutical preparations. Potentiometric measurements were based on
iodobismuthite-drug ion-pair as novel electroactive materials incorporating
a plasticized PVC membrane with o-nitrophenyl octyl ether or dioctyl
phthalate. Each sensor was conditioned for at least 2 days in 0.1 M drug solu-
tion before use. It exhibited fast and stable Nernstian response for
pioglitazone over the concentration range of 1.0  1071.0  102 M,
pH range of 3.07.0 pioglitazone sensors. Results with an average recovery
not more than 100.4% and a mean standard deviation less than 1.0% of the
nominal were obtained for the two sensors.
Mandil et al. [51] described a method for the determination of
pioglitazone HCl as antidiabetic drug in its pure form and pharmaceutical
formulations. The proposed methods depend on the polarographic activity
of pioglitazone HCl in BrittonRobinson buffer over the pH range 212
using direct current (DC) and differential pulse polarography (DPP), and
it showed well-defined two cathodic peaks with high selectivity. Its electro-
chemical behavior at a dropping mercury electrode (DME) and stating mer-
cury drop electrode (SMDE) has been investigated. Polarograms of the drug
at DME & SMDE in BR buffer at pH 6.0 exhibited two two-electron
Pioglitazone 407

irreversible cathodic peaks, the first peak (Ep1) is in the range of potential
at 0.05 to 0.10 V, while the second peak (Ep2) is in the potential ranges
at 0.975 to 1.10 V vs Ag/AgCl. The first and second peaks may be attrib-
uted to the reduction of oxy group (peak 1) and C]N group (peak 2),
respectively.
The diffusion currentconcentration relationship was found to be recti-
linear over the range 1.6224 and 1.628 g/mL for Ep1 and over the
ranges 1.6256 and 1.632 g/mL for Ep2 using DME & SMDE, respec-
tively, with limit of quantifying pioglitazone HCl as 1.6 g/mL, and relative
standard deviation (RSD)
4.0% and
4.3% for Ep1 and Ep2 using DME
and
3.6% and
3.8% for Ep1 and Ep2 using SMDE. The peaks were char-
acterized as being irreversible and diffusion-controlled although adsorption
phenomenon played a limited role in the electrode process.

5.3.2 Potentiometric Titration

Mostafa et al. [52] described the construction and electrochemical response
characteristics of poly (vinyl chloride) membrane sensors for determination
of pioglitazone HCl in which the authors used polyvinyl chloride (PVC)
membrane sensors. These membrane sensors incorporate ion association
complexes of pioglitazone cation and sodium tetraphenylborate (NaTPB)
(sensor 1) or phosphomolybdic acid (PMA) (sensor 2) or phosphotungstic
acid (PTA) (sensor 3) as electroactive materials. The sensors display a fast,
stable, and near-Nernstian response over a relative wide pioglitazone con-
centration range (1  102 to 106 M). The direct determination of 2.5
3900.0 g/mL of pioglitazone show an average recovery
a mean relative
standard deviation of 98.5
1.6, 99.0
1.5, and 98.4%
1.7% and at
100.0 g/mL for sensors 1, 2, and 3, respectively. These sensors were
applied for direct determination of pioglitazone in some pharmaceutical
preparations and have been used as indicator electrodes for potentiometric
titration.
El-Ghobashy et al. [53] applied Polyvinyl chloride (PVC) membrane
sensors for the determination of pioglitazone hydrochloride pioglitazone
and metformin hydrochloride (MET) by using the ion association com-
plexes between these drugs with either sodium tetraphenyl-borate (TPB)
or ammonium reineckate (RNC) counter ions. The performance character-
istics of the sensors evaluated according to IUPAC recommendations reveal
a fast, stable, and linear response over the concentration range 3.162  105
1  102 M for PIO and 1  1031  101 M for MET.
408 A. Al-Majed et al.

Faridbod et al. [54] selected pioglitazonetetraphenyl borate as a suitable

ion-pair reagent in making pioglitazone potentiometric PVC membrane
sensor. The proposed method showed a wide linear range of 105
102 mol/L and detection limit of 6.0  106 mol/L.
Badawy et al. [55] applied carbon paste and polyvinyl chloride as
membrane electrodes for the determination of anti-diabetic drugs for
type 2 diabetic patients. The authors used these electrodes for the
potentiometric determination of rosiglitazonne, pioglitazone, glime-
piride, and glyburide in their standard forms and also as pharmaceutical
preparation. The prepared ion-selective electrodes showed a Nernstian
response with the limit of detection amounting to 106 M in a pH range
of 35.

5.3.3 Voltammetric Method

Al-Arfaj et al. [56] used square-wave adsorptive cathodic stripping
voltammetry for the determination of pioglitazone HCl in Britton Robin-
son buffer of pH 5. The adsorptive cathodic peak was observed at 1.5 V vs
Ag/AgCl. Under optimal conditions, the peak current is proportional to the
concentration of pioglitazone HCl, and linear calibration graphs were
obtained within the concentration levels of 108 and 104 M following dif-
ferent accumulation time periods (0300 s). The detection limit is
8.08  109 M (3.17 ng/mL) using 300 s preconcentration time, whereas
the quantitative limit is 2.45  108 M (9.63 ng/mL).
Al-Arfaj et al. [57] developed a flow injection chemiluminescent (FI-CL)
method for the determination of pioglitazone HCl. It is based on the sen-
sitizing effect of the drug on the oxidation reaction of sulfite with cerium
(IV). The method permits the determination of 0.053.0 mg/mL of
pioglitazone HCl with correlation coefficient r 0.9999. The lower limit
of detection (LOD) is 0.01 mg/mL (S/N 2) and the lower limit of quan-
titation (LOQ) is 0.05 mg/mL.
Wang et al. [58] developed a method that uses electrochemical imped-
ance spectroscopy (EIS) to quantitatively determine pioglitazone, a
thiazolidine-2,4-diones derivative. The method uses a silver electrode.
An increase in the pioglitazone concentration results in an increase
in the Faradaic electron-transfer resistance (Ret) obtained from the EIS
measurements. Pioglitazone is quantified from the linear variation of
the sensor response (Ret) as a function of the pioglitazone concentration
in solution.
Pioglitazone 409

Wang et al. [59] developed a method that uses flow-through

voltammetric sensor to quantitatively determine thiazolidine-2,4-diones
(TZDs) derivatives which are pioglitazone, rosiglitazone, and troglitazone.
The method uses a gold electrode. The dynamic range for determining
thiazolidine-2,4-diones (TZDs) is extended by more than two orders of
magnitude. The quantification limits are 0.10, 23, 15, and 0.005 ng for
thiazolidine-2,4-dione, pioglitazone, rosiglitazone, and troglitazone,
respectively. The method can be applied to the quantitatively determining
pioglitazone and rosiglitazone in anti-diabetic drugs. Findings using HPLC
with a flow-through voltammetric detector and UV detector are
comparable.

5.4 Chromatography
5.4.1 Thin-Layer Chromatography
Kucher et al. [60] established a thin layer chromatography method for sep-
aration and quantitative analysis of pioglitazone hydrochloride tablet. Meth-
anol was selected as solvent for pioglitazone according to physicalchemical
properties. Behavior of pioglitazone was investigated in different chromato-
graphic conditions. Chromatographic plate Sorbfi l, Armsorb, and Merck
were used as stationary phase. General systems of TLC screening of acid
and neutral agents and others were used as mobile phase. When using the
mobile phase chloroformmethanol (90:10) RF value of pioglitazone was
0.82, 0.7, 0.79; chloroformacetone (80:20): 0.55, 0.42, 0.42; toluene
acetonemethanol-25% solution of ammonia (50:20:10:0.02): 0.57, 0.57,
0.57; and chloroformtolueneacetate acid conc.ethanol (4.5:4.5:1:1):
0.45, 0.45, 0.51, respectively. The most acceptable defined system is
chloroformtolueneacetic acid conc.ethanol (4.5:4.5:1:1). Detection of
spots (areas of absorbance) of substances on the chromatogram was carried
out in two ways: irradiation by UV light, and action by general and
specific detecting reagents. The most optimal reagent was Dragendorff spray
modified on Munje (limit of detection 0.01 mg).

5.4.2 Capillary Electrophoresis

Calixto et al. [61] established an alternative electrophoretic method for
determination Pioglitazone and its main metabolites in rat liver microsomal
fraction. The electrophoretic analyses were performed using an uncoated
fused-silica capillary of 50 m i.d., 48 cm in total length and 40 cm in effec-
tive length, and 50 mmol/L sodium phosphate buffer solution (pH 2.5).
All experiments were carried out under the normal mode. The capillary
410 A. Al-Majed et al.

temperature was set at 35C and a constant voltage of +30 kV was applied
during the analyses. Samples were introduced into the capillary by hydrody-
namic injection (50 mbar, 15 s) and detection was performed at 190 nm.
The sample preparation procedure, based on hollow-fiber liquid-phase
microextraction, was optimized using multifactorial experiments. Next,
the following optimal condition was established: sample agitation at
1500 rpm, extraction for 15 min, 0.01 mol/L hydrochloric acid as acceptor
phase, 1-octanol as organic phase, and donor phase pH adjustment to 6.0.
The method demonstrated LOQs of 200 ng/mL. Additionally, it was linear
over the concentration range of 20025,000 ng/mL for Pioglitazone and
2002000 ng/mL for the metabolites.
Yin et al. [62] established a capillary zone electrophoresis method for chi-
ral separation of pioglitazone hydrochloride. Methods by optimizing factors
which affect the chiral separation, the kinds and concentration of cyclodex-
trin, the pH value and concentration of buffer, the voltage and temperature,
and the optimum conditions for chiral separation were selected. The results
of the optimal separation were conditioned as a phosphate buffer 40 mmol/
L containing hydroxypropyl--cyclodextrin (6 mmol/L), detection wave-
length at 200 nm, voltage at 18 kV and separation temperature at 20C,
by which the separation of pioglitazone hydrochloride enantiomers was
achieved. Conclusion The established method is convenient, which can
be applied for chiral separation of pioglitazone hydrochloride enantiomers.

5.4.3 HPLC Methods

Tahmasebi et al. [63] evaluated the applicability of hollow fiber-liquid phase
microextraction (HF-LPME) for extraction and preconcentration of trace
amounts of pioglitazone as an anti-diabetic drug in biological fluids, prior
to the determination by HPLC. In this technique, the target drug was
extracted into di-n-hexyl ether immobilized in the wall pores of a porous
hollow fiber from 10 mL of the aqueous sample (source phase, SP) with
pH 8.0, and then back extracted into the receiving phase (RP) with pH
2.2 located in the lumen of the hollow fiber. The extraction occurred
due to a pH gradient between the two sides of the hollow fiber. After
extracting for a prescribed time, 24 L of the RP solution was taken back
into the syringe and injected directly into an HPLC instrument for quanti-
fication. The Taguchi orthogonal array (OAD) experimental design with an
OA16 (45) matrix was employed to optimize the HF-LPME conditions.
Under the optimum conditions (di-n-hexyl ether as membrane impregna-
tion solvent, pHs of the SP and RP equal to 8.0 and 2.2, respectively,
Pioglitazone 411

extraction time of 30 min, stirring speed of 500 rpm and 10% (w/v) NaCl
for adjusting the ionic strength), preconcentration factor of 180, linear
dynamic range (LDR) of 2.5250 g/L with good correlation of determi-
nation(r2 > 0.998) and limit of detection (LOD) of 1.0 g/L were obtained
for the target drug.
Ravikanth et al. [64] developed a rapid high-performance liquid chro-
matography with UVvisible (HPLC-UV) detection method for the deter-
mination of Pioglitazone in rat serum. Rosiglitazone was used as internal
standard. Pioglitazone and Rosiglitazone are extracted from serum using a
liquidliquid extraction procedure using ethyl acetate. Isocratic separation
of Pioglitazone and Rosiglitazone is carried out using a reversed-phase
phenomenex C18 (250 mm  4.6 mm, 5 m) column with mobile phase
consisting of methanol and 30 mM ammonium acetate buffer (pH adjusted
to 5 with ortho-phosphoric acid) in the ratio of 60:40 (v/v) and quantified
by UV detection at 269 nm. Analytical run time was less than 10 min. Mean
recovery was 97.12% for 0.110 g/mL concentrations. The assay exhibited
good linear relationship. Quantification limit was at 50 ng/mL of
Pioglitazone and accuracy and precision were over the concentration range
of 0.110 g/mL.
Lakshmi et al. [65] developed reverse-phase HPLC method for the deter-
mination of Pioglitazone and Glimepiride on a Shimadzu Class vp series
HPLC system with a phenomenex C18 column (150  4.6 mm, 5 m) using
a mobile phase mixture containing methanol and ammonium acetate buffer
(pH 3.5) in the ratio of 55:45 with the flow rate was 0.5 mL/min. The efflu-
ents were monitored at 252 nm and eluted at 5.63 min Pioglitazone and
7.18 min glimepiride. Calibration curve was plotted with a range from 25
to 25,000 ng/mL for Pioglitazone and 10 to 10,000 ng/mL for glimepiride.
The drugs were extracted from rat plasma by simple liquidliquid extraction
using diethyl ether as an extraction solvent.
Islambulchilar et al. [58] developed a simple and rapid HPLC method
with UV detection for the determination of pioglitazone in human plasma.
The method was based on protein precipitation using perchloric acid on an
ODS column. The mobile phase consisted of a mixture of phosphate buffer,
methanol, acetonitrile, and 12 M perchloric acid (54:33:12:1, v/v/v/v).
The UV detector was set at 269 nm. Under these conditions, the retention
time of pioglitazone was 5.2 min. The standard curve was linear over the
range of 502000 ng/mL pioglitazone in human plasma. The within-day
and between-day precision studies showed high reproducibility, with CV
less than 5. The LOQ was 44.2 ng/mL. The method has been applied to
412 A. Al-Majed et al.

a bioequivalence study after administration of pioglitazone as 30 mg tablets

to 12 healthy volunteers.
Arayne et al. [66] developed, validated, and applied a reversed-phase
high-performance liquid chromatographic (RP-HPLC) method for the
simultaneous determination of gliquidone, pioglitazone hydrochloride,
and verapamil in tablets and human serum. Chromatographic separation
was achieved on a C18 column (5 m, 25  0.46 cm) with a mobile phase
consisting of methanolwateracetonitrile (80:10:10, v/v/v) with a flow
rate of 0.7 mL/min and pH adjusted to 3.50 with phosphoric acid at
230 nm. Glibenclamide was used as internal standard. The experimentally
derived limit of detection and limit of quantitation were determined to
be 0.24, 0.93, 0.40, and 0.80, 3.11, 1.36 g/mL for gliquidone,
pioglitazone, and verapamil, respectively.
Saber [67] developed a rapid and accurate HPLC method for the deter-
mination of pioglitazone hydrochloride in tablets. Chromatographic analysis
was performed on a Nova-Pak C18 column (3.9 mm  150 mm, 5 m)
with a mixture of ammonium formate buffer adjusted with formic acid to
pH 3 and acetonitrile (75:25, v/v) as mobile phase, at flow rate of
1.0 mL/min, and UV detection at 225 nm. The determination was com-
pleted in less than 12 min. Linearity 0.5 g/mL, accuracy 99.14%,
and precision 0.6% were found to be acceptable over the range 0.5
20 g/mL.
Jedlicka et al. [68] developed a reversed-phase gradient HPLC method
for the evaluation of pioglitazone hydrochloride (PG-HCl) in tablets.
The limit of detection for PG-HCl was found to be 42 ng/mL. Analyses
were performed on a C18 column (Symmetry C18, 5 m, 2504.6 mm)
and mobile phase was a mixture of ammonium formate buffer adjusted with
formic acid to pH 4.1 and acetonitrile. Shortened purity method was used as
the assay method.
Sane et al. [69] developed a rapid HPLC method for simultaneous
determination of pioglitazone and glimepiride. Chromatographic separa-
tion of the two pharmaceuticals was performed on a Cosmosil C18 column
(150 mm  4.6 mm, 5 mm) with a 45:35:20 (v/v) mixture of 0.01 m
triammonium citrate (pH adjusted to 6.95 with orthophosphoric acid),
acetonitrile, and methanol as mobile phase, at a flow rate of 1.0 mL/
min, and detection at 228 nm. Separation was complete in less than
10 min. The method showed a linear response for concentrations over
the ranges of 2.5030.00 g/mL for pioglitazone and 0.1010.00 g/
mL for glimepiride.
Pioglitazone 413

Swapna et al. [70] developed an RP-HPLC method for the estimation of

Metformin HCl (MET) and Pioglitazone (PIO) in pure and in pharmaceu-
tical dosage forms. A BDS Hypersil C18 column (250  4.6 mm, 5 m) was
used with a mobile phase containing a mixture of Acetonitrile and Potassium
dihydrogen ortho phosphate buffer (pH 3) in the ratio of 50:50. The flow
rate was 1 mL/min and effluents were monitored at 238 nm and eluted at
2.81 min (MET) and 4.57 min (PIO). Calibration curve was plotted with
ranges of 40-240 g/mL for MET and 1272 g/mL for pioglitazone.
Karthik et al. [71] developed a reverse-phase isocratic HPLC method for
the separation and quantification of pioglitazone and glimepiride in bulk
drug and pharmaceutical dosage form. The quantification was carried out
using Inertsil ODS (250  4.6 mm, 5 m) column and mobile phase com-
prised of acetonitrile and ammonium acetate (pH 4.5, 20 mM) in proportion
of 60:40 (v/v). The flow rate was 1.0 mL/min and the effluent was moni-
tored at 230 nm. The retention time of pioglitazone and glimepiride were
7.0
0.1 and 10.2
0.1 min, respectively. Linearity of pioglitazone and
glimepiride were in the range of 2.0200.0 and 0.550 g/mL, respectively.
The percentage recoveries of both the drugs were 99.85% and 102.06% for
pioglitazone and glimepiride, respectively from the tablet formulation.
Jain et al. [72] developed an RP-HPLC method for the simultaneous
estimation of metformin hydrochloride (MET), pioglitazone hydrochloride
(PIO), and glimepiride (GLP) present in multicomponent dosage forms.
Chromatography is carried out isocratically at 25C
0.5C on an Inertsil
ODS-3 (C18) column (250  4.60 mm, 5 m) with a mobile phase com-
posed of methanolphosphate buffer (pH 4.3) in the ratio of 75:25 (v/v)
at a flow rate of 1 mL/min. Detection is carried out using a UV-PDA detec-
tor at 258 nm. The retention times for MET, PIO, and GLP are 2.66 + 0.5,
7.12 + 0.5, and 10.17 + 0.5 min, respectively. The linearity range and per-
centage recoveries for MET, PIO, and GLP are 105000, 10150, and
110 g/mL and 100.4%, 100.06%, and 100.2%, respectively.
Sarat et al. [73] developed and validated HPLC method for the analysis of
Sexaliptin and Pioglitazone. Chromatographic separation achieved
isocratically on a C18 column (Use Inertsil C18, 5 m, 150 mm  4.6 mm)
utilizing a mobile phase of acetonitrile/phosphate buffer (60:40, v/v, pH
7.0) at a flow rate of 0.8 mL/min with UV detection at 260 nm. Aceclofenac
was used as an internal standard. The retention time of Sexagliptin,
pioglitazone, and aceclofenac was 2.48, 4.45, and 6.34 min, respectively.
Kalyankar et al. [74] developed a reversed-phase liquid chromatographic
method for simultaneous determination of pioglitazone HCl in combination
414 A. Al-Majed et al.

with glimepiride. This method uses a Eurosphere-100 C18 (250 A

4.6 mm, 5 m) analytical column, a mobile phase of acetonitrile and buffer
containing 0.01 M potassium dihydrogen orthophosphate in the ratio 50:50
(v/v), and pH adjusted to 6.2 with orthophosphoric acid. The instrumental
settings are at the flow rate of 1.4 mL/min and a detector wavelength of
225 nm. The retention times for pioglitazone HCl and glimepiride are
4.34 and 6.33 min, respectively. The linearity range for pioglitazone HCl
and glimepiride were in the range of 0.92.4 and 0.120.32 g/mL,
respectively.
Havaldar et al. [75] developed a gradient RP-HPLC method and subse-
quently validated for the determination of glimipiride, rosiglitazone, and
pioglitazone hydrochloride. Separation was achieved with a nucleodur
C18 column having 250  4.6 mm i.d. with 5 m particle size and water
HPLC grade adjusted to pH 3.0 using diluted orthophosphoric acid and ace-
tonitrile (80:20, v/v) with gradient program as eluent at a constant flow rate
of 0.8 mL/min. UV detection was performed at 215 nm. The retention time
of glimipiride, rosiglitazone, and pioglitazone hydrochloride were about
17.9, 6.31, and 8.24 min, respectively.
Shankar et al. [76] developed two methods of analysis to determine
pioglitazone hydrochloride pioglitazone and metformin hydrochloride in
combined dosage forms using second-derivative spectrophotometry and
reversed-phase liquid chromatography (LC). In the LC method, analysis
was performed on a Hypersil ODS C18 column with 5 m particle size using
the mobile phase acetonitrilewateracetic acid (75:25:0.3), adjusted to pH
5.5 with liquor ammonia, at a flow rate of 0.5 mL/min. Measurement was
made at a wavelength of 230 nm. Both the drugs were well resolved on the
stationary phase, and the retention times were 8.5 min for pioglitazone and
16.0 min for metformin. The calibration curves were linear in the concen-
tration range of 420 g/mL for pioglitazone and metformin.
Nirupa et al. [77] developed a reverse-phase HPLC method for the
separation and estimation of three drugs glimepiride, pioglitazone, and
metformin in bulk drug mix and pharmaceutical dosage forms. The
estimation was carried out using Inertsil ODS-3V (250 mm  4.6 mm,
5 m) column; mobile phase consisting of acetonitrile, tetrahydrofuran,
and buffer at pH 5; the flow rate of 1.7 mL/min and UV detection at
228 nm. All the three drugs were properly resolved having run time of
5, 3.9, and 1.3 min for glimepiride, pioglitazone, and metformin, respec-
tively. The validated method applied to the commercially available
pharmaceutical dosage form.
Pioglitazone 415

Sakuntala et al. [78] developed an RP-HPLC method for the determi-

nation of Glimepiride (GLM) and Pioglitazone HCl (PIO) on shimadzu
HPLC systems with Inertsil ODS C18 column (150  4.6 mm, 5 m) and
using a mobile phase mixture containing mixed phosphate buffer and ace-
tonitrile in the ratio of 40:60. The flow rat e was 1.5 mL/min and the efflu-
ent was monitored at 225 nm. The retention time of Glimepiride and
Pioglitazone HCl were 3.06 and 1.97 min, respectively. Linearity of
Glimepiride and Pioglitazone HCl were in the range of 1.327.92 and
1060 g/mL. The percentage recoveries of both the drugs were 99.78%
and 100.10% for GLM and PIO, respectively from the tablet formulations.
The proposed method is suitable for simultaneous determination of
Glimepiride and Pioglitazone HCl for routine quality control of drugs in
bulk drug and formulation.
Havele et al. [79] developed and validated an RP-HPLC method for
simultaneous analysis of metformin hydrochloride, gliclazide, and
pioglitazone hydrochloride in a tablet dosage form. Chromatography was
performed on a 25 cm  4.6 mm i.d., 5 m particle, C18 column with
85:15 (v/v) methanol:20 mM potassium dihydrogen phosphate buffer as
mobile phase at a flow rate of 1.2 mL/min. UV detection at 227 nm; met-
formin hydrochloride, gliclazide, and pioglitazone hydrochloride were
eluted with retention times of 2.15, 3.787, and 4.57 min, respectively. Cal-
ibration plots were linear over the concentration ranges 50250 g/mL for
metformin hydrochloride, 3.015.0 g/mL for gliclazide, and 210 g/mL
for pioglitazone hydrochloride. Limits of detection were 0.20, 0.04, and
0.10 g/mL and limits of quantification were 0.75, 0.18, and 0.30 g/mL
for metformin hydrochloride, gliclazide, and pioglitazone hydrochloride,
respectively.
Souri et al. [80] developed a new, simple, and reproducible HPLC
method for the determination of pioglitazone in human plasma. After
liquidliquid extraction with diethyl ether, samples were quantitated on a
Nova-Pak C8 column using a mixture of acetonitrile140 mM K2HPO4
(40:60, v/v, pH 4.45) as mobile phase with UV detection at 269 nm.
The flow rate was set at 1.4 mL/min. Ethylparaben was used as internal stan-
dard and the total run time of analysis was approximately 7 min. The method
was linear over the range of 251500 ng/mL of pioglitazone in plasma
(r2 > 0.999). The within- and between-day precision values were in the
range of 2.46.8%. The limit of quantitation of the method was 25 ng/mL.
Radhakrishna et al. [81] developed HPLC and Micellar Electrokinetic
Chromatographic (MEKC) methods for the determination of pioglitazone,
416 A. Al-Majed et al.

a new englycemic antidiabetic agent. Pioglitazone and its unsaturated impu-

rity were separated by MEKC in less than 7 min using a 43 cm  50 m i.d.
uncoated fused-silica capillary with extended light path for better sensitivity
(25 kV at 30C) and a background electrolyte (BGE) consisting of 20% ace-
tonitrile (v/v) in 20 mM sodium borate buffer pH 9.3 containing 50 mM
sodium dodecyl sulfate (SDS). The HPLC method is capable of detecting
all process-related compounds, which may be present at trace levels in fin-
ished products.
Yamashita et al. [82] developed an HPLC method for the simultaneous
determination of pioglitazone and its metabolites (M-I to M-V) in human
serum and urine. The method for serum involved the solid-phase and
liquidliquid extraction. Urine with and without enzymatic hydrolysis using
-glucuronidase was treated with liquidliquid extraction. The compounds
in the extract were analyzed using HPLC with UV detection at 269 nm. The
detection limits of pioglitazone, M-I, M-II, M-III, M-IV, and M-V in
serum were 0.010.05 g/mL; those in urine were 0.10.5 g/mL; and
those in urine after enzymatic hydrolysis were 0.30.5 g/mL, respectively.
Sripalakit et al. [83] developed an analytical method based on HPLC-UV
detection (269 nm) for the determination of pioglitazone in human plasma.
Rosiglitazone was used as an internal standard. Chromatographic separation
was achieved with a reversed-phase Apollo C18 column and a mobile phase
of methanolacetonitrile mixed phosphate buffer (pH 2.6, 10 mM)
(40:12:48, v/v/v) with a flow rate of 1.2 mL/min. The calibration curve
was linear over the range of 502000 ng/mL and the lower limit of quan-
tification was 50 ng/mL.
Vinod et al. [84] developed a method for the simultaneous estimation of
Pioglitazone and glimepiride in a combined tablet dosage form by using RP-
HPLC method and UV spectrophotometric method. Both methods were
validated and compared for sensitivity and linearity. The RP-HPLC method
utilized Gliburide (Glibenclamide) as an internal standard and the mobile
phase composition was methanol and water that gave a retention time of
4.34 and 5.19 min for Glimepiride and Pioglitazone, respectively. The lin-
earity range was between 115 and 345 g/mL for glimepiride and
pioglitazone, respectively. The accuracy of the method was found to be
in the range of 98102%. Both these methods applied to pharmaceutical
dosage formulation and were validated according to ICH guidelines.
PremAnand et al. [85] developed and validated simple, rapid, fast, and
precise RP-HPLC method for the simultaneous estimation of Telmisartan
and Pioglitazone in tablet dosage form. The quantification was carried out
Pioglitazone 417

using Phenomenex C8 (250  4.6 mm, 5 m) column and mobile phase

comprised of acetonitrile and ammonium dihydrogen phosphate (pH 4.5,
20 mM) in proportion of 65:35 (v/v). The flow rate was 1.0 mL/min
and the effluent was monitored at 210 nm. The retention time of
Telmisartan and Pioglitazone were found to be 2.38 and 3.16 min, respec-
tively. Linearity of Telmisartan and Pioglitazone were in the range of 1050
and 7.537.5 g/mL, respectively. The percentage recoveries of both the
drugs were 99.85% and 102.06% for telmisartan and pioglitazone, respec-
tively from the tablet formulation. The proposed method is suitable for
simultaneous determination of Telmisartan and pioglitazone in pharmaceu-
tical dosage form and bulk drug.
Lakshmi et al. [86] developed an HPLC method and a UV derivative
spectrophotometric method for the simultaneous determination of metfor-
min (MFN), pioglitazone (PLZ) and glimepiride (GLM), in tablets. HPLC
was carried out by using the reversed-phase technique on an phenomenex
RP-18 column (150  4.6 mm, 5 m) with a mobile phase consisting of a
cetonitrile and phosphate buffer (pH 3) in the ratio of 65:35. The flow rate
was fixed at 0.5 mL/min and the drugs were monitored at 245 nm with UV
dual absorbance detector and the elution time was found less than 10 min,
indicating shorter analysis time.
Lakshmi et al. [87] developed a rapid RP-HPLC method for the
estimation of Metformin HCl and Pioglitazone in pure and in pharma-
ceutical dosage forms. A Gemini C18 column (150  4.6 mm, 5 m) was
used with a mobile phase containing a mixture of acetonitrile and ammo-
nium acetate buffer (pH 3) in the ratio of 42:58. The flow rate was
0.3 mL/min and effluents were monitored at 255 nm and eluted at
5.17 min Metformin and 8.1 min Pioglitazone. Calibration curve was plot-
ted with a range from 0.5 to 50 g/mL for metformin and 0.3 to 30 g/mL
for Pioglitazone.
Sharma et al. [88] reported a liquid chromatographic procedure that uses
micellar mobile phase containing only Tween-20 and n-butanol, for the
simultaneous determination of Atorvastatin Calcium (ATV) and
Pioglitazone (PIO) in tablet dosage form. The estimation was carried out
on Luna C18column (5 m  25 cm  4.6 mm) with a mixture of Tween-
20 and n-butanol phosphate buffer, pH 4.2 (50:25:25, v/v) at flow rate
of 1.5 mL/min at 25C temperature. Quantitation was achieved by
UV detection at 322 nm over lain spectra and the concentration range
was 5210 g/mL for both the drugs with mean recoveries of 99.01%
0.12 and 100.64% 0.20 for ATV and PIO, respectively.
418 A. Al-Majed et al.

Rashmithaa et al. [89] developed an RP-HPLC method for Pioglitazone

hydrochloride in the presence of its impurities and degradation products,
generated from forced degradation studies. The drug substance was sub-
jected to stress conditions of hydrolysis, oxidation, photolysis and thermal
degradation. The separation of the drug from the process-related impurities
and degradation products formed under stress conditions was achieved on an
Inertsil ODS-3V (150  4.6 mm), 5 m column. The gradient LC method
employs solution A and solution B as mobile phase. The solution A contains
phosphate buffer pH 3.1 and Solution B contains acetonitrile.
Madhukar et al. [90] developed an isocratic RP-HPLC method and sub-
sequently validated the determination of Pioglitazone Hydrochloride. Sep-
aration was achieved with a Symmetry-Extend-C18 HPLC column
(150 mm  4.6 mm). A mobile phase comprising 0.01 M buffer: Methanol
in the volume ratio of 40:60 was developed. The detection was carried out
using a UV detector set at a wavelength of 240 nm. The method was linear
over the concentration range of 1200 g/mL and can be used for quality
control assay of Pioglitazone Hydrochloride.
Abro et al. [91] developed a rapid and reliable analytical method based on
HPLC with UV detection (221 nm) for the determination of the
antihyperglycemic agent Pioglitazone in pharmaceutical formulations and
biological fluids (serum and urine) after clean up with solid-phase extraction.
Chromatographic separation was achieved with a Chromolith Perfor-
mance RP-18e (100  4.6 mm) column using mobile phase composition
of acetonitrile: mixed phosphate buffer (pH 2.5, 10 mM) (30:70, v/v) with
a flow rate of 2.0 mL/min. The total run time was only 2 min under opti-
mized conditions. The calibration curve was found to be linear in the range
of 110 g/mL with regression coefficient of 0.9996, and the lower limit of
detection was 72 ng/20 L.
Kumar et al. [92] developed and validated an HPLC-UV detection for
simultaneous determination of Pioglitazone and Clopidogrel. Separation
was performed on a C18 column by isocratic elution with a mobile phase
of methanol:acetonitrile:water (80:10:10) at pH 4.6. The UV detection
was set at 230 nm. The method proved to be specific, accurate, precise,
and linear over the concentration ranges of 20120 ppm for both
Pioglitazone and Clopidogrel with correlation coefficients always >0.999
for both drugs. The intra- and inter-day precision and accuracy were less
than 2 for both analytes.
Srinivasulu et al. [93] developed and validated specific and stability-indi-
cating reversed-phase gradient liquid chromatographic method for
Pioglitazone 419

determination of Pioglitazone Hydrochloride along with its impurities

in bulk samples. Drug substance was subjected to stress conditions of
hydrolysis (acid and base), oxidation, photolytic, humidity, and thermal
degradation as per International Conference on Harmonization (ICH) to
show the stability-indicating power of the method. Significant degrada-
tion was observed with alkali and hydrogen peroxide. The impurities
were characterized using spectral techniques like IR, 1H NMR, and
MS. Successful separation of impurities was achieved on C18 ODS
(150  4.6 mm) 3.5 m column using mobile phase consisting of Solvent
A: Ammonium acetate buffer and Acetonitrile in the ratio (57:43, v/v)
for 07 min and Solvent B: Ammonium acetate buffer and Acetonitrile
in the ratio (20:80, v/v) at a flow rate of 1.0 mL/min from 7 to 20 min
followed by Solvent A from 20 to 21 min. The retention times of impurity
A, impurity B, impurity C, and Pioglitazone were 3.44, 10.65, 17.95, and
8.32 min, respectively. The detection wavelength was set at 254 nm with
column temperature at 45C.
Pallapolu et al. [94] described a simple, economic, selective, accurate,
precise RP-HPLC method for the simultaneous estimation of metformin
and pioglitazone in pure and pharmaceutical dosage forms. Metformin
and pioglitazone were well separated using a Hypersil BDS C18 column
of dimension 250  4.6, 5 m and Mobile phase consisting of Phosphate
buffer:Methanol (Adjusted with Ortho phosphoric acid to pH 4.5) in the
ratio of 70:30 (v/v) at the flow rate 1 mL/min and detection was carried
out at 240 nm with PDA detector. The retention time for Metformin
and Pioglitazone were found to be 1.945 and 3.595 min, respectively.
Prava et al [95] developed and validated an RP-HPLC method for the
determination of process-related impurities in pioglitazone hydrochloride.
High-quality separation was achieved on a Luna C18 column (150 mm 
4.6 mm, 3 m) using gradient elution at a flow rate of 1 mL/min and a
column temperature of 45C. UV detection was performed at 254 nm.
The method gives satisfactory separation of impurities of pioglitazone
hydrochloride and so it is suitable for quantification of the process-related
impurities as well as for the assay of the active compound.
Madhukar et al. [96] developed a precise reverse-phase isocratic HPLC
method for the separation and quantification of pioglitazone and glimepiride
in bulk drug and pharmaceutical dosage form. The quantification was car-
ried out using X-Bridge ODS (150  4.6 mm, 5 m) column and mobile
phase comprised of Acetonitrile and Ammonium Acetate (pH 4.3,
20 mM) in proportion of 40:60 (v/v). The flow rate was 1.0 mL/min
420 A. Al-Majed et al.

and the effluent was monitored at 235 nm. The retention time of
Pioglitazone and Glimepiride were found to be 2.61 and 3.50 min, respec-
tively. Linearity of pioglitazone and glimepiride were in the range of 1.5
225 and 0.2030 g/mL, respectively. The percentage recoveries of both
the drugs were 98.95101.22% and 98.46100.98% for Pioglitazone and
Glimepiride, respectively, from the tablet formulation.
Shaik et al. [97] developed and validated an HPLC-UV method for the
determination of pioglitazone hydrochloride. It is an oral antidiabetic agent
belonging to the class of thiazolidinediones. Isocratic separation of
Pioglitazone is carried out using a reversed-phase Intersil ODS C18 column
(150 mm  4.6 mm, 5 m) with mobile phase consisting of Ammonium
acetate buffer with Acetonitrile and Glacial acetic acid in the ratio of
50:50:1 (v/v) and quantified by UV detection at 269 nm with flow rate
of 0.7 mL/min.
Najma et al. [98] achieved a method for the simultaneous quantification
of four NIDDM drugs (metformin, glimepride, glibenclamide, and
pioglitazone) on a Purospher Start C18 (5 m, 25  0.46 cm) and Supelco
C18 column in 2, 3, 7, 9 min, respectively. The optimized method involves
a C18 column thermostated at 30C, UV detection at 235 nm, at a flow rate
of 1 mL/min. Good separation of the analytes was achieved by gradient
HPLC-UV/visible detector in API, pharmaceutical dosages, and serum;
The mobile phase was a mixture of methanol:water (70:30, v/v) and the
pH of which was adjusted to 3.0 by phosphoric acid.
The method exhibited consistent, high-quality recoveries of the four
analytes which ranged from 93.8
2.1 to 99.8
1.5 (mean
RSD) with
a high precision for the drug and impurities. Validation under Food
and Drug Administration (FDA) guideline of the analytical parameters
includes: linearity (r2 > 0.9996), LLODs (0.315, 2.3, 0.2,0.1 ng/mL),
LLOQs (0.95, 0.7, 0.59,0.32 ng1), intra-day precision (0.001), and
inter-day precision 0.9 expressed as relative standard deviation (RSD),
and robustness parameters (less than 1.98%) with accuracies between
98% and 102%.
Sharma et al. [99] developed the method of RP-HPLC coupled with
a diode array detector (DAD) for the pharmacokinetic interaction study
of atorvastatin with pioglitazone and cholestyramine, respectively, in
Wistar rats. Atorvastatin and pioglitazone were resolved on a C18 column
with a mobile phase composed of 48% methanol, 19% acetonitrile, and
33% 10 mM ammonium formate (v/v/v; pH 3.5
0.3, by formic acid)
and a 260 nm detection wavelength on the diode array detector. The
Pioglitazone 421

method was validated according to international standards with good

reproducibility and linear response; mean (r) 0.9987 and 0.9972 to Ator-
vastatin and pioglitazone, respectively. The coefficients of variation of intra-
and inter-assay precision ranged between 4.958.12 and 7.299.67,
respectively.
Neelima et al. [100] developed a new stability-indicating RP-HPLC
method for estimation of Alogliptin and Pioglitazone in bulk and pharma-
ceutical dosage form. To optimize the mobile phase, various combinations
of buffer and organic solvents were used on Hypersil BDS C18 column.
Then the mobile phase containing a mixture of phosphate buffer:Acetoni-
trile in the ratio of 45%:55% (v/v) was selected at a flow rate of 1.0 mL/min
for developing the method and the peaks with good shape and resolution
were found resulting in short retention time, baseline stability, and mini-
mum noise. The retention times of Alogliptine and Pioglitazone were found
to be 3.42 and 5.24 min, respectively. Quantitative linearity was obeyed in
the concentration range of 31187 and 75450 g/mL of Alogliptin and
Pioglitazone, respectively. The limit of detection and limit of quantitation
were found to be 0.399 and 1.21 g/mL for Alogliptine and 0.516 and
1.565 g/mL Pioglitazone, respectively, which indicates the sensitivity of
the method.
Mohamed et al. [101] developed HPLC method for determination of
both metformin hydrochloride and pioglitazone hydrochloride in tablet
dosage form. The chromatographic separation was conducted on Shimadzu
(Prominence LC 20 UFLC XR) connected with PDA detector, using
mixed column ODS/Cyano; ACE (100  4.6 mm, 5 m). The mobile
phase was isocratic and consisted of Acetonitrile:Phosphate buffer in the
ratio of (50:50, v/v) (buffer was composed of 3.55 gm disodium hydrogen
phosphate per liter, adjusted by 85% phosphoric acid to pH 5) and was deliv-
ered to the system at a flow rate of 1.2 mL/min. An injection volume of
20 L was used for pioglitazone hydrochloride and 5 L for metformin
hydrochloride. The detection wavelength (max) was 235 nm for metformin
HCl and 266 nm for pioglitazone hydrochloride. All assays were performed
at ambient conditions. The calibration curve of metformin hydrochloride in
mobile phase was linear with correlation coefficient (r2) 0.99995; over a
concentration range of 30750 mg/L; with a retention time of 1.07 min.
While the calibration curve of pioglitazone HCl in mobile phase was
linear with correlation coefficient (r2) 0.99859; over a concentration
range of 125 mg/L; with a retention time of 1.85 min. The percentage
recoveries of metformin hydrochloride and pioglitazone hydrochloride
422 A. Al-Majed et al.

were 100.13% and 100.22%, respectively. The relative standard deviation

(RSD) was found to be <2.
Du et al. [102] developed and validated a selective chiral HPLC method to
separate and quantify the pioglitazone enantiomers in rat plasma. After extrac-
tion of the plasma samples with ethyl acetate, the separation of pioglitazone
enantiomers and internal standard (IS, dexamethasone acetate) was achieved
on a cellulose tris(3,5-dichlorophenylcarbamate) column known as Chiralpak
IC with a mobile phase of hexaneisopropanol (70:30, v/v) at a flow rate
of 1.0 mL/min. The UV detection wavelength was set at 225 nm. Baseline
separation of pioglitazone enantiomers and IS, free from endogenous inter-
ferences, was achieved in less than 25 min. Ratio of peak area of each enan-
tiomer to IS was used for quantification of plasma samples. Linear calibration
curves were obtained over the range of 0.2550 g/mL in plasma for both
enantiomers (r2 > 0.9990) with quantitation limit of 0.25 g/mL. The mean
extraction recoveries were 82.3791.38% for pioglitazone enantiomers and
95.76% for IS from rat plasma. The mean relative error (RE %) of accuracy
and the mean relative standard deviation (RSD %) of intra- and inter-day
precision for both enantiomers were <10%. The method was validated
with accuracy, precision, recovery, and stability and used to determine the
pharmacokinetics of pioglitazone enantiomers, after a single oral administra-
tion of racemic pioglitazone (30 mg/kg). The differences between the phar-
macokinetic parameters Cmax, AUC024, AUC01, CL/F of (+)-pioglitazone
and ()-pioglitazone were significant, suggesting that the disposition of
pioglitazone in rats may be enantio-selective. Moreover, the plasma levels
of (+)- and ()-pioglitazone in female rats were apparently higher than that
in male rats, respectively.
Malichetti et al. [103] developed a method for the determination of Met-
formin and Pioglitazone by using RP-HPLC in pharmaceutical dosage
form. Chromatographic separation was carried out by using mobile phase
0.02 M potassium dihydrogen ortho phosphate:acetonitrile (55:45, v/v,
pH 5.64 adjusted with Orthophosphoric acid) on Agilent Thermo Scientific
Hypersil, C18 column (250  4.6 mm, 5 m) at a flow rate 1.0 mL/min with
UV detection at 228 nm. The retention times for Metformin and
Pioglitazone were 2.579 and 5.633 min, respectively, and both drugs
showed good linearity in the range of 5002000 and 30120 g/mL. The
percentage recovery for Metformin and Pioglitazone was found between
99.48100.85% and 99.48100.89%, respectively, indicating the proposed
method was accurate and precise.
Sachin et al. [104] developed an isocratic RP-HPLC method and
validation for the determination of Pioglitazone and Glimepiride.
Pioglitazone 423

The separation was achieved using a Inertsil, Phenyl C18 column

(25 cm  4.6 mm i.d. and 5 m) with a mobile phaseComposition of
Buffer (1.15 g of diammonium hydrogen orthophosphate, 1.36 g of
potassium dihydrogen orthophosphate, and 1 mL of triethylamine in
1000 mL distilled water adjust pH 5.0 with orthophosphoric acid) and
Acetonitrile in the ratio of 50:50. Detection was carried out at
230.0 nm and the flow rate 1.0 mL/min. The method was capable of
resolving Pioglitazone and Glimepiride having resolution 6.76 between
Pioglitazone and Glimepiride.

5.4.4 HPLCTandem Mass Spectrometry Methods

Xue et al. [105] developed and validated a direct-injection HPLCtandem
mass spectrometry method (LCMS/MS) for the quantitation of
pioglitazone in human serum. After mixing the internal standard with a sam-
ple, a 10 L portion of the mixture was directly injected into a high-flow
LCMS/MS system, which included an extraction column, an analytical
column and a six-port switching valve. The online extraction was achieved
on an Oasis HLB column (1 mm  50 mm, 30 m) with a 100% aqueous
loading mobile phase containing 5 mM ammonium acetate (pH 4.0) at a
flow rate of 4 mL/min. The extracted analyte was eluted by a mobile phase
which contained 5 mM ammonium acetate and acetonitrile. The analytical
column was a Luna C18 column (4.6 mm  50 mm, 5 m). Detection was
achieved by positive ion electrospray tandem mass spectrometry. The lower
limit of quantitation of the method was 9 ng/mL. The standard curve,
which ranged from 9 to 1350 ng/mL, was fitted by a weighted (1/x2) qua-
dratic regression model. This method was used to analyze pioglitazone con-
centrations in human serum samples from a bioequivalence study of a
blinded Actos formulation (encapsulated 15 mg tablet) and an Actos
15 mg tablet. The blinded formulation was shown to be bioequivalent to
an Actos 15 mg tablet.
Surendra et al. [106] developed ultra-performance liquid chromatogra-
phy (UPLC) method and validated for the simultaneous determination of
Metformin and pioglitazone in bulk and pharmaceutical dosage forms.
The chromatographic separation of the drug components were achieved
on Waters Acquity BEH C18, 50  2.1 mm, 1.7 m UPLC column using
0.2% triethylamine in water:acetonitrile(70:30); pH adjusted with 3.8 with
o-phosphoric acid as mobile phase 0.5 mL/min and detection wavelength
was 243 nm. Within a short runtime of 5.0 min the linearity range of met-
formin and pioglitazone were found to be 300700 ppm and 1050 ppm
respectively, with correlation coefficient is 0.999.
424 A. Al-Majed et al.

Lin et al. [107] developed and validated a liquid chromatography

tandem mass spectrometry (LCMS/MS) method for the simultaneous
determination of pioglitazone (PIO) and its two metabolites: M-III
(keto-derivative) and M-IV (hydroxyl derivative) in human plasma. Human
plasma samples of 0.2 mL were extracted by a single step liquid/liquid
extraction procedure and analyzed using an HPLC electrospray tandem mass
spectrometer system. The compounds were eluted isocratically on a C18
column, ionized using a positive ion atmospheric pressure electrospray
ionization source and analyzed using multiple reaction monitoring mode.
The ion transitions monitored were m/z 357 ! 134 for PIO, m/z
371 ! 148 for M-III, m/z 373 ! 150 for M-IV, and m/z 413 ! 178 for
the internal standard. The chromatographic run time was 2.5 min per injec-
tion, with retention times of 1.45, 1.02, and 0.95 min for PIO, M-III, and
M-IV, respectively. The calibration curves of pioglitazone, M-III and M-IV
were well fit over the range of 0.52000 ng/mL by using a weighted (1/x2)
quadratic regression. The proposed method applied to sample analysis for
clinical studies.
Kumari et al. [108] developed and validated an LCMS/MS assay
method for simultaneous quantification of pioglitazone and candesartan in
human plasma. Irbesartan was used as an internal standard. The analytes were
extracted from human plasma samples by solid-phase extraction technique
using a Strata-X 33 mm polymeric sorbent. The reconstituted samples
were chromatographed on a C18 column by using a 80:20 (v/v) mixture
of acetonitrile and 0.1% formic acid as the mobile phase at a flow rate of
0.8 mL/min. The calibration curves obtained were linear over the concen-
tration range of 153000 ng/mL for pioglitazone and 5608 ng/mL for
candesartan. A run time of 2.7 min for each sample made it possible to
analyze more than 300 plasma samples per day. The proposed method
was found to be applicable to clinical studies.
Kawaguchi-Suzuki et al. [109] developed a simple and rapid HPLC
tandem mass spectrometry (LCMS/MS) method for the simultaneous
determination of pioglitazone and its active metabolites hydroxypiogli-
tazone and ketopioglitazone in human plasma. Samples were processed
by protein precipitation with acetonitrile and selective phospholipid deple-
tion in a 96-well plate format. The method used deuterated internal stan-
dards for each analyte. Chromatographic separation was achieved with
gradient elution on a Hypersil GOLD C18 column. The mass spectrometer
was operated in electrospray positive ion mode with detection by selected
reaction monitoring using the transitions m/z 357.1 > 134.0 for
Pioglitazone 425

pioglitazone, m/z 373.1 > 150.0 for hydroxypioglitazone, and m/z

371.0 > 148.0 for ketopioglitazone. A linear standard curve was established
for the range of 101800 ng/mL for all three analytes. Intra- and inter-run
precision and accuracy (relative error) were less than 15%, and the mean
extraction recoveries of all analytes were more than 87.8%. The validated
method is sensitive and selective and was successfully applied to analyze
clinical samples obtained from patients with nonalcoholic fatty liver disease
taking pioglitazone.
Tengli et al. [110] developed and validated an UPLCtandem mass
spectrometry method for simultaneous estimation and validation of tablet
dosage form containing glimepiride, metformin, and pioglitazone using
Tolzamide as an internal standard (IS). The chromatography separation
was achieved with Waters ACQUITY HSS C18 column, 1.8 m,
2.1  50 mm, with mobile phase containing acetonitrile (A) and 1% ammo-
nium acetate buffer (B) (pH 2.5 adjusted with trifluoro acetic acid) with
gradient mode Gradient mode [2 min: 20 A: 80% B, 24 min: 70% A:
30% B, 45 min: 80% A: 20% B, 810 min: 90% A: 10% B]. The flow rate
was 0.4 mL/min column maintained at 25C and the injection volume was
2 L. The selected chromatographic condition was found to effectively
separate glimepiride, metformin, and pioglitazone with retention time of
3.17, 0.425, 2.3 min, respectively. The proposed method was found to
be rectilinear over the range of 212, 5003000, and 1590 ng/mL for
glimepiride, metformin, and pioglitazone, respectively. The signal intensi-
ties obtained in both positive and negative ion mode for all drugs including
internal standard found to be much higher in positive ion mode (M+H)+
parent ions at m/z 491.11, m/z 129.87, m/z 357.04, and m/z 312.04,
respectively, in QUATTROZQ full scan mass spectra.
Gananadhamu et al. [111] developed an LCMS/MS-based method for
the simultaneous monitoring plasma levels of Sitagliptin and Pioglitazone for
applicability to pharmacokinetic studies. The method was based on HPLC
separation on the reversed-phase Phenomenex Synergy C18 column
(30 mm length, 4.6 mm internal diameter, and 4.0 m particle size) at a
temperature of 40C using a binary gradient mobile phase consisting of
methanol and 2 mM ammonium acetate buffer pH adjusted to 4.5 with
acetic acid, at a flow rate of 1 mL/min. Tolbutamide was used as an internal
standard. Detection of analytes was achieved with LCMS/MS system in
Multiple Reaction Monitoring (MRM) mode. The method was validated
over concentration range of 10.982091.77 ng/mL for SIT and
8.251571.63 ng/mL for PIO, and lower limit of quantification was
426 A. Al-Majed et al.

10.98 and 8.25 ng/mL for Sitagliptin and Pioglitazone, respectively. Recov-
eries from spiked controls were within acceptance criteria for all the analytes
and internal standard at all QC levels. Within-batch and between-batch
accuracy for Sitagliptin was found within 96.9100.3% and for Pioglitazone
was found within 100.0104.3%. Within-batch and between-batch preci-
sion for Sitagliptin was less than 3.1% CV (coefficient of variation) and
for Pioglitazone was less than 5.3% CV at all concentrations levels.
Jafari et al. [112] described a method based on ESI ion mobility spectrom-
etry as a detection technique after treatment with a molecularly imprinted
polymer for the analysis of pioglitazone. In addition to the molecularly
imprinted polymer separation methodology, the positive ion mobility spec-
trum and the reduced mobility values for pioglitazone are reported for the
first time. The method was exhaustively validated in terms of sensitivity,
imprinting factor, enrichment factor, and sorption capacity. A linear
dynamic range of 0.1020.00 g/mL and an RSD below 6% were obtained
for the analysis of this compound. The average recovery for the analysis of
spiked samples was calculated to be about 91%.

5.4.5 High-Performance Thin-Layer Chromatography Methods

Singh et al. [113] developed and validated a simple high-performance thin-
layer chromatographic (HPTLC) method for simultaneous determination of
pioglitazone and glimepiride in bulk and tablet dosage form. The method
employed TLC aluminum plates precoated with silica gel 60 F254 as the sta-
tionary phase. The mobile phase used was a mixture of Benzene:Ethyl ace-
tate:Diethyl ether 6:3:1 (v/v). The detection of spot was carried out at
254 nm. The calibration curve of pioglitazone was found to be linear
between responses of 6003600 ng/mL with regression coefficient 0.9984
and calibration curve of glimepiride was found to be linear between 200
and 1200 ng/mL for glimepiride with regression coefficient of 0.9991.
The limit of detection was 57.22 and 16.67 ng/mL and the quantification
limit was 190.73 and 55.58 ng/mL for pioglitazone and glimepiride, respec-
tively. The proposed method is applicable to routine analysis of Pioglitazone
in bulk and pharmaceutical formulations.
Sharma et al. [114] developed an HPTLC method for the simultaneous
estimation of Atorvastatin Calcium (ATV) and Pioglitazone (PIO) in tablet
dosage formulation. In this method, standard and sample solutions of Ator-
vastatin Calcium and Pioglitazone in tablet dosage form were separated on
the stationary phase used was precoated silica gel 60 F254. The mobile phase
used was a mixture of chloroform:methanol:toluene (6:3:4, v/v). The
Pioglitazone 427

detection of spot was carried out at 259 nm. The calibration curve was found
to be linear between 100 and 400 ng/spot for Atorvastatin Calcium and
Pioglitazone. The proposed method can be used to determine the drug
content of marketed formulations.
Kale et al. [115] developed and validated an HPTLC method for the
simultaneous determination of pioglitazone, metformin, and glimepiride
in multicomponent pharmaceutical preparations. Pioglitazone, metformin,
and glimepiride from the formulations were separated on silica gel 60 F254
HPTLC plates with acetonitrile, methanol, propyl alcohol, and ammonium
acetate solutions in the proportion of 7:2:1:1 (v/v) as mobile phase.
Densitometric quantification was performed at 240 nm. Well-resolved
bands were obtained with RF values 0.83, 0.21, and 0.89 for pioglitazone,
metformin, and glimepiride, respectively. The calibration curve was found
to be linear in the concentration range of 0.31.2, 1040, and 0.040.16 g
per band by area for pioglitazone, metformin, and glimepiride, respectively.
The method is selective and specific, with potential application in pharma-
ceutical analysis of these drugs in bulk and formulations.

6. STABILITY
Reddy et al. [116] developed and validated a stability indicating RP-
HPLC method for the determination of pioglitazone drug substance. Chro-
matographic separation was achieved on a Prontosil C8 SH colunm
(250  4.6 mm, 5 m), using a mobile phase consisting of 550 mL of pH
4.0 phosphate buffer, 300 mL acetonitrile, and 150 mL methanol at a flow
rate of 1.5 mL/min. The detection was made at 254 nm. The retention time
of pioglitazone peak was 5.9 min. The method was found linear over the
range of 50150%. The proposed method was validated as per the ICH
and USP guidelines.
Sharma et al. [117] developed an RP-HPLC method for the deter-
mination and stability indicating of pioglitazone hydrochloride in pure
and tablet forms. The recovery of the drug was calculated to be in the range
of 100.45100.53% from a mixture of degradation products. The method
was specific to drug and also selective to degradation products. The method
showed a linear response for concentrations in the range of 1065 g/mL
using 0.01 M potassium dihydrogen phosphate buffer (pH 3.5):methanol
[55:45] as the mobile phase with detection at 241.0 nm and a flow rate of
1.5 mL/min resulting in a retention time of 6.15 min.
428 A. Al-Majed et al.

Wanjari et al. [118] developed a stability indicating simple, rapid, and

precise RP-HPLC method for the quantitation of pioglitazone in tablet
on a Hypersil C8 column (250  4.6 mm) using a mobile phase consisting
of acetonitrile:0.15% (v/v) triethylamine (40:60, v/v) adjusted to pH 4.6
with orthophosphoric acid at a flow rate of 1.5 mL/min and detection at
220 nm. The retention time of pioglitazone has been found to be
7.6 min and recoveries were between 99% and 101%.
Sriram et al. [119] described an RP-HPLC method for the quantitation
of pioglitazone hydrochloride in the presence of its impurities. The active
pharmaceutical ingredient (API) of pioglitazone hydrochloride was sub-
jected to stress conditions viz., hydrolysis, oxidation, photolysis, and thermal
degradation. The drug was found to be sensitive under basic and oxidation
environment. Successful separation of the drug from the degradation prod-
ucts were achieved on Gemini C18 column (250  4.6 mm, 5 m) using a
mobile phase consisting of 50:50 (v/v) acetonitrile:0.05 M potassium
dihydrogen orthophosphate buffer of pH 3.0 and a flow rate of 1.0 mL/
min. Column oven temperature was kept at 40C and quantitation was
achieved with UV detection at 225 nm. The proposed method can be
employed as a stability-indicating method for studying stability of
pioglitazone hydrochloride.
Navaneethan et al. [120] developed and validated an RP-HPLC method
for the simultaneous estimation and stability indicating of pioglitazone,
glimepiride, and glimepiride impurities, ie, related compound B and related
compound C from combination drug product containing pioglitazone,
glimepiride, and metformin HCl. The chromatographic separation was
achieved on a cyano stationary phase (250  4.6 mm, 5.0 m particles) with
simple mobile phase combination delivered in gradient mode at a flow rate
of 0.8 mL/min at 230 nm.

7. CLINICAL APPLICATIONS
7.1 Pharmcodynamics
Clinical studies demonstrated that pioglitazone improves insulin sensitivity
in insulin resistant. Pioglitazone enhances cellular responsiveness to insulin,
increases insulin-dependent glucose disposal, and improves dysfunctional
glucose homeostasis. In patients with type II diabetes, the decreased insulin
resistance produced by pioglitazone has resulted in lowering plasma glucose
concentration, plasma insulin levels, and HbA1c values. Based on the results
from an open-label extension clinical, the glucose lowering effect of
Pioglitazone 429

pioglitazone appear to persist for at least 1 year. In controlled clinical trials,

pioglitazone in combination with sulfonylurea, metformin, or insulin had an
additive effect on glycemic control [121,122].
Patients with lipid abnormalities were included in clinical trials with
pioglitazone. Overall, patients treated with pioglitazone had mean decreases
in triglycerides, mean increases in HDL cholesterol, and no consistent mean
changes in LDL and total cholesterol [121,122].

7.2 Mechanism of Action

Insulin resistance may occur in type II diabetes mellitus in which the normal
levels of insulin do not activate the signal for glucose absorption.
Thiazolidinediones such as pioglitazone are potent synthetic peroxisome
proliferator-activated receptor (PPAR) ligands that have been shown effec-
tive in the treatment of diabetes [121]. Their glucose lowering effect is medi-
ated mainly through improved insulin sensitivity and therefore, facilitating
glucose uptake and utilization. Thiazolidinediones can enter the nucleus
where they bind to PPAR. PPAR is expressed most abundantly in adipose
tissue but is also found in pancreatic beta cells, vascular endothelium, and
macrophages [122]. Its discovery as the target for thiazolidinediones was
followed by large number of clinical trials of several agents [123].
Pioglitazone is a potent and highly selective agonist for PPAR. This recep-
tor regulates the expression of more than 100 genes, which cluster together
but are not identical. In addition, insulin secretory responses have been
reported to increase in subjects with impaired glucose tolerance and type
II diabetes, even after an improvement in insulin sensitivity. Other possible
mechanisms of thiazolidinediones may involve a reduction in adipocyte
cytokines and hormones that are involved in the pathogenesis of insulin
resistance [123].

7.3 Pharmacokinetics
Serum concentrations of total pioglitazone (pioglitazone plus its active
metabolites) remain elevated 24 h after once daily dosing. Steady-state
serum concentrations of both pioglitazone and total pioglitazone are
achieved within 7 days. At steady-state, two of the pharmacologically active
metabolites of pioglitazone, metabolites III (M-III) and IV (M-IV), reach
serum concentrations equal to or greater than pioglitazone. In both healthy
volunteers and patients with type II diabetes, pioglitazone comprises
approximately 3050% of the total pioglitazone serum peak concentrations
430 A. Al-Majed et al.

and 2025% of the total area under the serum concentrationtime curve
(AUC). Maximum serum concentration (Cmax), AUC, and trough
serum concentration for both pioglitazone and total pioglitazone increase
proportionally at doses of 15 mg and 30 mg/d. There is a slightly less than
proportional increase for pioglitazone and total pioglitazone at a dose of
60 mg/d [123].

7.4 Absorption
Following oral administration, in the fasting state, Eckland et al. [124] found
that the first measurable pioglitazone serum concentration was within
30 min, with peak concentrations observed within 2 h. Food slightly delays
the time to peak serum concentration to 34 h, but does not alter the extent
of absorption [125].

7.5 Distribution
The mean apparent volume of distribution (Vd/F) of pioglitazone following
single-dose administration is 0.63
0.41 (mean
SD) L/kg of body weight
[124]. Pioglitazone is extensively protein bound (>99%) in human serum,
principally to serum albumin. Pioglitazone also binds to other serum pro-
teins, but with lower affinity. Metabolites M-III and M-IV also are exten-
sively bound (>98%) to serum albumin [124].

7.6 Metabolism
Pioglitazone is extensively metabolized in the liver, with the majority
excreted as inactive metabolites in the feces. Pioglitazone undergoes signif-
icant hepatic metabolism by hydroxylation of aliphatic methylene groups to
form three metabolites M-I, M-II, and M-IV (hydroxy derivatives of
pioglitazone), by the oxidation of the methyl group to form an additional
metabolite (M-V), and by oxidation of metabolite M-IV to metabolite
M-VI [124]. Three of the metabolites, M-III, M-IV, and to a lesser extent
M-II (keto derivative of pioglitazone), were shown to have pharmacological
activity in diabetic animal models. In rats, the relative hypoglycaemic
potency (ED50) of these metabolites was 4060% of that of pioglitazone.
The potency of the triglyceride-lowering effect of M-II is nearly twice that
of the parent compound, while the potency of metabolites M-III and M-IV
is slightly less than that of pioglitazone [124]. The major active metabolites
M-III and M-IV have considerably longer terminal half-lives than the parent
compound (approximately 2628 h) [124]. Multiple CYP isoforms were
Pioglitazone 431

reported to contribute to pioglitazone metabolism with CYP2C8 and

CYP3A4 as the major pioglitazone metabolizing enzymes [124].

7.7 Excretion
Following oral administration, approximately 1530% of the pioglitazone
dose is recovered in the urine. Renal elimination of pioglitazone is negligi-
ble, and the drug is excreted primarily as metabolites and their conjugates. It
is presumed that most of the oral dose is excreted into the bile either
unchanged or as metabolites and eliminated in the feces [124].

7.8 Elimination Half-Life

The mean serum half-life (t1/2) of pioglitazone and its metabolites (M-III
and M-IV) range from 3 to 7 h and 16 to 24 h, respectively. Pioglitazone
apparent clearance, CL/F, has been calculated to be 57 L/h [124,126].

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 CHAPTER TWO

Calcium Carbonate
M.M.H. Al Omari*, I.S. Rashid*, N.A. Qinna, A.M. Jaber{,
A.A. Badwan*
*The Jordanian Pharmaceutical Manufacturing Co., Amman, Jordan

Petra University, Amman, Jordan

{
Philadelphia University, Amman, Jordan

Contents
1. Description 32
1.1 Nomenclature 32
1.2 Formulae 33
1.3 Elemental Analysis 33
1.4 Appearance 34
2. Methods of Preparation 34
2.1 Existence in Nature 34
2.2 Preparation of Crystalline Form 38
2.3 Preparation of Amorphous Form 46
2.4 Factors Affecting Preparation 47
2.5 Inhibitors of Precipitation 53
3. Physical Characteristics 54
3.1 Ionization Constant 54
3.2 Solubility Characteristics 54
3.3 Partition Coefficient 56
3.4 Optical Property 56
3.5 Polymorphism 56
3.6 Particle Morphology 57
3.7 Hygroscopicity 59
3.8 Molecular Modeling 59
3.9 Crystallographic Properties 61
3.10 Thermal Analysis 68
3.11 Spectroscopy 72
4. Methods of Analysis 79
4.1 Compendial Methods 79
4.2 Titrimetric Methods 87
4.3 Gravimetric Method 95
4.4 Spectroscopic Methods 95
4.5 Electrochemical Methods 99
4.6 Calcimetric Method 102
4.7 Chromatographic Methods 102

Profiles of Drug Substances, Excipients, and Related Methodology, Volume 41 # 2016 Elsevier Inc. 31
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/bs.podrm.2015.11.003
32 M.M.H. Al Omari et al.

5. Stability 104
5.1 Crystal Phase Transformation 104
5.2 Solid-State Stability 105
5.3 Stability in Solution 106
5.4 Interaction with Complexing Agents 106
6. Uses, Applications, and Pertinent History 107
7. Pharmacology 108
7.1 Pharmacokinetics 108
7.2 Mechanism of Action 109
7.3 Pharmacodynamics 110
7.4 Toxicities 110
7.5 Drug Interactions 111
References 112

1. DESCRIPTION
1.1 Nomenclature
1.1.1 Systematic Chemical Names
Calcium carbonate [1,2].
Carbonic acid calcium salt (1:1) [1].

1.1.2 Nonproprietary Names

Recommended international nonproprietary name: Calcium carbonate
[3].
Synonyms: E 170, calcite, aragonite, vaterite, chalk, CI pigment white 18
[4], drop chalk, prepared chalk, whiting, English white, Paris white [5].

1.1.3 Proprietary Names

Caltrate (Pfizer Consumer Healthcare), Maalox Quick Dissolve (Novartis
Consumer Healthcare), Maalox Regular Strength (Novartis Consumer
Healthcare), Os-Cal (Glaxo SmithKline), Alka-Seltzer (Bayer), Alcalak
(Medique Products), Oyster Shell Calcium (Swanson Health Products),
Oysco (Rugby), Cal-Gest (Rugby), Icar Prenatal Chewable Calcium (Haw-
thorn Pharmaceuticals), Oyster Shell Calcium (Swanson Health Products),
Childrens Pepto (Procter & Gamble), Rolaids Soft Chew (Johnson & John-
son), Adcal 1500 Chewable (Biokirch), Ostocal (Hikma Pharm.) [6], Cal-
cichew (Takeda), Calcichew 500 mg purutabletti (Takeda), Calcichew
Spearmint 500 mg purutabletti (Takeda), Calcimagon 500 mg (Takeda),
Calcioral (Takeda), Mastical (Takeda), Calcitugg (Takeda) [3], Boots
Calcium Carbonate 33

Indigestion Relief (The Boots Company Plc), Cacit (Warner Chilcott UK

Ltd), Remegel (SSL International Plc), Setlers Antacid (Thornton & Ross
Ltd), Tums Assorted Fruit Antacid (GlaxoSmithKline Consumer
Healthcare) [7], Calcidia (Bayer Healthcare) [5], Adcal 500 (JPM), Oystercal
(United Pharma) [8].

1.2 Formulae
1.2.1 Empirical Formula, Molecular Weight, Identification Numbers
Information of empirical formula, molecular weight, and different identifi-
cation numbers of CaCO3 are listed in Table 1 [4].

1.2.2 Structural Formula

The chemical structure of CaCO3 is shown in Fig. 1.

1.3 Elemental Analysis

The theoretical values of elemental compositions of CaCO3 are listed in
Table 2 [5].

Table 1 The Empirical Formula, Molecular Weight, and Identifications Numbers of

CaCO3
Empirical Molecular
Name Formula Weight CAS No. CI No. EEC No. INS No.
Calcium CaCO3 100.09 [471-34-1] [77220] [207-439-9] 170(i)
carbonate

O
Ca2+

O O

Figure 1 Chemical structure of CaCO3.

Table 2 The Theoretical Elemental Compositions of CaCO3

%
Compound Molecular Formula C Ca O
Calcium carbonate CaCO3 12.00 40.04 47.96
34 M.M.H. Al Omari et al.

1.4 Appearance
According to the United States Pharmacopeia (USP), CaCO3 is fine, white,
odorless, tasteless, and microcrystalline powder [9], while it is described as a
white or almost white powder in the European Pharmacopeia (Eur. Ph.) [2].

2. METHODS OF PREPARATION
2.1 Existence in Nature
CaCO3 is one of the most abundant materials found in earths crust and
forms the rock types like limestone and chalk [10]. Moreover, it is the most
abundant chemical sediment in modern and most ancient oceans, making up
roughly 10% of sediments [11]. CaCO3 can be a dominant sedimentary con-
stituent in virtually any environment, at any latitude, and in any depth of
water. However, it is most prevalent in warm, tropical, and subtropical seas
where the organisms that produce carbonate sediments can thrive [12]. On
the other hand, carbonates are forming extensively in many regions in the
western margins of large oceans, in both the southern and northern hemi-
spheres, at seawater temperatures ranging from about
2 to 40C [13,14].
Nearly all the CaCO3 that makes up carbonate platforms is derived from
marine organisms. CaCO3 is also an important component in biological sys-
tems, such as shells of marine organisms, pearls, and egg shells [15]. Some of
these systems, eg, Oyster shells, have enjoyed recent recognition as a source
of dietary Ca, but are also a practical industrial source [16]. While not prac-
tical as an industrial source, dark green vegetables such as Broccoli and Kale
contain dietarily significant amounts of CaCO3 [17].
Carbonates are largely made up of skeletal remains and other biological
constituents that include fecal pellets, lime mud (skeletal), and microbially
mediated cements and lime muds. Chemical constituents, including coated
grains such as ooids and pisoids, cements, and lime mud, are common in
carbonates but are absent in most siliciclastics. Clastic grains exist in carbon-
ates, as they do in siliciclastics. In carbonates, however, these grains are
mainly clasts of intraformational, lithified sediment (intraclasts) or of
reworked, older rock (lithoclasts). Carbonates have four main components:
(1) grains, (2) matrix, (3) cement, and (4) pores.
There are many classification schemes for carbonate rocks [1823].
Classifications for detrital carbonates were developed by Folk [18,19] and
Dunham [20]. Classifications for reef rocks were developed by Embry
and Klovan [21] and Riding [22]. A scheme to include depositional,
Calcium Carbonate 35

Figure 2 Dunham's classification of carbonate rocks.

diagenetic, and biological aspects of carbonates in one classification system

was proposed by Wright [23]. Fig. 2 represents Dunhams classification,
where it includes detrital carbonates as mudstones through grainstones, bio-
genic (reef ) carbonates as boundstones, and diagenetically altered carbonates
as crystalline carbonate [20].
Furthermore, there is a genetic classification of porosity linked to the com-
plete geological history of reservoir rocks as an alternative. It is based on the
idea that there are three end-member pore types in carbonate reservoirs:
depositional, diagenetic, and fracture pores. These different processes impart
distinctive characteristics to both rock matrix and pores. Because the distinc-
tive characteristics were imparted to pores and rocks at the same time and by
the same processes, key rock properties may act as markers or proxies for
pore types that can be identified and traced at stratigraphic scale. To the extent
that the proxies are identifiable and mappable, so will be the accompanying
pore types and, of capital importance, their petrophysical attributes [24].
In nature, exposure of CaCO3 (calcite or aragonite) to water containing
high Mg/Ca ratio and high absolute concentration of Mg may produce
dolomite (CaMg(CO3)2) [11]. Also the use of calcite and aragonite for shell
production and other metabolic needs in animal decreases the carbonate ions
in ocean leading to increase in its acidity [25]. Caldeira and Wickett showed
through their geochemical model that with no reduction in fossil fuel
emission of CO2 into the atmosphere, there may be a rapid decline in
pH in surface ocean water over the next 100 years of as much as 0.4 units
(a doubling of H+ ion concentration) [26]. Andersen et al. studied the effects
of ocean acidification on embryos and unfed larvae of the great scallop
36 M.M.H. Al Omari et al.

(Pecten maximus Lamarck), at various pCO2 until 7 days after fertilization.

They showed that ocean acidification affected both survival and shell growth
negatively [27]. Such high sensitivity of bivalve larvae may be connected to
the carbonate composition of the larval shell as the larval shell contains
mainly aragonite, the most soluble form of crystalline CaCO3 [2830].
The predicted undersaturation of aragonite in the worlds oceans may have
a great negative impact on these calcifying organisms [31,32].
Furthermore, it was assumed that diagenesis [33] alters aragonite-rich
sediments into calcitic limestones and elevated CO2 level initiates aragonite
dissolution and reprecipitation of the CaCO3 as calcite cement [34]. Diagen-
esis of carbonates in the deep sea almost exclusively involves the dissolution
of CaCO3, where only 2030% of the flux to the seafloor is preserved [35].
The major factors influencing CaCO3 preservation can be summarized as
follows: weight percent of CaCO3 in deep-sea sediments, the input of ter-
rigenous material which leads to more rapid burial and to better preservation
of carbonates [36], higher productivity in overlying water, often in upwell-
ing areas, can also lead to more rapid carbonate deposition and to higher car-
bonate concentrations in sediments, and the relative CaCO3 to organic
carbon rain rate ratio.
Generally, oxidation of sedimentary organic matter can decrease the sat-
uration of pore water with respect to CaCO3 by increasing pCO2 [37]. The
approach that has generally been used is to estimate carbonate dissolution
rates via diagenetic models [38], incorporating organic matter oxidation
by oxygen (assuming a C:O ratio) that produces undersaturated conditions
considered. A relationship is then established between the calcite dissolution
rate and the pore-water saturation state. The general kinetic equation for
calcite introduced by Morse and Berner for calcite dissolution in seawater
has been most commonly used [39]. Many investigators, eg, Morse [40],
Keir [41], and Hales and Emerson [42], have modeled rates of carbonate dis-
solution from deep-sea sediment. It is clear that diagenetic factors that con-
trol the partial or complete dissolution of deep-sea carbonates are dependent
on scale [43]. At the scale of the major ocean basins, it is clear that the pri-
mary variable is the saturation state of water at a given depth. This is largely
controlled by its potential pCO2.
Over 60 different minerals have been identified in organisms [44]. Living
organisms like photosynthetic microalgae can induce the precipitation
of CaCO3 through biomineralization. CaCO3 precipitation in the ocean
is almost exclusively due to an enzymatically controlled intracellular biomin-
eralization process [45], and even the carbonate rocks and caves are mainly
Calcium Carbonate 37

formed biogenically, by microorganisms and plants [46]. The level of bicar-

bonate in solution is crucially important for the rate of carbonate mineral
precipitation [47]. Bicarbonate is reversibly formed by hydration of CO2,
a reaction which represents the rate-limiting step in the process of CaCO3
precipitation in the presence of Ca cations. The conversion of CO2 and
water to HCO3 can be drastically upregulated by the carbonic anhydrase
(CA), a Zn-containing enzyme (EC 4.2.1.1). The cofactor in this enzyme
is involved in the attack of bound OH to CO2 molecule that is loosely
bound in the active center of the enzyme, a reaction during which the coor-
dinated HCO3 ion is displaced from the metal ion by water [48]. The CA,
one of the fastest catalyzing enzymes for the fixation of CO2, allows an
increased deposition of CaCO3 in this biomineralization process [49].
Fig. 3 shows the pathway of CaCO3 formation for CA-mediated enzy-
matic synthesis of calcitic spicular elements in calcareous sponges as proposed

Figure 3 The light microscopic images show (A) the initial calcitic pat-like deposits, (B)
the maturated calcitic crystals, (C) the organized piling up of the calcitic crystals on S.
raphanus spicules (Sr-s), and (D) the tight interaction of three cells within the tissue of S.
raphanus; into which the gaps between the cells the spicules are formed.
38 M.M.H. Al Omari et al.

by Muller et al. [50]. It is outlined that the CA reversibly interconverts CO2

and water into HCO3, a reaction which is followed by CaCO3 mineraliza-
tion in the presence of Ca. Finally, amorphous CaCO3 is synthesized and is
transformed to the different crystal forms of calcite.
Gebauer et al. described stable prenucleation CaCO3 clusters [51], and
Pouget et al. revealed the initial stages of template-controlled CaCO3
formation by cryo-TEM [52]. Jacob et al. found that membrane-coated
amorphous CaCO3 was deposited on the organic layer in pearls and
suggested a new growth mechanism beginning from amorphous CaCO3
[53]. All these investigations support the proposition that amorphous
CaCO3 is initially formed as a precursor and then transforms into calcite
or aragonite [54]. Finally, it is sketched that the growing spicules are formed
by a layer-by-layer apposition of calcitic bricks into a preformed collagenous
mold [50].

2.2 Preparation of Crystalline Form

CaCO3, upon precipitation, is capable of forming an amorphous phase com-
prising colloidal systems of amorphous primary particles. The colloidal sta-
bility of these systems is not sufficient to prevent aggregation. Due to the
high number density of primary particles and the high ionic strength of
the solution, the aggregation process leads to a gelation of the reaction mix-
tures. The gel is bound by van der Waals forces only. Therefore, the gel is
colloidally less stable and undergoes a quick morphological collapse. The
recrystallization to vaterite, aragonite, or calcite takes place simultaneously
with the dissolution of the gel (Fig. 4) [55].
The sequence of reactants directly affects the morphological structure of
CaCO3 upon precipitation. For example, if Na2CO3 solution is first intro-
duced into the reactor and then CaCl2 solution is fed to this solution under
standard conditions, the product consists almost entirely of vaterite. The
increase in particle size is due to spherulitic growth mechanism of vaterite
caused by multiple passages of the particle through the region of maximum
supersaturation at the feed inlet [56]. For the reversed sequence case (ie, the
CaCl2 solution is first introduced into the reactor and then Na2CO3 is fed to
this solution) a larger percentage of calcite (c.10% is formed). The calcite
cubes are aggregated to form larger irregular particles and are partly over-
grown by clusters of vaterite. This may be due to the same mechanism as
in the standard experiment (first case), with the vaterite nucleating on the
calcite surface. When MgCl2 is introduced into CaCl2 solution to avoid
Calcium Carbonate 39

Educt A Mixing Educt B

Supersaturation

Nucleation

CaCO3: Growth
Van der Waals
bonds Primary
particles
Gelation
Aggregation
Reorganization

Fragmentation

Recrystallization

Growth

Vaterite Aragonite Calcite

Figure 4 The formation mechanisms of CaCO3 under high supersaturation conditions.

premature precipitation, some calcites are formed but all the particles are
mainly vaterite. The vaterite particles of various shapes are aggregated and
of various shapes. When KOH is introduced into Na2CO3 solution for
the same purpose, large calcite particles and smaller aragonite needles, which
are attached to the calcite cubes, are formed [55].
The kinetics of CaCO3 precipitation is a significant tool to understand
because they relate the CaCO3 saturation, pH, and the alkalinity to the
CaCO3 precipitation potential. All of the aforementioned parameters rep-
resent key aspects which specifically affect the morphology of CaCO3 crys-
tals [57,58]. Colfen and Qi noted that the HCO3/CO3 rate, coupled with a
change in the solution supersaturation, leads to a change in the nucleation
rate of CaCO3 [59].
Generally, the precipitation of CaCO3 is accompanied by a drop in pH
and a reduction in hardness and in total alkalinity for each mole of precip-
itated CaCO3. The CaCO3 precipitation potential increases with saturation
index and buffer intensity. Buffer intensity in turn is a function of pH and
total alkalinity. Because buffer intensity decreases with increasing pH, the
CaCO3 precipitation potential also decreases as pH is increased [60]. The
detailed kinetics of such precipitation dependence is described by
Wojtowicz [60] and Plummer and Busenberg [61].
40 M.M.H. Al Omari et al.

It has been found that supersaturation is critical in determining the pre-

cipitation of CaCO3 [62]. At high degrees of supersaturation, where spon-
taneous precipitation occurs, vaterite forms predominantly even at 25C.
On the other hand, organic matrices are considered to play a principal role
in biomineralization.
It is known that many fresh water algae build calcite crystals when they
live in an environment supersaturated with Ca [63,64]. This is a result of the
influence of the algaes metabolism on their aqueous environment through
photosynthesis, whereby the algae assimilate CO2 and/or HCO3, and
release OH [65,66]. This leads to an increase in the pH and the concentra-
tion of carbonate within the solution [67,68]. With increasing pH, more
functional groups, such as COOH or NH groups, are exposed, which
increases the biosorption of metal ions on the negatively charged cell walls.
In the first step, Ca binds to the cell wall, followed by accumulation of car-
bonate anions and extracellular precipitation of CaCO3 [65].

2.2.1 Calcium Acetate and Carbon Dioxide

The hierarchical monodispersed aragonite microspheres can be prepared by
carbonating Ca(CH3COO)2 aqueous solution with CO2 gas at a high pres-
sure of 40 bar and a high temperature of 80C after 60 min of reaction [69].
Ca(CH3COO)2 aqueous solution is introduced into a reactor under atmo-
spheric pressure. Pure CO2 gas is then continuously introduced until it
reaches the setting pressure. During the experiments, the CO2 pressure in
the reactor is kept constant by continuous supplying of CO2 from the cyl-
inder using a pressure controller. After a given time, the stirring is stopped.
The obtained precipitates are collected from the man-made filter and
washed several times with water as well as anhydrous ethanol then air dried.
The reaction between CO2 and Ca(CH3COO)2 in an aqueous solution
was first proposed by Kakizawa et al. [70] for the fixation of the greenhouse
gas CO2 into solid CaCO3. Generally, CaCO3 can be dissolved in an
CH3COOH solution at atmosphere conditions [71,72]. Therefore,
CH3COOH is replaced by H2CO3 in this carbonation crystallization pro-
cess, resulting in precipitating CaCO3 particles. Moreover, it is reported that
the reaction between CO2 and Ca(CH3COO)2 is endothermic and occurs
spontaneously at temperatures above 45C at atmospheric pressure [73].

2.2.2 Calcium Hydroxide and Carbon Dioxide

CaCO3 can be precipitated by mixing Ca(OH)2 and H2CO3 solutions, the
latter solution being prepared by bubbling a high-grade CO2 stream into
Calcium Carbonate 41

water until saturation was achieved [74]. Using the aforementioned princi-
ple, fine particles of calcite with controlled morphology can be synthesized
by using hydrothermal carbonation of Ca(OH)2 at high CO2 pressure (initial
pCO2 55 bar) and at moderate and high temperatures (30 and 90C). A
specific volume of high-purity water with electrical resistivity of
18.2 M cm containing Ca(OH)2-portlandite material (7.4 g/100 mL)
with purity of 96% is placed in a titanium reactor (autoclave with internal
volume of 2 L). The Ca(OH)2 particles are immediately dispersed with
mechanical agitation. The dispersion is then heated to 90C with a heating
system adapted to the reactor. When the dispersion temperature is reached,
CO2 gas is injected in the reactor and the total pressure in the system is
adjusted to 90 bar by Ar injection. Under these preparation conditions,
the vapor phase consists mainly of an Ar and CO2 mixture with the CO2
in a supercritical state. At the end of the experiment, the autoclave is
removed from heating system and immersed in cold water. The reaction cell
is depressurized during the water-cooling period. After water cooling at
35C (about 15 min), the autoclave is disassembled and the solid product
is carefully recovered and separated by centrifugation. Finally, the solid
product is dried directly in the centrifugation flasks for 48 h at 60C and
consecutively for 12 h at 110C in order to eliminate the adsorbed water.
The metastable crystalline phases of CaCO3 (initial P 90 bar, T 90C
after 4 h), such as vaterite and aragonite, cannot be identified during the Ca
(OH)2 carbonation process, except when the reactor is depressurized after
the water-cooling stage at 35C. For this case, crystalline aragonite can be
also detected. However, the carbonation of Ca(OH)2 in the presence of
supercritical or gaseous CO2 led to the precipitation of submicrometric iso-
lated particles (<1 m) and micrometric agglomerates (<5 m) of calcite.

2.2.3 Calcium Chloride and Sodium Bicarbonate

The preparation of calcite hollow microspheres, at room temperature, onto
which aragonite needles are attached using Mg as a crystal growth modifier
has been reported [75]. 0.5 M CaCl2 and 2.0 M MgCl2 solutions are added
into water at the Mg/Ca molar ratio of 2 and the pH is adjusted to 7. Then
0.5 M Na2CO3 solution is injected quickly into the above mixed solution
under vigorous stirring by using a magnetic stirrer. This produces a solution
containing a final CaCO3 concentration of about 8 mM. The mixed disper-
sion is stirred for 1 min, and then the solution is kept under static conditions
at room temperature for 24 h before the precipitates are collected and dried.
The obtained product mainly consists of mixed phases of calcite and
42 M.M.H. Al Omari et al.

aragonite in addition to a small amount of vaterite. It consists of well-defined

spheres with attached bundles of needles on their surfaces. The spheres with
needle bundles can be broken by a simple ultrasonic method, clearly reveal-
ing the hollow nature of the spheres. It is important to note that the molar
ratio [Mg]/[Ca] and pH value of the solution play an important role in the
formation of special superstructure. A dissolutiongrowth mechanism could
account for the formation of hierarchical assemblies of calcite hollow spheres
and aragonite bundles.
Furthermore, different CaCO3 phases were prepared using a self-
designed crystallization apparatus [76,77]. Calcite was prepared by rapidly
mixing 2 M CaCl2 (pH 2.7) and 0.05 M Na2CO3 (pH 11.4) at 40C,
followed by stirring for 6 h. Aragonite was prepared by rapidly mixing
0.1 M CaCl2 (pH 4) and 0.1 M Na2CO3 (pH 11.4) at 80C, followed by
immediate filtration. Vaterite was prepared by rapidly mixing 2 M CaCl2
(pH 2.7) and 0.05 M Na2CO3 (pH 11.4) at 1C, followed by immediate
filtration. Calcite, aragonite, and vaterite were all dried at 70C in the air.
Monohydrocalcite (CaCO3H2O) was prepared according to the precip-
itation from artificial seawater (containing NaCl, MgCl2, CaCl2, SrCl2, and
K2CO3) and dried at room temperature in the air [78].

2.2.4 Calcium Acetate and Ammonium Carbonate

The most common crystallization method to produce CaCO3 is carried out
by preparing 0.5 M solutions of (NH4)2CO3 and Ca(CH3COO)2 followed
by mixing [79]. (NH4)2CO3 solution is tightly sealed with parafilm paper to
minimize the loss of NH3 and the introduction of CO2 from the atmo-
sphere. All solutions are stirred moderately until the reactants are completely
dissolved. The measured pH values before and subsequent to mixing are
8.39 for the (NH4)2CO3 solution and 8.26 for the Ca(CH3COO)2 solution.
After mixing the two solutions together, the pH equilibrated to 8.36. The
(NH4)2CO3 is then poured into beakers containing Ca(CH3COO)2 solu-
tion while being stirred, producing CaCO3. The precipitated CaCO3 slurry
is filtered using filter paper in a filtration system operated under vacuum.
Immediately after the precipitate is recovered, it is rinsed in water and dried
in a furnace at 105C.

2.2.5 Calcium Chloride, Gelatin, and Urea

Wang et al. prepared nonagglomerated calcite (trigonal), vaterite (spherical),
and aragonite (needlelike) particles by the decomposition of urea in CaCl2-
containing aqueous solutions at a temperature range of 5090C [80]. CaCl2
Calcium Carbonate 43

is added to water to prepare a solution containing 0.4 M Ca. Gelatin powder

is then dissolved in the Ca solution until reaching a gelatin content of 0.2 g/
100 mL. Finally, the urea powder is added to the aforementioned solution
up to a concentration of 0.5 M. For preparation of trigonal prismatic CaCO3
(calcite) crystals, portion of the solution is taken into a bottle in which one
piece of microscope cover glass is dropped. The bottle should be tightly
capped and placed in a microprocessor-controlled oven preheated to 70
C, and kept undisturbed for 24 h. The bottle is then opened; the white-
coated cover glass is removed, and washed with an ample supply of water,
followed by rinsing with ethanol. The cover glass is dried in an oven at 37C
overnight in air.
For the preparation of CaCO3 microtablets, a portion of the Ca-con-
taining gelatinurea solution is refrigerated. The pH of the refrigerated solu-
tion is measured to be 6.5 (at 6C). One piece of microscope cover glass is
dropped into the bottle. The bottle is capped and placed in a microproces-
sor-controlled oven preheated to 70C, and kept undisturbed for 24 h. At
the end of 24 h, the bottle is opened and the white-coated cover glass is
removed, and washed with an ample supply of water, followed by rinsing
with ethanol. The cover glass is dried in an air atmosphere oven at 37C,
overnight. For further analyses, the white powder material coating the cover
glass is gently scraped off using a clean and sharp razor blade [80].
Prerefrigerated solutions, on the other hand, produce vaterite microtablets
contaminated with a minor amount of the calcite phase [81].

2.2.6 Carbon Dioxide Sequestration

Carbon sequestration involves the intake/injection of carbon CO2 emitted
from stationary sources by deep underground reservoirs instead of being
released to the atmosphere. A geological seal, or caprock, prevents the
CO2 from leaking out of the reservoir and migrating toward the surface.
Modeling studies [8284] and laboratory and field-scale experiments [85
87] on CO2 injection show that carbonate minerals are the first minerals
to react. Precipitation can be abiotic (independent of biological activity)
or biologically induced (ie, bioremediation). Precipitation reactions involve
Ca, Mg, and CO2 and occur at the mixing interface between two advecting
fluids. Relevant scenarios in the subsurface include the mixing interface
between a plume of injected CO2 and the surrounding brine, or the mixing
interface between injected nutrients and surrounding groundwater that is
contaminated. In these cases, mixing is controlled by diffusion in the direc-
tion of and transverse to flow. Different carbonate may precipitate along this
44 M.M.H. Al Omari et al.

mixing zone in the forms of CaCO3, ie, calcite, aragonite, and vaterite; mag-
nesite (MgCO2); and dolomite (CaMg(CO)3)2.
Precipitation of carbonate minerals can cause pore blockage and lead to
decreased porosity and permeability. This is beneficial within the caprock
and may increase the integrity of the injection. Moreover, carbonate precip-
itation can impact in situ remediation of groundwater contaminants (Sr, Pb,
Cd, Cu, and UO2) through coprecipitation of carbonates and contaminants
[8890].

2.2.7 Cockle Shell and Dodecyl Dimethyl Betaine

Initially, micron-sized cockle shell powders (Anadara granosa) are extracted
by washing, boiling, and then are cooled at room temperature, washed thor-
oughly with water and dried in the oven for 7 days, ground, and finally
sieved. A slurry is formed by adding and mixing water. CaCO3 nanoparticles
are prepared by adding dodecyl dimethyl betaine into the slurry under
vigorous stirring at 1000 rpm at room temperature for 90 min using a
mechanical hot plate and magnetic stirrer bar. The obtained sample is sep-
arated from the mother liquid using a double ring filter paper of 18.0 cm size.
The final product is dried at 100C and packed in a polyethylene plastic bag
[91]. Pure crystalline aragonite nanoparticles were produced in the presence
of dodecyl dimethyl betaine which has most likely acted as a biomineraliza-
tion catalyst. The obtained crystals were small sized, agglomerated, clumped,
and homogenized.

2.2.8 Dolomite and Sucrose

Dolomite (CaMg(CO3)2) can be used to prepare precipitated CaCO3 by the
separation of Ca and Mg components. One of the difficulties in the separa-
tion of Ca and Mg components in dolomite is the low solubility of both
materials [92]. However, usage of sucrose solution to separate these compo-
nents from dolomite has been proven to be an exceptionally successful
method [93]. When calcined dolomite (CaOMgO) is dissolved in a sucrose
solution, CaO will be converted into soluble calcium sucrate [94], while
MgO remains unreacted and presents in the precipitated form [93]. The
purified MgO by-product provides an additional value to dolomite in many
applications, whereas the calcium sucrate solution can be used to prepare
precipitated CaCO3.

2.2.9 Quicklime or Hydrated Lime

Limestone is converted to CaO by heat, followed by formation of milk
of lime (Ca(OH)2) either by slaking quicklime or by dispersing hydrated
Calcium Carbonate 45

lime in water. Finally, Ca(OH)2 is combined with CO2 to form precipitated

CaCO3 [95,96].

2.2.10 Wollastonite
The direct dry (gassolid phase) carbonation of wollastonite (CaSiO3) can be
carried in a continuously stirred tank reactor at 25C and atmospheric pres-
sure for 0600 h. The main drawback of this method is the slow rate at ther-
modynamically set temperatures [97].
CaCO3 can be prepared by direct wet carbonation by: (1) leaching
of Ca, (2) dissolution of CO2 and subsequent conversion of bicarbonate
species, and (3) nucleation and growth of CaCO3. The main disadvantage
related to aqueous carbonation is the high energy consumption and cost
[97].
CaCO3 can be also prepared indirectly by dissolving wollastonite in HCl
to form CaCl2. The Ca(OH)2 produced (via CaCl2) is then dissolved in
water and then reacted with CO2 to produce CaCO3. The major drawbacks
of such method are the energy demand for the acid recycling stage and the
very large water demand for the carbonation stage [97].
Another indirect method to prepare CaCO3 is by extracting Ca ions
from wollastonite using CH3COOH [70,97]. CO2 is then injected into
the solution, which causes CaCO3 to crystallize.

2.2.11 Dolomite
The thermal decomposition of dolomite occurs in air in two steps from
CaCO3 and CaO, respectively [98]. The process can be controlled precisely
only at a temperature of 800C due to the two-step reaction of dissociation.
The main drawback of this method is the adsorption and precipitation of
AsCO3 and AsO.

2.2.12 Gypsum Waste

The gypsum waste can be thermally reduced into CaS, which is then
subjected to a direct aqueous carbonation step for the generation of
H2S and CaCO3 [99]. CaS can be successfully converted into CaCO3;
however, the reaction may yield low-grade carbonate products (<90%
as CaCO3) which comprise a mixture of calcite and vaterite, as well as
trace minerals originating from the starting material. Thus, indirect aqueous
CaS carbonation processing for the production of high-grade CaCO3
(>99% as CaCO3) or precipitated CaCO3 can be developed and
optimized.
46 M.M.H. Al Omari et al.

2.3 Preparation of Amorphous Form

2.3.1 Calcium Chloride and Sodium Carbonate
CaCl2 and Na2CO3 solutions, equimolar in Ca and carbonate ions, are rap-
idly mixed [100]. For example, 106 mg of solid Na2CO3 is added to 20 mL
of a 0.5 M NaOH solution and 30 mL of water. This solution is combined
with 50 mL of a 20 mM CaCl2 solution and stirred rapidly, followed imme-
diately by vacuum filtration and rinsing with acetone to dry the solid.

2.3.2 Calcium Chloride and Dimethyl Carbonate

Amorphous CaCO3 can be prepared by initially preparing dilute solutions
(0.01 M) of CaCl2 in water and an excess of (CH3)2CO3. When a 0.5 M
solution of NaOH is added, the solution starts to become turbid after 90 s at
25C. The reaction solution is stirred for 1 min at room temperature and pH
of the solution is about 12. The resulting precipitate is separated rapidly by
centrifugation (4000 rpm) and washed with ethanol and acetone once sep-
arately, and dried under vacuum at 35C for 48 h [101].

2.3.3 Dolomitic Marble with the Aid of Poly(Acrylic Acid)

Amorphous CaCO3 can be prepared using extensively distributed impure
dolomitic marbles and poly(acrylic acid) as stabilizer, whereby a bubbling
column can be used to enhance the yield. The best conditions are temper-
ature of 40C, pH of 4.5, and poly(acrylic acid) concentration of 10
2 M.
The prepared amorphous CaCO3 nanoparticles with high purity and quality
are suitable for industrial applications [102].

2.3.4 Calcium Hydroxide and Carbon Dioxide

Precipitates of pure amorphous CaCO3 can be obtained by bubbling CO2
through a saturated Ca(OH)2 solution at 0C. The precipitation is stopped
when the pH reaches 8 and the precipitate is filtered and rinsed with cold
acetone. The precipitate dried under high vacuum at 0C for 36 h and stored
under phosphorous pentoxide in a desiccator. Ikaite (CaCO36H2O) is pre-
pared in the same way as the amorphous, but the precipitate is not dried in a
vacuum after washing with acetone, instead, it is immediately placed in a
freezer at
20C [103].

2.3.5 Calcium Chloride and Sodium Bicarbonate by Sono Atomization

Amorphous CaCO3 can be prepared by sono atomization upon reactive
mixing whereby aqueous equimolar solutions of CaCl2 and Na2CO3 along
with a 20% methanol aqueous solution are inserted in containers each having
Calcium Carbonate 47

a small window opening. An ultrasonic generator of 2.4 MHz frequency is

immersed in each of the solutions. At this frequency, the droplet size is about
3 m. The fine droplets are formed by atomization of the suspension, and a
hollow material is obtained by subsequent removal of the water from the
formed droplet. In this method, droplets produced by atomization are used
as a reaction field for synthesis of CaCO3 [104].

2.4 Factors Affecting Preparation

2.4.1 Temperature, pH, and Pressure
The influence of temperature on CaCO3 polymorph formed from
(NH4)2CO3Ca(CH3COO)2 system was investigated. At 25 and 80C,
the predominant form was found to be vaterite, while calcite form was
obtained at 50C. Less than 5% aragonite was observed at 70C [79]. In
CaCl2Na2CO3 system, vaterite with specific morphologies was formed
at 235C, whereas needle-shaped aragonite crystals were obtained at 50
70C [105].
The effect of pH on the polymorphic phase and crystal growth upon
precipitation of CaCO3 was investigated by using gas diffusion method.
At pH 0.5, calcite and aragonite crystals are obtained. When the pH value
was higher than 5.5, the precipitates are calcite crystals with different mor-
phologies and particle sizes [105]. It was also reported that calcite preci-
pitation in water was favored in alkaline conditions [106]. Han et al. also
reported that higher pH value tended to induce calcite crystals [107]. Cheng
et al. [57] and Yu et al. [58] also reported that greater morphology control can
be afforded by low supersaturation as pH is decreased. This means that the
induction time of CaCO3 precipitation increases considerably with de-
creasing pH [105].
The effect of pressure on transition of calcite forms was first studied by
Bridgman [108]. It was observed a transition to a slightly denser phase (cal-
cite II) at 1.44 GPa and a transition to a significantly denser phase (calcite III)
at 1.77 GPa. Singh and Kennedy [109] and Merrill and Bassett [110] placed
the calcite Icalcite II transformation at 1.45 and 1.5 GPa and the calcite II
III transformation at 1.74 and 2.2 GPa, respectively. Moreover, the phase
transition of metastable calcite IIIpostcalcite III initiates at a pressure of
15.5 GPa and is completed between 25 and 30 GPa [111]. In addition to
the aforementioned calcite form, there exists a denser calcite VI form that
can be formed using shock compression experiments [112]. The phase dia-
gram of calcite is further complicated by the appearance of the calcite II and
calcite III intermediates at high-pressure phases in the range from 1.0 to
48 M.M.H. Al Omari et al.

2.5 GPa [108,113]. At higher pressures, calcite is known to undergo yet

another phase transition; this high-pressure phase, known as calcite IV,
occurs at pressures in excess of 10 GPa and is usually observed in impact
experiments in the form of a somewhat anomalous rarefaction shock.
None of the intermediate phases of calcite is stable under equilibrium con-
ditions [114116]. Kerley developed a theoretical model for the equation
of state of CaCO3 that includes phase transitions and melting. As noted,
calcite IV is assumed to have the same properties as aragonite except for
a shift in the energy [117]. Different CaCO3 phases have been deter-
mined by using a combination of advanced ab initio simulation techni-
ques and high-pressure experiment. The crystal structure of postaragonite
phase in CaCO3 at a pressure of 40 GPa was identified in addition to a
number of energetically competitive structures (stable phase I and meta-
stable phases IIIV). Above 137 GPa, phase I with a pyroxene-type
structure with chains of CO4
4 tetrahedra becomes more stable than pos-
taragonite [118].

2.4.2 Calcium and Carbonate Ion Concentration

The effect of Ca/CO3 concentration ratio on CaCO3 particles size and
shape, prepared from CaCl2 and Na2CO3 system, was investigated [119
121]. It was found that particles shape changed from squared, spherical,
to oblong by increasing the concentration (g/L) ratio of Ca/CO3 from
0.21 to 1.22. On the other hand, the average particle size changed from
3.5 to 1.8 m [119,120]. It was explained by formation of soluble complexes
Cax(CO3)y at excess CO3 anions, which lowers the supersaturation and,
accordingly, the primary nucleation rate [121].

2.4.3 Magnesium
It has been reported that the precipitated CaCO3 polymorphs are related
to the Mg/Ca ratio [122124] whereby low-Mg calcite, then high-Mg
calcite and aragonite, then aragonite, are the sequence of phases com-
monly precipitated from fluids with increasing Mg/Ca ratio. Furthermore,
Mg/Ca ratio of about five was considered as the threshold of the forma-
tion of low-Mg calcite to high-Mg calcite and aragonite [123]. Sandberg
[125] and Milliken and Pigott [126] attributed the inferred differences
in cortical mineralogies in Phanerozoic seas to the variations in marine
Mg/Ca ratio.
With respect to CaCO3 morphology, calcite progresses from angular
to spherical with increasing Mg concentration in solution, while by
Calcium Carbonate 49

decreasing the concentration, aragonite progresses from needlelike to spher-

ical [127]. It has been reported that the presence of Mg ions at high concen-
trations leads to a transformation process with multiple-step self-assembly
from amorphous into aragonite [128]. In both natural and most synthetic
monohydrocalcite, the presence of high concentrations of Mg relative to
Ca in the precipitating solution (0.17  Mg/Ca < 65) is a prerequisite for
its formation [129].

2.4.4 Organic and Inorganic Additives

Organic modifiers decrease the size of CaCO3 crystalline particles (<1 m)
upon preparation. Inorganic modifiers (eg, NH4Cl, PbCl2) favor an increase
in the size of CaCO3 crystals by a factor of 1.52 [119].

2.4.5 Antiscalants
Synthetic antiscalants including pentasodium salt of aminotrimethylene
phosphonic acid, and hepta-sodium salt diethylenetriamine pentamethylene
phosphonic acid, in addition to a copolymer containing acrylic acid,
methacrylic acid, and itaconic acid were found to be better suited for adsorp-
tion and prevention of CaCO3 precipitation [130].

2.4.6 Polysilicic Acid

The effect of polysilicic acid on the precipitation of CaCO3 from supersat-
urated solutions was investigated under pH 9 and room temperature. The
decrease in turbidity of the CaCO3 solution in the presence of polysilicic
acid indicated that the polysilicic acid inhibits the precipitation of CaCO3
[131].

2.4.7 Polyelectrolytes
Anionic polyelectrolytes were found to lengthen the induction time and
to reduce the size of CaCO3 nanocrystals precipitated from super-
saturated solution. The extent of time depends on the interaction efficiency
between the polymer anionic repeating units and the Ca ions [132]. Further,
nanocrystals having vaterite structure give spherical CaCO3, while nano-
crystals having calcite structure lead to either acicular or flower shapes
of CaCO3.
Poly(allylamine HCl) as a cationic polyelectrolyte strongly influences
CaCO3 precipitation and induces the formation of calcite thin films and
fibers by counterion-induced phase separation [133]. Furthermore, CaCO3
50 M.M.H. Al Omari et al.

is precipitated, in the presence of poly(allylamine HCl) and Mg ions, as thin-

ner and smoother films, together with fibers with more polycrystalline, gran-
ular structures.

2.4.8 Metal Ions

Aragonite appears to be a favored form in the presence of cationic impu-
rities such as Mg, Ni, Co, Fe, Zn, and Cu, whereas Cd ions showed no
significant effect [134]. It appears that the presence of such impurities can
result in a prolonged induction period leading to slower precipitation
[135]. It has been reported that the trace amounts of Zn ions can slow down
the nucleation rate of CaCO3 and also encourage its crystallization in
aragonite form, even under conditions favoring calcite [136]. Meyer
suggested that Zn ions can significantly reduce the crystal growth of calcite
[137]. Cu ions are much less effective than Zn ions [138,139]. Only a high
concentration of Cu ions in comparison to Zn ions is necessary to achieve a
similar effectiveness.
Divalent cations have been given some attention in CaCO3 precipitation
(eg, Mg, Mn, Cu, Sr, Cd) [140]. When vaterite and calcite crystals are pre-
pared by spontaneous precipitation under defined conditions, followed by
doping with foreign cations, all cations, except Cu, are incorporated in
vaterite. The uptake of Mn is about the same in the presence or absence
of cations [141,142]. Furthermore, the presence of Mn and Mg stimulates
the incorporation of Pb and the uptake of Mn, Cd, and Pb by vaterite is
much larger than that by calcite.

2.4.9 Surfactants
In the presence of cetyltrimethylammonium bromide (CTAB), crystal
agglomeration of CaCO3 increases and the irregular calcite rhombohedral
crystals are numerous. At high concentrations of CTAB (8.5 mM), the
number of spherical particles is smaller and the majority of the particles is
rhombohedral and consists of calcite crystals [143].
In the case of sodium dodecyl sulfate (SDS), at low concentrations
(5 mM), which is greater than the critical micellar concentration of SDS
in 0.1 M NaCl (1.5 mM at 25C) [144], the crystals are primarily flower
shaped and only few rhombohedral and spherical particles are obtained.
When the concentration is increased, eg, 10 times, very small crystals result.
At very high SDS concentration, the aspect of the crystals and their magni-
tude are changed dramatically, but not the polymorphism of CaCO3 because
even in the presence of SDS the form obtained is calcite [143].
Calcium Carbonate 51

It has been reported that addition of sodium dodecylbenzene-

sulfonate improves the formation of vaterite, while SDS enhances the
formation of calcite and the presence of poly(N-vinyl-1-pyrrolidone)
affects the crystal shape [145,146]. When trichloroacetic acid and
hydroxyethylidene-1,1-phosphonic acid are present in the sonically
mixing solutions at 95C, vaterite is stably formed [147].
2.4.10 Media Viscosity
Gels, in general, can effectively reduce the ion activities and diffusion rates so
that the crystallization process in the gel is totally different from that occur-
ring in aqueous solution [148,149]. It has been reported that the morphol-
ogy of CaCO3 can be controlled in the presence of double-hydrophilic
block polymers in a mixture of solvents [150152]. These studies suggest
that the reaction medium has a great effect on the crystallization process.
It was reported that the transformation of amorphous CaCO3 to crystal-
line form is affected by solvent viscosity [54]. The solid transition from
amorphous to calcite is only observed in ethanol/water and the reaction
is suppressed in both ethylene glycol/water and diethylene glycol/water.
Furthermore, the resulting crystals always have different morphologies
and size distributions although they all have the calcite structure.
2.4.11 Proteins
CaCO3 crystal forms, the chemical composition, morphology, and texture
of the deposited mineral can be easily controlled by the type of proteins
[153]. For example, in the presence of glycine, L-alanine, or L-aspartic acid,
vaterite are produced when mixing the two salt solutions, CaCl2 and
Na2CO3 [154157].
Furthermore, CaCO3 form selectivity in organisms is controlled by spe-
cific glycoproteins in the presence of particular substrates [158]. For exam-
ple, oriented crystallization of vaterite occurs in uniaxially deformed gelatin
films containing poly-L-aspartate [159]. No orientation of the mineral phase
can be observed with entrapped poly-L-glutamate, suggesting that the ori-
ented crystallization is controlled by the -sheet structure assumed by poly-
L-aspartate in the presence of Ca ions.
Nucleation of calcite instead of aragonite in situ preparation in
immobilized bovine serum albumin was obtained due to the difference in
the charge distribution on the surface and faster reaction kinetics at the inter-
face [160].
Belcher et al. showed that a controlled phase transition between arago-
nite and calcite in nacre could be obtained in the laboratory with the use of
52 M.M.H. Al Omari et al.

soluble polyanionic proteins [161]. This was directly observed by Thomp-

son et al. through atomic force microscopy [162]. Mann et al. explained the
role of soluble proteins as effective agents to the reduction of interfacial ener-
gies on the surface of the inorganic. An increase in hydrophobicity of the
additive reduces its ability to control morphology and phase transition dur-
ing crystallization [163].

2.4.12 Sucrose/Bovine Serum Albumin System

The sucrose/bovine serum albumin system in biological organisms has been
investigated on changes in the form and morphology of CaCO3 [164]. The
results revealed that low concentration of sucrose (5%) leads to the formation
of calcite and vaterite, while high concentration (20%) leads to the formation
of vaterite.
Moreover, the activation energy of nucleation is decreased by interfacial
molecular recognition leading to form easily vaterite [165]. Also, glucan has
been used as a template to control preparation of aragonite CaCO3 [166] and
its morphology has been controlled by -cyclodextrin [167].

2.4.13 AlgaeMetal Ions System

The effect of metal ions bound to cell surfaces of living organisms on the
precipitation of CaCO3 has been investigated [168]. Calcite and aragonite
are formed in the absence or presence of low Zn amount (3.27 mg/L); how-
ever, at higher Zn concentration (6.53 mg/L), pure aragonite crystals can be
formed. In contrast, in the inorganic, algae-free solutions without Zn, pure
calcite is precipitated. Both inorganic solutions with Zn show major calcite
precipitation.

2.4.14 Mixing Equipment

When CaCO3 is prepared by slowly pouring CaCl2 solution into an equi-
molar solution of K2CO3, using a double-cylinder-type homogenizer or an
ultrasonic homogenizer to another solution agitated using the homogenizer,
vaterite particles are produced. On the other hand, the same particle type is
produced when one of the two salt solutions is added at once to another
solution agitated using the ultrasonic homogenizer [169].

2.4.15 Ultrasound Waves

The nucleation of crystals, from type, particle size, and shape of CaCO3, can
be affected by the presence of ultrasound waves [170173]. Sonawane et al.
[170] obtained only calcite form using sonochemical carbonization method.
Calcium Carbonate 53

Kojima et al. [174] have also investigated the effect of frequency and
amplitude on the morphological of vaterite at high supersaturation without
additives. Kirboga et al. [175] showed that the higher amplitude, the higher
relative vaterite fraction. Moreover, specific surface area increases gradually
with the increase in the sonicator amplitude in the presence of biopolymer
carboxymethyl inulin. The presence of biopolymer affects the specific sur-
face area of CaCO3 crystals prepared not only with magnetic stirring [176]
but also with ultrasound waves [175].

2.5 Inhibitors of Precipitation

There are many inhibitors for CaCO3 precipitation reported in the litera-
ture. A summary list of these inhibitors is presented in Table 3. A list of var-
ious inhibitors for the crystal growth of CaCO3 with their affinity constants
(Kd/Ka) is also given in Table 4. The inhibitory effect of these substrates is
mainly due to adsorption and subsequent blocking of the active growth site
[189,190]. The variation on the affinity constant values for the same inhib-
itor (eg, sodium alginate or phosphate) is most probable due to the difference
in the experiments parameters such as the form of CaCO3 used, pH, con-
centration of inhibitor, and ions (Table 4).

Table 3 List of Inhibitors for CaCO3 Precipitation

Name of Inhibitor References
Synthetic polyacrylic acid [177]
Carboxymethyl inulin [178]
Modified palygorskite by maleic anhydride and styrene sulfonic sodium [179]
Perlinhibin (19.5% cysteine-, 17% histidine-, and 14.6% arginine-rich [180]
miniprotein from Abalone (Haliotis Laevigata) Nacre)
Fulvic acid and Mg ions [181]
Cysteine-rich Mdm2 peptide [182]
Aqueous extract of Paronychia argentea [183]
Xanthan gum [184]
Acrylic acid/2-acrylamido-2-methylpropanesulfonic [185]
acid/2-hydroxypropyl acrylate and orthophosphate
A synthetic polyacrylic acid-allyloxy poly(ethylene glycol) polyglycerol [186]
carboxylate block copolymer
54 M.M.H. Al Omari et al.

Table 4 Comparative Data on the Inhibition of CaCO3 Precipitation

CaCO3 Kd/Ka 1024
Form Inhibitor (mol/dm3) References
Calcite Mg 0.42 [187]
Calcite Glycerophosphate 6 [188]
Vaterite Sodium alginate 19.3 [189]
Calcite Sodium alginate 999.8 [190]
Calcite Phosphate 50 [188]
Calcite Phosphate 5842 [191]
Calcite Mellitic acid (benzenehexacarboxylic acid) 200 [192]
Calcite Ethylenediamine-tetra-bis-methylene 1000 [193]
phosphonic acid
Vaterite 2-Dihydroxyphosphonyl-2-hydroxyl- 1350 [194]
propionic acid
Vaterite 1,3-Bis[(1-phenyl-1-dihydroxy-phosphonyl) 1588 [194]
methyl]-2-imidiazolidenon
Ka and Kd are the specific rate constants for adsorption and desorption, respectively.

3. PHYSICAL CHARACTERISTICS
3.1 Ionization Constant
Chemical equilibria of CaCO3 in aqueous solution can be described as
H2CO3 undergoing dissociation to give H+, HCO
2

3 , and CO3 ionic spe-
cies [195]. The corresponding dissociation constants pKa1 and pKa2 are 6.35
and 10.32 at 25C, respectively [61]. Fig. 5 shows the distribution of carbon-
ates species (H2CO3, HCO
2

3 , and CO3 ) as a function of pH. However, the
presence of Ca ions leads to the formation of CaHCO+3 , CaCO3, and
CaOH+ in solution with association constants pKCaHCO3 + , pKCaOH + , and
pKCaCO3 of 1.26, 1.49, and 3.22 at 25C, respectively [61,196].

3.2 Solubility Characteristics

According to the USP, it is practically insoluble in water. Its solubility in
water is increased by the presence of any ammonium salt or of CO2. The
presence of any alkali hydroxide reduces its solubility. It is insoluble in
Calcium Carbonate 55

1.00

0.75
Mole fraction

CO32
0.50 H2CO3
HCO3

0.25

0.00 pK1 pK2

2 4 6 8 10 12 14
pH
Figure 5 The distribution of carbonate species as a fraction of total dissolved carbonate
in relation to solution pH.

alcohol. It dissolves with effervescence in 1 N CH3COOH, in 3 M HCl,

and in 2 M HNO3 [9]. The solubility of CaCO3 (conventional) in water
is 16.6 mg/L (20C, pH 99.4), while that of the aragonite and calcite forms
is 6.6 mg/L (20C) and 11 mg/L (20C) for the vaterite form. The amor-
phous form of CaCO3, with particles predominately in the nanoscale range,
is approximately 10 times more soluble than crystalline CaCO3 [4].
The solubility products (
log pKsp) of various solid forms and hydrate
forms are listed in Table 5. In general, the solubility of the hydrate forms
of CaCO3 was found to be higher than those of anhydrous forms calcite,
aragonite, and vaterite [197].
The pH solubility of CaCO3 in water was measured as a function of pH
of solution. The pH was adjusted with HCl or NaOH depending on the pH
needed [198]. As shown in Fig. 6, the solubility decreases with the pH of

Table 5 Solubility Products (
log pKsp) for the Various Forms of CaCO3 at 25C [197]
Form Structure 2log pKsp
Amorphous (monohydrate) 6.40
Ikaite (hexahydrate) Monoclinic 6.62
Vaterite (anhydrous) Hexagonal 7.91
Aragonite (anhydrous) Orthorhombic 8.34
Calcite (anhydrous) Rhombohedric 8.48
56 M.M.H. Al Omari et al.

80

Solubility as elemental Ca (mg/mL)

Calculated
Measured
60

46
40

20

3.8 0.005
0.13
0
2 4 6 8 10 12 14
pH
Figure 6 The calculated and measured solubility of CaCO3 as a function of pH of solu-
tion open to the atmosphere at room temperature. The pH of aqueous solutions
adjusted with HCl or NaOH (0.110 M) depending on the pH needed.

solution [198,199]. The measured solubility [198] is in agreement with that

calculated using the dissociation and solubility product constants [199].

3.3 Partition Coefficient

The octanolwater partition coefficient (log P) of CaCO3 at pH 7 and 20C
was reported as
0.81 [200].

3.4 Optical Property

Carbonates can easily be recognized in soil thin sections, as their birefrin-
gence is extreme resulting in upper-order creamy white interference colors.
They are also characterized by high relief generally from strongly negative to
strongly positive depending upon their orientation. Table 6 represents some
optical properties of different forms of CaCO3 [201]. As shown in the table,
the optical properties of the various CaCO3 forms are quite similar.

3.5 Polymorphism
CaCO3 exists naturally in six different forms: three crystalline polymorphs,
calcite, aragonite, and the metastable vaterite; two hydrate phases,
monohydrocalcite and ikaite; in addition to amorphous CaCO3 [202].
Calcium Carbonate 57

Table 6 Optical Properties of Different Forms of CaCO3

Form Crystal System Refractive Index Birefringence () Uniaxial/Biaxial
Calcite Trigonal ne: 1.486; 0.172 U

no: 1.658
Aragonite Orthorhombic nx: 1.530; ny: 0.156 B

1.682; nz: 1.686
Vaterite Hexagonal ne: 1.550; no: 1.650 0.100 U+

3.6 Particle Morphology

Calcite typically displays the rhombohedral habit. Aragonite typically
appears as prisms or needlelike crystals, and vaterite tends to form polycrys-
talline spherulites. They are metastable phases and, especially the
latter, may play a role as a precursor in calcite formation. The amorphous
CaCO3 typically appears as small spheres less than 1 m in diameter [203].
The morphological modifications in the absence and presence of L-
aspartic acid as organic template that induce the nucleation of CaCO3
were investigated [157]. In the absence of L-aspartic acid in the reaction
system, only calcite can be observed and the precipitate is bulky amor-
phous crystal with irregular shape (Fig. 7A). While in the presence of
the acid with a concentration of 0.25 mg/mL, the precipitate contains
calcite which appears layered and rhombohedral shape (Fig. 7B). At high
acid concentration (>1 mg/mL), nearly all the precipitate becomes
spherical vaterite crystal (Fig. 7C). Beck and Andreassen described differ-
ent morphologies of the calcite form [204]. For example, the plate-like
morphology with hexagonal features for calcite could only be obtained
under the influence of significant levels of Li ions at 45C. The spheru-
litic, cube-shaped crystals of calcite are produced at 10C. Precipitation at
room temperature resulted in smooth calcite polyhedra, but the
unwanted extent of agglomeration is much higher at that temperature.
Needle-shaped particles (Fig. 7D) could be obtained at low supersatura-
tion and high temperature (90C). In this experiment, low supersatura-
tion during the crystallization process was obtained by a slow addition of
Ca ions.
The different crystalline polymorphic phases of CaCO3 possess different
particle morphology, shape, porous structure, and density (Table 7). The
foregoing properties are further dependent on whether CaCO3 exists as nat-
ural or synthetically prepared via the various synthetic routes.
58 M.M.H. Al Omari et al.

A B

C D

Figure 7 The SEM images of CaCO3 forms: (A) amorphous calcite, (B) layered and rhom-
bohedral calcite, (C) spherical vaterite, and (D) needle aragonite.

Table 7 Reported Particle Size, Specific Surface Area, and Pore Size of CaCO3
Specific Surface
Form Particle Size (m) Area (m2/g) Pore Size (cm3/g) References
Calcite 0.99 0.068 [205,206]
0.1 30 [207]
Aragonite 3.6 0.163.8 nm [208,209]
Vaterite 5 77 0.068 [206]
35 915 10 nm [62,210]
Amorphous 0.04 65 [104]
0.0860.31 1242 [205]
0.190.41 919 [205]
Natural 13 410 [211]
Precipitated 0.07 1725 2.5 [212,213]
Calcium Carbonate 59

With regard to specific gravity and Mohs scale of CaCO3, all crystalline
forms showed similar values recorded as 2.542.95 and 34, respectively
[214,215].

3.7 Hygroscopicity
Amorphous and ikaite forms of CaCO3 exist as mono- and hexahydrate,
respectively, while vaterite, aragonite, and calcite exist as anhydrous.
According to USP and Eur. Ph. [1,2], CaCO3 does not loss more than
2% upon drying at 200C, while in Japanese Pharmacopeia (JP) [216], it does
not loss more than 1% upon drying at 180C.

3.8 Molecular Modeling

Molecular modeling studies of calcite and aragonite have been carried by
molecular dynamics (MD) simulations approach [217]. In this study, con-
stant volume and constant stress simulations were performed using the
force-field parameters of Dove et al. [218]. The reported crystallographic
data were used to build the initial configurations of calcite and aragonite
[219,220]. The calcite simulation cell was composed of 20 unit cells, four
unit cells along the a-axis, five along b, and one along c, while the aragonite
simulation cell included 27 unit cells, three along each of the a, b, and c axes.
In the case of calcite, the constant volume MD simulations show little
difference between the initial (Fig. 8A) and final structures (Fig. 8B), apart
from some thermally induced positional and rotational disorder. For arago-
nite, there is apparently very little rearrangement of the structure after the

A B C

Figure 8 The molecular dynamic simulation of calcite: (A) the initial, (B) the final with
a constant volume simulation (298K and density of 2.71 g/cm3), and (C) the final with a
constant stress simulation (298K and 1 bar pressure). Carbonate ions are shown with
a stick representation for the bonds between carbon and oxygen atoms. The projection
is along the b-axis.
60 M.M.H. Al Omari et al.

A B C

Figure 9 The molecular dynamic simulation of aragonite: (A) the initial, (B) the final
with a constant volume simulation (298K and density of 2.944 g/cm3), and (C) the
final with constant stress simulation (298K and 1 bar pressure). Carbonate ions are
shown with a stick representation for the bonds between carbon and oxygen atoms.
The projection is along the a-axis.

simulation (Fig. 9B) in comparison with the initial configuration (Fig. 9A).
The agreement between experimental and simulated pair distribution func-
tions would suggest that the force field reproduces the structural features of
calcite and aragonite when using constant volume simulations. However,
the constant volume/shape constraints may have had the effect of stabilizing
metastable structures [217].
Fig. 8C shows the final system configuration of the constant stress sim-
ulation for calcite. Comparing this with the starting configuration (Fig. 8A),
it is evident that the calcite structure is not greatly altered by relaxation in the
simulation constraints. The final and initial configurations of aragonite
(Fig. 9C and A, respectively) do show some differences. The equilibrated
structure from the constant stress simulation indicates that the carbonate ions
had rotated through an angle of c.60 degree, although the Ca and carbon
center-of-mass positions did not change appreciably. The differences in
the simulation outcomes can be associated with differences in the carbonate
anion geometries between the calcite and aragonite natural crystalline forms.
Furthermore, the equilibrium unit cell parameters of calcite calculated from
the constant stress simulation show a close agreement with the X-ray crys-
tallographic values. Aragonite, however, shows greater differences in the
unit cell dimensions. All three lengths, a, b, and c, were longer so that the
density was lower, in fact quite close to the calcite, suggesting that the ara-
gonite phase is tending toward this structure in an attempt to relieve stress on
the system [217].
Calcium Carbonate 61

Atomistic simulation techniques, based on the Born model of solids,

have been used to investigate the effect of molecular adsorption of water
on the low-index surfaces of calcite, aragonite, and vaterite [221]. For
calcite, the results showed that {1014} surface is the most stable surface
of calcite and exhibits the same 1  1 symmetry and structural features of
the surface oxygen ions. Also the presence of partially hydrated surfaces
has equivalent surface carbonate groups, while the {1011} surface and also
the {1120} plane both exhibit bulk-like termination of the surface with only
some rotation of surface carbonate groups. In the case of aragonite, the sta-
bility and regularity of the hydrated {010} surface confirms the needs to be
stabilized by an ordered water layer to be able to fit well onto hydrated gyp-
sum (010). In the case of vaterite, the calculated equilibrium morphology of
the hydrated vaterite crystals has a disk shape, while the unhydrated and
growth morphologies are elongated and perhaps more needlelike.
The bulk lattice energies of the three forms (Table 8) reflect their ther-
modynamic stability, in that the stability of calcite over aragonite is due to its
higher entropy content at elevated temperatures rather than its bulk lattice
energy, which favors aragonite. The relative surface contributions of the
forms show that in the crystal nucleation stage, calcite is the most stable form,
while aragonite becomes more stable when the crystal is large enough for the
bulk lattice energy to outweigh the surface energy terms [219].

3.9 Crystallographic Properties

3.9.1 Single-Crystal Structure
3.9.1.1 Calcite
Calcite has a rhombohedral crystal structure as determined by Bragg [222].
The hexagonal unit cell of calcite, with space group R3c, has
a b 4.990 A, c 17.061 A, 90 degree, and 120 degree
(Fig. 10) [219,223,224].

Table 8 Calculated Lattice Energies of Calcite, Aragonite,

and Vaterite
Form Bulk Lattice Energy 108 (kJ/m3)
Calcite
1.6867
Aragonite
1.7851
Vaterite
1.6577
62 M.M.H. Al Omari et al.

A B

Figure 10 The hexagonal representation of a calcite unit cell: (A) side view and (B) top
view (Ca green (light gray in the print version), O red (dark gray in the print version),
C gray).

A B

Figure 11 The orthorhombic double cells of aragonite: (A) side view and (B) top view
(Ca green (light gray in the print version), O red (dark gray in the print version),
C gray).

3.9.1.2 Aragonite
Aragonite has an orthorhombic crystal structure with space group Pmcn. The
experimental structure parameters found by Dickens and Bowen are:
a 4.960 A, b 7.964 A, c 5.738 A, and 90 degree, containing
four CaCO3 molecules [220]. The theoretical crystal parameters a
(5.0192 A), b (8.0393 A), and c (5.7952 A), as obtained by USPEX simula-
tion method are almost close to the experimental values [118].
The Ca ion (green (light gray in the print version) spheres in Fig. 11) is
very close to the limiting value for transition making aragonites
orthorhomobic structure fairly easy to shift into the thermodynamically
Calcium Carbonate 63

stable rhombohedral structure, though depending on approach the activa-

tion energy ranges from 159 to 452 kJ/mol [225227].

3.9.1.3 Postaragonite and Phases IIV

It was reported by Santillan and Williams that CaCO3 postaragonite phase
has a trigonal structure [228], while Ono et al. showed that it has an ortho-
rhombic structure [229]. Furthermore, a combination of advanced ab initio
simulation techniques (USPEX) and high-pressure experiment was used to
determine the crystal structure of the postaragonite phase in CaCO3, in
addition to different stable (phase I) and metastable phases (phases IIIV)
at high-pressure conditions [118]. Fig. 12 shows the best structure of these
phase produced by USPEX, where the structure optimizations were per-
formed at a series of pressures between 0 and 100 GPa.
It was found that the postaragonite structure is characterized by the pres-
ence of 12-coordinate Ca atoms (in aragonite Ca is 9-coordinate) and can be
viewed as hexagonal close packing of Ca2+ and O2
ions with small C4+ ions
in triangular voids (Fig. 12A). Furthermore and for postaragonite, theoret-
ical and experimental lattice parameters are in excellent agreement with each
other. Also it was found that at 50 GPa, all crystal structures contain

A B

C D E

Figure 12 The crystal structures of CaCO3: (A) postaragonite, (B) phase I, (C) phase II, (D)
phase III, and (E) phase IV (Ca red (dark gray in the print version), O green (gray in the
print version), C blue (dark gray in the print version)).
64 M.M.H. Al Omari et al.

triangular CO2
3 carbonate ions, whereas at 80 GPa, many structures also

contained CO4
4 tetrahedra (eg, phase I, Fig. 12B), indicating that such
structures become competitive well within the experimentally reachable
pressure range. The most stable structure identified at both 50a and
80 GPa is the postaragonite structure, but several metastable structures turn
out to be almost stable in this pressure range [118].
The orthorhombic crystal structure with space group C2221
(a 5.544 A, b 6.926 A, and c 3.213 A) pyroxene-type structure (phase
I, Fig. 12B) appeared as metastable at 80 GPa, but at 150 GPa it became as
the most stable one. Thus, the limit for the enormous stability field of pos-
taragonite was obtained at the pressure range of 42137 GPa. Additionally,
several energetically competitive structures, but always metastable phases II
(triclinic, space group P1, 10 atoms in the unit cell), III (trigonal, space
group R3m, 5 atoms in the rhombohedral cell), and IV (monoclinic, space
group P21, 10 atoms in the cell), all contain carbon only in the form of CO2

3
ions (Fig. 12CE) [118].

3.9.1.4 Vaterite
Its crystal structure differs from those of calcite and aragonite in terms of
symmetry, orientation of CO3 ions, and coordination environment of Ca
ions. There is general agreement that the carbonate planes are parallel to
the c-axis and that the Ca atoms are in eightfold coordination with oxygen
atoms. In contrast, the CO3 ions in calcite and aragonite are perpendicular to
the c-axis, and the Ca atoms have sixfold coordination in calcite and ninefold
in aragonite [81,230,231].
Meyer first reported an orthorhombic unit cell with a space group Pbnm
and Z 4 (Fig. 13A) for vaterite crystal structure derived from single-crystal
X-ray diffraction experiments [232]. On the other hand, Kamhi [81] found a
structure with hexagonal symmetry and space group P63/mmc using the
same XRD technique (Fig. 13B).
Later, Meyer [232] provided a structure mainly consistent with Kamhis
structure [81], which has the same space group but different carbonate site
symmetry. Furthermore, Lippmann [225] developed an idealized structure
from the vaterite-type high-temperature phase of YbBO3 [233] with space
group P6322. Spectroscopic methods have been applied to resolve the con-
troversy on the vaterite structure and focused on space group and site sym-
metry. However, the results are inconsistent, often due to impurities in the
samples, mode assignments, and differences of group theory analysis. For
example, Sato and Matsudas infrared spectra [234] support Kamhis
Calcium Carbonate 65

A B

Figure 13 Crystal structures of vaterite: (A) orthorhombic structure with space group
Pbnm and (B) hexagonal structure with space group P63/mmc (Ca black, O light gray,
C gray).

structure [81]. Behrens et al. [235] argued that none of the proposed struc-
tures, P63/mmc or P6322, is consistent with the Raman spectra, while
Gabrielli et al. [236] show that Meyers structure [232] is consistent with
the Raman spectra. On the other hand, Anderson [237] favors both Kamhis
[81] and Lippmanns [225] structures. Deer et al. [223] reported that vaterite
is hexagonal with space group P63 and a b 4.13 A, c 8.48 A, 90
degree, and 120 degree.
Wang and Becker used MD simulation with temperature annealing to
study the structure and carbonate orientation of vaterite, in addition to esti-
mate the relative stability of the structures proposed in the literature [238].
The investigation revealed that the orthorhombic crystal structure of vaterite
proposed by Meyer [232] cannot be confirmed by quantum-mechanical cal-
culations and by the experiments by Kamhi [81]. Thus, partial occupancy
and disordering are unavoidable for correctly describing the vaterite
structure.
Subsequently, the temperature-annealing MD simulation of vaterite
structure at the end of the annealing cycle at room temperature (300K) shows
that the orientations of CO3 ions are disordered (Fig. 14A) and there are three
preferred orientations with an angle of 120 degree between CO3 planes.
However, the structure lacks long-range orientational order [238]. This
disordered structure (Fig. 14A), when taking a space average, is consistent with
Kamhis structure [81], in which each lattice site of the CO3 ion is partially
occupied and all atoms of the CO3 ions are randomly distributed among three
positions. An ordered hexagonal superstructure with space group P6522 or
P3221 appeared at the end of about 7 ns of simulated annealing with a final
66 M.M.H. Al Omari et al.

A B

Figure 14 Snapshots of the molecular dynamic computations projected onto (001)

plane for vaterite: (A) at the end of the annealing cycle at 300K, where CO3 ions are dis-
ordered and (B) at the end of the annealing cycle at 1500K, where they are ordered
(Ca black, O light gray, C gray).

A B

Figure 15 The structure of monohydrocalcite (CaCO3H2O) viewed down {001}: (A) the
P3121 subcell structure showing the disordered carbonate groups and (B) the super-
structure P31 model (Ca large gray, O large black, C small black, H small gray).

annealing temperature of 1500K (Fig. 14B). The different crystal structures of

vaterite energetically follow the order: the ordered superstructure > the disor-
dered superstructure > orthorhombic structure [238].

3.9.1.5 Monohydrocalcite
The generally accepted structure of monohydrocalcite is that of Effenberger
et al. [219]. The structure from single-crystal diffraction is solved in P3121
(Fig. 15A) with cell parameters a 6.0931 A and c 7.5446 A, which
requires orientationally disordered carbonate groups. A superstructure that
determines the orientation of water oxygen, one carbonate oxygen, and the
hydrogen atoms is solved in P31 (Fig. 15B) with a 10.5536 A and
c 7.5446 A, on the basis of weak super-lattice reflections, in which the car-
bonate groups are orientationally ordered. The positions are mapped from
Calcium Carbonate 67

the P3121 substructure, and the ordered orientations of the carbonate groups
are refined using rigid bodies.
Monohydrocalcite consists of eightfold coordinated Ca2+ ions, in which
some of the oxygen coordination is direct to carbonate groups and some
to water molecules. The eightfold Ca coordination consists of bonding to
four neighboring carbonate groups and two water molecules. Two of the
carbonate groups are involved in two bonds from Ca to two separate O
atoms, and two others are involved in one bond from Ca.

3.9.1.6 Ikaite
Ikaite tends to form very steep or spiky pyramidal crystals, often radially
arranged, of varied sizes from thumbnail size aggregates to gigantic salient
spurs. Upon synthesis, CaCO3H2O crystallizes in well-defined rhombohe-
dral crystals in the size range 1040 mm. It crystallizes in the monoclinic
crystal system in space group C2/c with lattice parameters a  8.87 A,
b  8.23 A, c  11.02 A, and  110.2 degree [239]. The structure of ikaite
consists of an ion pair of (Ca2+CO2
0
3 ) surrounded by a cage of hydrogen-
bonded water molecules which serve to isolate one ion pair from another
(Fig. 16) [240].

3.9.2 X-Ray Powder Diffraction Pattern

Different anhydrous and hydrate CaCO3 forms were prepared and tested by
a Siemens D500 diffractometer operating with Cu K radiation
( 1.5406 A) at 40 kV and 20 mA in Bragg-Brentano mode with a step size

Figure 16 Ion pair (Ca2+CO2
0

3 ) and hydration cage. Ca is in dodecahedral coordination
with O atoms of the carbonate and water molecules, while hydrogen bonds (dotted)
between H atoms of the water molecules to the O atoms of the carbonate ion exist
(Ca blue (light gray in the print version), O red (dark gray in the print version),
CO3 black planar, H yellow (white in the print version)).
68 M.M.H. Al Omari et al.

Figure 17 XRPD patterns of different CaCO3 forms including calcite, vaterite, aragonite,
monohydrocalcite (CaCO3H2O), ikaite (CaCO36H2O), and amorphous.

of 0.02 degree 2 and a counting time of 1 s per step [76,77]. XRPD patterns
of these prepared CaCO3 were shown in Fig. 17 and their corresponding
crystallographic data including 2, d-spacing, hkl indices [241,242], and %
intensity are listed in Tables 9 and 10. The interplaner d-spacing is calculated
from the Bragg equation (2d sin n), where (1.5406 A) is the wave-
length of the X-ray (Cu K radiator).
As shown in Fig. 17, the XRPD patterns of the three crystalline forms of
CaCO3 (calcite, aragonite, and vaterite), prepared according to the above
conditions, represent their pure phases with no other crystalline phases
detected. Also monohydrocalcite can be clearly identified by XRPD in a
phase-pure form. Ikaite form has only one major and sharp peak at 2 of
about 17 and 2 minors around 35. However, amorphous can be distin-
guished from other forms by a halo broad peak (Fig. 17).

3.10 Thermal Analysis

3.10.1 Melting Point
The reported melting points of CaCO3 are 825 and 1339C for aragonite
and calcite, respectively [243].

3.10.2 Differential Scanning Calorimetry

The differential scanning calorimetry (DSC) thermogram of CaCO3 calcite
form was recorded using an STA S-1500 instrument at a heating rate of
10C/min in an air flow (Fig. 18A) [102]. From the DSC results, calcite
has an endothermic peak at 726C, which reveals the decomposition of
Calcium Carbonate 69

Table 9 The Crystallographic Data from the X-Ray Powder Diffraction Patterns of
Anhydrous Crystalline CaCO3 Forms Calcite, Aragonite, and Vaterite
Scattering Angle (2) d-Spacing () hkl Indices Relative Intensity (%)
Calcite
23.0 3.8637 {012} 10.0
29.4 3.0356 {104} 100.0
35.9 2.4995 {110} 15.0
39.5 2.2796 {113} 20.0
43.1 2.0971 {202} 17.5
47.5 1.9126 {024} 17.5
48.5 1.8755 {018} 20.0
57.5 1.6015 {112} 7.5
Aragonite
21.1 4.2071 {110} 12.2
26.3 3.3834 {111} 100.0
27.3 3.2618 {021} 51.2
31.2 2.8689 {002} 4.9
33.2 2.6931 {012} 29.3
36.2 2.4821 {200} 34.1
37.3 2.4076 {031} 34.1
37.9 2.3708 {112} 22.0
38.5 2.3394 {130} 39.0
41.3 2.1833 {221} 17.1
43.0 2.1036 {220} 36.6
46.0 1.9722 {221} 87.8
48.3 1.8821 {202} 41.5
50.2 1.8159 {132} 24.4
52.5 1.7425 {113} 22.0
53.1 1.7233 {231} 17.1
Continued
70 M.M.H. Al Omari et al.

Table 9 The Crystallographic Data from the X-Ray Powder Diffraction Patterns of
Anhydrous Crystalline CaCO3 Forms Calcite, Aragonite, and Vateritecont'd
Scattering Angle (2) d-Spacing () hkl Indices Relative Intensity (%)
Vaterite
21.0 4.2269 {004} 11.4
24.9 3.5730 {110} 75.0
27.0 3.2997 {112} 100.0
32.7 2.7364 {114} 84.1
44.5 2.0343 {211} 79.5
49.0 1.8575 {304} 25.0
50.0 1.8227 {300} 45.5
56.0 1.6408 {224} 22.7

Table 10 The Crystallographic Data from the X-Ray Powder Diffraction Patterns of
Hydrate CaCO3 Forms Monohydrocalcite (CaCO3H2O) and Ikaite (CaCO36H2O)
Scattering Angle (2) d-Spacing () Relative Intensity (%)
Monohydrocalcite
20.8 4.2671 46.7
29.0 3.0765 46.7
32.0 2.7946 100.0
36.0 2.4927 13.3
38.0 2.3660 20.0
42.0 2.1495 40.0
45.5 1.9919 60.0
47.0 1.9318 53.3
52.0 1.7572 20.0
56.0 1.6408 20.0
Ikaite
17.5 5.0637 100.0
34.0 2.6347 40.0
34.5 2.5976 40.0
Calcium Carbonate 71

A
10

Heat flow (mV)

10

20

30

40

50
400 500 600 700 800
Temperature (C)
B
100

90
Weight (%)

80

70

60

50

40
200 400 600 800 1000
Temperature (C)
Figure 18 (A) The DSC and (B) TGA thermograms of CaCO3 calcite form recorded at a
heating rate of 10C/min in an air flow.

CaCO3 into CaO. Both aragonite and vaterite forms show almost similar
DSC patterns to calcite with endothermic peaks at around 800 (heating rate
of 10C/min per Ar gas) and 775C (heating rate of 5C/min per air flow),
respectively [244,245]. In the case of vaterite, two additional exothermic
peaks obtained at 481 and 488C are unambiguously attributed to the trans-
formation of vaterite into calcite [245].

3.10.3 Thermogravimetric Analysis

The thermogravimetric analysis (TGA) thermogram of CaCO3 calcite form
was recorded using an STA S-1500 instrument at a heating rate of 10C/min
in an air flow (Fig. 18B) [102]. From the TGA results gave 43% weight loss
in the temperature range of 680780C, which attributed to the
72 M.M.H. Al Omari et al.

100 Exo 15

99 10

98 5
0

Heat flow (mV)

97
Weight (%)

5
96
10
95
15
94
20
93 25
92 Step 1 Step 2 30

50 100 150 200 250 300 350

Temperature (C)
Figure 19 TGA and DSC thermograms of amorphous CaCO3, showing the steps of water
loss prior to crystallization to form calcite.

decomposition of CaCO3 to CaO. Both aragonite and vaterite forms show

almost similar TGA patterns to calcite [244,245]. Fig. 19 represents the
TGADSC of amorphous CaCO3 prepared by bubbling of CO2 and by
mixing of Ca and carbonate solutions [103]. Water is lost in two distinct
steps. From 50 to 100C, a first step is assigned to loosely bound water.
In the second step from 100 to 250C, structural water is lost with a begin-
ning crystallization to calcite as indicated by DSC.

3.11 Spectroscopy
3.11.1 Ultraviolet/Visible Spectroscopy
The ultraviolet/visible (UV/VIS) spectrophotometry was used for direct
measurement of carbonate ions (CO2
3 ) concentration. For example,
CO2
3 absorbs light at wavelengths of less than 250 nm, this facilities
acidimetric titration with UV detection of most carbonate-containing nat-
ural waters and observe an increase in % transmittance [246]. Ariponnammal
reported that CaCO3 has three characterized wavelengths at 233.42, 254.91,
and 356.52 nm [247]. Furthermore, Nangare described direct UV/VIS
method for simultaneous determination of CaCO3 and aspirin in tablet dos-
age form [248]. Fig. 20 represents the UV/VIS spectrum of CaCO3 in 0.1
NaOH, recorded by a Shimadzu model 1700 double beam UV/VIS spec-
trophotometer, which shows a maximum at about 240 nm.
Calcium Carbonate 73

Figure 20 The UV/VIS absorption spectrum of CaCO3 (20 g/mL) in water.

3.11.2 Vibrational Spectroscopy

3.11.2.1 Fourier Transform Infrared Spectroscopy
The Fourier transform infrared spectroscopy (FTIR) absorption spectra of
different CaCO3 forms were recorded using Shimadzu IRAffinity-1 FTIR
spectrophotometer (Fig. 21) [128]. Carbonate ions and similar molecules
have four normal modes of vibration peaks: 1 symmetric stretching, 2
out-of-plane bending, 3 doubly degenerate planar asymmetric stretching,
and 4 doubly degenerate planar bending [249]. As shown in Fig. 21, ara-
gonite has two characteristic absorption bands at 2 856 cm
1 and 4
713 cm
1 along with a weak 700 cm
1 absorption peak, and also a 3
absorption band at 1490 cm
1, and these can be considered as the com-
mon characteristic features of the CO2
3 ions in CaCO3 and are the funda-
mental modes of vibration for this molecule [250]. Vaterites characteristic
absorption bands are 2 875 cm
1 and 4 745 cm
1 and a split peak of 3 at
1440 and 1490 cm
1. For calcite, there are two absorption bands at 2
875 cm
1 and 4 713 cm
1 and an absorption peak at 1420 cm
1.
Monohydrocalcite shows weak absorption bands at 4 700 and
727 cm
1, at 2 873 cm
1, at 1 1068 cm
1, and a split peak of 3 at
1418 and 1488 cm
1 [78,251]. Amorphous CaCO3 has a characteristic
broad 2 absorption band at 866 cm
1, and a split peak at 1418 and
1475 cm
1.
From the earlier results, the characteristic carbonate 2 band of aragonite
is at 856 cm
1; whereas that of both calcite and vaterite is at 875 cm
1. The
74 M.M.H. Al Omari et al.

1490 856

713 700 Aragonite

1490 875
1440

1487 745 Vaterite

1408
875
Monohydrocalcite
700

1420 875

713 Calcite

1475 1418
866 Amorphous

00 00 0 0 0 0 0 0 0 0
90 85 80 75 70 65 60 55 50
0
20 15
Wavenumber (cm-1)
Figure 21 The FTIR spectra of different CaCO3 forms.

characteristic carbonate 4 band of aragonite is at 700 and 713 cm
1,

whereas that of calcite is at 713 cm
1 and vaterite at 744 cm
1 [252].

3.11.2.2 Raman Spectroscopy

The Raman absorption spectra of CaCO3 were obtained with a Jobin-Yvon
T64000 Raman spectrometer. The Raman spectra were excited by the laser
line having a wavelength of 514.5 nm, provided by an Ar laser [253]. The
Raman spectra of three crystalline forms of CaCO3 (calcite, aragonite, and
vaterite) are shown in Fig. 22 and the assignments of distinctive Raman
bands are given in Table 11 [236]. Three zones can be used to distinguish
the three forms. The selection of the appropriate bands for quantitative anal-
ysis is a difficult task since there is a strong overlapping. Unfortunately, the
strongest bands of CaCO3 forms at 1000 cm
1 overlap and cannot be used
for analytical purposes. Vibration modes 700 cm
1 are very weak, and par-
ticularly undetectable for vaterite. The most intense Raman bands are
observed in the low frequencies region (50400 cm
1), corresponding to
the lattice mode vibrations. For this reason, the range chosen for the quan-
titative analysis is situated between 50 and 400 cm
1.
The Raman spectra of monohydrocalcite are shown in Fig. 23A. The
spectrum is dominated by the symmetric stretching vibration (1) of
the CO3 group, which occurs at 1069 cm
1. The only other features in
Calcium Carbonate 75

10,000

Raman intensity (a.u.)

Calcite

Aragonite

Vaterite

200 400 600 800 1000 1200 1400 1600

Wavenumber (cm-1)

B
Raman intensity (a.u.)

Calcite

Aragonite

Vaterite

50 100 150 200 250 300 350 400

Wavenumber (cm-1)
Figure 22 The Raman spectra of different CaCO3 forms in the ranges of (A) 501600 and
(B) 50400 cm
1.

the spectrum are a weak lattice vibration at 212 cm
1 and traces of 1 at
880 cm
1 [254]. For amorphous CaCO3, the typical Raman spectrum is
shown in Fig. 23B. The amorphous character is initially confirmed by the
low intensity for all Raman bands when compared to other crystalline forms.
Furthermore, the main Raman band of the carbonate ion (1 at 1085 cm
1)
is shifted toward lower wavenumbers (1079.87 cm
1) and is broader than
for well-crystallized forms [255,256].
76 M.M.H. Al Omari et al.

Table 11 The Wavenumbers (cm
1) and Vibrational Mode of the Raman Absorption
Bands of Different CaCO3 Forms
Form Wavenumber (cm21)
1 2 3 4
Lattice Symmetric Out-of-Plane Asymmetric In-Plane
Mode Stretching Bending Stretching Bending
Calcite 284 1086 1434 1747
Aragonite 151, 206, 275 1085 853, 910 1460, 1570
Vaterite 267, 300, 325 1074, 1090 874 1445, 1485, 1749
1550, 1595

Figure 23 The Raman spectra of (A) monohydrocalcite (CaCO3H2O) and (B) amorphous
CaCO3. The wavenumbers at 914.90 and 1371.26 cm
1 correspond to Ne emission lines,
(C and D) ikaite (CaCO3 6H2O) at pressures from 0.14 to 4.08 GPa in a diamond anvil cell
in the ranges of 2001100 and 28004000 cm
1, respectively.
Calcium Carbonate 77

The Raman spectra of a single crystal of ikaite over a range of pressures in

a diamond anvil cell are shown in Fig. 23C and D [257]. The spectrum at
1.32 GPa shows eight peaks, four corresponding to Eg external vibrations of

1
CO2
3 ions at 228, 240, 280, and 308 cm , two corresponding to internal
vibrations of CO2

3 ions, namely, the Eg (internal) and A1g (internal) vibra-
tions at 729 and 1081 cm
1, respectively, and two peaks related to water at
3207 and 3403 cm
1 [258]. Ikaite has a spectrum similar to the other CaCO3
forms [259,260], but with water peaks visible between 3000 and 3600 cm
1.
The peaks become more intense as the pressure increases and the pressure
shifts become larger for the external (lattice) modes than for the internal
vibrations.

3.11.3 Nuclear Magnetic Resonance Spectrometry

3.11.3.1 1H MAS NMR Spectrum
Solid-state proton NMR spectroscopy was performed on a Bruker ASX 400
spectrometer with a 400.132 MHz resonance frequency. For all solid-state
spectra, magic angle spinning (MAS) was applied at a frequency of
15,000 Hz in a 4-mm rotor and direct excitation of the protons was induced
by a single 90 degree pulse [76].
The use of 1H MAS NMR technique is mainly aimed to analyze the
incorporated molecular water. Vaterite, monohydrocalcite, and amorphous
forms contain molecular water as the main hydrogen component and only
traces of hydroxide. A signal at around 5 ppm is observed for the aforemen-
tioned forms (Fig. 24). Such signal is assigned to incorporated molecular

* * Vaterite
Intensity

* *
Monohydrocalcite

* * Amorphous

100 80 60 40 20 0 20 40 60 80
Chemical shift (ppm)
Figure 24 1H MAS NMR spectra of vaterite, monohydrocalcite (CaCO3H2O), and amor-
phous forms. *Spinning side bands.
78 M.M.H. Al Omari et al.

water. The peaks between 1.5 and 2.5 for the amorphous are due to traces of
a mobile water fraction containing hydroxide ions.

13
3.11.3.2 C MAS NMR Spectrum
Solid-state carbon NMR spectroscopy was performed on a Bruker ASX 400
spectrometer with a 100.623 MHz resonance frequency. For all solid-state
spectra, MAS was applied at a frequency of 4000 Hz in a 7-mm rotor and
direct excitation of 13C nuclei was induced by a single 30 degree pulse of
5.75 s duration [76].
The 13C MAS NMR spectra of all hydrated and anhydrous CaCO3
forms are shown in Fig. 25. All samples show carbonate or hydrogen carbon-
ate peaks in the range of 156174 ppm. The crystalline, water-free phases
calcite and aragonite show very narrow NMR peaks, whereas the water-
containing phases all showed broad peaks [76,261]. The linewidth of the
amorphous peak is large due to the disordered structure. There is a clear dis-
tinction between the regions of carbonate peaks (about 166174 ppm) and
hydrogen carbonate peaks (below 166 ppm). The foregoing indicates that
the majority of the carbon atoms in amorphous are present as carbonate
and not as hydrogen carbonate, with some similarity of the chemical envi-
ronment to the highly hydrated phase ikaite.

Calcite
* *

* * Aragonite

* * Vaterite
PTFE
Monohydrocalcite
* *
Intensity

Ikaite
*
Amorphous
* *

250 200 150 100 50 0

Chemical shift (ppm)
Figure 25 13C MAS NMR spectra of anhydrous forms of CaCO3 (calcite, aragonite,
and vaterite) and hydrated forms (monohydrocalcite, ikaite, and amorphous). *Spinning
side bands.
Calcium Carbonate 79

For aragonite, there is a single peak at 169.9 ppm; for calcite, there is a
single peak in the range of 167.4167.9 ppm with a full width at half max-
imum (fwhm) of 11.1 ppm; for vaterite containing 9% calcite, there is a
single peak at 168.7 ppm (fwhm 1.9 ppm). Ikaite shows a slow decomposi-
tion during the NMR experiment, even if it is carried out at
20C as it
partially converts to calcite (confirmed by X-ray powder diffraction) [76].

4. METHODS OF ANALYSIS
4.1 Compendial Methods
4.1.1 Calcium Carbonate
CaCO3 monograph is listed in the Eur. Ph. [2], United States Pharmaco-
peiaNational Formulary (USPNF) [1], and JP [216]. Table 12 shows a
summary of its specifications and methods of analysis.

4.1.2 Calcium Carbonate Tablets

CaCO3 tablets monograph is listed in the USP-NF [262]. Table 13 shows a
summary of its specifications and methods of analysis.

4.1.3 Chewable Calcium Carbonate Tablets

Chewable CaCO3 tablets monograph is listed in the British Pharmacopeia
(BP) [263]. Table 14 shows a summary of its specifications and methods of
analysis.
Combination of CaCO3 with heavy MgCO3 in chewable tablets, as ant-
acid, is also listed in BP [264].

4.1.4 Calcium Carbonate Oral Suspension

CaCO3 oral suspension monograph is listed in the USP-NF [265]. Table 15
shows a summary of its specifications and methods of analysis.

4.1.5 Calcium Carbonate Lozenges

CaCO3 lozenges monograph is listed in the USP-NF [266]. Table 16 shows
a summary of its specifications and methods of analysis.
Different combinations with aluminum hydroxide and magnesium
hydroxide [267], with magnesium hydroxide and simethicone [268], with
magnesium hydroxide [269], with aluminum hydroxide, magnesium
hydroxide and simethicone [270], and with magnesium carbonate
[271,272] monographs are listed in USP.
Table 12 The Summary of the Compendial Methods of CaCO3
Test Eur. Ph. USP-NF JP

Definition CaCO3 CaCO3 Precipitated CaCO3

98.5100.5% (dried substances)
Characters A white or almost white powder. It is practically
insoluble in water
Identification It gives the reaction of carbonates
A (carbonate) (see Section 2.3.1)
Identification 0.2 mL of solution S gives the reactions of Ca
B (Ca) (see Section 2.3.1)
Solution S: Dissolve 5.0 g in 80 mL of dilute
acetic acid R. When the effervescence ceases,
boil the solution for 2 min, allow to cool, dilute
to 100 mL with dilute acetic acid R and filter, if
necessary, through a sintered-glass filter (see
Section 2.1.2)
Insoluble Wash any residue obtained during the
substances preparation of solution S with four quantities,
each of 5 mL, of hot water R, and dry at 100
105C for 1 h (NMT 0.2%)
98.5100.5% (dried substances)
A fine, white, odorless, tasteless, microcrystalline powder.
It is stable in air. Practically insoluble in water. Its solubility
in water is increased by the presence of ammonium salt or
of CO2. The presence of any alkali hydroxide reduces its
solubility. Insoluble in alcohol. Dissolves with
effervescence in 1 N acetic acid, in 3 N HCl, and in 2 N
HNO3. NF category: Diluent; pH modifier (acidifying
agent/alkalizing agent/buffering agent); coating agent;
wet binder
The addition of acetic acid to it produces effervescence
The resulting solution in identification A, after boiling,
meets the requirements of Ca test h191i
Mix 5.0 g with 10 mL water and add HCl, dropwise, with
agitation, until it ceases to cause effervescence, then add
water to make 200 mL, and filter. Wash the insoluble
residue with water until the last washing shows no
chloride, and ignite and weigh the residue (NMT 0.2%)
Not less than 98.5% (dried substances)
A white, fine crystalline powder. It is
odorless and tasteless. It is practically
insoluble in water, but its solubility is
increased in the presence of CO2. It is
practically insoluble in ethanol (95%) and
in diethyl ether. It dissolves with
effervescence in dilute acetic acid, in
dilute HCl, and in dilute HNO3
It responds to the Quantitative test h1.09i
for carbonate
Dissolve 0.5 g in 10 mL of dilute HCl,
boil, then cool, and neutralize with
ammonia TS (the solution responds to
the Quantitative test h1.09i (1) for
carbonate.)
To 5.0 g add 50 mL of water, then 20 mL
of HCl dropwise with stirring, boil for
5 min, cool, add water to make 200 mL,
and filter. With filter paper for
quantitative analysis. Wash the residue
until the last washing shows no turbidity
with silver nitrate TS, and ignite the
residue together with the filter paper and
weigh (NMT 0.2%)
Chloride Dilute 3 mL of solution S to 15 mL with
distilled water R (see Section 2.4.4) (NMT
330 ppm)
Sulfates 1.2 mL of solution S diluted to 15 mL with
distilled water R complies with the limit test for
sulfates (see Section 2.4.13) (NMT 0.25%)
Arsenic 5 mL of solution S (see Section 2.4.2,
Method A) (NMT 4 ppm)
Barium To 10 mL of solution S add 10 mL of calcium
sulfate solution R. After at least 15 min (any
opalescence in the solution is not more intense
than that in a mixture of 10 mL of solution S and
10 mL of distilled water R)
Iron Dissolve 50 mg in 5 mL of dilute HCl R and
dilute to 10 mL with water R (see Section 2.4.9)
(NMT 200 ppm)
Lead
Slowly dissolve 1.0 g in 15 mL of HCl and dilute with Moisten 0.40 g with 1 mL of water, then
water to 55 mL (arsenic, method I, omit the addition of dissolve in 4 mL of dilute HCl, use this
20 mL of 7 N H2SO4) (NMT 3 ppm) solution as the test solution, and perform
the Arsenic test h1.11i (NMT 5 ppm)
A platinum wire, dipped in the filtrate obtained in the test Mix 1.0 g with 10 mL of water, add
for insoluble substances and held in a nonluminous flame dropwise 4 mL of HCl with stirring, boil
(does not impart a green color) for 5 min, cool, add water to make
40 mL, and filter. With the filtrate,
perform the Flame Coloration test h1.04i
(1) (no green color appears)
40 mg in 5 mL of 2 N HCl. Transfer to a beaker with the
aid of water and dilute with water to 10 mL (sample
solution). Transfer 4.0 mL of the standard iron solution
h241i to a beaker and dilute with water to 10 mL (standard
solution). Add separately to the sample solution and
standard solution 2 mL of citric acid solution (1 in 5) and 2
drops of thioglycolic acid, adjust with ammonia TS to a pH
of 9.5  0.1, dilute with water to 20 mL, and allow to stand
for 5 min. Dilute with water to 50 mL. Measure the
absorbances of the solutions from the sample solution and
the standard solution at 530 nm h851i (NMT 0.1%; the
absorbance of the solutions from the sample solution does
not exceed that of the standard solution)
To 1.0 g in 5 mL of water, slowly add 8 mL of 3 N HCl,
evaporate on a steam bath to dryness, and dissolve the
residue in 5 mL of water h251i (NMT 3 ppm)
Continued
Table 12 The Summary of the Compendial Methods of CaCO3cont'd
Test Eur. Ph.
Magnesium Dissolve 1.0 g in 12 mL of dilute HCl R. Boil
and alkali the solution for about 2 min and add 20 mL of
metals water R, 1 g of ammonium chloride R and
0.1 mL of methyl red solution R. Add dilute
ammonia R1 until the color of the indicator
changes and then 2 mL in excess. Heat to
boiling and add 50 mL of hot ammonium
oxalate solution R. Allow to stand for 4 h, dilute
to 100 mL with water R, and filter through a
suitable filter. To 50 mL of the filtrate add
0.25 mL of H2SO4 R. Evaporate to dryness on a
water bath and ignite to constant mass at 600C.
The residue weighs not more than 7.5 mg
(NMT 1.5%)
Heavy metals 12 mL of solution S complies with limit test A
for heavy metals. Prepare the standard using lead
standard solution (1 ppm Pb) R
(see Section 2.4.8, Test A) (NMT 20 ppm)
USP-NF
Mix 1.0 g with 35 mL of water. Carefully add 3 mL of
HCl, heat the solution, and boil for 1 min. Rapidly add
40 mL of oxalic acid TS and stir vigorously until
precipitation is well established. Add immediately to the
warm mixture 2 drops of methyl red TS and then 6 N
ammonium hydroxide, dropwise, until the mixture is just
alkaline. Cool to room temperature, transfer to a 100-mL
graduated cylinder, dilute with water to 100 mL, mix, and
allow to stand for 4 h or overnight. Filter, and to 50 mL of
the clear filtrate in a platinum dish add 0.5 mL of H2SO4,
and evaporate the mixture on a steam bath to a small
volume. Carefully heat over a free flame to dryness and
continue heating to complete decomposition and
volatilization of ammonium salts. Finally, ignite the
residue to constant weight (NMT 1.0%; the weight of the
residue is NMT 5 mg)
Mix 1.0 g with 5 mL of water, slowly add 8 mL of 3 N
HCl, and evaporate on a steam bath to dryness. Dissolve
the residue in 20 mL of water, filter, and add water to the
filtrate to make 25 mL h231i (NMT 20 ppm)
JP
Dissolve 1.0 g in 20 mL of water and
10 mL dilute HCl, boil, neutralize with
ammonia TS, and add ammonium
oxalate TS until precipitation of calcium
oxalate is completed. Heat the mixture
on a water bath for 1 h, cool, dilute with
water to 100 mL, shake well, and filter.
To 50 mL of the filtrate add 0.5 mL of
H2SO4, evaporate to dryness, and ignite
at 600C to constant mass (the mass of the
residue is not more than 5 mg)
Mix 2.0 g with 5 mL of water, slowly add
6 mL of dilute HCl, and evaporate on a
water bath to dryness. Dissolve the
residue in 50 mL of water, and filter. To
25 mL of the filtrate add 2 mL of dilute
acetic acid, 1 drop of ammonia TS and
water to make 50 mL, and perform the
test using this solution as the test solution.
Prepare the control solution as follows:
evaporate 3 mL of HCl on a water bath
to dryness, add 2 mL of dilute acetic acid,
2.0 mL of standard lead solution and
water to make 50 mL h1.07i
(NMT 20 ppm)
Mercury Mercury stock solution and Standard Mercury solution: Proceed
as directed in Mercury h261i
Standard solution: Proceed as directed in Mercury h261i,
except use 3 mL of HCl instead of 3 mL of H2SO4
Sample stock solution: 4.0 g in a 100-mL beaker, and
cautiously dissolve in 14 mL of 6 N HCl
Sample solution: Proceed as directed in Mercury h261i
using the Sample stock solution, except use 3 mL of HCl
instead of 3 mL of H2SO4
Analysis samples: Standard solution and sample solution
proceed as directed in Mercury h261i method IIa
(NMT 0.5 ppm)
Fluoride [Prepare and store all solutions in plastic containers]
Solution A: 294 mg/mL of sodium citrate dentate in water
Standard solution: Combine 20.0 mL of the standard stock
solution (1.11 mg/mL of USP NaF RS in water) with
50.0 mL of solution A, and dilute with water to 100.0 mL
Electrode system: Use a fluoride-specific ionindicating an
electrode and a silversilver chloride reference electrode
connected to a pH meter capable of measuring potentials
with a minimum reproducibility of 0.2 mV h791i
Standard response line: Transfer 50.0 mL of solution A and
4.0 mL of HCl to a beaker, and add water to make
100 mL. Add a plastic-coated stirring bar, insert the
electrodes into the solution, stir for 15 min, and read the
potential (mV). Continue stirring, and at 5-min intervals
add 100, 100, 300, and 500 L of the standard solution,
reading the potential 5 min after each addition. Plot
the logarithms of the cumulative fluoride ion
concentrations (0.1, 0.2, 0.5, and 1.0 g/mL)
vs potential (mV)
Analysis: Transfer 2.0 g to a beaker containing
a plastic-coated stirring bar, add 20 mL of
water and 4.0 mL of HCl, and stir until dissolved.
Continued
Table 12 The Summary of the Compendial Methods of CaCO3cont'd
Test Eur. Ph. USP-NF JP

Add 50.0 mL of solution A and sufficient water to make

Loss on Determined on 1.000 g by drying in an oven at
drying 200C (NMT 2.0%)
Assay Dissolve 0.150 g in a mixture of 3 mL of dilute
HCl R and 20 mL of water R. Boil for 2 min,
allow to cool, and dilute to 50 mL with water R.
Carry out the complexometric titration of Ca.
1 mL of 0.1 M EDTA is equivalent to 10.01 mg
of CaCO3
Storage
100 mL of test solution. Rinse and dry the electrodes,
insert them into the sample solution, stir for 5 min, and
read the potential (mV). From the standard response line
(NMT 50 ppm)
Dry the sample at 200C for 4 h h731i (NMT 2.0%)
Transfer 0.200 g, previously dried at 200C for 4 h to a
250-mL beaker. Moisten thoroughly with a few milliliters
of water, and add, dropwise, sufficient 3 N HCl to
dissolve. Add 100 mL of water, 15 mL of 1 N NaOH, and
300 mg of naphthol blue. Titrate with 0.05 M EDTA VS
to change the color to distinct blue h541i. 1 mL of 0.05 M
EDTA disodium is equivalent to 5.005 mg of CaCO3
Preserve in well-closed containers
Dry the sample at 180C for 4 h h2.41i
(NMT 1.0%)
Transfer 0.12 g, previously dried, and
dissolve in 20 mL of water and 3 mL of
dilute HCl. Add 80 mL of water, 15 mL
of KOH (1 in 10), and 0.05 g of NN
indicator, and titrate with 0.05 mol/L
EDTA VS to change the color from red-
purple to blue h2.50i. 1 mL of 0.05 mol/
L EDTA is equivalent to 5.004 mg of
CaCO3
Tight containers
Calcium Carbonate 85

Table 13 The Summary of the USP-NF Compendial Methods of CaCO3 Tablets

Test USP-NF
Definition CaCO3 tablets contain NLT 90.0% and NMT 110.0% of the
labeled amount of CaCO3. For tablets labeled for any indication
other than, or in addition to, antacid use, the tablets contain NLT
90.0% and NMT 115.0% of the labeled amount of CaCO3
Identification Ca h191i: The addition of 6 N acetic acid to the tablets produces
effervescence, and the resulting solution, after being boiled to
expel CO2 and neutralized with 6 N ammonium hydroxide,
meets the requirements
Assay Sample solution: Finely powder NLT 20 tablets. Transfer a portion
of the powder, equivalent to 200 mg of CaCO3, to a suitable
crucible. Ignite to constant weight. Cool the crucible, add 10 mL
of water, and dissolve the residue by adding sufficient 3 N HCl,
dropwise, to achieve complete solution
Blank: 150 mL of water and 15 mL of 1 N NaOH
Titrimetric system (see Titrimetry h541i)
Mode: Direct titration
Titrant: 0.05 M EDTA VS
Indicator: 300 mg of hydroxy naphthol blue
Endpoint detection: Visual, change to distinct blue
Analysis: Transfer the sample solution completely to a suitable
container and dilute with water to 150 mL. Add 15 mL of
1 N NaOH and 300 mg of hydroxy naphthol blue. Titrate
with the titrant
Calculation: Calculate the percentage of CaCO3 in the sample
taken:
Result [(VS
VB)  M  F  100]/W. VS volume of the
titrant consumed by the sample solution (mL). VB volume
of the titrant consumed by the blank (mL). M titrant molarity
(mmol/mL). F equivalency factor, 100.09 mg/mmol.
W weight of CaCO3 taken (mg)
Dissolution [Note: For tablets labeled for any indication other than, or
h711i in addition to, antacid use]
Medium: 0.1 N HCl; 900 mL, Apparatus 2: 75 rpm, time: 30 min
Lanthanum chloride solution: 50 mg/mL of lanthanum chloride
in 0.1 N HCl
Standard stock solution: 100 g/mL of Ca in 0.1 N HCl
Standard solutions: Into separate 100-mL volumetric flasks
containing 10.0 mL of lanthanum chloride solution, pipet 3-, 4-,
5-, and 6-mL portions of standard stock solution and dilute each
with 0.1 N HCl to volume to obtain solutions with Ca
concentrations of 3, 4, 5, and 6 g/mL, respectively
Continued
86 M.M.H. Al Omari et al.

Table 13 The Summary of the USP-NF Compendial Methods of CaCO3 Tabletscont'd

Test USP-NF
Sample solution: Filter a portion of the solution under test. Pipet a
volume of the filtrate, estimated to contain 1 mg of Ca, into a
250-mL volumetric flask. Add 25.0 mL of lanthanum chloride
solution and dilute with 0.1 N HCl to volume
Instrumental conditions: (See Spectrophotometry and Light
Scattering h851i)
Mode: Atomic absorption spectrophotometry
Analytical wavelength: 422.8 nm
Lamp: Ca hollow cathode
Flame: Air-acetylene
Blank: Lanthanum chloride solution and 0.1 N HCl (1:9)
Samples: Standard solutions and sample solution
Concomitantly determine the absorbances of the standard
solutions and the sample solution against the blank. Construct
a standard curve by plotting absorbances vs Ca concentrations
of the standard solutions, then from it obtain the
concentration, C (g/mL) of Ca, of the sample solution
Calculation: Calculate the percentage of the labeled amount of
CaCO3 (CaCO3) dissolved:
Result (Mr/Ar)  (C  D  V/L)  100. Mr molecular
weight of CaCO3, 100.09. Ar atomic weight of Ca, 40.08.
C measured concentration of Ca in the sample solution
(mg/mL). D dilution factor for the sample solution.
V volume of medium, 900 mL. L label claim (mg/tablet)
Tolerances: NLT 75% (Q) of the labeled amount of CaCO3
(CaCO3) is dissolved
Acid- [For tablets labeled for antacid use]
neutralizing Analysis: Proceed as directed in the chapter h301i
capacity h301i Acceptance criteria: NLT 5 mequiv. of acid is consumed by the
minimum single dose recommended in the labeling, and NLT
the number of mequiv. calculated as follows:
Result (C  ANC)  F. C quantity of CaCO3 in the sample
(mg), based on the labeled amount. ANC theoretical
acid-neutralizing capacity of CaCO3, 0.02 mequiv./mg.
F acceptance factor for the lower limit of the required
acid-neutralizing capacity, 0.9
Uniformity Meets the requirements
of dosage units
h905i
Storage Preserve in well-closed containers
Calcium Carbonate 87

Table 14 The Summary of the BP Compendial Methods of Chewable CaCO3 Tablets

Test BP
Definition Chewable CaCO3 tablets contain NLT 95.0% and NMT 105.0%
of the stated amount of Ca. The tablets may be flavored. They
comply with the general requirements stated under tablets
Identification A. Ca: The powdered tablets yield reaction B characteristic
of Ca salts
B. The powdered tablets yield the reactions characteristic
of carbonates
Disintegration The requirement for disintegration does not apply to chewable
CaCO3 tablets
Assay Weigh and powder 20 tablets. To a quantity of the powder
containing the equivalent of 0.25 g of Ca, add 50 mL of water
and 5 mL of HCl. Heat the dispersion gently to boiling and
continue to boil for about 2 min. Allow to cool, add sufficient water
to produce 250 mL, and filter, if necessary. To 50 mL of this
solution add 50 mL of 0.05 M EDTA VS. Neutralize the solution
using 2 N NaOH and add 10 mL of ammonia buffer pH 10.9 and
50 mL of water. Titrate the excess of EDTA with 0.05 M zinc
chloride VS using mordant black 11 solution as indicator. 1 mL
of 0.05 M EDTA is equivalent to 2.004 mg of Ca
Storage Chewable CaCO3 tablets should be protected from moisture

The analytical methods reported for Ca and carbonate ions, summarized

in the following sections, are not necessary used for CaCO3, but they are
general methods used for measuring Ca and carbonate individually. It is
worth mentioning that these methods may require some verification prior
to their use for analyses of CaCO3 (eg, sample treatment, using suitable sol-
vents, pH adjustment, and changes in method parameters).

4.2 Titrimetric Methods

4.2.1 Aqueous AcidBase
About 500 mg of CaCO3, accurately weighed, is dissolved in exactly 50 mL
of 0.5 N HCl VS and gently swirled. The sample was heated gently to boil
for exactly 5 min to remove the H2CO3. The sample was then removed
from the heat and allowed to cool. Then, about two to three drops of phe-
nolphthalein indicator solution and 75 mL of water were added and swirled.
Then, the exact amount of CaCO3 was calculated using back titration with
0.5 N NaOH VS. Each milliliter of 0.5 N HCl is equivalent to 25.02 mg of
CaCO3 [273].
88 M.M.H. Al Omari et al.

Table 15 The Summary of the USP-NF Compendial Methods of CaCO3 Oral

Suspension
Test USP-NF
Definition CaCO3 oral suspension contains NLT 90.0% and NMT 110.0%
of the labeled amount of CaCO3
Identification Ca h191i: The addition of acetic acid to it produces effervescence
(presence of carbonate). The resulting solution, after boiling,
meets the requirements
Assay Sample solution: Transfer a portion of oral suspension, equivalent
to 1 g of CaCO3, previously well shaken in its original container,
to a beaker with the aid of 25 mL of water. Add 20 mL of 1 N
HCl. Heat on a steam bath for 30 min. Allow to cool, and transfer
with the aid of water to a 100-mL volumetric flask. Dilute with
water to volume. Mix and filter
Blank: 100 mL of water, 15 mL of 1 N NaOH, and 5 mL of
triethanolamine
Titrimetric system (see Titrimetry h541i)
Mode: Direct titration
Titrant: 0.05 M EDTA VS
Indicator: 100 mg of hydroxy naphthol blue
Endpoint detection: Visual, change to distinct blue
Analysis: Transfer 20.0 mL of the sample solution to a suitable
container. Dilute with water to 100 mL. Add 15 mL of 1 N
NaOH, 5 mL of triethanolamine, and 100 mg of hydroxy
naphthol blue. Titrate with the titrant
Calculation: Calculate the percentage of the labeled amount of
CaCO3 in the sample taken:
Result [(VS
VB)  M  F  100]/W. VS volume of the
titrant consumed by the sample solution (mL). VB volume of
the titrant consumed by the blank (mL). M titrant molarity
(mmol/mL). F equivalency factor, 100.09 mg/mmol.
W nominal amount of CaCO3 taken
Fluoride [Note: Prepare and store all solutions in plastic containers]
Solution A: 294 mg/mL of sodium citrate dihydrate in water
Standard stock solution: 1.1 mg/mL of USP sodium fluoride RS in
water
Standard solution: Combine 20.0 mL of the standard stock
solution with 50.0 mL of solution A, and dilute with water to
100.0 mL. [Note: Each milliliter of this solution contains 100 g
of fluoride ion]
Sample solution: Transfer a portion of oral suspension, equivalent
to 2.0 g of CaCO3, to a beaker containing a plastic-coated
stirring bar. Add 20 mL of water and 4.0 mL of HCl. Stir until
Calcium Carbonate 89

Table 15 The Summary of the USP-NF Compendial Methods of CaCO3 Oral

Suspensioncont'd
Test USP-NF
dissolved. Add 50.0 mL of solution A and sufficient water to
make 100.0 mL.
Electrode system: Use a fluoride-specific ionindicating an
electrode and a silversilver chloride reference electrode
connected to a pH meter capable of measuring potentials with a
minimum reproducibility of 0.2 mV (see pH h791i)
Standard response line: Transfer 50.0 mL of solution A and 4.0 mL
of HCl to a beaker. Add water to make 100.0 mL. Add a
plastic-coated stirring bar, insert the electrodes into the solution,
and stir for 15 min. Read the potential (mV). Continue stirring,
and at 5-min intervals add 100, 100, 300, and 500 L of the
standard solution, reading the potential 5 min after each addition.
Plot the logarithms of the cumulative fluoride ion concentrations
(0.1, 0.2, 0.5, and 1.0 g/mL) vs potential (mV)
Analysis: Rinse and dry the electrodes, and insert them into
the sample solution. Stir for 5 min and read the potential (mV).
From the measured potential and the standard response line,
determine the concentration, C (g/mL), of fluoride ion in the
sample solution.
Calculation: Calculate the content of fluoride in the sample taken:
Result (V  C)/W. V volume of the sample solution (mL).
C determined concentration of fluoride in the sample solution
(g/mL). W nominal weight of CaCO3 taken (g)
Acceptance criteria: 50 g/g, with respect to the labeled amount
of CaCO3
Arsenic h211i, Test preparation: Slowly dissolve a portion of oral suspension
Method I equivalent to 1.0 g of CaCO3 in 15 mL of HCl. Dilute with
water to 55 mL
Analysis: Proceed as directed in the chapter h211i, except omit
the addition of 20 mL of 7 N H2SO4 specified under Procedure
Acceptance criteria: NMT 3 g/g, with respect to the labeled
amount of CaCO3
Lead h251i Test preparation: Mix a portion of oral suspension equivalent
to 1.0 g of CaCO3 in 5 mL of water
Analysis: To the test preparation slowly add 8 mL of 3 N HCl.
Evaporate on a steam bath to dryness, and dissolve the residue
in 5 mL of water
Acceptance criteria: NMT 3 g/g, with respect to the labeled
amount of CaCO3
Continued
90 M.M.H. Al Omari et al.

Table 15 The Summary of the USP-NF Compendial Methods of CaCO3 Oral

Suspensioncont'd
Test USP-NF
Heavy metal Test preparation: Mix a portion of oral suspension equivalent
h231i to 1.0 g of CaCO3 with 5 mL of water. Slowly add 8 mL of 3 N
HCl, and evaporate on a steam bath to dryness. Dissolve the
residue in 20 mL of water. Filter and add water to the filtrate
to make 25 mL.
Acceptance criteria: NMT 20 g/g, with respect to the labeled
amount of CaCO3
Microbial The total aerobic microbial count is NMT 102 cfu/mL. It meets
enumeration the requirements of the tests for the absence of Escherichia coli
tests h61i and and Pseudomonas aeruginosa
tests for specified
microorganisms
h62i
pH 7.58.7 h791i
Storage Preserve in tight containers, and avoid freezing

Previously, back-titration technique has been used for the analysis of Ca

in the presence of strontium in blood serum [274], Mg, phosphate, and sul-
fate in urine [275]. Pretreatment of the samples was done by precipitating Ca
as the oxalate, followed by converting to the carbonate or oxide by heating.
The content of the later is then determined by back titration using methyl
red as indicator. In the presence of relatively large amounts of Mg, calcium
oxalates are isolated by double precipitation instead of a single precipitation
to reduce the interference [275].
Bundy and Bremner described a simple titration method of determining
inorganic carbon in soils [276]. The sample is treated with 2 N HCl at room
temperature for 1624 h in a stoppered bottle containing 2 N KOH in a
small beaker, and the CO2 released from carbonates is determined by titra-
tion of the KOH solution with standard HCl. El Mahi et al. reported that the
acid neutralization method suffers from the reaction of the acid with soil
constituent other than carbonates and the consumption of protons by the
exchange complex. The latter error was corrected by assuming that protons
occupied the entire exchange complex [277,278]. The values of carbonate
equivalent estimated by acid neutralization were corrected for cation
exchange capacity (CEC) as
CaCO3 equivalent acid neutralization % CaCO3
0:05 CEC
Calcium Carbonate 91

Table 16 The Summary of the USP-NF Compendial Methods of CaCO3

Lozenges
Test USP-NF
Definition CaCO3 lozenges contain NLT 90.0% and NMT 110.0% of the
labeled amount of CaCO3
Identification Ca h191i: The addition of 6 N HCl to a lozenge produces
effervescence, and the resulting solution, after being boiled to
expel CO2 and then neutralized with 6 N ammonium
hydroxide, meets the requirements of the tests
Assay [Note: The standard solutions and the sample solution may be
modified, if necessary, to obtain solutions of suitable
concentrations adaptable to the linear or working range of the
instrument]
Lanthanum chloride solution: Transfer 10 g of potassium chloride
and 20 g of lanthanum chloride to a 2000-mL volumetric flask.
Add 1000 mL of water and 40 mL of HCl, mix, and allow to
cool. Dilute with water to volume
Standard stock solution: Transfer 250 mg of chelometric standard
CaCO3, previously dried at 110C for 2 h and then cooled in
a desiccator, to a 500-mL volumetric flask. Add 100 mL of
water and 12 mL of 1 N HCl, swirl to dissolve the CaCO3, and
allow to cool. Dilute with water to volume. This stock
solution contains about 500 g/mL of CaCO3
Standard solutions: To three separate 100-mL volumetric flasks
add 2.0, 3.0, and 4.0 mL of the standard stock solution, and dilute
each with lanthanum chloride solution to volume. These
standard solutions contain 10, 15, and 20 g/mL of CaCO3,
respectively
Sample stock solution: Transfer the equivalent to 3000 mg of
CaCO3, from powdered lozenges, to a 1000-mL volumetric
flask. Add 100 mL of 1 N HCl and 300 mL of water, and sonicate
to dissolve the powder. Dilute with water to volume
Sample solution: Transfer 5.0 mL of sample stock solution to a
1000-mL volumetric flask, and dilute with lanthanum chloride
solution to volume
Instrumental conditions: (See Spectrophotometry and Light
Scattering h851i) Mode: Atomic absorption spectrophotometry
Lamp: Ca hollow cathode, Flame: Nitrous oxide-acetylene,
Analytical wavelength: Ca emission line at 422.7 nm
Blank: Lanthanum chloride solution
Samples: Standard solutions, sample solution, and blank
Continued
92 M.M.H. Al Omari et al.

Table 16 The Summary of the USP-NF Compendial Methods of CaCO3

Lozengescont'd
Test USP-NF
Plot the absorbances of the standard solutions vs their
concentrations of CaCO3 (g/mL), by drawing a straight line
best fitting the three plotted points. From the graph determine
the concentration, C (g/mL), of CaCO3 in the sample
solution.
Calculation: Calculate the percentage of label claim of CaCO3 in
the portion of lozenges taken:
Result (C/CU)  100. C and CU measured and nominal
concentrations of CaCO3 in the sample solution (g/mL)
Sodium Standard stock solution: Transfer 2.542 g of sodium chloride,
(If so labeled) previously dried at 105C for 2 h, to a 1000-mL volumetric flask.
Dissolve in and dilute with water to volume. Transfer 10.0 mL
of this solution to a 100-mL volumetric flask, and dilute with
water to volume
Standard solutions: To three separate 100-mL volumetric flasks,
add 1.0, 3.0, and 5.0 mL of the standard stock solution, and dilute
each with water to volume. These standard solutions contain 1.0,
3.0, and 5.0 g/mL of sodium, respectively
Sample stock solution: Prepare as directed in the Assay. Pass a
portion of it, if necessary, through a filter of 0.5 m or finer
pore size, and use the clear solution
Sample solution: Transfer 10.0 mL of the sample stock solution
to a 25-mL volumetric flask, and dilute with water to volume
Instrumental conditions: (See Spectrophotometry and Light
Scattering h851i)
Mode: Atomic absorption spectrophotometry, Lamp: Sodium
hollow cathode, Flame: Air-acetylene, Analytical wavelength:
Sodium emission line at 589.6 nm, Blank: Water
Samples: Standard solutions, sample solution, and blank
Plot the absorbances of the standard solutions vs their contents
of sodium (g/mL) by drawing a straight line best fitting the
three plotted points. From the graph determine the quantity,
C (g), of sodium in each milliliter of the sample solution
Calculation: Calculate the percentage of label claim of sodium in
the portion of lozenges taken:
Result (C/CU)  100. C measured concentration of sodium
in the sample solution (g/mL), as calculated earlier.
CU nominal concentration of sodium in the sample solution
(g/mL)
Acceptance criteria: NMT 115.0% of the labeled amount
Calcium Carbonate 93

Table 16 The Summary of the USP-NF Compendial Methods of CaCO3

Lozengescont'd
Test USP-NF
Uniformity Meet the requirements
of dosage units
h905i
Acid- Analysis: The acid consumed by the minimum single dose
neutralizing recommended in the labeling is NLT 5 mequiv. of acid and NLT
capacity h301i the number of mequiv. calculated by: Result (FC  C)  0.9.
FC theoretical acid-neutralizing capacity of CaCO3,
0.02 mequiv. C quantity of CaCO3 in the sample tested (mg),
based on the labeled quantity
Storage Preserve in well-closed containers

Maulood et al. used back titration for CaCO3 determination in soil sam-
ples using 0.5 N HCl to dissolve the samples, followed by back titration with
0.2 N NaOH [279]. The results were found to be comparable with those
obtained using calcimetric method.

4.2.2 Complexometry
Ethylenediaminetetraacetic acid (EDTA) is used as a complexing agent to
determine CaCO3 in its pure form [1,2,216] or in different dosage forms
including tablets [262], chewable tablets [263], and oral suspension [265].
Full detailed procedures are mentioned in Tables 1215.
Previously, micro- and macrodeterminations of serum Ca by direct titra-
tion with EDTA with ammonium purpurate as the indicator [280,281]. The
end point is determined by changing the indicator color to purple [280] or
graphically from spectrophotometric readings at 620 nm taken during the
titration [281]. Also Beale and Bostrom used a microtitration of Ca in the
presence of Mg in serum and urine, with EDTA as titrant and Corinth
Ca (Plasmocorinth B) as indicator [282].
Garvey et al. analyzed Ca in dietary supplements using complexometric
titration with EDTA and then, following ion exchange of the Ca ion present
for hydronium ion, by acidbase titration with NaOH [283]. Also statistical
comparison of both methods was adopted.
94 M.M.H. Al Omari et al.

4.2.3 Oxidometry
In direct oxidometric determination of Ca in solution containing also Mg,
phosphates and small amounts of Fe, or in the ash of food or feces has been
reported [284]. The method is based upon the formation of calcium oxalate,
followed by immediate titration of oxalate in acid media with standard
K2MnO4. The microdetermination of Ca in whole blood, plasma, serum,
urine, and stools was also performed by direct precipitation of calcium oxa-
late and then titration with 0.01 N K2MnO4 [285,286]. Furthermore, an
improvement in the method by using new washing solution for calcium
oxalate precipitation of 2% NH3 in equal parts of alcohol, ether, and water
is introduced to prevent flotation and permit washing of the precipitate
without appreciable loss of Ca [287].

4.2.4 Amperometry
Indirect amperometric titration of Ca using dropping mercury electrode has
been reported [288]. The method is based upon the precipitation of Ca as
picrolonate and followed by back titration of excess picrolonate with meth-
ylene blue.

4.2.5 Spectrophotometry
Sweetser and Bricker were the first to use spectrophotometric measurements
to determine the end points of EDTA titrations, which they applied to Ca
and Mg in two stages of analysis [289].

4.2.6 Coulometry
Caughey and Barcelona used this technique for the determination of total
inorganic carbon (TIC) using 2 N HClO4 with the UIC 5130/5011 mod-
ules coulometer [290].
Coulometry is still used, more than 25 years later, by the Integrated
Ocean Drilling Program for shipboard analysis of CaCO3 concentration
[291]. M orth and Backman described a practical approach for acquiring
accurate measurements of the carbonate content in sediments by using an
UIC Inc. coulometer [292]. The coulometer readings are absolute; ie, the
total amount of available carbon is converted by HCl to CO2 gas and the
output reading (counts) of the instrument is in g C. In this work, they
investigated the effect of sample weight, sampling tool, preparation proce-
dure, and use of a multipoint regression analysis on the precision of the
method.
Calcium Carbonate 95

Dabke et al. used the coulometric back-titration method for the deter-
mination of CaCO3 in antacid tablets [293]. The sample was dissolved in
excess acid and the remaining acid was back titrated against the coulomet-
rically generated OH ions. The amount of acid neutralized by CaCO3 was
determined from the difference in the anodic and cathodic charge.

4.3 Gravimetric Method

Determination of Ca in solution containing also Mg, phosphates, and small
amounts of Fe, or in the ash of food or feces, has been reported [284]. The
method is based upon the formation of calcium oxalate, followed by burning
in a platinum crucible to CaO and brought to constant weight by heating in
a blast lamp.
Another direct gravimetric analysis of Ca has been also reported by
complete precipitation at pH 3 as calcium oxalate at pH 3, followed by
simple drying of the precipitate at 105C and weighing [294]. Another
approach for quantitative determination of CaCO3 is based on the reac-
tion of HCl with carbonate and then the loss of CO2 is measured gravimet-
rically [295].

4.4 Spectroscopic Methods

4.4.1 UV/VIS Spectrophotometry
Nangare described direct UV/VIS method for simultaneous determination
of CaCO3 and aspirin in tablet dosage form [248]. The determination is
based on the use of simultaneous equations and Q-absorbance ratio method
by using 240 and 230 nm as absorbance maxima for CaCO3 and aspirin,
respectively, and 290.5 nm (isoabsorptive point). A 0.1 M NaOH was used
as solvent. Linearity was observed in the concentration range of 224 g/mL
for CaCO3 and 525 g/mL for aspirin.
Direct spectrophotometric method for the measurement of CaCO3 sat-
uration states in seawater has been reported [296,297]. Easley et al. used the
measurements of pH and carbonate ion concentrations in the seawater to
determine CaCO3 saturation states. The spectrophotometric method is
based upon the measurement of the ratio of absorbances at 250 and
234 nm using Pb(II) as a complexing agent [297].

4.4.2 Colorimetry
An indirect colorimetric method for measuring blood Ca has been devel-
oped, which is based upon the precipitation of Ca as phosphate, and the
determination of the latter by the MoO3 colorimetric procedure [298].
96 M.M.H. Al Omari et al.

Further improvement of the proposed method, microlevel determination,

and minimizing the interference of other cations such as Mg were carried
out [299].
A colorimetric method using N-hydroxy-naphthalene-1,8-dicarboxylic
acid imide in the presence of ethylenediaminetetraacetic acid has been
developed for convenient and rapid estimation of 515 g Ca in 0.1 mL
of blood serum at wavelength of 338 nm [300]. Tartaric acid, low concen-
trations of citric acid, phosphate, Mg, zinc, and ferrous or ferric ions will not
interfere in the determinations of Ca. However, higher concentrations of
citric acid as well as the presence of Mn, Sr, and Ba must be avoided.
Radin and Gramza described a colorimetric method using Eriochrome
Blue SE to measure the concentration of Ca in serum and urine [301]. The
study indicated that at pH value above 13.7, Ca will complex and cause a
change of dye absorbance, while Mg does not complex with the dye.
Prokopov described a direct colorimetric method for determination of
Ca in the range of 2 mg to 0.002 mg/mL by using sodium rhodizonate as
reagent. The proposed procedure is not laborious and easily eliminates all
interfering ions [302]. A modified flow-through colorimeter for the deter-
mination of Ca, Mg, and phosphate in a pmol level has been described by
Adkinson and Evans [303]. Methylthymol and 8-quinolinol, which is the
Mg-complexing agent, polylectrolite, and a monoethanolaminesodium
sulfite buffer were used in the analysis of Ca.
Blanco et al. have described a simultaneous flow injection spectrophoto-
metric method for Ca and Mg with Arsenazo III based on the use of diode-
array detector and merging zones [304]. Quantitation is based on the normal
absorbance and first-derivative absorbance spectra. The method is applied to
0.21.5 g/mL for Ca and 0.11.0 g/mL for Mg. Another simultaneous
determination of Ca and Mg in different types of water by colorimetric
method has been reported [305]. The determination is based on the formation
or their complexes with 4-(2-pyridylazo)resorcinol and measuring the absor-
bances over the wavelength range 470650 nm (max 490 and 502 nm for
Mg and Ca, respectively). To minimize the overlapping, the absorbance band
yielded by the mixture was resolved by applying a computer multilinear
regression program to the corrected, standardized spectrum of each metal
ion as standard. The proposed method is straightforward and rapid and
provides a linear determination range of 0.104.0 g/mL for Ca and 0.15
2.5 g/mL for Mg. Shanahan and Kapustin measured the level of Ca in blood
samples by using o-cresolphthalein as complexing agent. The Ca concentra-
tion is quantified by measuring the absorbance at 450 nm [306].
Calcium Carbonate 97

Colorimetric methods for determination of Ca and Mg in mineral water

[307] and pharmaceuticals [308] were proposed. The determination in both
methods is based on the blue color developed by the reaction of Ca and Mg
with methylthymol blue (MTB) at pH 11. The first proposed method used
the multivariate partial least-squares regression and adapted as a portable
(static mode) or automatic (flow injection) method [307]. The JOB method
was used in the second method to determine the ratio combination of com-
plex ions Ca-MTB and Mg-MTB [308].

4.4.3 Atomic Absorption Spectrophotometry

In 1950, Severinghaus and Ferrebee determined Ca in serum, urine, and
other fluids by flame photometry [309]. In the case of serum, proteins are
precipitated in 4% trichloroacetic acid, and the supernatant solution, effec-
tively a 1:10 dilution of the serum electrolytes, is nebulized into a constant
flame. The intensity of the flame light at 556 nm is compared with that pro-
duced by standard solutions containing Ca.
Welch et al. study the effect of phosphate on the determination of Ca in
urine with lanthanumair/acetylene and potassiumnitrous oxide/acetylene
methods [310]. The 20 g/L La-air/acetylene method was the most nearly
accurate, followed by the 2 g/L K-nitrous oxide/acetylene method, 10 g/
L La-air/acetylene, and finally 5 g/L La-air/acetylene.
The determination of Ca in cereal with flame atomic absorption spec-
troscopy has been reported [311]. A cereal sample is crushed and dry-ashed
at 600C in a silica crucible. The residue is treated with 5 mL of 6 M HCl,
diluted to volume, and aliquots taken and prepared for analysis using the
two-increment standard addition approach. The prepared solutions are aspi-
rated into a nitrous oxideacetylene flame and the absorbance measurements
are made using the resonance line of Ca at 422.7 nm, 0.5-nm slit width,
and 5.0-mA lamp current. The results for the determination of Ca in two
brands of cereal showed good precision with the majority (72%) of the data
having RSD  5%.
Furthermore, atomic absorption spectroscopy was used to measure the
Ca content in analgesic tablets [312], in hydroethanolic extracts of propolis
[313], and in dissolution test for CaCO3 tablets [262], CaCO3 lozenges
[266], and CaCO3 gallstones [314].

4.4.4 Fluorometry
Lerga and OSullivan reported a fluorometric method for a simultaneous
combined determination for Ca and Mg (water hardness) using a
98 M.M.H. Al Omari et al.

double-labeled synthetic oligonucleotide as a fluorescent molecular aptamer

beacon [315]. The fluorescent emission of the beacon was measured
(ex. 494 nm; em. 518 nm). Interference of different cations (eg, K)
was eliminated by increasing the temperature beyond the melting point
of the potassium-stabilized quadruplex. The detection limit of the aptamer
beacon is 0.04 mmol/L, with a dynamic linear range of 00.5 M, and is
very reproducible, with an RSD 8%, n 3. An excellent correlation
was obtained when the performance of the proposed method compared
to that of the standard method of complexometric titration and atomic
absorption spectroscopy.

4.4.5 FTIR Spectroscopy

Analysis of ternary mixtures of CaCO3 forms (calcite, aragonite, and
vaterite) was quantitatively performed using the FTIR spectra of pure cal-
cite, aragonite, and vaterite powders with KBr [316]. The absorptivities, a,
of the absorption bands at 713 cm
1 for calcite, 745 cm
1 for vaterite, and
713 and 700 cm
1 for aragonite were determined. Analysis of a known ter-
nary mixture of CaCO3 forms tested the validity of the method.
oke et al. used the same technique for the quantitative analysis method
B
for mixtures of CaCO3, calcium sulfite hemihydrate (CaSO3 1/2H2O),
and gypsum (CaSO4 2H2O). The method involves the FTIR analysis of
powder mixtures of several compositions on KBr disc specimens. Inten-
sities of the resulting absorbance peaks for CaCO3, CaSO31/2H2O, and
CaSO42H2O at 1453, 980, and 1146 cm
1, respectively, were used in
the analysis [317].
Recently, Changwen et al. applied the infrared photoacoustic spec-
troscopy as an alternative to conventional infrared reflectance spectroscopy
for rapidly estimating a wide array of soil properties [318]. Principal com-
ponent analysis, partial least-squares regression, and generalized regression
neural network (GRNN) models were used to calibrate and validate soil car-
bonate analysis. Significant relationships were observed between carbonate
content and FTIRPAS spectral components, particularly in the range of
10002000 cm
1.
Attenuated total reflectanceFourier transform infrared spectroscopy
(ATRFTIR) has been used to determine the content of CaCO3 and
styrenebutadiene (SB) latex in the coating layer of coated paper [319].
The GRNN model can be used to estimate the CaCO3 and SB latex con-
tents in coatings of coated papers. The maximum errors for CaCO3 and SB
latex were only 3.32% and 3.39%, respectively.
Calcium Carbonate 99

4.4.6 LA-ICP Mass Spectroscopy

A precise and accurate laser ablation inductively coupled plasma mass spec-
trometry (LA-ICP-MS) microanalysis of speleothems and biogenic analysis
of CaCO3 to improve the understanding of past climatic conditions has been
reported [320]. Isotopes for interference-free measurements at low (M/M
300) and medium (M/M 4000) mass resolution have been identified.
Analytical reproducibility (RSD) is a factor of 2 better using the 193 nm
laser than the 213 nm laser. The LA-ICP-MS method was applied for the
determination of trace element concentrations in calcite and aragonite layers
of a stalagmite and found large variations for Mg, Zn, Sr, and U. In ostracod
shells, the concentrations of some trace elements (eg, Sr and Ba) vary signif-
icantly, indicating the potential for paleoclimate research.

4.4.7 Raman Spectroscopy

Quantitative analysis by Raman spectroscopy has been mainly reported for
binary mixtures [236,321,322]. When three forms are present in the mix-
ture, treatment of the spectra becomes difficult due to overlapping bands.
Dandeu et al. investigated the possibility of using the Raman spectroscopy
for quantitative analysis of CaCO3 forms (calcite, aragonite, and vaterite) in
ternary mixtures. It was found that quantitative analysis is a difficult task
since there is a strong overlapping, where the strongest bands of CaCO3
forms at around 1000 cm
1 overlap and the vibration modes around
700 cm
1 are very weak, and particularly undetectable for vaterite. The
low frequencies region (50400 cm
1), which corresponds to the lattice
mode vibrations, was chosen for the quantitative analysis. The partial least
squares (PLS) method was used for mathematical treatment, which takes into
account the intensity of the whole spectral range, and not the intensity of a
specific wavelength [253]. Wang et al. used this technique to measure Mg
content in amorphous CaCO3 [256].

4.5 Electrochemical Methods

4.5.1 Ion-Selective Electrode
Deoxy-3,12-bis(TFAB)CA-based electrode was used as a carbonate-selec-
tive electrode to measure CO2 in seawater [323]. The method was evaluated
and compared with Severinghaus-type CO2 gas sensor and the traditional
potentiometric titration methods. The results showed that the carbonate-
selective electrode provides accurate measurement for CO2 comparable
to that obtainable with the other two methods. The proposed method does
not require any sample pretreatment and extra reagents other than the
100 M.M.H. Al Omari et al.

standard calibration solutions, while providing the measured results directly

and immediately.
Ca ion-selective electrode method was used to study Casoy protein
interactions [324]; to determine Ca-binding constants of caseins, pho-
sphoserine, citrate and pyrophosphate [325], and phytase-aided release of
bound Ca in soymilk [326]; and to study the complexation of Ca ions with
[(UO2)(CO3)3]4
[327] and the transport of Ca ions across the hydrogel
membrane [328]. Kabagambe et al. described an ultrasensitive ion-selective
electrode of subnanomolar level measurements based on stripping
voltammetry [329]. An acrylic acid-grafted PVC [330], polyindole-cam-
phorsulfonic acid composite [331], PVC containing attapulgite/
thioacetamide as ionophore [332], graphene as ionophore [333], schiff base
[334], lactate enzyme [335], plasticized PVC incorporating ester as an ion-
ophore [336], vinyl acetic acid-grafted PVC [337], and PVC with and with-
out ETH129 as Ca ionophore [338,339]-based membrane ion-selective
electrodes containing dioctyl phthalate (plasticizer) and sodium tetraphenyl
borate (anion excluder) were used as an indicator electrode in potentiomet-
ric titration of Ca with EDTA [340] and determination of Ca in milk [341]
and water samples. Stoodley et al. used Ca ion-selective electrode for field
Ca measurement in seawater and compared the other techniques such as
potentiometric and colorimetric titration with EDTA [342]. Kuwamoto
et al. proposed a system to perform pretreatment before measuring Ca ion
in a sample solution with a Ca ion-selective electrode by an acid and
base-adding mechanism [343].

4.5.2 Polarography
Cohn and Kolthoff used picrolonic acid to precipitate Ca as calcium
picrolonate and then the excess of the acid is determined polarographically
without filtering the solutions. The method yields good result in the pres-
ence of relatively large amounts of Na, K, NH3, Mg, sulfate, and phosphate
in the solutions [344]. Another indirect polarographic determination of
Ca by chloranilic acid has been also reported [345]. Ca was determined
by precipitating as chloranilate complex, followed by measuring the polar-
ographic diffusion current of the residual chloranilic acid without need of
separation of the solid complex. The interference of different cations as a
function of their concentration has been investigated. Fleet et al. also
reported indirect polarographic method based on the decrease in the height
of the anodic polarographic waves of EDTA and ethylene glycol-bis-(-
aminoethylether)-NNN 0 N 0 -tetraacetic acid [346]. Different complexing
Calcium Carbonate 101

agents were investigated in a DC polarographic determination of Ca in

aqueous solutions [347]. A dropping mercury electrode was applied to get
peak-shaped waves for Ca in tetrabutylammonium hydroxide supporting
electrolyte. The square root of the peak height is directly proportional to
the Ca concentration over the range 0.11.0  10
3 M. In the presence of
the sequestrants EDTA, nitrilotriacetic acid (NTA), and tripolyphosphate
(TP), indications of distinct Ca complexes were seen by noting the positions
of slope changes in plots of peak height vs mole ratio of Ca to sequestrant. The
species found were CaEDTA, Ca(3)(NTA)(2), Ca(2)(TP)(3), and Ca(3)TP.

4.5.3 Voltammetry
In direct measurement of Ca by differential pulse stripping voltammetry has
been achieved by using hanging electrolyte drop electrode [348]. The
method is based on the transfer of Ca ions from water to nitrobenzene facil-
itated by the complex formation with the macrocyclic polyether
diamide,7,19-dibenzyl-2,3-dimethyl-7,19-diazo-1,4,10,13,16-pentaoxacy-
cloheneicosane-6,20-dione. Wang et al. described a sensitive adsorptive
stripping procedure for trace measurement Ca using their chelates with
the dihydroxyazo dye solochrome violet RS [349].
Kim used a water-soluble calix[4]arene-diquinone-diacid (CDA) to
quantify Ca in aqueous solution by forming a complex with Ca ions
[350]. Fig. 26 shows the redox changes of CDA as a function of Ca concen-
tration in aqueous buffered solution of pH 7.4.

Potential/V vs Ag/AgCl
0.6 0.4 0.2 0.0 0.2 0.4 0.6

2 A

Increase of [Ca2+]

Figure 26 Squarewave voltammograms of calix[4]arene-diquinone-diacid as a function

of Ca concentration (0.011.5 mM).
102 M.M.H. Al Omari et al.

Furthermore, Almeida et al. used a squarewave voltammetry and a glassy

carbon electrode in a solution containing EDTA to determine Ca ions in real
biodiesel samples [351].

4.6 Calcimetric Method

This method depends upon the conversion of carbonate ions into CO2 gas
by adding diluted HCl to the sample and the pressure of the released CO2
was measured by manometer [352]. Dreimanis described a quantitative
determination of calcite and dolomite by measuring the content of the
CO2 evolved in the Chittick gasometric apparatus [353].
The analysis of CO2 by pressure-calcimetric method is also described by
Loeppert and Suarez [354]. It is a direct, accurate, and inexpensive method,
but the complexity of the pressure calcimeter apparatus makes large sample
runs impractical [355]. Wagner et al. described a modified volumetric analysis
system of the pressure-calcimeter apparatus by utilizing a pressure transducer,
which is monitored by a data acquisition card connected to a personal com-
puter [356]. Furthermore, modifications of the pressure-calcimetric method
by using Wheaton serum bottles (20 and 100 mL) sealed with butyl rubber
stoppers and aluminum tear-off seals as the reaction vessel and a pressure
transducer monitored by a digital voltmeter [355]. This method was used
for CaCO3 determination in soil samples [279]. The results were found
to be comparable with those obtained using back-titration method.

4.7 Chromatographic Methods

4.7.1 High-Performance Liquid Chromatography
A sensitive and selective high-performance liquid chromatography (HPLC)
method for the separation of Mg and Ca in complex saline matrices has been
reported [357]. The mobile phase contains the selective metallochromic
chelating ligand, o-cresolphthalein complexone, and the separation is done
at a reversed-phase porous graphitic carbon column using a spectrophoto-
metric detector at 575 nm. Detection limits of 0.05 mg/L for Mg and
0.10 mg/L for Ca were obtained in samples containing in excess of
2300 mg/L of Na, without interference.

4.7.2 Ion-Exchange Chromatography

Fritz and Waki described an anion exchange chromatographic separation
for Ca and Mg using 0.5 M HNO3 in 90% of water-miscible isopropyl
alcohol as the eluent. The separation was performed using Amberlyst
XN-1002 resin [358]. Moreover, Fritz and Story proposed a forced-flow
Calcium Carbonate 103

chromatography on partially sulfonated macroreticular resin beads to rapid

separate several metal ions using strong acid of moderate concentration as
the eluent [359]. Separation of Ca and Mg from each other and from
several other cations by ion-exchange chromatography was proposed
also by Argiiello and Fntz [360]. They used a sulfonated macroporous resin
of 1.82.0 mequiv./g capacity and 1 M NH4Cl or 0.03 M ethylenediam-
monium chloride as the eluent. A sensitive continuous metal ion detection
system based on the use of 2-(pyridylazo)resorcinol-Zn-EDTA was devel-
oped and employed in a liquid chromatographic separation [361].
Trace enrichment and HPLC have been examined for the determination
of metal ions in the low pg/mL range. The metal ions are enriched on a short
bonded-phase ion exchanger and then separated on a 13 m styrene
divinylbenzene resin [362,363]. The use of ion-exchange chromatography
was also used to determine Ca in dietary supplement tablets [364].

4.7.3 Gas Chromatography

Amundson et al. reported a rapid and sensitive method of soil carbonate
analysis, utilizing gas chromatography [365]. The results of analysis by gas
chromatography and the Chittick methods are comparable (r 0.96,
slope 1.11). The proposed method was the most sensitive and had the
lowest possible detection limit (0.008 mg CaCO3). However, low and high
concentrations should be avoided to get accurate results.
A continuous-flow isotope ratio mass spectrometry (CF-IRMS) coupled
online with gas chromatography (GasBench II) sample preparation and with
multiloop injection procedures was applied for the analysis of carbonates
(calcite and aragonite). The method showed precise and accurate measure-
ments at high sample throughput [366]. Ishimura et al. developed a CF-
IRMS system to determine stable isotopic compositions (13C and 18O)
of submicrogram quantities of CaCO3 for the purpose of analyzing individ-
ual foraminiferal shells [367]. The system consists of a microvolume CaCO3
decomposition tube, stainless steel CO2 purification vacuum line with a
quantity-regulating unit, helium-purged CO2 purification line, gas chro-
matograph, and a CF-IRMS system. Fiebig et al. described a method for
the precise measurement of carbonate samples in the range 1030 g for
the GasBench II [368]. However, their method requires a modification of
the hardware and the use of liquid nitrogen, which increases the costs
and the complexity of the measurements. Skrzypek and Paul used the same
IRMS technique coupled online with gas chromatography (GasBench II)
and an elemental analyzer system to measure stable carbon isotopic
104 M.M.H. Al Omari et al.

composition (13C) of carbonates or carbonate-rich soils [369]. The 13C

analyses of CaCO3 samples by using both techniques showed high precision.
However, the results suggested that the 13C of pure CaCO3 samples can
also be analyzed using the elemental analysis technique. Technical mo-
dification of the conventional method for the 13C and 18O analysis of
1030 g carbonate samples was described by Velivetskaya et al. [370].
The method has been used successfully for the analyses of the oxygen and
carbon isotopic composition of the planktonic and benthic foraminifera
in detailed paleotemperature reconstructions of the Okhotsk Sea. Also
the method has been used to determine the content and isotopic composi-
tions of minor amounts of carbonate in silicate rocks [371]. Further technical
improvements have been achieved in carbonate samples analysis, as small as
20 g can be analyzed routinely, using a GasBench II continuous-flow
IRMS setup, with standard deviations smaller than 0.07% for both 18O
and 13C [372].

5. STABILITY
5.1 Crystal Phase Transformation
Calcite, stable anhydrous form of CaCO3, undergoes a series of structural
transitions toward denser of calcite IIIV phases with increasing pressure
[108,110,111]. In addition to the aforementioned calcite forms, there exists
a denser form calcite VI that can be formed using shock compression exper-
iments [112]. At even higher pressures (>100 kbar), calcite is known to
undergo yet another phase transition, known as calcite IV [114116].
However, monohydrocalcite, the hydrate form of calcite, is not stable
thermodynamically and will transform into other crystal phases upon the loss
of crystalline water (eg, calcite and aragonite) [78,240]. In addition, low con-
tent of Mg in aqueous solution will lead to its transformation to aragonite
over 25 days at ambient temperatures [373,374].
Aragonite, metastable anhydrous form of CaCO3, will remain unaltered
for tens of millions of years in dry conditions at temperatures below 400C.
If water is present, however, aragonite will convert to calcite in a matter of
months due to its greater solubility in water. The difference in solubility is
one of the reasons why aragonite is not as common in geological beds and is
rarely found outside of organically controlled systems [225]. At standard
temperature and pressure, aragonite is thermodynamically unstable and
tends to alter to calcite [375]. At high pressure, it becomes the stable phase
[117]. A postaragonite phase in CaCO3 at a pressure of 40 GPa and a number
Calcium Carbonate 105

of energetically competitive structures (stable phase I and metastable phases

IIIV) were identified [118]. Above 137 GPa, phase I with a pyroxene-type
structure with chains of CO4
4 tetrahedra becomes more stable than
postaragonite.
Vaterite is also a metastable anhydrous form of CaCO3 at ambient con-
ditions, and once it is exposed to water, it converts to aragonite or calcite
[376]. It is a rare mineral in geologic settings, though it may be an important
precursor in several carbonate-forming processes [238].
The amorphous form of CaCO3 is unstable solid phase, which undergoes
a rapid transformation to more stable anhydrous forms, while the two
hydrated forms monohydrocalcite and ikaite can be kept unchanged for a
few days at temperatures below 0C before they undergo transformation
into calcite. As a result, preparation of ikaite was carried out at temperatures
below
10C [62]. Mg ions were noticed to further enhance the form trans-
formation of amorphous CaCO3 into aragonite [128]. Bentov et al.
improved the stability of amorphous CaCO3 by phosphate-rich organic
matrix proteins and by single phosphoamino acids [377]. Such stable form
showed an improvement in solubility and bioavailability in comparison with
the crystalline form [378,379].
Ikaite, hydrate form of CaCO3, is thermodynamically stable at moderate
pressures near the earths surface. It has been found in deep sea and conti-
nental shelf sediments, and in groundwater discharge sites in lakes, glacial
environments, coastal waters, and sea ice [380383]. It decomposes rapidly
by losing most of its water content once removed from near-freezing water
[239]. Both amorphous and ikaite forms are metastable in the environment
and change easily to the more stable form calcite [223,375].

5.2 Solid-State Stability

CaCO3 decomposes upon heating (>650C) to CaO and CO2. This endo-
thermic process is called calcination [384,385]:
CaCO3s ! CaOs + CO2g H + 182:1kJ=mol
The reaction is favored by higher temperatures and will proceed only if
the partial pressure of CO2 in the gas above the solid surface is less than the
decomposition pressure of the CaCO3 [384]. Furthermore, evaluation of
the kinetics of calcination is complicated by: (1) CO2 atmosphere and its
concentration, which inhibits the reaction; (2) sample weight and particle
size, which may introduce both thermal and mass transfer limitations;
106 M.M.H. Al Omari et al.

(3) catalysis/inhibition by impurities; (4) the applying pressure and inert

atmosphere; (5) the use of isothermal or nonisothermal method; and (6)
the rate of temperature increase [384390].
Lvov studied the mechanism and kinetics of CaCO3 decomposition and
showed that the effect of self-cooling of reactant is of primary importance in
the explanation of many controversial results obtained by different researchers
in investigations of the kinetics of carbonate decomposition [391].
Gabal et al. also studied the effect of different atmosphere (air, N2, and
N2H2 mixture) on the thermal decomposition of CaCO3. It was found that
changing of the applied atmosphere affects the carbon content of the ash,
which results in different thermal decomposition behaviors [392]. Effect
of tartaric, succinic, and citric acids on the decomposition of CaCO3 has
been studied [393]. The decomposition temperature of CaCO3 is not
decreased, and at the same time, particle size distribution and morphology
of CaO are changed.
Rodriguez-Navarro et al. studied the mechanism of CaO nanocrystals
formation and their texture during the decomposition of calcite [394]. It
was found that the thermal decomposition of calcite is homogeneous and
topotactic in nature, and does not depend on the experimental conditions
(eg, CO2 pressure, crystal size, or type of energy used for the activation
of the reaction). Their findings may help establishing conditions to achieve
the best properties in terms of crystal size, surface area, porosity, and
reactivity. The proposed model may also help establishing crystallographic
constraints for possible relationships between reactant and product, in order
to interpret textural relationships found in natural samples (eg, Martian
meteorites).

5.3 Stability in Solution

CaCO3 has a very low solubility in water, but in saturated with CO2, its
solubility increases due to the formation of more soluble calcium bicarbon-
ate (Ca(HCO3)2). However, like all metal carbonates, CaCO3 reacts with
acidic solutions to produce CO2 gas [395].

5.4 Interaction with Complexing Agents

Interaction of some complexing agents such as EDTA, oxalate, phosphate,
picrolonic, rhodizonic, arsenazo, and resorcinol derivative with Ca is widely
used for analysis of pharmaceutical and biological samples. These studies are
fully discussed in Section 4.
Calcium Carbonate 107

6. USES, APPLICATIONS, AND PERTINENT HISTORY

CaCO3 is authorized as a food color in the European Union (EU)
under Directive 94/36/EC and is also authorized as an additive generally
permitted in foodstuffs under Directive 95/2/EC. It is also included in
Directive 2001/15/EC on substances that may be added for specific nutri-
tional purposes in foods for particular nutritional uses, and in Directive
2002/46/EC relating to food supplements, and it can be used in fortified
foods according to Regulation 1925/200626 on the addition of vitamins
and minerals and of certain other substances in food. CaCO3 has been eval-
uated by Joint FAO/WHO Expert Committee on Food Additives ( JECFA)
in 1965, when the Committee established an acceptable daily intake (ADI)
not limited. The EU Scientific Committee of Food (SCF) evaluated CaCO3
as part of a group of carbonates and assigned a group ADI not specified.
CaCO3 together with other carbonates has also been reviewed by
TemaNord, who concluded that there was no need for a reevaluation.
The SCF allocated a tolerable upper intake level (UL) for Ca of
2500 mg/person per day as a nutrient and also established a population ref-
erence intake of 700 mg Ca/day (range 4001200 mg/day depending on
age and physiological status) [4].
CaCO3 is included in Commission Decision 2006/257, establishing an
inventory of ingredients in cosmetic products. In pharmaceuticals, CaCO3
is used as an excipient and as an active ingredient of antacids. It is also in-
cluded in Directive 91/41428 concerning the placing of plant products on
the market. CaCO3 has been registered under the Reach Regulation
1907/2006 [4].
CaCO3 is included in the Food and Drug Administration (FDA) list of
food additives that are generally recognized as safe (GRAS) for use in
nutrient and dietary supplements, and is also certified by the FDA for use
in amounts consistent with good manufacturing practice to color drugs gen-
erally [396].
CaCO3, both natural and precipitated, is widely used as a major filler in
paper, paint, adhesives and sealants, and polymers. Also CaCO3 meets the
pharmacopeia requirements as a therapeutic source in antacids, as Ca supple-
ments, and as a tableting excipient [397,398]. As a pharmaceutical excipient,
it is mainly used as diluents, coating agent, and wet binder in solid dosage
forms, as a base for medicinal and dental preparations, and as buffering
and dissolution aid in dispersible tablets, as well as food additive [9,398].
108 M.M.H. Al Omari et al.

CaCO3 is also used as a fire extinguisher foam filler, as an abrasive in

household cleaners, as a flux in welding rod coatings, as a diluent in agricul-
tural pesticide dusts, and as a dusting agent in mines, in the manufacture of
Portland cement, lime, glass, and metallurgical fluxes, as well as in flue gas
desulfurization processes and as a soil amendment [397].

7. PHARMACOLOGY
7.1 Pharmacokinetics
7.1.1 Absorption
Ca is endogenously occurring substance within the body. It is actively
absorbed into the body and its level is controlled by various Ca homeostasis
mechanisms [399]. After oral administration, 1840% of Ca is absorbed from
the small intestine by active transport and passive diffusion. Active absorp-
tion of Ca is highly dependent on vitamin D, and vitamin D deficiency
decreases the absorption of Ca [399,400]. Absorption of Ca is dose depen-
dent, with fractional absorption being highest when at doses up to 500 mg.
Absorption of Ca is also dependent on pH (reduced in alkaline), body size,
estrogen status, vitamin D status, age, and genetic polymorphisms. The
absorption of Ca from CaCO3 is increased when taken with food [399,400].
Different Ca salts show different levels of absorption. For example,
calcium citrate is more bioavailable than CaCO3 [401403]. Hanzlik et
al. showed that calcium formate is clearly superior to both CaCO3 and cal-
cium citrate in ability to deliver Ca to the blood stream after oral adminis-
tration [404].
Zhao et al. compared the Ca bioavailability from CaCO3-fortified
soymilk (CCSM) and tricalcium phosphate-fortified soymilk (TCPSM)
with cows milk in young healthy women using ICP-MS technique
[405]. They found that the fractional Ca absorption in CCSM did not differ
from that of cows milk, but both were higher than that of TCPSM.
Ayed and Thannoun studied the effect of phosphorus on the bioavail-
ability of Ca [406]. They concluded that CaCO3-based diet containing
0.19% Ca with 1.5:1 Ca to phosphorus ratio may give high Ca bioavailability
for growing rats which was considered as standard (control) diet for other
diet supplement. Kressel et al. showed that calcium lactate citrate and cal-
cium lactate malate may offer a very good choice for the fortification
of beverages to increase the daily Ca intake comparing with CaCO3 and
calcium gluconate [407]. The two former salts have higher water
solubility with a satisfactory Ca content and availability comparing with
the later salts.
Calcium Carbonate 109

Mueller et al. showed that daily intake of 1200 mg of Ca, as CaCO3, and
800 IU of vitamin D3, with a new chewable tablet increased the intestinal
Ca absorption compared to the results from the placebo [408].
Meiron et al. compared the solubility and fractional absorption of a
stabilized amorphous CaCO3 without and with the presence of chitosan
and crystalline CaCO3 [378]. The results demonstrated that the amorphous
is more soluble than the crystalline form. Fractional absorption was evaluated
by intrinsically labeling CaCO3 preparations with 45Ca, orally administrated
to rats using gelatin capsules. The results revealed that Ca absorption
from the amorphous preparations is up to 40% higher than from the crys-
talline form.
It was reported that Ca from nanoparticulate CaCO3 is more readily
absorbed than the microparticulate form in mice study. A slight increase
in bioavailability (by 38%) of the nanosized CaCO3 by comparison with
the micronized form in humans indicates that the absorption levels of
Ca from both forms are almost similar [4].

7.1.2 Distribution
Skeletal Ca accounts for 99% of the Ca in the body. Of the remaining 1%,
4045% is bound to proteins, primarily albumin. About 510% is
complexed to phosphate, citrate, or other anions. Approximately 50% of
Ca in the serum is in the physiologically active ionized form [399,400].

7.1.3 Metabolism
As an endogenously occurring substance, Ca is not metabolized in the
traditional pharmacokinetic sense [399].

7.1.4 Elimination
Unabsorbed Ca from the small intestine is excreted in the feces. Renal
excretion depends largely on glomerular filtration and Ca tubular
reabsorption with more than 98% of Ca reabsorbed from the glomerular
filtrate, with only 2% lost as obligatory Ca loss. This process is regulated
by active vitamin D and parathyroid hormone (PTH) [399,400]. Excess
carbonate is excreted as CO2 via respiration [4].

7.2 Mechanism of Action

Antacid: Neutralizes gastric acidity.
Dietary supplement: Prevents or treats negative Ca balance; oral Ca sup-
plements may protect against renal calculi formation by chelating with
oxalate in gut and preventing its absorption.
110 M.M.H. Al Omari et al.

Phosphate binder: Binds with dietary phosphate to form insoluble calcium

phosphate, which is excreted in feces [409].

7.3 Pharmacodynamics
Ca administration decreases the elevated rate of bone turnover typically
seen in postmenopausal women with osteoporosis. In randomized, pla-
cebo-controlled studies in postmenopausal women, Ca administration
(5001600 mg) decreased biochemical markers of bone turnover, including
urine N-telopeptide, urine-free pyridinoline (markers of bone resorption),
alkaline phosphatase, and osteocalcin (markers of bone formation) relative to
placebo-treated women. Ca administration may transiently increase levels of
serum Ca with compensatory reductions in serum PTH and an increase in
urinary Ca. However, urinary and serum Ca levels usually remain within the
normal reference range [400].

7.4 Toxicities
Different toxicity studies have been carried out with CaCO3 in rats, mice,
and cats. They have overall not demonstrated any evidence of toxicity attrib-
utable to CaCO3 [4].
Recommendations for daily dietary Ca intake that range from 400 to
1200 mg/day depending on age and gender have been issued by governmen-
tal and nongovernmental organizations in many countries [4,399,400,404].
Total daily intake of Ca above 1500 mg has not demonstrated additional bone
benefits, while daily intake above 2000 mg has been associated with increased
risk of adverse effects, including hypercalcemia and kidney stones [399,400].
However, intake of dietary Ca equivalent to 250 or 500 mg/kg bw/day
in rats leaded to nephrocalcinosis, while in Beagle dogs at the same doses did
not show any signs of nephrocalcinosis [4]. Nephrocalcinosis was also not
observed in a recent combined repeat dose oral toxicity/reproduction/
developmental toxicity screening study with CaCO3 (having a particle size
of 60100 nm) carried out in Wistar rats at dose levels of up to 1000 mg/kg
bw/day for up to 48 days. The only changes seen in this study were slight but
statistically significant hematological and biochemical effects in males receiv-
ing 1000 mg/kg bw/day, and significant reductions in plasma phosphate
levels in all male-treated groups. No evidence of toxicity was reported in
a study in which mice were administered CaCO3 (described as nano
CaCO3) by oral gavages at dose levels up to 1300 mg/kg bw/day [4].
Calcium Carbonate 111

Evidence of fetotoxicity of Ca (as CaCO3) was observed when admin-

istered during pregnancy at levels in the diet greater than 1500 mg/kg bw/
day CaCO3. Overall, it was noted that in rodents high doses of CaCO3
(>1500 mg/kg bw/day) causing hypercalcemia during gestation can result
in adverse effects on reproduction, fetotoxicity, and elemental imbalances in
the offspring [4].
No data are available indicating that CaCO3 has allergenic properties or
can invoke sensitivity or intolerance reactions in exposed individuals [4].
CaCO3 (including CaCO3 having a particle size of 60100 nm) has
given negative results in a range of in vitro genotoxicity assays. No data
are available on the chronic toxicity or carcinogenicity of CaCO3. How-
ever, it is very unlikely that CaCO3 has carcinogenic potential, given that
both Ca and carbonate are natural constituents of the body and normal
metabolites of man, animals, and plants and have a long history of safe
use as a source of Ca supplementation for humans [4].
Furthermore, there are reports which indicated health associated
problems with Ca intake upon the presence of other compounds. For
example, Picolos and Orlander reported the presence of Ca with antacids
can lead to milk-alkali syndrome, which is a common cause of hypercalce-
mia [410].

7.5 Drug Interactions

Ca may reduce the absorption of bisphosphonates (such as risedronate,
alendronate, etidronate, ibandronate, pamidronate), thyroid hormones
(levothyroxine), fluoroquinolones (such as ciprofloxacin, moxifloxacin,
and ofloxacin), tetracyclines (such as doxycycline, minocycline, and tetracy-
cline), and omeprazole [399,400,410]. There is a report, which describes
antagonism of the antiarrhythmic effects of oral verapamil due to the use
of oral Ca and calciferol [399].
Ca absorption is reduced when CaCO3 is taken concomitantly with
systemic glucocorticoids and thiazide diuretics reduced urinary excretion
of Ca during concomitant use with CaCO3 [400].
Absorption of Ca may be increased when CaCO3 is given concomitantly
with vitamin D analogues (such as calcitriol, doxercalciferol, and
paricalcitol). Also Ca may interfere with the absorption of iron. Patients
being treated for iron deficiency should take iron and Ca at different times
of the day [400]. Certain foods (eg, those containing oxalic acid, phosphate,
or phytanic acid) may reduce the absorption of Ca [399].
112 M.M.H. Al Omari et al.

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 CHAPTER ONE

Bupropion Hydrochloride
S.R. Khan*, R.T. Berendt*, C.D. Ellison*, A.B. Ciavarella*,
E. Asafu-Adjaye*, M.A. Khan, P.J. Faustino*
*
Division of Product Quality Research, US Food and Drug Administration, Center for Drug Evaluation
and Research, Office of Testing and Research, Silver Spring, MD, United States

Rangel College of Pharmacy, College Station, TX, United States

Contents
1. Description 2
1.1 Nomenclature 2
1.2 Formula 3
1.3 Elemental Analysis 3
1.4 Appearance (Smell, Documented Taste) 3
1.5 Uses and Applications 3
2. Method of Preparation 4
2.1 Synthesis 4
3. Physical Properties 5
3.1 Dissociation Constant 5
3.2 Solubility 5
3.3 pH 5
3.4 Partition Coefficient 5
3.5 Hygroscopicity 5
3.6 Crystallographic Properties 6
3.7 Thermal Analysis 7
3.8 Spectroscopy 8
4. Methods of Analysis 20
4.1 Electrochemical Analysis 20
4.2 Chromatographic Analysis 22
5. Stability 24
5.1 Solution Stability 24
5.2 Solid-State Stability 25
5.3 Stability in Biological Medium 25
6. Biological Properties 26
6.1 Toxicity 26
6.2 Drug Metabolism and Pharmacokinetics 26
Acknowledgments 28
References 28

Profiles of Drug Substances, Excipients, and Related Methodology, Volume 41 # 2016 Elsevier Inc. 1
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/bs.podrm.2015.12.001
2 S.R. Khan et al.

1. DESCRIPTION
Bupropion belongs to the chemical class of aminoketones and is
known also by the generic name amfebutamone. It is a norepinephrine-
dopamine disinhibitor (NDDI), which promotes the release of norepineph-
rine and dopamine, and is administered for the treatment of depression and
smoking cessation [1]. It is a second-generation antidepressant approved in
the United States and in the European Union. Bupropion is a trimethylated
monocyclic phenylaminoketone compound that differs both structurally
from most first-generation tricyclics and second-generation SSRI-
antidepressants, and is part of a novel mechanistic class of antidepressants that
has no direct action on the serotonin system [1,2].
Bupropion has a single chiral center, giving rise to two enantiomeric
forms. Although pure bupropion enantiomers have been synthesized suc-
cessfully, rapid racemization in solution is observed [3,4]. Therefore, this
drug is marketed as a racemate, with equimolar ratios of both enantiomers
being present in Wellbutrin and Zyban [5].
The active pharmaceutical ingredient (API) in marketed bupropion drug
products is either bupropion hydrochloride (HCl) or bupropion hydro-
bromide (HBr). The HCl salt is the more common API, and thus is the focus
of the profile reported herein. Because the crystal structures of these two salt
forms may significantly differ, certain solid-state physicochemical properties
(eg, dissolution rate, number of polymorphic forms, stability, etc.) also may
differ. However, upon dissolution (and dissociation of the chloride and bro-
mide ions from the bupropion), bupropion from either salt form will have
the same properties.

1.1 Nomenclature
1.1.1 Systematic Chemical Name
1-(3-Chlorophenyl)-2-[(1,1-dimethylethyl)amino]-1-propanone; synonym:
(
)-2-(tert-butylamino)-30 -chloropropiophenone

1.1.2 Nonproprietary Names

Bupropion, Amfebutamone

1.1.3 Proprietary Names

Wellbutrin, Zyban, Voxra, Budeprion, Prexaton, Elontril, or Aplenzin
Bupropion Hydrochloride 3

1.2 Formula
1.2.1 Empirical Formula, Molecular Weight, CAS Number
Molecular formula: C13H18ClNOHCl
Molecular weight: 276.20
CAS Number: 31677-93-7

1.2.2 Structural Formula

See Fig. 1.

1.2.3 Stereochemical Description

Bupropion has a single chiral center that gives rise to two enantiomers. Pure
enantiomers have been synthesized, and pharmaceutical uses of the pure
enantiomers of bupropion, (+)- and ()-bupropion, are reported [3,4,6].
However, in aqueous solution, the enantiomers rapidly interconvert to exist
as a 50:50 racemic mixture [3,4]. Thus, the drug is marketed as a racemate.
Racemic bupropion is the API of Wellbutrin and Zyban (marketed by
Glaxo Smith Kline).

1.3 Elemental Analysis

C 56.53%, H 6.93%, Cl 25.67%, N 5.07%, O 5.79%.

1.4 Appearance (Smell, Documented Taste)

Crystallization of bupropion hydrochloride from isopropanol and absolute
ethanol results in white crystals that possess a bitter, anesthetizing taste.

1.5 Uses and Applications

Bupropion is second-generation antidepressant indicated for smoking cessa-
tion [7,8]. In clinical trials, bupropion is being tested as a candidate treatment
for psycho-stimulant drug abuse, attention-deficit hyperactivity disorder
(ADHD), and obesity. Bupropion is available in three bioequivalent oral

Figure 1 Molecular structure of bupropion hydrochloride. The asterisk denotes the

chiral center.
4 S.R. Khan et al.

formulations: immediate release (IR), sustained release (SR), and extended

release (XL). In 2003, the FDA approved the first XL formulation,
Wellbutrin XL 300. In 2006, the FDA approved a generic version of the
XL bupropion formulation, Budeprion XL, 300 mg [9]. Budeprion XL
was voluntarily removed from the market in 2013 [10].

2. METHOD OF PREPARATION
See Scheme 1.

2.1 Synthesis
The synthesis of bupropion hydrochloride is reported by Mehta et al. [3].
The ketone 1 was converted to 2-bromo-30 -chloropropiophenone 2 by a
reaction of bromine with ketone in dichloromethane. The SN2 displace-
ment of bromine by t-butylamine in N-methylpyrrolidinone (NMP) yields
3 as a noncrystalline oil. This was converted into the crystalline ammo-
nium hydrochloride salt 4 by reaction with hydrochloric acid. The yield
improved as a result of using N-methylpyrrolidinone (NMP, also called
2-methyl-2-pyrrolidinone) in place of dimethylformamide (DMF) as a sol-
vent for the amination of 2. In DMF, the reaction can take 34 h, whereas in
NMP, it is complete in less than 10 min at 5060C. The secondary amine 3
reacts with hydrochloric acid to produce 4 in good yield.

Cl Cl Cl
H3C

Br2 NH2 C CH3

H3C
H
Br N
C(CH3)3
O
O O

1 CH3 2 CH3 CH3 3

N O

CH3
Cl HCl

H.HCl
N
C(CH3)3
O

CH3
4
Scheme 1 Synthesis of bupropion hydrochloride.
Bupropion Hydrochloride 5

3. PHYSICAL PROPERTIES
3.1 Dissociation Constant
Bupropion is a weak base. The pKa of bupropion is 7.9 at 25C [5].

3.2 Solubility
See Table 1.

3.3 pH
The pH of subsaturated solutions of bupropion hydrochloride are 4.8
(10 mg/mL) and 4.1 (50 mg/mL) in DI water.

3.4 Partition Coefficient

See Table 2.

3.5 Hygroscopicity
Bupropion is very hygroscopic and sensitive to decomposition [14].
Bupropion hydrochloride is slightly hygroscopic [15]. According to the
United States Pharmacopeia, bupropion hydrochloride should be stored
in a closed container under refrigerated conditions and should not require
drying if stored properly.

Table 1 Solubility of Bupropion HCl at Room Temperature

Solvent Solubility (mg/mL) [11,12]
Water 312
Alcohol 193
0.1 N HCl 333

Table 2 n-Octanol/Water Partition Coefficient (log Po/w) [13]

System log Po/w
n-Octanol/distilled water 1.32
n-Octanol/distilled water (pH 1.2) 0.60
n-Octanol/distilled water (pH 6.0) 0.91
n-Octanol/distilled water (pH 7.4) 1.54
6 S.R. Khan et al.

3.6 Crystallographic Properties

3.6.1 Polymorphism
Two crystalline polymorphs of bupropion HCl have been reported in the
literature [16,17]. These polymorphs were shown to be enantiotropically
related, where Form 2 is most stable at room temperature, and Form 1
is more stable at elevated temperatures (demonstrated conversion at
190C) [17].

3.6.2 Single-Crystal Structures

A single-crystal structure of the ethanol hemisolvate of racemic bupropion
HCl exists in the literature [18]. However, due to an apparent difficulty in
preparing a single crystal of adequate size and quality, the reported crystal
structure for desolvated racemic bupropion HCl is based on a powdered
crystalline sample, not a single-crystal sample [16]. For desolvated racemic
bupropion HCl, the authors obtained the crystal structure by applying
the ab initio X-ray powder diffraction (XRPD) technique and a global opti-
mization strategy, adopting the single-crystal structure of the solvate form as
a starting point for molecular simulations. Table 3 lists the literature-
reported crystallographic parameters for Form 1 and its corresponding

Table 3 Published Crystal Data for Bupropion Hydrochloride

Bupropion HCl Bupropion HCl Bupropion HCl Ethanol
(Form 1) [16] (Form 2) [17] Hemisolvate [18]
Space
group Monoclinic Orthorhombic Triclinic
P21/c Pbca P1
Z 4 8 2
a 14.3406(3) A 27.2853(5) A 7.571(1) A
b 8.7564(2) A 8.7184(3) A 9.310(1) A
c 11.8801(2) A 12.0422(3) A 11.687(1) A
94.58(1)
78.025(2) 101.49(1)
V 1459.34(5) A3 2864.7(1) A3 804.5(2) A3
1.5418 A 1.5418 A 1.54178 A
Temp (K) 293 298 293
Bupropion Hydrochloride 7

ethanol hemisolvate. Crystallographic parameters for bupropion HCl Form

2 are not reported in the literature.

3.6.3 XRPD Pattern

Figure 2 shows an experimental XRPD pattern of anhydrous bupropion
HCl. The most intense peaks from the experimental pattern are listed in
Table 4. This pattern is consistent with the bupropion HCl Form 1
polymorph [16].

3.7 Thermal Analysis

3.7.1 Melting Behavior
The melting range of bupropion hydrochloride (polymorph unknown) is
reported to be 233234C [11]. The melting range of bupropion hydro-
chloride (polymorph unknown) was experimentally determined to be
230.9231.8C using a Kruss M5000 melting point apparatus (Hamburg,
Germany). Decomposition was observed at the melting range.

Figure 2 Experimental XRPD pattern of bupropion HCl, collected at a wavelength of

1.54060 nm under ambient laboratory conditions.
8 S.R. Khan et al.

Table 4 List of the Most Intense Peaks (>5% Rel. Intensity) in the Experimental XRPD
Pattern (Fig. 2) of Bupropion HCl
Scattering
Angle d-Spacing Relative Scattering Angle d-Spacing Relative
(Degrees 2) () Intensity (%) (Degrees 2) () Intensity (%)
11.920 7.41887 37.60 24.325 3.65612 7.50
12.639 6.99818 10.90 25.454 3.49649 8.90
13.433 6.58602 5.90 25.981 3.42678 8.30
14.847 5.96191 19.00 26.996 3.30018 34.50
15.264 5.80017 14.10 27.408 3.25147 19.80
16.201 5.46663 7.20 27.924 3.19261 13.70
16.756 5.28662 14.90 29.967 2.97937 11.70
17.703 5.00596 5.00 31.906 2.80262 39.60
18.996 4.66814 99.80 32.455 2.75647 5.80
20.420 4.34569 19.70 32.740 2.73313 19.50
21.539 4.12241 48.70 32.811 2.72739 15.80
23.066 3.85283 6.00 39.783 2.26397 5.50
24.180 3.67780 24.80 39.816 2.26217 6.20

3.7.2 Differential Scanning Calorimetry and Thermogravimetric Analysis

Differential scanning calorimetry (DSC) and thermogravimetric analysis
(TGA) of bupropion HCl Form 1 are shown in Fig. 3. The DSC and
TGA thermograms were both collected at a ramp rate of 10C/min, using
Q2000 and Q5000 instruments (TA Instruments, New Castle, Delaware),
respectively. The TGA weight loss that begins at approximately 140C
can be attributed to sublimation of the substance. This observation may sug-
gest that the DSC endotherm at 241C is due to the sublimation process, not
the melting of bupropion HCl [16,17].

3.8 Spectroscopy
3.8.1 Electronic Spectroscopy
3.8.1.1 UV/VIS Spectroscopy
UV/VIS spectra were acquired over the spectral range of 200700 nm on
an Agilent Technologies 8453 photodiode array spectrophotometer.
A spectrum of the sample solvent was used as the reference. Bupropion spec-
tra in various solvents are shown in Fig. 4 with peak assignments in Table 5.
Bupropion Hydrochloride 9

20
190 241.60C
DSC 630.7 J/g
170
0
150

130 20

Heat flow (mW)

140.65C
Weight (%)

110 TGA

40
90
241.88C
70
60
50

30
236.87C 80
10

10 100
0 50 100 150 200 250 300
Exo Up Universal V4.7A TA Instruments
Temperature (C)
Figure 3 DSC and TGA thermograms of bupropion HCl Form 1.
1.0
0.8
Absorbance (AU)
0.6
0.4
0.2
0.0

200 220 240 260 280 300 320 340

Wavelength (nm)
Figure 4 The UV spectra of 10 g/mL bupropion HCl in methanol (solid line), ammo-
nium acetate aqueous phase pH 4.0 (dashed line), and 0.01 M HCl (dotted line).
10 S.R. Khan et al.

Table 5 UV/VIS Peak Assignments for Bupropion in Methanol

Wavelength Maxima (nm)a Chromophore
210 Aromatic ring
248251 Aromatic ring
295298 Ketone
a
Consistent with literature data [11].

The molar absorptivity in methanol is 22,278 L/mol cm at 210 nm. No

spectral peaks were observed above 360 nm in the visible spectral region.

3.8.2 Vibrational Spectroscopy

3.8.2.1 NIR
The NIR spectrum (11002500 nm) of bupropion HCl Form 1 was
acquired on a Foss NIR spectrometer equipped with a diffuse reflectance
apparatus over the range of 11002500 nm. The spectrum shown in
Fig. 5 represents the average of seven spectral acquisitions, which were per-
formed by scanning the powdered sample directly through the bottom of the
borosilicate-glass sample vial (transparent to NIR), with rotations of the
sample vial between each acquisition Table 6.

3.8.2.2 Fourier Transform IR

The Fourier transform IR (FTIR) spectrum of bupropion HCl Form 1 is
shown in Fig. 6. The spectrum was acquired on a Thermo Nicolet Nexus
670 FTIR equipped with an attenuated total reflectance accessory. The
structural assignments are provided in Table 7.

3.8.2.3 Raman
The Raman spectrum of bupropion HCl Form 1 is given in Fig. 7. The
spectrum was acquired on a Bruker MultiRAM Raman with a liquid-
nitrogen-cooled germanium diode detector Table 8.

3.8.3 Nuclear Magnetic Resonance Spectrometry

All NMR spectra were collected on a Varian NMR spectrometer (Agilent,
Santa Clara, CA) operating at a proton frequency of 399.82 MHz and a car-
bon frequency of 100.54 MHz. All data processing and analyses, including
multiplets analysis, were performed using Mnova NMR software (version
8.1.0-11315, Mestrelab Research S.L., Spain). Implemented Varian pulse
sequences included s2pul (for proton and carbon spectral acquisitions),
Bupropion Hydrochloride 11

C-H aromatic or CH3 stretch

CH aromatic or CH3 stretch

CH aromatic or CH3 stretch

2nd overtone aromatic CH stretch

CH aromatic
CH aromatic
CH aromatic
0.6

0.5
1st overtone symmetric CH3 stretch
1st overtone asymmetric CH3 stretch

1st overtone NH stretch

0.4 CH aromatic

CH aromatic
Absorbance

0.3
CH aromatic

0.2

0.1

1100 1158 1216 1274 1332 1390 1448 1506 1564 1622 1680 1738 1796 1854 1912 1970 2028 2086 2144 2202 2260 2318 2376 2434 2492
Wavelength (nm)

Figure 5 NIR spectrum of bupropion HCl Form 1.

gCOSY (for COrrelation SpectroscopY), NOESY (nuclear Overhauser

enhancement spectroscopy), HSQCAD (for heteronuclear single-quantum
correlation spectroscopy), gHMBCAD (for heteronuclear multiple-bond-
correlation spectroscopy), DEPT (for distortionless enhancement by polar-
ization transfer-135), tancpx (for 13C CP-MAS NMR spectral acquisition),
and tancpxt1 (for 13C T1 relaxation measurements).

3.8.3.1 1H
See Fig. 8 and Table 9.

3.8.3.2 COSY
See Fig. 9.

3.8.3.3 NOESY
See Fig. 10.
12 S.R. Khan et al.

Table 6 NIR Peak Assignments for Bupropion HCl Form 1

Wavelength (nm) Assignments [19]
2460 CH aromatic
2414 CH aromatic
2398 CH aromatic
2366 CH aromatic or CH3 stretch
2340 CH3 stretch
2296 CH aromatic or CH3 stretch
2260 CH aromatic or CH3 stretch
2150 Second overtone aromatic CH stretch
2096 NH
1996 First overtone C]O stretch
1756 First overtone NH stretch
1730 CH aromatic
1684 CH aromatic
1672 First overtone CH stretch
1374 CH aromatic
1178 First overtone symmetric CH3 stretch
1136 First overtone asymmetric CH3 stretch

90

85

80
% Transmittance

75

70

65

60

55

50

3500 3000 2500 2000 1500 1000

Wavenumbers (cm1)

Figure 6 The FTIR spectrum of bupropion HCl Form 1.

Bupropion Hydrochloride 13

Table 7 FTIR Peak Assignments for Bupropion HCl Form 1

Frequency (cm21) Assignments
3093 Aromatic CH stretch
2982 Aliphatic CH stretch
28402450 NH stretch, salt of secondary amine
1688 C]O stretch
1558 Aromatic CH stretch
CH3, asymmetric bending
1382 CH3, symmetric bending
1079 CCl, aryl chloride
Complete list of infrared peaks for bupropion HCl Form 1: 3363, 3093, 2982, 2840,
2745, 2670, 2607, 2453, 1688, 1558, 1458, 1427, 1403, 1382, 1281, 1237, 1211, 11-
70, 1134, 1079, 1021, 1005, 903, 864, 799, 780, 753, 738, 706, 671, and 530 cm1.
3067.91
2988.59
2933.09

1688.92
1592.16

994.32

740.87
649.94

199.72
146.11
5
4
Raman intensity
3
2
1
0

3500 3000 2500 2000 1500 1000 500

Wavenumber (cm1)
Figure 7 The Raman spectrum of bupropion HCl Form 1.

13
3.8.3.4 C
See Fig. 11 and Table 10.

3.8.3.5 HSQC
See Fig. 12.
14 S.R. Khan et al.

Table 8 Most Intense Raman Peak Positions for Bupropion HCl Form 1
Frequency (cm21) Assignments [20]
3068 Aromatic CH, amine
2989 CCH3
2933 CCH3
1689 Ketone
1592 Aromatic ring
994 Aromatic ring
741 CCl
650 CCl
200 Lattice vibrations
146 Lattice vibrations

O
8 HCl
13, 14, 15
Cl 6 7 NH CH3
16 1 5 9 10 12 15
CH3
2 4 CH3 CH3 14
3 11 13

3 120
6
100
80
4 2
60

10 10 40
20

9.5 9.0 8.5 8.0 7.5

ppm

11
4
6 3
2
10 10 9

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
ppm

Figure 8 1H NMR spectrum of bupropion HCl (400 MHz, DMSO-d6). Chemical shifts
reported in Table 9.
Bupropion Hydrochloride 15

Table 9 1H NMR Chemical Shifts (ppm) for Bupropion HCl at 400 MHz
CDCl3 (Spectrum D2O (Literature)
Predictionsa DMSO-d6 (from Fig. 8)b Not Shown)c [21,22]
9.77 (d, J 12.6 Hz, 1H) 12.11 (s, 1H)
8.60 (dd, J 12.8, 8.017.95 (m, 1H) 7.958
6.9 Hz, 1H)
7.89 8.24 (t, J 1.9 Hz, 1H) 7.90 (ddd, J 7.8, 1.7, 7.810
1.0 Hz, 1H)
7.85 8.14 (ddd, J 7.8, 1.7, 7.70 (ddd, J 8.0, 2.1, 7.450
1.0 Hz, 1H) 1.0 Hz, 1H)
7.67 7.82 (ddd, J 8.0, 2.1, 7.597.50 (m, 1H) 7.368
1.0 Hz, 1H)
7.47 7.63 (t, J 7.9 Hz, 1H) 6.95 (s, 1H)
4.30 5.355.23 (m, 1H) 4.83 (t, J 7.2 Hz, 1H) 4.242
1.32 1.51 (d, J 7.0 Hz, 3H) 1.93 (d, J 7.3 Hz, 3H)
1.27 1.29 (s, 9H) 1.50 (s, 9H)
a
ChemBioDrawUltra, version 12.0, CambridgeSoft.
b
Consistent with literature values [4,23].
c
Consistent with literature values [24].

O
8 HCl
Cl 6 7 NH CH3
16 1 5 9 10 12 15
CH3
2 4 CH3 CH3 14
3 11 13

10 6 23 9 11 0
4 13,14,15
1
13,14,15
11
2

4
f1 (ppm)

5
9

64 2 3 7
7.5
3 3
f1 (ppm)

2
4 2 8
6
8.0
4 9
6

10
10 8.5 8.0 7.5 10
ppm
11
11 10 9 8 7 6 5 4 3 2 1 0
ppm

Figure 9 NMR COSY contour plot of bupropion HCl (400 MHz, DMSO-d6).
 O
8 HCl
Cl 6 7 NH CH3
16 1 5 9 10 12 15
CH3
2 4 CH3 CH3 14
3 11 13

10 6 23 9 11 0
4 13,14,15
1
13,14,15
11
2

f1 (ppm)
5
9
6 4 2 3
7.5 6
3

f1 (ppm)
2 7
8.0
3 4
2
6 8
64

8.0 7.5 9
ppm
10
10

10 9 8 7 6 5 4 3 2 1 0
ppm

Figure 10 NMR NOESY contour plot of bupropion HCl (400 MHz, DMSO-d6) demon-
strates 1H1H through-space interactions (typically 4.5 or less).

O
8 HCI
Cl 6 7 NH CH3
16 1 5 9 10 12 15
CH3
2 4 CH3 CH3 14
3 11 13

1
5 6
4
2 3

12
134 132 130 128
ppm

5
1
7 6
13,14,15

4
11
9
3
2

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
ppm

Figure 11 13C NMR spectrum of bupropion HCl (100 MHz for carbon, DMSO-d6). Chem-
ical shift values reported in Table 10. Assignments confirmed by HSQC, HMBC, and DEPT
experiments.
Bupropion Hydrochloride 17

Table 10 Solution and Solid-State 13C NMR Chemical Shift and Relaxation Values
Solution NMR Chemical Shifts (ppm) Solid-State NMR
13
ChemDraw DMSO-d6 (from CDCl3 (Spectrum Chemical Shifts C T1 (s)
Predictionsa Fig. 11)b Not Shown) c
(ppm) (from Fig. 15) Relaxation
195.00 195.51 194.45 199.01 29.8
138.10 134.80, 134.71 136.20 136.80 76.7
(broad, split)
134.20 134.33 135.57, 135.52, 135.20 51.9
135.48
133.20 134.13 133.35 134.15 48.3
130.00 131.36, 131.28 131.07, 131.04, 131.72 60.7
(broad, split) 130.97
128.80 128.65 129.12, 129.06 129.35 75.4
126.90 127.81, 127.78 127.13, 127.10, 128.48 69.8
127.06
62.10 58.14 59.48 58.82 4.1
58.60 52.99, 52.87 53.79, 53.65 53.13 14.3
d
29.70 26.10, 26.03, 26.86, 26.78, 27.37
25.98, 25.93 26.75, 26.66
d
17.40 18.09, 18.06 18.58, 18.55, 19.58
18.51, 18.49
a
ChemBioDrawUltra, version 12.0, CambridgeSoft.
b
Consistent with literature values [23,24].
c
Consistent with literature values [24].
d
Very rapid relaxation; not observed under the experimental conditions.

3.8.3.6 HMBC
See Fig. 13A and B.

3.8.3.7 DEPT
See Fig. 14.

13
3.8.3.8 C Solid-State
See Fig. 15.
 O
8 HCI
Cl 6 7 NH CH3
16 1 9 10 12 15
5
CH3
14
2 4 CH3 CH3
3 11 13

6 2 3 9 11 10
11 4 13,14,15 20
13,14,15
30
40
50
9
11 13,14,15 10 60

6 4 2 3 70

f1 (ppm)
15
4 11 80
130 20
6
90
3 25
13,14,15 100
135 30
2 110
1.8 1.6 1.4 1.2
120
8.0 7.5
4
36 130
2
140
150

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
ppm

Figure 12 NMR HSQC contour plot of bupropion HCl (400 and 100 MHz for 1H and 13C,
DMSO-d6). Cross peaks are observed for all one-bond CH couplings.

O
A 8 HCl
Cl 6 7 NH CH3
16 1 5 9 10 12 15
CH3
2 4 CH3 CH3 14
3 11 13

6 4 2 3 9 11 13,14,15 15

11
20

25
13,14,15

30
50

9
f1 (ppm)

55

12
60

4
6
130
3

51 135

195
7

8.6 8.4 8.2 8.0 7.8 7.6 7.4 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4240 1.8 1.6 1.4 1.2 1.0
ppm

Figure 13 (A) NMR heteronuclear multiple bond correlation (HMBC) contour plot of
bupropion HCl (400 and 100 MHz for 1H and 13C, DMSO-d6). Cross peaks are observed
for two-bond and some three-bond CH couplings. Several spinning sidebands (spin
speed of 20 Hz) are observed. All regions of the contour plot with cross peaks.
Bupropion Hydrochloride 19

O
B 8 HCl
Cl 6 7 NH CH3
16 1 5 9 10 12 15

CH3
2 4 CH3 CH3 14
11 13
3

6 4 2 3
127

128
4

6 129

130

131

f1 (ppm)
3
132

133

134
1
5 135

136

137

8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5
ppm

Figure 13Cont'd (B) NMR HMBC contour plot of bupropion HCl (400 and 100 MHz for
1
H and 13C, DMSO-d6). Expansion of the aromatic region, which was used for the assign-
ment of C1 and C5 peaks.

3.8.4 Mass Spectrum

A Waters Corporation SQD 3100 mass spectrometer interfaced with a
Waters Acquity UPLC was operated in the positive ion mode with
electrospray ionization source temperature of 150C, desolvation tempera-
ture 350C, desolvation gas flow 700800 L/h, and Cone Voltage of 22.3
20 S.R. Khan et al.

1
25 3 6 4 O
8
HCl
Cl 6 7 NH CH3
16 1 5 9 10 12 15
6
51 4 CH3
2 3
2 4 CH3 CH3 14
3 11 13

136 135 134 133 132 131 130 129 128 127
ppm

4
9
2 13,14,15
DEPT spectrum 53
11
7 1 12

12
5
7 1 6
13,14,15
Unedited spectrum 4
9 11
2 3

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 30 20 10 0
ppm

Figure 14 13C NMR of bupropion HCl (100 MHz, DMSO-d6). The lower spectrum is a
standard 13C spectrum, and the upper spectrum is a DEPT-135 spectrum in which
the quaternary carbons are suppressed, tertiary and primary carbons (CH/CH3) are pos-
itive, and secondary carbons (CH2, none present in bupropion) would be inverted.

(analyte-specific parameter). Mass spectra were collected on a Waters Cor-

poration SQD 3100 single quadrupole mass spectrometer in the full scan ES
positive mode. Figure 16 illustrates the optimized cone voltage for creating
the bupropion product ions at m/z 240.17 and m/z 242.17 (bupropion chlo-
rine isotope). Minor fragmentation peaks were also observed at m/z 262 and
m/z 365. All data processing was performed using Waters Empower 2 soft-
ware (Waters Corporation, Milford, MA).

4. METHODS OF ANALYSIS
4.1 Electrochemical Analysis
Bupropion hydrochloride has been analyzed by potentiometric methods in
the pharmaceutical formulation matrix. Ganjali et al. have reported the use of
a modified carbon paste electrode and ionic liquid to monitor bupropion
hydrochloride in tablet formulations [25]. They also reported a second
Bupropion Hydrochloride 21

13,14,15

11

12
1,2,3,4,5,6 9

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
ppm

Figure 15 Experimental 13C CP-MAS NMR spectrum of crystalline bupropion hydrochlo-

ride. 1H T1 0.505 (
0.029) s. The asterisk denotes a spinning sideband of the carbonyl-
carbon peak ( 199.0), and peaks below 10 ppm correspond to spinning sidebands of
the aromatic peaks ( 133), spin speed 13 kHz. The spectrum was externally
referenced to 3-methylglutaric acid (methyl peak at 18.84 ppm). The assignment of
C9 was confirmed through interrupted-decoupling experiments (not shown). Chemical
shift values reported in Table 10.

Combined -19 09 2.5 ug/mL SQD - SQ 1: MS scan 1: 150.00 650.00 ES+, Centroid, CV = 24
3.0 106 240.17

2.5 106

2.0 106
Intensity

1.5 106

242.17
1.0 106

5.0 105

0.0
200.00 300.00 400.00 500.00 600.00
m/z
Figure 16 Mass spectrum of bupropion hydrochloride.
22 S.R. Khan et al.

potentiometric method that used a PVC membrane electrode with

bupropion hydrochloride embedded as an ionophore. Both potentiometric
methods were reported to be based on ion-exchange using an ion-pair com-
plex of bupropion hydrochloride and sodium tetraphenyl borate.

4.2 Chromatographic Analysis

4.2.1 Thin Layer Chromatography
Yeniceli and Dorrukol-Ak have reported a sensitive thin layer chromatog-
raphy (TLC) method for bupropion hydrochloride in pharmaceutical
tablets [26]. High performance silica gel 60 plates were used with ethanol-
chloroform-glacial acetic acid 30:10:1 (v/v). The development length was
8 cm and the Rf was 0.56. Densitometry was performed at 256 nm.
A novel use of amino acids was reported by Batra and Bhushan for use as
chiral selectors for enantiomeric separation of bupropion using chiral ligand-
exchange TLC [27]. The United Stated Pharmacopeia (USP) previously
reported in USP 29NF 24, a TLC method for the related impurities of
bupropion contained in the analytical monograph for bupropion [28].

4.2.2 Gas Chromatography

Rohrig and Ray first reported the use of gas chromatography with nitrogen-
phosphorus detection to evaluate bupropion in human tissue [29]. Sane et al.
reported a gas chromatography method for the determination of bupropion
in a pharmaceutical formulation using a nitrogen carrier gas and flame ion-
ization detection [30].

4.2.3 High-Performance Liquid Chromatography

Bupropion has been analyzed by high-performance liquid chromatography
(HPLC). These HPLC methods have been applied to pharmaceutical char-
acterization [31,32], plasma level determinations [3335], and pharmacoki-
netics [36,37]. Bupropion and its synthetic impurities can also be analyzed
using HPLC. A modified USP method was used to chromatographically
resolve bupropion from USP-related impurity compounds using an Agilent
series 1100 HPLC system (Wilmington, DE), equipped with a quaternary
pump, a vacuum solvent degasser, a thermostated autosampler, a
thermostated column compartment and a diode array detector (DAD). Sep-
aration was achieved on a Zorbax SB reverse phase C18 column
(150 mm  4.6 mm i.d., 3.5 m) maintained at 30C. The mobile phase
consisted of 90% H2O: 10% ACN: 0.04% TFA (A), and 5% H2O: 95%
ACN: 0.03% TFA (B). The following gradient was used: 0 min, 87%
Bupropion Hydrochloride 23

Compound C
Compound F
Absorbance

3-Chlorobenzoic acid
Bupropion
0 1 2 3 4 5 6 7 8 9 10
Time (min)
Figure 17 Typical chromatogram of bupropion and USP-related compounds:
0.3 mg/mL bupropion hydrochloride detected at 250 nm (retention time 6.42 min),
0.0018 mg/mL USP-related compound F, 1-(3-chlorophenyl)-1-hydroxy-2-propanone
(7.14 min), 0.0018 mg/mL USP-related compound C (7.71 min), 1-(3-chlorophenyl)-2-
hydroxy-1-propanone, and 0.045 mg/mL 3-chlorobenzoic acid (8.54 min).

A13% B; 10 min, 75% A-25% B; 10.1 min, 0% A-100% B; 13.2 min,

87% A-13% B; 18 min, 87% A-13% B for sample elution. The flow rate
was set at 1 mL/min and the injection volume was 2 L. DAD detection
was achieved in the range of 190400 nm and the detection wavelength was
set at 226 nm for quantitative analysis. The chromatogram is shown in Fig. 17.

4.2.4 Ultra Performance Liquid Chromatography

Bupropion was chromatographically resolved from its major metabolites
using a Waters Acquity Series ultra performance liquid chromatography
(UPLC) system equipped with binary solvent pump, autosampler, photo-
diode array detector, thermostated column compartment, and Empower 2
chromatographic software. Separation was achieved on an Acquity ethyl-
ene bridged hybrid [BEH] C18 (100  2.1 mm i.d., 1.7 m), placed in
series with an Acquity BEH C18 guard column (20  2.1 mm i.d.,
1.7 m). Analytes were detected at 212 nm. All experiments were carried
out at 35C and at a flow rate of 0.5 mL/min mobile phase with an injec-
tion volume of 10 mL under full loop conditions. Mobile phase was
degassed with on-line degasser and delivered isocratically containing
24 S.R. Khan et al.

0.007

Bromobupropion (IS)
0.006

Bupropion
Hydroxybupropion

Threo-bupropion
0.005

Erythro-bupropion
0.004
AU

0.003

0.002

0.001

0.000

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes
Figure 18 UPLC chromatogram of bupropion and its metabolites and the
bromobupropion analog. The limit of detection of bupropion and bromobupropion
is at least 2 ng/mL at 250 nm. The limit of detection for hydroxybupropion, erythro-,
and threo-bupropion is at least 5 ng/mL at 212 nm.

4.0 mM ammonium formate buffer at 7% ACN and 3.1% THF (pH 4.02).
The chromatogram is shown in Fig. 18.

5. STABILITY
5.1 Solution Stability
The chemical stability of bupropion hydrochloride in 0.01 N HCl was
tested under long-term storage at 4C. Solutions of approximately 1 mg/mL
were prepared and analyzed by UPLC/MS. Bupropion hydrochloride was
stable under these conditions. Results for the amount of bupropion measured
when compared to a freshly prepared solution are shown in Table 11.
Bupropion was found to be less stable with increasing pH in the
range of 513. Bupropion has increasing stability at pH 7.4 with the follow-
ing buffers: Borate < TRIS < Phosphate < Citrate [53]. The half-life of
bupropion in isotonic phosphate medium at pH 7.4 was found to be
9.9 d [38]. Bupropion was also found to be less stable with increasing pH
in formalin solutions in the pH range of 3.09.5. Increasing formalin con-
centration in the range of 520% in water resulted in lower bupropion
stability [39].
Fang et al. studied the rate of racemization of enantiopure (S)-bupropion
hydrochloride in phosphate buffer (pH 7.4, 25C) using chiral-HPLC
Bupropion Hydrochloride 25

Table 11 Stability of Bupropion in 0.01 N HCl at 4C

65 Days 97 Days 188 Days
Amount bupropion found 100.0% 102.3% 101.4%

analysis [6]. The authors found that racemization readily took place under
these conditions: 42% in 2 h, 62% in 4 h, and >94% in 24 h. Additionally,
the authors note that the enantiopure hydrochloride salts were prepared in
an ethyl ether solution, presumably to minimize racemization during the
synthesis.

5.2 Solid-State Stability

Physical stability: Two crystalline polymorphs (Form 1 and Form 2) of
bupropion HCl have been reported in the literature. Maccaroni et al.
reported that the crystal forms are enantiotropically related, where Form
2 is stable at room temperature but converts to Form 1 at temperatures
greater than 190C [17]. Form 2 was discovered by Maccaroni et al. due
to the physical instability of Form I, the powder of which underwent a
solidsolid transformation to Form 2 upon storage (approximately 1 year)
at ambient laboratory conditions.
Chemical stability: No studies on the solid-state chemical stability of
bupropion HCl have been reported in the literature.

5.3 Stability in Biological Medium

Bupropion was found to be unstable in plasma, while shielded from light at
room and physiological temperatures and over the pH range of 510 [40,41].
The half-lives were 54.2 and 11.4 h at 22 and 37C, respectively. A second
patient had a half-life of 41.9 h at 22C. Degradation appeared to occur in a
log linear fashion. However at pH 2.5 there was no significant change in the
amount of bupropion in plasma up to 48 h. Bupropion was stable at 17C
and pH 7.4 for up to 326 d [41]. Bupropions three major metabolites
hydroxybupropion, threo-bupropion, and erythro-bupropion were stable
under all the above conditions [40,41].
Bupropion was also found to be unstable in serum at room temperature
and 4C [40]. There was 24% loss of bupropion at 4 h and 91% loss after 4 d
in serum exposed to light at room temperature. Degradation of bupropion
occurred to a lesser extent in serum stored in the dark. There was 8% loss of
bupropion at 4 h and 80% loss after 4 d at room temperature. Bupropion was
stable in serum at 4C for 24 h, but after 4 d there was 16% loss.
26 S.R. Khan et al.

Hydroxybupropion was stable under all the above conditions. Appropriate

storage and handling of bupropion samples in biological medium is impor-
tant for reliable results

6. BIOLOGICAL PROPERTIES
6.1 Toxicity
Bupropion is associated with mild to moderate toxicity. Development of
seizures occurs most often at the higher doses with XL [42]. Bupropion is
considered moderately dangerous: overdosage (greater than 5 g) may lead
to severe neurological and cardiovascular toxicity. Unwanted effects (side
effects) of bupropion use in humans include: severe blistering, peeling,
and red skin rash, fever, swollen glands, rash or itching, joint pain, or general
ill feeling, confusion, trouble concentrating, or hallucinations, and unusual
thoughts or behavior [42]. The toxicity of bupropion was determined in
man, rats, and mice. The fatal toxicological dose has been estimated to be
329 mg/kg (LDLo, oral. human). For animals, the following LD50 values
have been reported: 482 mg/kg (oral, rat), 210 mg/kg (intraperitoneal,
rat), 544 mg/kg (oral, mouse), and 230 mg/kg (intraperitoneal, mouse)
[12,42,43].

6.2 Drug Metabolism and Pharmacokinetics

6.2.1 Absorption
Bupropion is rapidly absorbed in the gastrointestinal tract after oral admin-
istration of IR formulations [44]. In man, absolute bioavailability of
bupropion has not been determined because an IV formulation is currently
not available. However, intestinal absorption has been reported to be nearly
100% [45]. Systematic bioavailability is less than 100% because of extensive
first pass metabolism, and studies in rats and dogs suggest that the absolute
bioavailability may range between 5% and 20% [45,46]. Urine excretion
data from drug-product labeling indicate that approximately 87% of the dose
of bupropion is absorbed [9,47]. According to pharmacokinetic studies in
healthy volunteers, bupropion and its metabolites appear to be absorbed
throughout the gastrointestinal tract, with diminished absorption near the
colon [48].

6.2.2 Distribution
Bupropion is extensively distributed throughout the body. In addition,
highly bound (>80%) to human plasma proteins over a wide concentration
Bupropion Hydrochloride 27

range (up to 200 g/mL) [44]. The extent of protein binding of the hydro-
xybupropion metabolite is similar to that for bupropion, whereas the protein
binding of the threo-bupropion metabolite is about half that of bupropion.

6.2.3 Metabolism
Bupropion is extensively metabolized in humans by hepatic enzymes
(primarily CYP2B6) to three metabolites: (2S,3R)- and (2S,3S)-hydro-
xybupropion, (R,R)- and (S,S)-threo-bupropion, and (R,S)-, and (S,R)-
erythro-bupropion (Fig. 19) [44,48,49]. The approximate Tmax value for
bupropion is 1.55 h depending on the drug-product formulation (IR,
SR, and XL, respectively), for hydroxybupropion it is 7 h, and for threo-
bupropion and erythro-bupropion it is 8 h. The Cmax values of hydro-
xybupropion and threo-bupropion are four- to eightfold and three- to
fivefold greater, respectively, than that of bupropion [45,50].
Bupropion and its metabolites exhibit linear kinetics following single
doses and chronic administration of bupropion dose strengths of
150450 mg/d [44,48,49]. Following daily dosing, bupropion and its
metabolites generally reach steady state in 58 d [47]. The metabolic profile
is shown in Fig. 19.

O
H
N
Cl
C(CH3) 3

CH3

Bupropion
OH OH
H H
N N
Cl Cl
C(CH3) 3 C(CH3) 3

P-450 CYP2B6
CH3 CH3
CH3

Threo-bupropion Erythro-bupropion
O CH3
Cl NH

OH
CH3

Hydroxybupropion
Figure 19 Metabolism of bupropion.
28 S.R. Khan et al.

6.2.4 Elimination
Bupropion is metabolized extensively so that less than 10% of the parent
drug is eliminated in urine or feces [51]. Only 1% of bupropion is eliminated
in the urine as unchanged drug with the remaining bupropion and metab-
olites eliminated in the urine as glycine conjugates [46]. The mean half-life
of bupropion is 3.5 h, and the terminal elimination half-life is approximately
18 h [52]. A single dose of 150 or 300 mg of bupropion generally requires
approximately 67 d for complete elimination from the body [46].

ACKNOWLEDGMENTS
The authors would like to acknowledge Mr. Alan Carlin for the collection NIR spectra and
verification of the IR, NIR, and Raman data. The authors would like to thank Mr. Arthur
Bryant and Mr. Bryan Lowry for determining the melting point range and Ms. Gretchen
Whitesell for determining the pH of the bupropion hydrochloride solutions.

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 CHAPTER FOUR

Dacarbazine
Abdullah A. Al-Badr, Mansour M. Alodhaib
King Saud University, Riyadh, Saudi Arabia

Contents
1. Description 323
1.1 Nomenclature 323
1.2 Formulae 324
1.3 Elemental Analysis 324
1.4 Appearance 324
2. Uses and Applications 324
3. Methods of Preparation 325
4. Physical Characteristics 326
4.1 Ionization Constant 326
4.2 Solubility Characteristics 326
4.3 X-ray Studies 327
4.4 Thermal Methods of Analysis 336
4.5 Spectroscopy 336
4.6 Mass Spectrometry 341
5. Methods of Analysis 343
5.1 Compendial Methods of Analysis 343
5.2 Reported Methods of Analysis 355
6. Metabolism 363
7. Pharmacokinetics 368
8. Stability 370
9. Reviews 373
Acknowledgments 374
References 374

1. DESCRIPTION
1.1 Nomenclature
1.1.1 Systematic Chemical Names
5-(3,3-Dimethyl-1-triazenyl)-1H-imidazole-4-carboxyamide.
5-(or 4)-(Dimethyltriazeno)-imidazole-4(or 5)carboxamide.
1H-Imidazole-4-carboxamide, 5-(3,3-dimethyl-1-triazenyl).

Profiles of Drug Substances, Excipients, and Related Methodology, Volume 41 # 2016 Elsevier Inc. 323
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/bs.podrm.2015.12.002
324 Abdullah A. Al-Badr and Mansour M. Alodhaib

1H-Imidazole-4-carboxamide, 5-[(1E)-3,3-dimethyl-1-triazen-1-yl].
4-[(1E)-3,3-Dimethyl-1-triazen-1-yl]-1H-imidazole-5-carboxyamide.
5-(3,3-Dimethyltriaz-1-en-1-yl)-1H-imidazole-4-carboxamide.
5-[(1E)-3,3-Dimethyltriaz-1-en-1-yl]-1H-imidazole-4-carboxamide
[1,2].

1.1.2 Nonproprietary Names

Dacarbazina, Dacarbazinum, Dakarbatsuni, Dacarbazin, Dacarbazine, DIC,
DTIC, and NSC 45388 [1,2].

1.1.3 Proprietary Names

Dacarbazina, Dacarbazine, Dacarbazin, Deticene, DTIC, Oncocarbil,
Dacatic, and Detimedac [1,2].

1.2 Formulae
1.2.1 Empirical Formula, Molecular Weight, and CAS Number
Empirical formula is C6H10N6O, molecular weight is 182.20; and CAS
number is 4342-03-4.

1.2.2 Structural Formula

O H O
N NH2 N NH2

N N CH3 N N CH3
N N
N N
H
CH3 CH3
1.3 Elemental Analysis
C: 39.56%, H: 5.53%, N: 46.13%, and O: 8.78%.

1.4 Appearance
A white or slightly yellowish, crystalline powder [2].

2. USES AND APPLICATIONS

Dacarbazine is a cell cycle nonspecific antineoplastic agent that func-
tions as an alkylating agent after activation in the liver. The drug is used
in the treatment of metastatic malignant melanoma. It is also given to
patients with Hodgkins disease, notably with doxorubicin, bleomycin,
and vinblastine. Dacarbazine is used with other drugs in the treatment of soft
Dacarbazine 325

tissue sarcoma and may be given in neuroblastoma, Kaposis sarcoma, and

other tumors. Dacarbazine is given by the intravenous route. Injections
may be given over 1 min. The reconstituted solution can be further diluted
with up to 300 mL of glucose 5% or sodium chloride 0.9% and given by
infusion over 1530 min; doses of 200 mg/m2 and over are usually infused.
Dacarbazine is licensed for use as a single agent for metastatic melanoma in
doses of 24.5 mg/kg daily for 10 days, repeated at intervals of 4 weeks, or
200250 mg/m2 daily for 5 days, repeated at intervals of 3 weeks. The drug
can also be given in a dose of 850 mg/m2 by intravenous infusion at 3-week
intervals. In the treatment of Hodgkins disease, doses of 150 mg/m2 daily
for 5 days repeated every 4 weeks or 375 mg/m2 every 15 days have been
given with other agents. In the treatment of soft tissue sarcoma, dacarbazine
250 mg/m2 is given daily for 5 days repeated every 3 weeks; it is usually
given with doxorubicin [24].

3. METHODS OF PREPARATION
Shealy et al. [5] prepared dacarbazine by a coupling reaction of
4-diazoimidazole-5-carboxamide 6 as follows: 4-aminoimidazole-5-
carboxamide 6 was allowed to react with sodium nitrite in acidic solution
to produce the 5-diazonium chloride derivative 7 which on treatment
with dimethylamine gives dacarbazine 8 (Scheme 1). The synthesis of
4-aminoimidazole-5-carboxamide 6 is described by Shaw and Woolley
[6] and by Iradyan et al. [7].
Shaw and Woolley [6] prepared 4-aminoimidazole-5-carboxamide 6 as
follows:
The imino ethyl ether derivative 2 of ethyl cyanoacetate 1 was treated
with alcoholic ammonia, and simultaneous introduction of the amidine
and amide groups is taking place. The resultant malonamamidine 3 was
coupled with benzene diazonium chloride to yield the phenylazo derivative
4. The phenylazo derivative 4 was reduced with zinc dust in 98% formic

O O O
NH2 NH2 NH2
HN HN HN
NaNO2/HCl HN(CH3)2 CH3
NH2 N N Cl N N N
N N N
CH3
6 7 8
Scheme 1 Synthesis of dacarbazine [5].
326 Abdullah A. Al-Badr and Mansour M. Alodhaib

OC2H5 OC2H5 NH2

O O NH3 O NH2 N N Cl
CH2 C N CH2 C NH CH2 C
OC2H5 NH

1 2 3

NH2 NH2
O NH2 O
O NH2
CH C H2N NH2
CH C
N NH HN NH
HCl HN N
N HCl O
H
4 5 6
Scheme 2 Synthesis of 4-aminoimidazole-5-carboxamide HCl [6].

acid, and the formamidoamidine 5 was obtained. Ring closure of the for-
mamidoamidine 5 to form 4-aminoimidazole-5-carboxamide 6 was
achieved most conveniently merely by melting the formamidoamidine 5
as the hydrochloride (Scheme 2).
Iradyan et al. [7], in a comprehensive review on the antitumor activity of
imidazole derivatives, dacarbazine and imidazine, outlined a scheme for the
preparation of 4-aminoimidazole-5-carboxamide 6 from which dacarbazine
8 was produced: treatment of ethyl cyanoacetate 1 with ethanol and
hydrochloric acid provided the imino ethyl ether derivative 2. Compound
2 on treatment with alcoholic ammonia produced the amide 3. Compound
3 when allowed to react with benzene diazonium chloride produced the
phenylazo compound 4. Compound 4 was reduced with zinc and formic
acid and the formamide product 5 was formed. Compound 5 was cyclized
and 4-aminoimidazole-5-carboxamide 6 was obtained. Treatment of com-
pound 6 with sodium nitrite in acidic solution produced the 4-diazonium
chloride derivative 7. Treatment of compound 7 with dimethylamine pro-
duced dacarbazine 8 (Scheme 3).

4. PHYSICAL CHARACTERISTICS
4.1 Ionization Constant
pKa value: 4.4.

4.2 Solubility Characteristics

Slightly soluble in water and in anhydrous alcohol. Practically insoluble in
dichloromethane [2].
Dacarbazine 327

O O
NH3
N C CH2 C OC2H5 C2H5OH, HCl HCl.HN C CH2 C OC2H5
OC2H5
1 2

O O
N N Cl
HCl.HN C CH2 C NH2 HCl.HN C CH C NH2 Zn, HCOOH
NH2 NH2
N N

3 4

O
NH2
O N
t NaNO2/HCl
HCl.HN C CH C NH2 NH2
N
NH2 HN C H H
O
5 6

O O
NH2 NH2
N HN(CH3)2 N CH3
N N Cl N N N
N N
H H CH3

7 8
Scheme 3 Synthesis of dacarbazine [7].

4.3 X-ray Studies

4.3.1 X-ray Powder Diffraction Pattern
Bei et al. [8] investigated the physicochemical properties of dacarbazine-
loaded cubosomes (Fig. 1). The drug-loaded cubosome nanocarriers were
prepared by a fragmentation method and then freeze dried. They were then
characterized for size, morphology, thermal behavior, and crystallography
using dynamic light scattering, transmission electron microscopy, differen-
tial scanning calorimetry, and powder X-ray diffraction. The drug loading
and encapsulation efficiency were determined by ultraviolet spectropho-
tometry. The results showed that the prepared dacarbazine-loaded cub-
osomes had mean diameters ranging from 86 to 106 nm. In addition to
the transmission electron microscopy, the characteristic peaks from powder
X-ray diffraction data suggested that the freeze-dried nanoformulations
were cubic in nature. Differential scanning calorimetry and powder
X-ray diffraction analysis suggested that the 0.06% or 0.28% (w/w) actual
drug loaded inside cubosomes was in the amorphous or molecular state.
328 Abdullah A. Al-Badr and Mansour M. Alodhaib

Figure 1 The X-ray powder diffraction pattern of native dacarbazine [8].

These physicochemical characteristics would affect the nanoformulation

shelf-life, efficacy, and safety.

4.3.2 Crystal Structure

Freeman and Hutchinson [9] determined the crystal structure of the anti-
tumor agents: 5-(3,3-dimethyl-l-triazenyl)-imidazole-4-carboxamide
(NSC-45388) from three-dimensional X-ray data. The crystals are mono-
clinic, space group P21/n, with a 14.042 (2), b 10.661 (2), c 11.914
(4) A, 91.49 (8) degree, V 1783.0 (8) A3, Z 8. The structure was
solved by direct methods and refined using block-diagonal least-squares
calculations. The final R for 1350 independent observed reflections is
0.042. There are two molecules in the asymmetric unit. In one molecule
the protonated N in the imidazole ring is adjacent to the triazene group;
and in the other, it is adjacent to the carboxamide group. Each molecule
is approximately planar and contains an internal NdHN hydrogen bond.
Intermolecular hydrogen bonding produces sheets of molecules lying
approximately perpendicular to the b axis.
The title compound (DTIC) is used in the chemotherapy for malignant
melanoma. The structure of the cation HDTIC+ in crystals grown at very
low pH has been determined by Edwards et al. [10]. The structure of neutral
DTIC, as part of a study of the drug and its interactions with transition metal
ions, is reported [11].
Dacarbazine 329

A crystal exhibiting the forms {100} and {011}, with dimensions

0.30  0.12  0.12 mm, was selected from a sample of DTIC supplied by
the Drug Development Branch, National Cancer Institute, Bethesda,
Maryland. Diffraction data were recorded on an Enraf-Nonius CAD-4/F
automatic diffractometer using graphite-monochromated Mo Ka radiation
[(Mo Ka1) 0.70926, (Mo Ka2) 0.71354 A]. The 2 angle of the mono-
chromator was 12.18 degree and the crystal-to-detector distance was
173 mm. Unit-cell dimensions were obtained by least-squares refinement
of 2 values for 23 automatically centered reflections ( > 17 degree).
Crystal data
Molecular formula C6H10N6O, Mr 182.20, monoclinic, a 14.042 (2),
b 10.661 (2), c 11.914 (4) A, 91.49 (8) degree, V 1783.0 (8) A3;
space group P21/n from systematic absences (h0l absent for h + l odd,
0 k 0 absent for k odd). Dx 1.357 Mg/m3 for Z 8 (2 molecules per
asymmetric unit).
Profile analysis of a representative reflection indicated that the conditions
for the measurement of integrated intensities would be optimized by -(S)
2 scans, where S 1/2. The -scan angle and the horizontal counter aper-
ture, both reduced as much as possible so as to minimize the effect of thermal
diffuse scattering [12], were (1.5 + 0.35 tan ) degree and (1.8 + 0.35 tan )
mm, respectively. The scan speeds were determined by a required precision
(I) < 0.005 I, subject to a maximum scan time of 180 s per reflection. Each
reflection was scanned in 96 steps. The peak count P was recorded over the
central 64 steps, with 16 steps at each end to measure the backgrounds B1 and
B2. The intensity I was calculated as I [P - 2(B1 + B2)] with standard devi-
ation (I) { [P + 4(B1 + B2)]}1/2, where is a factor to account for the
differences in scan speeds.
Three reference reflections were measured after every 250 min of X-ray
exposure. The orientation of the crystal was checked after every 200 reflec-
tions. No decomposition or movement of the crystal was detected. Inten-
sities were recorded for 2183 (hkl)  reflections and 1655 equivalent (hkl) 
reflections ( < 22 degree).
The data were corrected for the Lorentz and polarization factors.
Absorption corrections were not applied ( 0.110 mm1). There were
732 pairs of equivalent reflections Pwith I >P 3(I). The unweighted discrep-
ancy factor RD, defined as ( jFj2/ jFavj2)1/2, was 0.027, where
F jF hkl  j  jF hkl
 and Fav [jF(hkl)j
 + jF(hkl)j]/2.

An analysis of the errors in the data was made by dividing the data into
22 ranges of jFavj and plotting the mean [(F)2  2stat(F)] values vs the mean
330 Abdullah A. Al-Badr and Mansour M. Alodhaib

jFavj values. Here 2stat (F) was the variance of an observed structure factor
from counting statistics alone. The function Vs(F) l + mjFj + njFj2 + pjFj3,
representing the contributions of systematic errors to the variances [13], was
fitted to the above plot. The coefficients were l 1.69  101,
m 3.86  102, n 1.14  103, and p 9  106. (By coincidence, these
coefficients were on an approximately absolute scale. The factor subse-
quently required to convert the arbitrary Fs to an absolute scale was
1.057.) A new variance 2(F) for each reflection was calculated as the
sum of 2stat (F) and Vs (F). The data were then reduced to a single list of
2183 F values by averaging F(hkl) and F(hkl) whenever both had been mea-
sured. There were 833 F values derived from intensities I < 3(I). The
remaining 1350 F values were used in the structure analysis.

Structure Determinations and Refinement

The structure was solved by means of the direct methods program package
MULTAN [14]. The starting data were the 400 reflections with jEj > 1.3.
The set of phases with the highest figure of merit led to an E map in which
all the nonhydrogen atoms could be located. Scattering factors for O, N, C,
and H were taken from International Tables for X-ray Crystallography
(1974) [15]. Initially the structure was refined by Pfull-matrix least-squares
calculations. The function minimized was w(jFoj  sjFcj)2, where
w 2(F) and s is a scale factor. After several cycles of refinement in which
the nonhydrogen atoms had anisotropic thermal parameters, the H atoms
were located in an (Fo  Fc) synthesis. In the final refinement cycles, the
H atoms were included but were given a fixed thermal parameter
(Uiso 0.059 A2). At this stage the matrix was partitioned into two blocks each
containing the parameters for one molecule of the asymmetric unit. A final
3
difference Fourier synthesis
P showed noP peaks larger than 0.30 e/AP. The final
residualsPwere R( jjFoj  sjFcjj/ jFoj) 0.042 and Rw{[ w(jFoj 
sjFcj)2/ wjFoj2]1/2} 0.030 for the 1350 reflections used in the refinement.
The atomic positional parameters are shown in Table 1.

Description of the Structure

The asymmetric unit consists of two nonidentical molecules. Their
dimensions are shown in Fig. 2. In molecule 1, an H atom (located
by the structure analysis) is attached to the imidazole nitrogen N(2), adja-
cent to the triazene group. In molecule 2, the protonated imidazole
nitrogen is N(7), adjacent to the carboxamide group. The formal
nomenclature, in which the numbering starts at the imidazole N to
Dacarbazine 331

Table 1 Positional Parameters (Fractional Coordinates 104) with Estimated Standard

Deviations in Parentheses [9]
x y z
C(1) 8,337 (2) 8,195 (3) 3,686 (3)
C(2) 9,003 (2) 8,602 (3) 5,331 (2)
C(3) 9,689 (2) 8,263 (3) 4,581 (2)
C(4) 10,724 (2) 8,120 (3) 4,750 (3)
C(5) 9,470 (3) 9,668 (4) 8,496 (3)
C(6) 7,695 (3) 10,018 (5) 8,562 (4)
C(7) 6,009 (2) 8,072 (3) 6,451 (3)
C(8) 5,379 (2) 8,474 (3) 4,842 (2)
C(9) 4,652 (2) 8,151 (3) 5,530 (2)
C(10) 3,609 (2) 8,055 (3) 5,358 (3)
C(11) 4,978 (3) 9,451 (5) 1,618 (3)
C(12) 6,740 (3) 9,858 (4) 1,628 (4)
N(1) 9,264 (2) 8,007 (2) 3,542 (2)
N(2) 8,147 (2) 8,555 (3) 4,745 (2)
N(3) 11,065 (2) 8,417 (3) 5,775 (3)
N(4) 9,159 (2) 8,953 (2) 6,438 (2)
N(5) 8,391 (2) 9,285 (3) 6,921 (2)
N(6) 8,532 (2) 9,651 (3) 7,956 (2)
N(7) 5,073 (2) 7,894 (2) 6,563 (2)
N(8) 6,238 (2) 8,428 (2) 5,424 (2)
N(9) 3,281 (2) 8,296 (3) 4,329 (2)
N(10) 5,235 (2) 8,808 (2) 3,720 (2)
N(11) 6,013 (2) 9,130 (2) 3,252 (2)
N(12) 5,891 (2) 9,466 (3) 2,201 (2)
0(1) 11,228 (1) 7,742 (2) 3,992 (2)
0(2) 3,098 (1) 7,745 (2) 6,139 (2)
H(1) 7,859 (20) 8,045 (28) 3,036 (24)
Continued
332 Abdullah A. Al-Badr and Mansour M. Alodhaib

Table 1 Positional Parameters (Fractional Coordinates 104) with Estimated Standard

Deviations in Parentheses [9]cont'd
x y z
H(2) 7,576 (20) 8,593 (29) 5,069 (28)
H(3) 10,606 (22) 8,821 (26) 6,393 (24)
H(4) 11,567 (23) 8,339 (34) 5,886 (28)
H(5) 9,769 (23) 8,903 (29) 8,439 (27)
H(6) 9,963 (20) 10,081 (28) 7,937 (26)
H(7) 9,364 (20) 10,089 (29) 9,199 (26)
H(8) 7,803 (22) 10,691 (32) 8,967 (27)
H(9) 7,546 (22) 9,402 (30) 9,152 (25)
H(10) 7,135 (21) 9,978 (30) 8,098 (26)
H(11) 6,449 (20) 7,928 (27) 7,137 (25)
H(12) 4,784 (22) 7,601 (30) 7,206 (24)
H(13) 3,616 (23) 8,585 (30) 3,842 (26)
H(14) 2,654 (21) 8,250 (30) 4,157 (26)
H(15) 4,583 (23) 8,842 (30) 1,869 (27)
H(16) 4,546 (21) 10,188 (29) 1,957 (26)
H(17) 5,092 (22) 9,522 (30) 890 (24)
H(18) 6,572 (22) 10,435 (32) 1,132 (27)
H(19) 6,912 (22) 9,230 (30) 1,073 (25)
H(20) 7,234 (22) 10,117 (30) 2,151 (27)

which the H is attached, is 5-(3,3-dimethyl-l-triazenyl)-imidazole-4-

carboxamide for molecule 1 and 4-(3,3-dimethyl-l-triazenyl)-imidazole-
5-carboxamide for molecule 2.
A comparison of the dimensions of molecules 1 and 2 in the orientations of
Fig. 2 reveals a significant difference (4.5 times its own standard deviation)
between the lengths of the bonds C(1)dN(2), 1.352 (4) A, and C(7)dN(8),
1.328 (4) A. In addition, every internal bond angle in the imidazole ring of
molecule 1 except N(1)dC(1)dN(2) is significantly different from the
internal angle at the corresponding atom of molecule 2. For example, in
molecule 1 the internal angle at the ring C to which the triazene group is
Dacarbazine 333

Figure 2 Molecular geometry and dimension (bond lengths () and angles () of DTIC):
(A) molecule 1 and (B) molecule 2. The estimated standard deviations of the bond
distances and angles are 0.004 and 0.3 degree, respectively. From figure 1 of H.C.
Freeman, N.D. Hutchinson, The crystal structure of the anti-tumor agent 5-(3,3-
dimethyl-1-triazenyl)imidazole-4-carboxamide (NSC-45388). Acta. Crystallogr. B35 (1979)
20512054.

attached is 106 degree, and the internal angle at the C to which the carbox-
amide group is attached is 110 degree. In molecule 2 the values of
these angles are interchanged (with concomitant changes in the bond
angles which are external to the ring). The differences between pairs of
corresponding dimensions in molecules 1 and 2 disappear if the
corresponding positions are defined not in relation to the substituents
on the rings, but in relation to the protonated imidazole N atoms. The angles
in the imidazole rings of both molecules then also become, within the limits
of precision, consistent with those in crystalline imidazole [16]. A similar
dependence of the internal bond angles in imidazole rings on the position
of the protonated N has been noted in 5-amino-4-carbamoyl-1H-imidazole
and 4-amino-5-carbamoyl-1H-imidazolewater (A. Kalman et al., personal
communication).
The sequences N(2)dC(2)dN(4)dN(5) and N(8)dC(8)dN(10)dN
(11) are in syn configurations. There are intramolecular hydrogen bonds N
(3)dHN(4) (2.868 A) and N(9)dHN(10) (2.908 A) between the car-
boxamide and triazene side chains. The imidazole rings and the carboxamide
groups in both molecules are planar within the limits of precision. The entire
334 Abdullah A. Al-Badr and Mansour M. Alodhaib

molecules, however, are not strictly planar. The bonds C(3)dC(4) and C(9)
dC(10) are bent by 1.4 degree and 0.5 degree, respectively, from the imid-
azole planes. The carboxamide groups are rotated by 2.5 degree and 1.0
degree, respectively, about the CdC bonds. Further deviations from planar-
ity are caused by out-of-plane bending of the bonds C(2)dN(4) (1.3 degree)
and C(8)dN(10) (0.2 degree), and by small rotations about the CdN and
NdN bonds within the triazene groups. The carboxamide and triazene
groups are bent and rotated to opposite sides of the imidazole plane in molecule
1, and to the same side in molecule 2.
A similar molecular configuration, an equivalent intramolecular hydro-
gen bond (2.974 A), and slightly greater deviations from planarity are found
in the HDTIC+ cation [10]. Differences between the bond lengths in DTIC
and HDTIC+ are probably not significant, but a number of marked differ-
ences do occur between corresponding bond angles. In HDTIC+ the inter-
nal bond angles of the imidazole ring are all close to 108 degree. In DTIC the
angles at C(1) in molecule 1 and at C(7) in molecule 2 are 112113 degree,
and the angles at N(1), C(2), N(8), and C(9) are 104106 degree. There are
similar differences between the protonated and neutral forms of the imidaz-
ole rings in L-histidine [17] and also of imidazole itself [18].
The molecular packing in the crystals of DTIC bears no resemblance
to that in HDTIC+C1H2O. Infinite DTIC chains in which molecules
1 and 2 alternate are formed by hydrogen bonds N(2)dHN(8) and
N(7)dHN(1) between the imidazole rings (Fig. 3 and Table 2). The
angle between the average planes of adjacent DTIC molecules in the chains
is 36.3 degree.
Similar strong hydrogen bonds occur in imidazole [16] where the
NdHN distance is 2.86 A compared with values of 2.83 and 2.81 A
in the present structure. Cross-linking between the chains of DTIC mole-
cules is provided by pairs of hydrogen bonds between amide groups
[N(3)dHO(2) and N(9)dHO(1)]. In the directions normal to the
planes of the imidazole rings there are no contacts shorter than 3.5 A with
neighboring molecules.
Results of the present work which may be relevant to the biological
effects of DTIC are that (i) the side-chain configurations are not affected
by changes in pH (since the same configurations are observed in crystals
of DTIC and HDTIC+ grown under quite different conditions and having
different intermolecular interactions), and (ii) the shape of the imidazole ring
undergoes subtle changes depending on whether one N(imidazole) atom or
the other or both are protonated.
Figure 3 Packing of DTIC molecules in relation to the unit cell. Molecules symmetry
related to molecules 1 and 2 have hollow and filled bonds, respectively. Hydrogen
bonds are shown as dashed lines. From figure 2 of H.C. Freeman, N.D. Hutchinson, The
crystal structure of the anti-tumor agent 5-(3,3-dimethyl-1-triazenyl)imidazole-4-
carboxamide (NSC-45388). Acta. Crystallogr. B35 (1979) 20512054.

Table 2 Hydrogen Bonding [9]

Superscripts refer to the following equivalent positions
None x y z
(i)  + x 1  y +z
(ii) +x 1  y  + z
(iii) 1+x y z
(iv) 1 + x y z
XdHY XdY () HY () LXdHY ()
N(2)dH(2)N(8) 2.824 (4) 1.94 (3) 165 (3)
N(3)dH(3)N(4) 2.868 (4) 2.04 (3) 131 (2)
iii
N(3)dH(4)O(2 ) 2.964 (3) 2.25 (3) 170 (4)
N(3iv)dH(4iv)O(2)
N(7)dH(12)N(1i) 2.813 (4) 1.88 (3) 177 (3)
N(7ii)dH(12ii)N(1)
N(9)dH(13)N(10) 2.908 (4) 2.29 (3) 132 (3)
iv
N(9)dH(14)O(l ) 2.960 (3) 2.08 (3) 166 (3)
N(9iii)dH(14iii)O(1)
336 Abdullah A. Al-Badr and Mansour M. Alodhaib

4.4 Thermal Methods of Analysis

4.4.1 Melting Behavior
250254C.

4.4.2 Differential Scanning Calorimetry

The differential scanning calorimetry thermogram of dacarbazine was
obtained using DuPont 2100 thermal analyzer system. The thermogram
shown in Fig. 4 was obtained at a heating rate of 10C/min and was run
over the range of 20300C. Dacarbazine was found to melt at 202C.
Bei et al. [8] used the differential scanning calorimetry as one of the physi-
cochemical studies that were used to investigate the physicochemical prop-
erties of dacarbazine-loaded cubosomes on dacarbazine.

4.5 Spectroscopy
4.5.1 Ultraviolet Spectroscopy
The ultraviolet absorption spectrum of dacarbazine in methanol shown in
Fig. 5 was recorded using a Shimadzu UVvis spectrophotometer 1601
PC. The compound exhibited maximum at 380 nm.

Figure 4 The differential scanning calorimetry thermogram of dacarbazine.

Dacarbazine 337

0.827

3
0.600

0.400
Abs.

4
5

0.200
9

2
1
0.000

6
0.083 7
190.00 300.00 400.00 500.00 600.00
nm
Figure 5 The ultraviolet absorption spectrum of dacarbazine in methanol.

4.5.2 Vibrational Spectroscopy

The infrared absorption spectrum of dacarbazine was obtained in a KBr pel-
let using a Perkin-Elmer infrared spectrophotometer. The infrared absorp-
tion spectrum is shown in Fig. 6. Assignments for the major infrared
absorption bands are listed in Table 3.
Gunasekaran et al. [19] recorded the FTIR and FT Raman spectra of
dacarbazine in the regions 4000400 and 3500100 cm1, respectively. The
optimized geometry, wave number, polarizability, and several thermodynamic
properties of dacarbazine were studied using ab initio HartreeFock, MP2,
and DFT methods. A complete vibrational assignment aided by the theoretical
harmonic wave number analysis was proposed. The calculated harmonic
vibrational frequencies were compared with experimental FTIR and FT
Raman spectra. Based on the comparison between calculated and experimental
results and the comparison with related molecules, assignments of funda-
mental vibrational modes were made. The X-ray geometry and experimental
frequencies were compared with the results of theoretical calculations.

4.5.3 Nuclear Magnetic Resonance Spectrometry

4.5.3.1 1H NMR Spectrum
The proton nuclear magnetic resonance (1H NMR) spectrum of
dacarbazine shown in Fig. 7 was obtained using a Bruker instrument oper-
ating at 500 MHz. The sample was dissolved in DMSO-d6 and all resonance
338 Abdullah A. Al-Badr and Mansour M. Alodhaib

Figure 6 The infrared absorption spectrum of dacarbazine (KBr pellet).

Table 3 Vibrational Assignments of the Infrared Absorption Bands

of the Dacarbazine Infrared Spectrum
Frequency (cm21) Assignments
3172, 3262, 3379 Amide NH2
2557, 2753 CdH aliphatic
1655 Amide C]O
1607 C]C
1474 CdH aromatic
629, 792, 881 CdC

bands were referenced to the internal standard, tetramethylsilane (TMS).

Standard Bruker software was used to execute the recording of the 1D
and 2D spectra of the drug. The positions of the various protons of
dacarbazine are listed in Table 4.

13
4.5.3.2 C NMR Spectrum
The carbon-13 NMR spectrum of dacarbazine was obtained using a Bruker
instrument operating at 125 MHz and is shown in Fig. 8. The sample was
Dacarbazine 339

7.520
7.430
7.297

3.512
3.349

3.136

2.507
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 ppm
3.11

9.32

2.17
Figure 7 The 1H NMR spectrum of dacarbazine in DMSO-d6.

Table 4 The Assignments of the Resonance Bands in the 1H NMR Spectrum

of Dacarbazine
O
NH2
6
1 5 7
HN N CH3
N N
4
2
N3
CH3
8

Chemical Shift Assignment

(ppm, relative Number (protons at carbon
to TMS) of Protons Multiplicity* number)
3.14 3 s 7 or 8
3.51 3 s 8 or 7
7.30 1 s NH2
7.43 1 s NH2
7.52 1 s 2
12.52 1 s dNHd
*s = singlet.
 161.172

149.286

135.477

115.433

42.988
40.083
39.995
39.916
39.828
39.750
39.661
39.494
39.327
39.160
38.993
36.010
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
13
Figure 8 The C NMR spectrum of dacarbazine in DMSO-d6.
Dacarbazine 341

dissolved in DMSO-d6 and TMS was used as the internal standard. The
COSY NMR spectrum of the drug is presented in Fig. 9. Figs. 10 and 11
show the HMBC and the HSQC spectra, respectively. Positions of the various
carbons of dacarbazine are shown in Table 5.

4.6 Mass Spectrometry

The electrospray ionization mass spectrum of dacarbazine obtained using an
Agilent 6410 QQQ mass spectrometer is shown in Fig. 12. Table 6 shows
the mass fragmentation pattern of the drug [20].
Fabrizi et al. [20] presented an innovative screening method based on the
use of the desorption electrospray ionization (DESI) interface coupled with a
hybrid quadrupole linear ion trap mass spectrometer. A rapid, simple, and
sensitive procedure was developed for the simultaneous surface monitoring

ppm
2

10

11

12

13

13 12 11 10 9 8 7 6 5 4 3 2 ppm
Figure 9 The COSY NMR spectrum of dacarbazine in DMSO-d6.
 ppm

20

40

60

80

100

120

140

160

180

200

13 12 11 10 9 8 7 6 5 4 3 2 ppm
Figure 10 The HMBC NMR spectrum of dacarbazine in DMSO-d6.

ppm
0

20

40

60

80

100

120

140

160

180

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 ppm
Figure 11 The HSQC NMR spectrum of dacarbazine in DMSO-d6.
Dacarbazine 343

13
Table 5 The Assignments of the Resonance Bands in the C NMR Spectrum
of Dacarbazine
O
NH2
6
1 5 7
HN N CH3
N N
4
2
N3
CH3
8

Carbon Number Assignment Carbon Number Assignment

36.01 7 or 8 42.99 7 or 8
115.43 5 135.48 4
149.29 2 161.17 6

of dacarbazine, cyclophosphamide, methotrexate, vincristine, gemcitabine,

and cytarabine. Since analytes were in the solid state, a novel approach
based on the use of passive samplers was combined with the direct analysis
of wipes. A PTFE-printed glass slide was used as a passive sampler, while
hydrophobic centers of Swiffer cloths were judged extremely efficient as
wipe samplers. After the sampling period, the CD collectors were directly
processed with the DESI-MS system without any further treatment.
MS/MS confirmatory analysis was conducted using selected reaction
monitoring in the positive-ion mode, and detection limits were evaluated.
Values were at the picograms per square millimeter levels on the passive
collector and at the picograms per square centimeter levels for the wipe ones.
Direct determination on solid-state samples combined with mass spec-
trometry selectivity provided a powerful tool so far unapplied to occupa-
tional hygiene.

5. METHODS OF ANALYSIS
5.1 Compendial Methods of Analysis
5.1.1 United States Pharmacopeia Methods [21]
5.1.1.1 Dacarbazine
Dacarbazine contains not less than 97.0% and not more than 102.0% of
C6H10N6O.
Caution. Great care should be taken in handling dacarbazine, as it is a
potent cytotoxic agent.
x105 + Scan (0.198 min) decarbazin0003.d

1.9
183.1
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7 166.1
0.6
0.5
0.4
0.3
0.2 123.1 138.0
0.1 205.0

0
115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250
Counts vs. Mass-to-Charge (m/z)

Figure 12 The mass spectrum of dacarbazine.

Dacarbazine 345

Table 6 Mass Spectral Fragmentation Pattern of Dacarbazine [20]

Fragment
m/z Relative Intensity (%) Formula Structure
183 100 C6H10N6O H2N
O CH3 H+
N N
N
N CH3
N
H

166 38 C6H8N5O O CH3

N
N CH3
N
N
N
H

138 9 C5H8N5 H3C

CH3
N
N
N N

N
H

123 12.5 C4H3N4O O

H+
N
N N

N
H

Packaging and storage. Preserve in tight, light-resistant containers, in

a refrigerator.
USP Reference standards h11i. USP Dacarbazine RS. USP
Dacarbazine-Related Compound A RS. USP Dacarbazine-Related Compound
B RS.
Identification. The IR absorption spectrum of a potassium bromide
dispersion of it exhibits maxima only at the same wavelengths as that of a
similar preparation of USP Dacarbazine RS.
Residue on ignition. Carry out this test as described in the general
method h281i: not more than 0.1%.
Related compounds. Dissolve an accurately weighed quantity of
dacarbazine in 0.1 N hydrochloric acid to obtain a solution having a con-
centration of 40 mg/mL, and apply 5 L of the solution to a suitable
thin-layer chromatographic plate (see Chromatography, carry out this test
as described in the general method h621i) coated with a 0.25-mm layer
346 Abdullah A. Al-Badr and Mansour M. Alodhaib

of chromatographic silica gel mixture. Apply, separately, 5 L of a

methanolic solution containing 0.40 mg of USP Dacarbazine-Related
Compound A RS per mL, and 5 L of an aqueous solution containing
0.40 mg of USP Dacarbazine-Related Compound B RS per mL. Develop
the chromatogram in a mixture of butanol, water, and acetic acid (5:2:1),
until the solvent front has moved about three-fourths of the length of the
plate. Remove the plate from the developing chamber, mark the solvent
front, and allow the solvent to evaporate. Locate the spots on the plate by
viewing under short-wavelength UV light: any spots obtained from the test
solution are not greater in size or intensity than the spots, occurring at the
respective RF values, produced by the Standard solutions, corresponding to
not more than 1.0% of dacarbazine-related compound A and not more than
1.0% of dacarbazine-related compound B.
Assay. [Note: throughout this procedure, avoid exposing dacarbazine
and its solutions to light.]
Standard preparations. Transfer about 30 mg of USP Dacarbazine RS,
accurately weighed, to a 50-mL volumetric flask, add 0.1 N hydrochloric
acid to volume, and mix (Standard stock solution). Dilute a portion of Standard
stock solution quantitatively and stepwise with 0.1 N hydrochloric acid to
obtain an Acidic standard preparation having a known concentration of about
6 g/mL. Dilute a portion of Standard stock solution quantitatively and step-
wise with pH 7.0 phosphate buffer (see Buffer Solutions in the section
Reagents, Indicators, and Solutions) to obtain a Neutral standard preparation hav-
ing a known concentration of about 6 g/mL.
Assay preparations. Prepare as directed under Standard preparations, except
to use about 30 mg of dacarbazine, accurately weighed.
Procedure. Concomitantly determine the absorbances of the Acidic stan-
dard preparation and the Acidic assay preparation in 1-cm cells at the wavelength
of maximum absorbance at about 323 nm, with a suitable spectrophotom-
eter, using 0.1 N hydrochloric acid as the blank. Concomitantly determine
the absorbances of the Neutral standard preparation and the Neutral assay prep-
aration in 1-cm cells at the wavelength of maximum absorbance at about
329 nm, using pH 7.0 phosphate buffer (see Buffer Solutions in the section
Reagents, Indicators, and Solutions) as the blank. Calculate the quantity, in mil-
ligram, of C6H10N6O in the portion of dacarbazine taken by the formula:
 
5C A323 + A329 U =A323 + A329 S

in which C is the concentration, in g/mL, of USP Dacarbazine RS in the

Standard preparations, and the parenthetic expressions are the sums of the
Dacarbazine 347

absorbances of the Assay preparations (U) and the Standard preparations (S),
respectively, measured at the wavelengths indicated by the subscripts.

5.1.1.2 Dacarbazine for Injection

Dacarbazine for Injection is a sterile, freeze-dried mixture of dacarbazine
and suitable buffers or diluents. It contains not less than 90.0% and not more
than 110.0% of the labeled amount of C6H10N6O.
Caution. Great care should be taken to prevent inhaling particles of Dacarbazine
for Injection and exposing the skin to it.
Packaging and storage. Preserve in single-dose or multiple-dose Con-
tainers for Sterile Solids as described under Injections, described in the general
method h1i, preferably of Type I glass, protected from light.
USP Reference standards h11i. USP Dacarbazine RS. USP
Dacarbazine-Related Compound B RS. USP Endotoxin RS.
Completeness of solution. When dissolved as directed in the labeling,
it yields a clear, pale yellow to yellow solution.
Constituted solution. At the time of use, it meets the requirements for
Constituted Solutions under Injections, described in the general method (1).

Identification
(A) Dissolve a suitable quantity of Dacarbazine for Injection in water to
obtain a solution having a concentration of 10 mg of dacarbazine per mL.
Apply separately 1 L of the freshly prepared solution and 1 L of an aque-
ous solution, containing 10 mg each of USP Dacarbazine RS and citric acid
per mL, to a suitable thin-layer chromatographic plate (see Chromatography,
carry out this experiment as described in the general method h621i) coated
with a 0.25-mm layer of chromatographic silica gel. Develop the chromato-
gram in a solvent system consisting of a mixture of isopropyl alcohol and 1 N
ammonium hydroxide (3:1) until the solvent front has moved about three-
fourths of the length of the plate. Remove the plate from the developing
chamber, mark the solvent front, and allow the solvent to evaporate. Spray
the plate evenly with a freshly prepared solution containing 1% of ferric
chloride and 1% of potassium ferricyanide (prepared by mixing 5 mL of a
10% aqueous solution of ferric chloride with 5 mL of a 10% aqueous solution
of potassium ferricyanide and diluting with water to 50 mL). Dacarbazine
appears as an intense blue spot on a light yellow background: the RF value
of the spot obtained from the test solution corresponds to that obtained from
the Standard solution.
(B) To 1 mL of a solution (1 in 100) in a test tube add a few crystals of
periodic acid and four drops of methanol. Shake, and after 1 min add 5 mL of
348 Abdullah A. Al-Badr and Mansour M. Alodhaib

a 0.2% acetylacetone reagent solution (prepared by mixing 15.0 g of ammo-

nium acetate, 0.30 mL of glacial acetic acid, and 0.20 mL of acetylacetone in
a 100-mL volumetric flask, adding water to volume, and mixing). Shake and
place in a water bath maintained at a temperature of 60C: an intense yellow
color develops in a few minutes (presence of mannitol).
(C) To two drops of an aqueous solution (1 in 100) in a 15-mL test tube
add 10 mL of a solution prepared by mixing 10 mL of acetic anhydride with
30 mL of pyridine: an intense yellow color is produced immediately and
after a few minutes becomes red-violet (presence of citric acid).
Bacterial endotoxins. This test should be carried out as described in
the general procedure h85iIt contains not more than 0.52 USP Endotoxin
Unit per mg of dacarbazine.
pH. This test should be carried out as described in the general procedure
h791i: between 3.0 and 4.0, in a solution containing an amount of
Dacarbazine for Injection equivalent to about 1 g of dacarbazine in
100 mL of water.
Water. Method I, carry out this test as described in the general method
h921i: not more than 1.5%.
Limit of 2-azahypoxanthine. [Note: the Mobile phase employed in this
procedure is corrosive. The system should be rinsed well with methanol fol-
lowing completion of analysis.]
Mobile phase. Transfer 2.2 g of docusate sodium to a 1000-mL volumetric
flask, dissolve in a mixture of 100 mL of water and 15 mL of glacial acetic
acid, and dilute with water to volume. Filter the solution through a 0.5-m
porosity filter. Prepare this solution fresh daily.
Standard solution. Prepare a solution of USP Dacarbazine-Related Com-
pound B RS to contain 0.04 mg/mL.
Test solution. Constitute the contents of one vial of Dacarbazine for Injec-
tion. Using the contents of the constituted vial, dilute quantitatively with
water to obtain a solution containing 4 mg of dacarbazine per mL.
Chromatographic system (see Chromatography, described in the general pro-
cedure, h621i). The liquid chromatograph is equipped with a 254-nm
detector and a 3.9-mm  30-cm column that contains packing Ll. The flow
rate is about 1.2 mL per minute. Chromatograph five replicate injections of
the Standard solution and record the peak responses as directed for Procedure:
the relative standard deviation is not more than 2.0%.
Procedure. Separately inject equal volumes (about 20 L) of the Standard
solution and the Test solution into the chromatograph by means of a suitable
sampling valve or high-pressure microsyringe. Measure the peak responses at
Dacarbazine 349

corresponding retention times obtained from the Standard solution and the
Test solution, and calculate the quantity, in mg, of 2-azahypoxanthine mono-
hydrate in the dacarbazine taken by the formula:

CV rU =rS

in which C is the concentration, in mg/mL, of USP Dacarbazine-Related

Compound B RS in the Standard solution; V is the final volume, in mL, of
the Test solution; and rU and rS are the peak responses obtained from the Test
solution and the Standard solution, respectively: not more than 1.0% is found.
Other requirements. It meets the requirements for Sterility Tests,
described in the general method h71i, Uniformity of Dosage Units, described
in the general method h905i and Labeling under Injections, described in the
general method h1i.
Assay. Dissolve the contents of not fewer than 10 containers of
Dacarbazine for Injection in 0.1 N hydrochloric acid. Transfer and combine
the solutions quantitatively rinsing as necessary with 0.1 N hydrochloric
acid. Dilute quantitatively, and stepwise if necessary, with 0.1 N
hydrochloric acid to obtain a solution containing about 0.4 mg/mL. Trans-
fer 2.0 mL of this solution to a 250-mL volumetric flask, dilute with 0.1 N
hydrochloric acid to volume, and mix. Dissolve an accurately weighed
quantity of USP Dacarbazine RS in 0.1 N hydrochloric acid, and dilute
quantitatively and stepwise with the same solvent to obtain a Standard solu-
tion having a known concentration of about 3.2 g/mL. Concomitantly
determine the absorbances of both solutions in 1-cm cells at the wavelength
of maximum absorbance at about 323 nm, with a suitable spectrophotom-
eter, using 0.1 N hydrochloric acid as the blank. Calculate the average quan-
tity, in mg, of C6H10N6O in each container of Dacarbazine for Injection
taken by the formula:

125VC=N AU =AS

in which V is the volume, in mL, of the solution produced by dilution

of the combined container contents to a concentration of 0.4 mg/mL
taking account of dilution factors in the case of stepwise dilution; C is
the concentration, in mg/mL, of USP Dacarbazine RS in the Standard
solution; N is the number of vials taken; and AU and AS are the absorbances
of the solution of Dacarbazine for Injection and the Standard solution,
respectively.
350 Abdullah A. Al-Badr and Mansour M. Alodhaib

5.1.2 British Pharmacopeia Methods [22]

5.1.2.1 Dacarbazine
Dacarbazine contains not less than 98.5% and not more than 101.0% of
C6H10N6O, calculated with reference to the dried substance.

Characteristics
A Colorless or pale yellow, crystalline powder. Slightly soluble in water and
in ethanol (96%).

Identification
(A) The infrared absorption spectrum, Appendix II A, is concordant with the
reference spectrum of dacarbazine (RS 082).
(B) The light absorption, Appendix II B, in the range 230350 nm of a
0.0006% (w/v) solution in 0.1 M hydrochloric acid exhibits a maximum at
323 nm and a pronounced shoulder at 275 nm. The absorbance at 323 nm
is about 0.64.

Tests
Clarity and color of solution. A 2.0% (w/v) solution in 0.1 M citric acid is
clear, Appendix IV A, and not more intensely colored than reference solution
BY6, Appendix IV B, Method II.
5-Aminoimidazole-4-carboxamide hydrochloride. Carry out the
following procedure protected from light and inject samples within 1 h of
preparation. Use low actinic glassware. Carry out the method for liquid
chromatography, Appendix III D, using two solutions in 0.1 M acetic acid
containing (1) 0.40% (w/v) of the substance being examined and (2)
0.0024% (w/v) of 5-aminoimidazole-4-carboxamide hydrochloride. The
chromatographic procedure described under Related substances may
be used but using 0.005 M dioctyl sodium sulfosuccinate in a mixture of 3 vol-
umes of glacial acetic acid, 87 volumes of water, and 110 volumes of methanol
as the mobile phase. The area of any peak corresponding to
5-aminoimidazole-4-carboxamide hydrochloride in the chromatogram
obtained with solution (1) is not greater than the area of the peak in the
chromatogram obtained with solution (2) (0.6%).
Related substances. Carry out the following procedure protected
from light and inject samples within 1 h of preparation. Carry out the
method for liquid chromatography, Appendix III D, using two solutions in
0.25 M acetic acid containing: (1) 0.40% (w/v) of the substance being exam-
ined and (2) 0.0040% (w/v) of 2-Azahypoxanthine BPCRS. The chromato-
graphic procedure may be carried out using (a) a stainless steel column
Dacarbazine 351

(20 cm  4 mm) packed with stationary phase C (10 m) (Nucleosil C18 is

suitable), (b) 0.005 M dioctyl sodium sulfosuccinate in a mixture of 1.5 vol-
umes of glacial acetic acid and 98.5 volumes of water as the mobile phase with
a flow rate of 1.5 mL per minute, and (c) a detection wavelength of
254 nm. After use the column should be thoroughly flushed with methanol
to remove dacarbazine which does not elute with the mobile phase. In the
chromatogram obtained with solution (1) the area of any secondary peak is
not greater than the area of the principal peak in the chromatogram
obtained with solution (2) (1%), not more than one such peak has an area
greater than half the area of the peak in the chromatogram obtained with
solution (2) (0.5%) and the sum of the areas of all such peaks is not greater
than three times the area of the peak in the chromatogram obtained with
solution (2) (3%).
Loss on drying. When dried to constant weight at 60C at a pressure
not exceeding 0.7 kPa, loses not more than 0.5% of its weight. Use 1 g.
Sulfated ash. Not more than 0.1%, Appendix IX A.

Assay
Dissolve 0.4 g in 10 mL of anhydrous acetic acid and carry out Method I for
nonaqueous titration, Appendix VIII A, determining the end point pot-
entiometrically. Each mL of 0.1 M perchloric acid VS is equivalent to
18.22 mg of C6H10N6O.

Storage
Dacarbazine should be protected from light and stored at a temperature
of 28C.

5.1.2.2 Dacarbazine Injection

Definition Dacarbazine injection is a sterile solution of Dacarbazine in
Water for Injections. It is prepared by dissolving Dacarbazine for Injection
in the requisite amount of Water for Injections.
The injection complies with the requirements stated under Parenteral
Preparations.

Storage
Dacarbazine Injection should be used immediately after preparation but, in
any case, within the period recommended by the manufacturer when pre-
pared and stored strictly in accordance with the manufacturers instructions.
352 Abdullah A. Al-Badr and Mansour M. Alodhaib

5.1.2.3 Dacarbazine for Injection

Definition Dacarbazine for Injection is a sterile material consisting of
Dacarbazine with or without excipients. It is supplied in a sealed container.
The contents of the sealed container comply with the requirements for Powders
for Injections stated under Parenteral Preparations and with the following
requirements.
Content of dacarbazine, C6H10N6O. 90.0110.0% of the stated
amount.

Characteristics
A white or very pale yellow powder.

Identification
(A) Dissolve a quantity of the contents of the sealed container containing
0.1 g of dacarbazine in 200 mL of 0.1 M mixed phosphate buffer pH 7.0, dilute
with the buffer solution to 250 mL, and dilute 3200 mL with the same
buffer solution. The light absorption of the resulting solution, Appendix II
B, in the range 230350 nm exhibits two maxima, at 237 nm and 330 nm.
(B) In the test for 5-aminoimidazole-4-carboxamide the principal peak
in the chromatogram obtained with solution (1) corresponds to that in the
chromatogram obtained with solution (2).

Tests
5-Aminoimidazole-4-carboxamide hydrochloride. Carry out the fol-
lowing procedure protected from light and inject samples within 1 h of
preparation. Use low actinic glassware. Carry out the method for liquid chro-
matography, Appendix III D, using the following solutions. For solution (1)
dissolve a quantity of the contents of the sealed container containing 0.20 g
of dacarbazine in 40 mL of 0.1 M acetic acid and add sufficient 0.1 M acetic acid
to produce 50 mL. For solution (2) dilute 1 volume of solution (1) to 100
volumes with 0.1 M acetic acid. Solution (3) contains 0.004% (w/v) of
dacarbazine BPCRS in 0.1 M acetic acid. Solution (4) contains 0.0024%
(w/v) of 5-aminoimidazole-4-carboxamide hydrochloride in 0.1 M acetic acid.
The chromatographic procedure described under Related substances
may be used but using 0.005 M dioctyl sodium sulfosuccinate in a mixture of
3 volumes of glacial acetic acid, 87 volumes of water, and 110 volumes of
methanol as the mobile phase. The area of any peak corresponding to
Dacarbazine 353

5-aminoimidazole-4-carboxamide hydrochloride in the chromatogram

obtained with solution (1) is not greater than the area of the peak in the chro-
matogram obtained with solution (4) (0.6%).
Related substances. Carry out the following procedure protected from
light and inject samples within 1 h of preparation. Carry out the method for
liquid chromatography, Appendix III D, using the following solutions. Solu-
tion (1) contains 0.0040% (w/v) of 2-azahypoxanthine BPCRS in 0.25 M
acetic acid. For solution (2) dissolve a quantity of the contents of the sealed
container containing 0.20 g of dacarbazine in 40 mL of 0.25 M acetic acid
and add sufficient 0.25 M acetic acid to produce 50 mL. The chromatographic
procedure may be carried out using (a) a stainless steel column
(20 cm  4 mm) packed with stationary phase C (10 m) (Nucleosil C18 is
suitable), (b) 0.005 M dioctyl sodium sulfosuccinate in a mixture of 1.5 volumes
of glacial acetic acid and 98.5 volumes of water as the mobile phase with a flow
rate of 1.5 mL per minute, and (c) a detection wavelength of 254 nm. After
use the column should be thoroughly flushed with methanol to remove
dacarbazine which does not elute with the mobile phase. In the chromato-
gram obtained with solution (2) the area of any secondary peak is not greater
than the area of the principal peak in the chromatogram obtained with solu-
tion (1) (1%), not more than one such peak has an area greater than half the
area of the peak in the chromatogram obtained with solution (1) (0.5%), and
the sum of the areas of all such peaks is not greater than three times the area of
the peak in the chromatogram obtained with solution (1) (3%).
Uniformity of content. Sealed containers containing 200 mg or less of
dacarbazine comply with the requirements stated under Parenteral Prepara-
tions, Powders for Injections. Use the individual results obtained in
the Assay.

Assay
Carry out the following procedure protected from light. Dissolve the con-
tents of one container in 0.1 M hydrochloric acid and dilute with sufficient
0.1 M hydrochloric acid to produce a final solution containing 0.0008%
(w/v) of Dacarbazine. Measure the absorbance of the resulting solution at
the maximum in 323 nm, Appendix II B. Calculate the content of
C6H10N6O in the sealed container taking 1090 as the value of A(1%,
1 cm) at the maximum at 323 nm. Repeat the procedure with a further nine
sealed containers and calculate the average content of C6H10N6O per con-
tainer from the 10 individual results thus obtained.
354 Abdullah A. Al-Badr and Mansour M. Alodhaib

Storage
The sealed container should be protected from light and stored at a temper-
ature of 28C.

5.1.3 International Pharmacopoeia Methods [23]

Storage. Dacarbazine should be kept in a tightly closed container, protected
from light, and stored at a temperature not exceeding 8C.
Additional information. Caution: dacarbazine must be handled with
care, avoiding contact with the skin and inhalation of airborne particles.
Requirements
Dacarbazine contains not less than 97.0% and not more than 102.0% of
C6H10N6O, calculated with reference to the dried substance.
Identity tests
Either test A alone or tests B, C, and D may be applied.
Test A. Carry out the examination as described under 1.7 Spectrophotometry
in the infrared region. The infrared absorption spectrum is concor-
dant with the spectrum obtained from dacarbazine RS or with
the reference spectrum of dacarbazine.
Test B. The absorption spectrum of a 6 g/mL solution in hydrochloric
acid (0.1 mol/L) VS, when observed between 230 and 350 nm,
exhibits a maximum at about 323 nm and a pronounced shoulder
at 275 nm. The absorbance of a 1-cm layer at the maximum wave-
length of 323 nm is about 0.64.
Test C. Dissolve 25 mg in 5 mL of water, add one drop of cobalt(II) chlo-
ride (30 g/L) TS and one drop of ammonia (100 g/L) TS; a
violet-red solution is produced.
Test D. Dissolve 25 mg in 5 mL of hydrochloric acid (70 g/L) TS, add
about 0.2 g of zinc R powder, and allow to stand for 5 min. Filter,
and to the filtrate add three drops of sodium nitrite (10 g/L) TS
and 0.5 mL of ammonium sulfamate (5 g/L) TS. After the reac-
tion has subsided add five drops of N-(1-naphthyl)eth-
ylenediamine hydrochloride/ethanol TS; a deep red solution is
produced.
Clarity and color of solution
A solution of 0.20 g in 10 mL of citric acid (20 g/L) TS is clear and not
more intensely colored than standard color solution Yw2 when com-
pared as described under 1.11 Color of liquids.
Sulfated ash. Not more than 1.0 mg/g.
Dacarbazine 355

Loss on drying. Dry at 60C to constant mass under reduced pressure

(not exceeding 0.6 kPa or about 5 mm of mercury); it loses not more than
5 mg/g.
Related substances. Carry out the test as described under 1.14.1 Thin-
layer chromatography, using silica gel R2 as the coating substance and five vol-
umes of 1-butanol R, two volumes of water, and one volume of acetic acid
(300 g/L) TS as the mobile phase. Apply separately to the plate 5 L of
each of the three following solutions in methanol R containing
(A) 0.04 g of dacarbazine per mL, (B) 0.4 mg of dacarbazine-related com-
pound A RS per mL, and (C) 0.4 mg of dacarbazine-related compound
B RS per mL. After removing the plate from the chromatographic chamber,
allow it to dry in air and examine the chromatogram in ultraviolet light
(254 nm).
Any spot obtained with solution A, other than the principal spot, is not
more intense or greater in size than that obtained with solution B (1%) and
solution C (1%).

Assay
The solutions must be protected from light throughout the assay.
Dissolve about 30 mg, accurately weighed, in sufficient hydrochloric acid
(0.1 mol/L) VS to produce 50 mL of stock solution. For solution S1, dilute
1.0 mL of the stock solution to 100 mL with hydrochloric acid (0.1 mol/L)
VS. For solution S2, dilute a further 1.0 mL aliquot of the stock solution to
100 mL with phosphate buffer, pH 7.0, TS. Measure the absorbance of a
1-cm layer of solution S1 at the maximum at about 323 nm against a solvent
cell containing hydrochloric acid (0.1 mol/L) VS. Measure the absorbance
of a 1-cm layer of solution S2 at the maximum at about 329 nm against a
solvent cell containing phosphate buffer, pH 7.0, TS. Calculate the percent-
age content of C6H10N6O.

5.2 Reported Methods of Analysis

5.2.1. High-Performance Liquid Chromatography
Fiore et al. [24] developed a high-performance liquid chromatographic
method for the simultaneous analysis of dacarbazine, its photolytic
degradation product, 2-azahypoxanthine, and the metabolite
5-aminoimidazole-4-carboxamide in plasma or urine. Plasma samples
were prepared by ultrafiltration, whereas urine samples were filtered and
diluted for analysis. Chromatography was done with a C18 Bondapak
356 Abdullah A. Al-Badr and Mansour M. Alodhaib

column along with gradient elution of the drug. The mobile phase con-
sisted of 100% 0.5 M sodium acetate (pH 7) and 25% acetonitrile in
0.05 M sodium acetate (pH 5.5) with detection at 280 nm. Linearity
was observed up to 500 g/mL for dacarbazine and up to 53 g/mL for
5-aminoimidazole-4-carboxamide and 2-azahypoxanthine. The assay
methodology was reproducible, with a lower limit of detection of 5,
0.5, and 0.5 g/mL for dacarbazine, 5-aminoimidazole-4-carboxamide,
and 2-azahypoxanthine, respectively. Intraday and interday coefficients
of variation ranged between 414% and 216%, respectively. The method
was applied to the analysis of plasma and urine samples resulting from the
isolation perfusion chemotherapy of an extremity with 57 mg of
dacarbazine per kg in a patient with melanoma.
Tate and Briele [25] described a reversed-phase high-performance liquid
chromatographic method for the separation and determination of
dacarbazine and metabolites on a single, reversed-phase phenyl column.
Initial conditions consisted of 0.1% ammonium formate buffer pH 5.5
methanolwater (90:5:5) (v/v) at 2 mL/min. At 0.5 min, a 1-min linear gra-
dient was used to change the composition to 40:30:30 (v/v) at a flow rate of
2 mL/min. Run time was 9 min, with an equilibrium delay of 4 min. Injec-
tion volume was 20100 L, depending on drug concentration. Buffer was
prepared from ammonium hydroxide and 88% formic acid.
A 30 cm  3.9 mm column with 10-m Bondapak phenyl silica-based
packing was used, and the absorbance detection was carried out at 254 nm.
King and Stewart [26] described a high-performance liquid chromato-
graphic method for the assay of dacarbazine, doxorubicin, and ondansetron
mixture in a 5% dextrose injection. The separation and quantitation are
achieved on a 22-cm underivatized silica column at ambient temperature
using a mobile phase of 60:40 (v/v) 6.25 mM phosphate buffer, pH
3acetonitrile at a flow rate of 1 mL/min with detection of all three analytes
at 216 nm. The separation was achieved within 10 min with sensitivity in
the ng/mL range for each analyte. The predominant mechanism of retention
for the analytes on silica was cation-exchange. The method showed linearity
for dacarbazine, in the 0.797.90 g/mL range. Accuracy and precision
were in the 07% range, for dacarbazine. The limit of detection for
dacarbazine was 12.5 ng/mL, based on a signal-to-noise ratio of 3 and a
50 L injection.
Orsatti et al. [27] studied a series of 3,3-dimethyl-l-(isoxazol-3-yl)
triazenes, the anticancer agents by electron impact ionization, and fast-atom
bombardment mass spectrometry. Their behavior was compared with that
Dacarbazine 357

of dacarbazine, which is employed in the treatment of several neoplastic

conditions. An interesting electron impact-generated decomposition
pathway was observed, consisting in the primary formation of [NH2CH3]+
cations, involved in the metabolic pathway of triazenes, as the alkylating
agent responsible for the anticancer properties of the drug. A higher ther-
modynamic stability of the examined compounds than dacarbazine was
observed, which reasonably reflects the higher chemical stability.
Haque and Stewart [28] separated dacarbazine and its two related impu-
rities 5-aminoimidazole-4-carboxamide and 2-azahypoxanthine by capillary
electrophoresis and reversed-phase high-performance liquid chromatogra-
phy. Baseline separation of dacarbazine and the impurities was achieved
by capillary electrophoresis on a fused silica capillary (70 cm  50 m) with
an electrolyte buffer of methanol:0.025 M phosphate buffer pH 4 (2:98 by
vol.) at an applied voltage of 15 kV and detection at 220 nm. The capillary
electrophoresis method could not be used for the quantitation of the impu-
rities because at high concentrations the dacarbazine peak overlapped the
nearest impurity peak. The initial high-performance liquid chromatography
method with an ODS column separated the impurities from dacarbazine,
but required a longer analytical time (50 min). Newer high-performance
liquid chromatography method with an avidin protein column separated
the drug and its impurities with a run time of 27 min and was chosen for
the development and validation of the method. Dacarbazine and the two
impurities were separated isocratically on the avidin column using a mobile
phase of isopropanol:0.02 M phosphate buffer pH 7 (4:96 by vol.) at a flow
rate of 0.6 mL/min and detection at 230 nm. Calibration curves were pre-
pared for each impurity at concentration ranges 1001000 ng/mL. The
detector response was linear for each individual impurity down to
100 ng/mL levels representing 0.5% (w/w) of the dacarbazine concentra-
tion. Accuracy and precision of the analysis for each impurity were 3% at
100 ng/mL. The limits of quantitation and detection for both impurities
were 100 and 50 ng/mL, respectively. Linearity for dacarbazine was in
the 1525 g/mL range, with a correlation coefficient of 0.998.
Safgren et al. [29] developed a reversed-phase high-performance
liquid chromatography method with UV detection for the simultaneous
determination of dacarbazine and its metabolites 5-(3-hydroxymethyl-
3-methyl-1-triazeno)imidazole-4-carboxamide and 5-(3-methyl-1-triazeno)
imidazole-4-carboxamide. The chromatographic separation was achieved
with a Zorbax SB-CN column and a mobile phase of 80% 50 mM
ammonium phosphate, pH 6.5, 20% methanol, and 0.1% triethylamine.
358 Abdullah A. Al-Badr and Mansour M. Alodhaib

The drug and its two metabolites were extracted from plasma with meth-
anol precipitation of the proteins. Recovery of dacarbazine and the metab-
olites from whole blood was greater than 92%. Rapid processing of whole
blood, methanol extraction, and storage at 70C substantially increased
the stability of the two metabolites from less than 15 min to 3 days. Pre-
cision of the two metabolites and the drug ranged from 3.7% to 16.3%
relative standard deviation. The accuracy ranged from 101% to 114%
for all three analytes. The validated assay was used to determine the phar-
macokinetic data for dacarbazine and its active metabolites for human
patients with recurrent glioma receiving dacarbazine intravenously.
Hartinger et al. [30] studied the complexation properties of dacarbazine
by comparison of the electrospray ionization mass spectra of isolated transi-
tion metal complexes and in situ-formed ones. Ferric chloride was reacted
with dacarbazine at a molar ratio of 1:1, while cobalt chloride, nickel chlo-
ride, and cis-[RuCl2(dmso)4] were mixed with the ligand at a 1:2 ratio. The
obtained dacarbazine complexes were isolated by precipitation and charac-
terized by NMR, ESI-MS, IR, and elemental analysis. In order to form
complexes in situ, reaction mixtures of metal salts and the ruthenium com-
plex with dacarbazine were prepared at molar ratios of both 1:1 and 1:2.
Comparison of the data for isolated and in situ-prepared complexes revealed
that the ferric forms slowly but exclusively a complex of the
[Fe(dacarbazine)2]-type (independent of the ratio between the iron salt
and dacarbazine), while incubation of the Ru(II)complex cis-
[RuCl2(dmso)4] with dacarbazine yields a mixture of [Ru(dacarbazine)]-
and [Ru(dacarbazine)2]-type complexes. The exchange of dmso ligands by
dacarbazine was found to proceed rather slowly. In contrast, the complexation
of Ni(II) and Co(II) toward dacarbazine was much faster and the reaction of
dacarbazine with cobalt chloride delivers only a [Co(dacarbazine)2]-type
complex, whereas coordination compounds with Ni(II) were identified to
be of [Ni(dacarbazine)]- and [Ni(dacarbazine)2]-types when being incubated
at molar ratios Ni:dacarbazine of 1:1 and 1:2, respectively.
Delmas et al. [31] installed a quantitative and qualitative high-
performance liquid chromatography control of cytotoxic preparations.
A 100 L sample of each preparation was assayed by high-performance liq-
uid chromatography with ultravioletvisible-diode array detection, which
enabled the identification of all cytotoxic agents by their characteristic ultra-
violet spectra. A rapid and specific high-performance liquid chromatogra-
phy assay method was developed and used to determine qualitatively and
quantitatively dacarbazine and 20 other cytotoxic agents in less than
Dacarbazine 359

3.5 min. A 15% tolerance from the theoretical concentration was chosen in
agreement with preparation and dosage bias, and a first period control of
more than 4400 preparations revealed that around 7.7% preparations did
not conform. The main objective of these controls was to avoid the admin-
istration of defective chemotherapies to patients and finally to use their
results to identify error factors. One flow path was used for the flow-
injection analysis, and the five other paths were connected to a reversed-
phase C18 column (AQ+ 15 cm  4.6 mm, 5 m pore size, ProntoSIL
Bischoff Chromatography, Leonberg, Germany): in this work only four col-
umn paths were necessary. Each column was dedicated to a range of mobile
phase concentration. Supragradient high-performance liquid chromatogra-
phy grade acetonitrile, formic acid, gradient high-performance liquid chro-
matography grade methanol, and microfiltered water were used as mobile
phase with flow rate of 1 mL/min. Every morning columns were
preequilibrated with their specific mobile phase and every evening all col-
umns were rinsed and conserved during night in acetonitrile or methanol
water 90:10 (v/v).
Malik et al. [32] developed a reversed-phase high-performance liquid
chromatographic method with ultraviolet detection at 230 nm with ODS
Hypersil C18 column (25 cm  4.6 mm, 5 m, particle size) for the simulta-
neous determination of dacarbazine in plasma of lymphoma patients using
mobile phase composition of 300 volumes of acetonitrile and 700 volume
of 0.05 M disodium hydrogen phosphate containing 0.5 mL triethylamine,
and pH of the mobile phase was maintained at 3.7 with 2 M phosphoric acid
at a flow rate of 0.75 mL/min with linearity ranges 0.0950 g/mL with
limit of detection and limit of quantitation of 0.060 and 0.090, respectively.
Recovery of dacarbazine was 99.09%.
Prasanth and Siddiraju [33] developed a simple, rapid, and precise sta-
bility indicating high-performance liquid chromatographic method for the
quantitative determination of dacarbazine in pharmaceutical dosage form.
The chromatographic separation of dacarbazine was achieved with an
Agilent Eclipse XDB C18, 150  4.6 mm, 5 m particle size analytical col-
umn using buffer and acetonitrile taken in 96:4 (v/v) and the response was
detected at 323 nm by using PDA detector. The retention time was found
to be 4.333. Dacarbazine drug substance was exposed to thermal, photo-
lytic, hydrolytic, and oxidative stress conditions, and the stressed samples
were analyzed by the method. Peak homogeneity data of dacarbazine are
obtained by photodiode array detector in the stressed sample chromato-
grams, demonstrating the specificity of the method for its estimation in
360 Abdullah A. Al-Badr and Mansour M. Alodhaib

the presence of degradation product. The described method shows excel-

lent linearity over a range 25150 g/mL. The correlation coefficient
for dacarbazine was found to be 0.9999. The relative standard deviation
for six measurements in two sets of dacarbazine in injection is always less
than 2%. The method was found to be suitable and accurate for quantita-
tive determination and stability study of dacarbazine in pharmaceutical
preparations.

5.2.2 Liquid ChromatographyMass SpectrometryMass Spectrometry

Chowdhury et al. [34] developed and validated a sensitive and selective
high-performance liquid chromatographicelectrospray ionization tandem
mass spectrometric method for the quantitative determination of 5-(3-N-
methyltriazen-1-yl)-imidazole-4-carboxamide, a pharmacologically active
hydrolysis product of temozolomide and a monodemethylated product of
dacarbazine. The method was validated over a linear range from 10 to
400 ng/mL in dog plasma and from 10 to 500 ng/mL in rat plasma. The
method utilized small plasma volumes (70 L), rapid sample processing,
and isocratic elution conditions to achieve sensitive and selective MS
MS detection. Samples were processed and analyzed one at a time
every 4.5 min in order to compensate for the inherent instability of
5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide. Both 5-(3-N-
methyltriazen-1-yl)-imidazole-4-carboxamide and the internal standard,
dacarbazine, were quantified in the positive ion, selected reaction moni-
toring mode. The lower limit of quantitation was 10 ng/mL in the plasma
from both species. Interassay accuracy and precision of all calibration standards
and quality control samples were within 11% and 12%, respectively, with
the exception of the lower limit of quantitation in rat plasma (17%). The val-
idated method was used to determine the time-dependent plasma concentra-
tion of 5-(3-N-methyltriazen-1-yl)-imidazole-4-carboxamide in rats and
dogs following a single oral dose of temozolomide. The standard curve and
the quality control data indicate that the method performed acceptably
throughout the sample analysis period.
Liu et al. [35] developed and validated a hydrophilic interaction high-
performance liquid chromatographytandem mass spectrometric method
for the simultaneous quantitation of dacarbazine and its terminal metabolite,
5-amino-4-imidazole carboxamide in human plasma. The plasma samples
are first extracted by a C8 + SCX mixed-mode 96-well plate to extend
the extraction stability of the drug and its terminal metabolite. The extracted
residues are further cleaned by a primary and secondary amine adsorbent for
Dacarbazine 361

the minimization of matrix effect. Analyses are done on an Amide-80 high-

performance liquid chromatography column coupled to a tandem mass
spectrometer fitted with an atmospheric pressure turbo ion spray ionization
interface in the positive-ion mode. Both the drug and its terminal metabolite
have reproducible retention times on the Amide-80 high-performance liq-
uid chromatography column. The column not only has an excellent column
life (over 4000 injections) but also has zero carryover effect. The injection
volume should be limited at 10 L or less to avoid the peak splitting. The
validated concentration ranges are from 0.5 to 500 ng/mL for the drug
and from 2 to 500 ng/mL for its terminal metabolite. The validated method
has been applied to determine the pharmacokinetic profiles for human
patients receiving dacarbazine infusions.

5.2.3 Polarographic Methods

Wasilewska [36] studied the reduction of dacarbazine in various basic elec-
trolytes and buffers within a pH range 2.211.5, by classical polarography. In
quantitative determinations, the electrolyte of choice was 0.1 N
hydrochloric acid with 0.005% gelatin, protected against light. Very good
results were noted for dacarbazine injection solutions containing 106
5  104 mol dacarbazine/L.
Kazemifard et al. [37] compared the polarographic and the colorimetric
methods for the determination of dacarbazine. The drug is easily reduced by
polarography, adding four electrons and four protons to cleave the azo group
and give the 5-amine. The polarography of the pure dacarbazine in meth-
anol buffer and methanol-0.1 M lithium chloride gave a well-defined reduc-
tion step, as did a dacarbazine-containing mixture in methanol0.1 M
lithium chloride and indicated that dacarbazine, pure or in pharmaceutical
mixtures, may be determined at 103105 M. A colorimetric method
using BrattonMarshall reagent, UV irradiation, and measurement at
540 nm was not specific and selective enough to allow a simple and
trouble-free dacarbazine determination, whereas the polarographic method
was quick and sufficiently sensitive.
Wasilewska and Deres [38] isolated dacarbazine, and its metabolite,
5-amino-4-carbamoylimidazole, from blood and urine samples on Dowex
W (H+) columns; upon dilution with 0.1 N hydrochloric acid, contami-
nants were eluted from the column with 0.1 N ethanolic hydrochloric acid
and dacarbazine and its metabolite, 5-amino-4-carbamoylimidazole, with
10% hydrochloric acid. Dacarbazine was assayed directly by polarography
in 10% hydrochloric acid, whereas 5-amino-4-carbamoylimidazole was
362 Abdullah A. Al-Badr and Mansour M. Alodhaib

first diazotized to azahypoxanthine. The sensitivity limit was 5 g/mL

blood for dacarbazine, 10 g/mL blood for 5-amino-4-
carbamoylimidazole, and 4 g/mL urine for both dacarbazine acid and
5-amino-4-carbamoylimidazole.
Ordieres et al. [39] investigated the electrochemical behavior of
dacarbazine by Tast and differential pulse polarography at the dropping mer-
cury electrode, by cyclic and differential pulse voltammetry at the hanging
mercury drop electrode, and by anodic voltammetry at the glassy carbon
electrode. Calibration graphs were obtained for 2  1082  105 M
dacarbazine by differential pulse polarography, for 5  1091  l05 M
by adsorptive stripping voltammetry at a hanging mercury drop electrode,
and for 110  105 M by high-performance liquid chromatography with
oxidative amperometric detection at a glassy carbon electrode. The methods
are compared and applied to determine dacarbazine added to blood serum
after a simple cleanup procedure.
Rodriguez et al. [40] studied the electrochemical behavior of dacarbazine
and its major metabolite 5-aminoimidazole-4-carboxamide on carbon paste
electrodes. Linear sweep voltammetry and differential pulse voltammetry
have been applied, showing that both molecules are active in oxidation.
Both compounds are oxidized in a two-electron process, presenting a charge
transfer coefficients () of 0.44  0.03 and 0.48  0.03 for dacarbazine
and 5-aminoimidazole-4-caroxamide, respectively, which indicate slow
process. The oxidation mechanisms proposed for each of these compounds
seem to yield the same final product, the 5-hydroxyimidazole-4-
carboxamide. Differential pulse voltammetry proved to be a valuable
analytical technique that is suitable for distinguishing and analyzing both
compounds when the most adequate medium (0.1 M perchloric acid) and
operating conditions are chosen.
Zhang et al. [41] explored the binding of dacarbazine to DNA bases in
the absence and presence of gold nanoparticles by electrochemical study.
The results indicate that the binding of purines (adenine and guanine) to
dacarbazine is stronger than of pyrimidines (thymine and cytosine) in the
order of A > G > C > T. It was also observed that the presence of gold
nanoparticles could facilitate the specific interaction between dacarbazine
with DNA bases.
Zhang et al. [42] studied the specific binding of dacarbazine to DNA
bases and oligonucleotides attached to gold nanoparticles by using electro-
chemical methods, and the results indicate that the presence of gold
nanoparticles could facilitate the binding of dacarbazine to specific DNA
Dacarbazine 363

bases and remarkably enhance the relative detection sensitivity. The results
of the study on interaction of dacarbazine with oligonucleotides also illus-
trate that dacarbazine could recognize some specific sequence in DNA chain
and sensitively detect single-base mismatch in DNA helix.
Temerk et al. [43] investigated the electrochemical reduction of
dacarbazineCu2+ complex using cyclic voltammetry and square wave
voltammetry at a hanging mercury drop electrode. The reduction of the
dacarbazineCu2+ complex is irreversible. A reduction mechanism compris-
ing a one-electron reduction of the Cu2+ directly within the complex is used.
The sharp peak of the absorbed dacarbazineCu2+ complex associated with an
effective interfacial accumulation facilitates the determination of dacarbazine
in pharmaceutical formulations and biological fluids. Detection limits for
dacarbazine of 6.12 1010 M, 1.57  1010 M, and 1.97  109 M were
achieved for the determination of the drug in vial, human urine, and serum,
respectively.

6. METABOLISM
Skibba et al. [44] reported that dacarbazine was N-demethylated
in vitro by rat liver microsomes resulting in the formation of
20.5 mol of formaldehyde/mg microsomal protein/30 min. 4(5)-
Aminoimidazole-5(4)-carboxamide was recovered in vitro as a major
metabolic product following N-demethylation resulting in the formation
of 14.9 mol/mg microsomal protein/30 min. The administration of
14
C-methyl-labeled dacarbazine intraperitoneally to rats was followed by
the recovery of 4% of the dose as 14CO2 within 6 h. When 14C-methyl-
labeled dacarbazine was administered intraperitoneally to rats pretreated
with prochlorperazine or phenobarbital, increased amounts (8.1% and
10.5%, respectively) of dacarbazine were N-demethylated to 14CO2 within
6 h. One patient given 14C-methyl-labeled dacarbazine orally expired
21.4% of the radioactivity as 14CO2 within 6 h. N-Demethylation appears
to be a major metabolic pathway of dacarbazine in rats and human. This
reaction is mediated by liver microsomal enzymes that can be induced by
barbiturates and perhaps prochlorperazine.
Beal et al. [45] determined the effects of dacarbazine and its metabolites
on the growth and macromolecular synthesis of Novikoff hepatoma cells in
culture. Dacarbazine (3.0 mM) in light decreased the viable cell count by
90% within 96 h. Dacarbazine protected from light, 2-azahypoxanthine,
dimethylamine, and 5-aminoimidazole-4-carboxamide, all at 3.0 mM,
364 Abdullah A. Al-Badr and Mansour M. Alodhaib

reduced the rate of cellular proliferation. 5-Diazoimidazole-4-carboxamide

(1.0 mM) and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (3.0 mM)
decreased the viable cell count by 99%. Effects on macromolecular synthesis
were determined by the rate of incorporation of the appropriate 3H-labeled
precursor. Results after 6 h are given as percentage of controls. Dacarbazine
(1.0 mM) in light inhibited DNA (8%), RNA (41%), and protein (63%) syn-
thesis. Dacarbazine (1.0 mM) protected from light inhibited DNA (12%) and
RNA (57%) synthesis. 5-Diazoimidazole-4-carboxamide (0.1 mM) inhibited
DNA (1%), RNA (9%), and protein (1%) synthesis. 5-(3-Methyl-1-triazeno)
imidazole-4-carboxamide (1.0 mM) inhibited DNA (72%) and protein (65%)
synthesis but stimulated RNA (127%) synthesis. 2-Azahypoxanthine
(1.0 mM) inhibited DNA (43%), RNA (82%), and protein (28%) synthesis.
5-Aminoimidazole-4-carboxamide (3.0 mM) stimulated DNA (354%) and
RNA (266%) synthesis. These data show that dacarbazine is able to generate
several toxic metabolites that may be responsible for its biological effects.
Larsson et al. [46] reported that dacarbazine was found to inhibit com-
petitively the low-Km cyclic AMP phosphodiesterase activity in an
ammonium-sulfate-precipitable fraction of the 2000  g supernatant of rat
liver. With substrate concentration at 0.25 M, I50 was 790 M for
dacarbazine and 350 M for theophylline. Dacarbazine at 2 mM more than
doubled the cAMP response to glucagon in hepatocytes and to adrenaline in
MH1C1 hepatoma cells, indicating that it also exerts its inhibitory effect on
the phosphodiesterase in intact cells. The possible contribution of the phos-
phodiesterase inhibition to the growth inhibitory and cytotoxic effects of
dacarbazine is discussed.
Kolar et al. [47] reported that dacarbazine is metabolized in rats to a
structurally related product which was detected by thin-layer chroma-
tography. The novel metabolite has a lower mobility and a color
reaction that is indistinguishable from the parent compound. The meta-
bolite is not retained on an anionic exchanger which is inconsistent with
the expected covalent binding of the drug to endogenous anionic sub-
strates (eg, glucuronic acid). Since both dacarbazine and the metabolite
yielded 5[(4-ethylamino-l-naphthyl)-azo]imidazole-4-carboxamide through
release of 5-diazoimidazole-4-carboxamide, followed by coupling with
N-ethyl-1-naphthylamine, no biotransformation (hydroxylation) of the
imidazole moiety of the injected dacarbazine had occurred. By corollary,
the lowered chemotherapeutic mobility of the metabolite was applicable
by the introduction of a polar but nonacidic function into the terminal
dimethylamino group on the triazene side chain. The metabolite
Dacarbazine 365

was identified as 5-(3-hydroxymethyl-3-methyl-1-triazeno)imidazole-4-

carboxamide by cochromatography with an authentic sample of 5-(3-
hydroxymethyl-3-methyl-1-triazeno)imidazole-4-carboxamide and by its
methylating capacity for nucleophilic substrates.
Meer et al. [48] studied the in vivo metabolism and reaction with DNA
of dacarbazine. Bioactivation of dacarbazine yields a methylating intermedi-
ate but the extent of interaction with cellular macromolecules has not pre-
viously been reported. Following a single i.p. injection of [14C-methyl]
dacarbazine, exhalation of 14CO2 occurred with a t1/2 max of approximately
2 h (0.95 mg/kg) and 2.5 h (95 mg/kg). Of the total radioactivity adminis-
tered, 8.5% was exhaled as 14CO2; 54% was excreted via the urine, predom-
inantly as unchanged dacarbazine. In liver, kidney, and lung, formation of
7-[14C]methylguanine in DNA and RNA was directly proportional with
dose. DNA methylation by a single dose of dacarbazine (9.8 mg/kg; 5 h sur-
vival time) was highest in liver (35 mol 7-methylguanine/mol guanine),
followed by kidney (25 mol) and lung (20 mol). The remainder tissues
showed 7-methylguanine concentrations approximately 50% of those in
liver DNA, with the exception of the brain which had a very low extent
of DNA modification (approximately 1 mol/mol guanine). At the
specific radioactivity used (48 mCi/mmol), the promutagenic base O6-
methylguanine was only detectable in liver, kidney, lung, and stomach
DNA (0.60.8 mol/mol guanine). Autoradiographic studies revealed
a diffuse distribution of reaction products in rat liver. In contrast,
N-nitrosodimethylamine and related carcinogens known to be bioactivated
by the hepatic cytochrome P-450 system show a predominantly
centrilobular distribution. This difference may be due to the greater stability
of proximate carcinogens generated by -C hydroxylation at one of the
methyl groups of dacarbazine.
Lewis et al. [49] described the application of homology modeling to gen-
erate CYPlAl mutants with enhanced activation of dacarbazine. Dacarbazine
is activated by CYPlAl and CYP1A2 catalyzed N-demethylation. Structural
features of these enzymes that confer dacarbazine N-demethylation have not
been characterized. A validated homology model of CYPlAl was employed
to elucidate structureactivity relationships and to engineer CYP1A1
enzymes with altered dacarbazine activation. In silico docking demonstrated
that dacarbazine orientates proximally to Serl22, Phel23, Asp313, Ala317,
Ile386, Tyr259, and leu496 of human CYP1A1. The site of metabolism
is positioned 5.6 A from the heme iron at an angle of 105.3 degree. Binding
in the active site is stabilized by H-bonding between Tyr259 and the
366 Abdullah A. Al-Badr and Mansour M. Alodhaib

N2 position of the imidazole ring. Twenty-seven CYPlAl mutants were

generated and expressed in Escherichia coli in yields ranging from 9 to
225 pmol P450/mg. Dacarbazine N-demethylation by the E161K,
E256K, and I458V mutants exhibited MichaelisMenten kinetics, with
decreases in Km (183249 M) that doubled the catalytic efficiency
(P < 0.05) relative to wild-type CYPlAl (Km, 408  43 M; Vmax,
28  4 pmol/min per pmol of P450). The generation of enzymes with cat-
alytically enhanced dacarbazine activation highlights the potential use of
mutant CYP1A1 proteins in P450-based gene-directed enzyme prodrug
therapy for the treatment of metastatic malignant melanoma.
Yagmagata et al. [50] studied the metabolism of dacarbazine by rat liver
microsomes and the contribution of CYP1A enzymes to the
N-demethylation of dacarbazine. The N-demethylation of dacarbazine in
liver microsomes was significantly increased by treatment of rats with
-naphthoflavone, dexamethasone, or phenobarbital. The extent of increase
in the N-demethylation observed in -naphthoflavone-treated rats was
much greater than that observed in dexamethasone-treated rats. A good cor-
relation between N-demethylation of dacarbazine and O-deethylation of
phenacetin was observed when a low concentration of phenacetin was used.
The activity of dacarbazine N-demethylase in rat liver microsomes was
highly correlated with the amounts of CYP protein immunochemically
determined with antirat CYP1A2 antibodies. Antibodies to rat CYP1A2,
and furafylline and -naphthoflavone, which are known inhibitors of CYP1A
enzymes, exhibited inhibitory effects on dacarbazine N-demethylation.
Results have indicated that CYP1A enzymes are responsible for the N-
demethylation of dacarbazine in rat liver microsomes.
Reid et al. [51] studied the role of CYP1A1, CYP1A2, and CYP2E1
on the metabolic activation of dacarbazine by human CYP 450. Dacarbazine
is inactive until metabolized in the liver by cytochromes P450 to
form the reactive N-demethylated species: 5-[3-hydroxymethyl-3-
methyltriazen-1-yl]imidazole-4-carboxamide and 5-[3-methyltriazen-1-yl]
imidazole-4-carboxamide (MTIC). The modest activity of dacarbazine in
the treatment of cancer patients has been attributed, in part, to lower activity
of cytochromes P450 (P450) in humans when compared with rodents. The
dacarbazine N-demethylation involved in MTIC formation by human liver
microsomes is catalyzed by CYP1A1, CYP1A2, and CYP2E1. The most
potent inhibitors of dacarbazine N-demethylation were -naphthoflavone
(CYP1A1 and CYP1A2), quercetin (CYP1A2), chlorzoxazone (CYP1A2
and CYP2E1), and disulfiram (CYP2E1). Antihuman CYP1A2 antiserum
Dacarbazine 367

also inhibited dacarbazine N-demethylation. Dacarbazine N-demethylation

in a panel of 10 human liver microsome preparations was correlated with
the catalytic activities for CYP1A2 (ethoxyresorufin O-deethylation and caf-
feine N3-demethylation) in the absence of -naphthoflavone and with the
catalytic activities for CYP2E1 (chlorzoxazone 6-hydroxylations) in the pres-
ence of -naphthoquinone. Dacarbazine metabolism was catalyzed by the
recombinant human CYP1A1, CYP1A2, and CYP2E1. The Km (Vmax)
values for metabolism of dacarbazine by recombinant human CYP1A1 and
CYP1A2 were 595 M (0.684 nmol/min per mg protein) and 659 M
(1.74 nmol/min per mg protein), respectively. The CYP2E1 Km value
exceeded 2.8 mM. It is concluded that: (a) CYP1A2 is predominant P450 that
catalyzes dacarbazine hepatic metabolism; (b) CYP2E1 contributes to hepatic
dacarbazine metabolism at higher substrate concentrations, and (c) CYP1A1
catalyzes extrahepatic metabolism of dacarbazine.
Rooseboom et al. [52] reported that dacarbazine is activated by hydrox-
ylation to produce 5-(3-hydroxymethyl-3-methyltriazen-1-yl)imidazole-4-
carboxamide (Scheme 4). Formaldehyde is subsequently eliminated from
5-(3-hydroxymethyl-3-methyltriazen-1-yl)imidazole-4-carboxamide non-
enzymatically, resulting in 5-(3-methyltriazen-1-yl)imidazole-4-carbox-
amide, which rapidly decomposes to aminoimidazole carboxamide, N2,
and CH 3 . P450-mediated dacarbazine bioactivation induces apoptosis

O O
CH3 H2N CH2OH
H2N
N N Hydroxylation N N
N CH3 N CH3
N N
NH NH

Darcarbazine HMMTIC

H2CO

O O
H2N H
H2N
N N NH2
N CH3
N
NH
N
NH
+ CH3+ + N2

MTIC AIC
Scheme 4 Activation of dacarbazine by cytochrome P450. Hydroxylation of
dacarbazine by cytochrome P450 results in the formation of HMMTIC. MTIC is generated
nonenzymatically by loss of formaldehyde. MTIC rapidly decomposes into amino-
imidazole carboxamide, CH+3 , and N2 [52].
368 Abdullah A. Al-Badr and Mansour M. Alodhaib

and mutagenicity via the formation of O6-alkylguanine-DNA adducts [53].

The limited antitumor effect of dacarbazine in humans compared with
rodents has been attributed to lower P450 activity levels in humans. Addi-
tionally, pretreatment of mice with the P450 inhibitor metyrapone inhibited
dacarbazine-induced apoptosis in intestinal tumors [54]. Dacarbazine
bioactivation by human liver microsomes, resulting in the formation of
5-(3-methyltriazen-1-yl)imidazole-4-carboxamide, was shown to be cata-
lyzed by CYP1A1, 1A2, and 2E1 [51]. Furthermore, dacarbazine
bioactivation was inhibited by 75% in human liver microsomes using human
anti-CYP1A2 antibody, indicating a predominant role for this P450
enzyme. Using cDNA-expressed human P450 isoenzymes, it has been
shown that CYP1A2 is the predominant P450 catalyzing dacarbazine
hepatic metabolism (kcat 14 min1; Km 0.66 mM), CYP2E1 contributes
to hepatic dacarbazine metabolism at higher substrate concentrations
(Km > 2.8 mM), and CYP1A1 activates dacarbazine extra hepatically
(kcat 10 min1; Km 0.60 mM) [51]. Human CYP2C9/19, CYP2D6,
and CYP3A4 were not capable of dacarbazine prodrug activation.

7. PHARMACOKINETICS
Breithaupt et al. [55] studied the pharmacokinetics of dacarbazine and
its main metabolite 5-aminoimidazole-4-carboxamide in eight patients with
malignant melanoma or sarcoma receiving 2.656.85 mg dacarbazine/kg
body weight by intravenous bolus injection or by continuous 0.56 h infu-
sions on 5 consecutive days. The plasma disappearance of dacarbazine was
biphasic, with a terminal half-life of 41.4 min (range 30.351.6 min). The
mean distribution volume of dacarbazine was 0.632 L/kg and the total clear-
ance was 15.4 mL/kg min (range 8.723.3 mL/kg min). The renal clear-
ance of dacarbazine was 5.210.9 mL/kg min, indicating that about 50%
of dacarbazine was eliminated by extrarenal mechanisms. The plasma decay
of 5-aminoimidazole-4-carboxamide was monoexponential with a half-life
of 43116 min. A renal clearance of 2.65.3 mL/kg min was calculated
for 5-aminoimidazole-4-carboxamide. The urinary recovery was 4652%
for dacarbazine and 918% for 5-aminoimidazole-4-carboxamide. The
plasma concentrations of dacarbazine observed during 0.56 h infusions
of dacarbazine (5.456.85 mg/kg) were 0.666.2 g/mL. Comparison
of various dosage schedules within the same patient did not reveal relevant
differences of the areas under the concentrationtime curves. Immunother-
apy with Bacillus Calmette-Guerin did not significantly influence the
Dacarbazine 369

pharmacokinetics of dacarbazine. During isolated extremity perfusion with

dacarbazine (75130 mg/kg extremity) for treatment of malignant
tumors of the extremities concentrations of dacarbazine ranged from
150 to 500 g/mL perfusate. There was no evidence of 5-amino-
imidazole-4-carboxamide formation. In isolated liver perfusion experiments
in anesthetized dogs, metabolic degradation of dacarbazine and 5-
aminoimidazole-4-carboxamide was demonstrated.
Buesa and Urrechaga [56] studied the pharmacokinetics of dacarbazine
given at a dose of 8501980 mg/m2 as a 1030-min infusion, in cancer
patients, and the plasma concentrationtime curves were adjusted to a
two-compartment model, with a mean tl/2 value of 0.17 h (range
0.10.26 h) and a mean tl/2 value of 2 h (range 1.52.7 h) being found.
The mean volume of the central compartment (Vc) and the apparent volume
of distribution (VB) were 0.42 L/kg (range 0.240.54 L/kg) and 1.49 L/kg
(range 0.881.74 L/kg), respectively. The mean total body clearance of
dacarbazine was 0.58 L/kg per h (range 0.260.82 L/kg per h) and the mean
renal clearance was 0.28 L/kg per h (range 0.170.49 L/kg per h).
Unchanged dacarbazine recovered from urine within 24 h varied from
11% to 63% of the delivered dose, with an inverse correlation being found
between the dacarbazine dose and the amount excreted. The metabolite
aminoimidazole carboxamide was detectable in plasma from the start of
dacarbazine infusion, and its concentrationtime curve showed a monoph-
asic decay, exhibiting a mean tl/2 value of 3. 25 h (range 1.775.82 h).
Mean aminoimidazole carboxamide renal clearance was 0.15 L/kg per h
(range 0.050.32 L/kg per h). The amount of aminoimidazole carboxamide
excreted in urine increased with increasing dacarbazine dose and varied from
1.2% to 13.6% of the delivered dacarbazine dose. Both dacarbazine distribu-
tion and disposition and aminoimidazole carboxamide production and renal
excretion seemed to be limited after high dacarbazine doses compared with
the pharmacokinetics of low-dose dacarbazine. Nonlinear pharmacokinetics
for high dose dacarbazine could not be clearly excluded.
Didolkar et al. [57] studied the pharmacokinetics of dacarbazine in
six patients with melanoma of an extremity who were undergoing hyper-
thermic isolation perfusion with dacarbazine in order to understand better
its clinical pharmacokinetics. Plasma was sampled from the arterial
and venous lines of extracorporeal pump during the perfusion with the
systemic vein and urine sampled postperfusion. Samples were analyzed
for dacarbazine, 2-azahypoxanthine, and aminoimidazole-4-carboxamide.
99m
Tc (Technetium) human serum albumin was used in the perfusion
370 Abdullah A. Al-Badr and Mansour M. Alodhaib

circuit to monitor the crossover of the perfusate into the systemic circulation
during the procedure. The data were analyzed using a compartmental model
of sampled body compartments incorporating the isolated extremity. High
tissue dacarbazine levels were maintained throughout the perfusion, whereas
in the systemic circulation, plasma dacarbazine concentrations, when
observed, were 40100-fold less than those in the perfusate. Almost 70%
of the dacarbazine administered was not recovered in the perfusate after
the washout of the extremity. High levels of dacarbazine can be maintained
in an extremity (arm or leg) during perfusion.
Rajkumar et al. [58] conducted a randomized phase II study to determine
the efficacy of dacarbazine in recurrent gliomas. Patients were randomly
assigned to receive either dacarbazine 750 mg/m2 intravenously day 1 every
28 days (Arm A) or dacarbazine 200 mg/m2 days 15 every 28 days (Arm B).
Pharmacokinetics were studied in six patients on each arm using high-
performance liquid chromatography analysis. Thirty-nine patients (30 males,
9 females), aged 2767 years (median 53), were entered on the study (20 on
Arm A, 19 on Arm B). No objective responses were seen. Median time to
progression was 3 months. Median survival was 8 months. Treatment was
generally well tolerated. Major toxicities were grade 12 nausea (33%), leth-
argy (28%), diarrhea (15%), alopecia (15%), and grade 3 neutropenia (8%).
Four patients on Arm A had mild self-limited episodes of intravascular hemo-
lysis occurring immediately after drug infusion, the mechanism of which is
unknown. Mean AUC for dacarbazine, hydroxymethyl dacarbazine, and
(5-[3-methyl-l-triazeno]imidazole-4-carboxamide) in Arm A were 14.8,
0.17, and 1.15 mM/min, respectively. Corresponding values for Arm
B (on day 1 of 5) were 1.7, 0.06, and 0.29 mM/min, respectively. The
predicted hydroxymethyl dacarbazine and 5-[3-methyl-l-triazeno]imidaz-
ole-4-carboxamide exposure over 5 days for Arm B, based on the day 1 data,
are higher than with Arm A. Dacarbazine is well tolerated but does not have
activity in patients with recurrent gliomas. The 5-day schedule appears less
toxic, and pharmacokinetic studied show that it provides greater exposure
to (5-[3-methyl-1-triazeno]imidazole-4-carboxamide) and hydroxymethyl
dacarbazine compared to 1-day schedule.

8. STABILITY
Benvenuto et al. [59] determined the stability of dacarbazine and other
antitumor agents in underfilled plastic and glass administration containers.
Drugs were reconstituted according to manufacturers instruction and added
Dacarbazine 371

to 5% dextrose injection 50 mL in both polyvinyl chloride bags and glass

partial-fill bottles. Mitomycin was added to 0.9% sodium chloride injection
50 mL in both polyvinyl chloride bags and partial-fill bottles. All admixtures
were stored at room temperature, not protected from light. Stability of
dacarbazine and a number of antitumor drugs were equally stable (10% or
less change in concentration over 24 h), in glass and plastic containers.
Shetty et al. [60] investigated the effect of the initial concentration
(0.055 mg/mL, 2.5  1040.025 M) (pH 113), buffer concentration
(0.010.075 M), light, antioxidants, and cosolvents on the degradation of
dacarbazine in aqueous solution at 37C. Liquid chromatography was used
to monitor the degradation of dacarbazine as well as the appearance of the
degradation products. The kinetics of hydrolysis of dacarbazine in the dark
were pseudo first order and independent of the initial concentration of the
drug. The degradation of dacarbazine was accelerated by light and at low
concentration, proceeded by pseudo zero-order kinetics. The pH-rate pro-
files showed that both the photolytic and the hydrolytic reactions were
dependent on the ionization state of the molecule. The main degradation
product for both hydrolysis and photolysis was detected by liquid chroma-
tography and confirmed by mass spectrometry to be 2-azahypoxanthine.
El-Aatmani et al. [61] studied the stability of dacarbazine in commercial
glass vials and polyvinyl chloride bags in various storage conditions and the
emergence of 2-azahypoxanthine, a major degradation product possibly
linked with some adverse effects. Triplicate samples of reconstituted
(11 mg/mL) and diluted (1.40 mg/mL) dacarbazine admixtures were pre-
pared and stored at 4C or at 25C in daylight, fluorescent light, or the dark.
The effect of several light-protective measures (amber glass vials, aluminum
foil wrapping and opaque tubing) on dacarbazine stability in a simulated i.v.
infusion system was evaluated. Dacarbazine quantitation and main degrada-
tion product determination were performed by high-performance liquid
chromatography. Stability was defined as the conservation of 90105% of
initial dacarbazine concentration without major variations in clarity, color,
or pH and without precipitate formation. Reconstituted dacarbazine solu-
tions were stable for 24 h at room temperature and during light exposure and
stable for at least 96 h at 26C when stored in the dark. After dilution in
polyvinyl chloride bags, stability time increased from 2 h in daylight to
24 h in fluorescent light and to 72 h when covered with aluminum foil.
After 2 h of simulated infusion, dacarbazine remained stable. Diluted
dacarbazine solutions, stored at 26C, were stable for at least 168 h. The
only degradation product found was 2-azahypoxanthine, which was
372 Abdullah A. Al-Badr and Mansour M. Alodhaib

detected in every sample. The storage and handling of dacarbazine should

take into account both the loss of the drug and the production of its poten-
tially toxic degradation product. Dacarbazine must be carefully protected
from light, administered using opaque infusion tubing, and refrigerated
before administration to reduce the formation of 2-azahypoxanthine.
Shukla and Pitre [62] studied the role of bio-metal Fe(III) in the anticancer
effect of dacarbazine. Physicochemical, Microbial, and Pharmacological stud-
ies on Fe(III)dacarbazine complex have been done in solid and aqueous
phase. On the basis of elemental analysis, polarographic studies, amperometric
titrations, and infrared spectral studies the probable formula for the complex
has been worked out to be 1:1, Fe(III)dacarbazine. The metalligand inter-
action has been studied using polarographic method at 25  1C and at ionic
strength of 1.0 (potassium chloride). Microbial studies on the complex
were done against various pathogenic bacteria, viz., Pseudomonas mangiferae,
Staphylococcus aureus, Salmonella typhi, and Vibrio cholerae and fungi, ie,
Trichothecium and Chrysosporium sp. using Rapers method. Mouse sarcoma cell
line 180 and Balb/C mice were used for the anticancer screening of solid com-
plex in vitro and in vivo, respectively. The observed polarographic data on
Lingane treatment revealed the formation of single (1:1) (M:L) complex with
Fe(III) and dacarbazine ligands. The results of amperometric titrations of
Fe(III) with dacarbazine in 1 M potassium chloride supporting electrolyte
pH 7.0  0.1 supported the above findings, the infrared data speaks of the
complex formation between the metal and the dacarbazine ligand through
the two nitrogen one each of primary amide and trizo groups. The results
of microbial and pharmacological studies with the M:Drug complex revealed
that the anticancer activity of the drugmetal complex is nearly doubled as
compared to the pure drug. As such Fe(III)dacarbazine complex may be rec-
ommended to the therapeutic experts for its possible use as more potent
anticancer drug.
Teimouri et al. [63] compared the effectiveness and side effects of
dacarbazine with those of temozolomide through a meta-analysis.
A thorough literature bibliography search was conducted up to 2012 to
gather and review all randomized clinical trials comparing the use of
dacarbazine with that of temozolomide in the treatment of malignant mel-
anoma. Three head-to-head randomized clinical trials comprising 1314
patients met the criteria and were included. Comparison of temozolomide
with dacarbazine yielded a nonsignificant relative risk of 0.83 [95% confi-
dence interval 0.262.64, P 0.76] for complete response, a nonsignifi-
cant relative risk of 1.05 (95% confidence interval 0.851.3, P 0.65)
Dacarbazine 373

for stable disease, and a nonsignificant relative risk of 2.64 (95% confidence
interval 0.971.36, P 0.11) for disease control rate. The relative risk for
nonhematologic side effects and hematologic side effects, such as anemia,
neutropenia, and thrombocytopenia, of temozolomide compared with
dacarbazine in patients with malignant melanoma was nonsignificant in all
cases, but the relative risk for lymphopenia of temozolomide compared with
dacarbazine was 3.79 (95% confidence interval 1.3810.39, P 0.01),
which was significant. Although it is easier to administer oral medication,
according to the results, there is no significant difference in the efficacy
and side effects of these two drugs. Owing to the higher cost of treatment
with temozolomide and the increased prevalence of lymphopenia on using
temozolomide, use of dacarbazine as the first choice treatment for malignant
melanoma is suggested.

9. REVIEWS
Nussbaumer et al. [64] reviewed the analytical methods used for the
determination of the most commonly used anticancer drugs including
dacarbazine. In the last decades, the number of patients receiving chemo-
therapy has considerably increased. Given the toxicity of cytotoxic agents
to humans (not only for patients but also for healthcare professionals), the
development of reliable analytical methods to analyze these compounds
became necessary. From the discovery of new substances to patient admin-
istration, all pharmaceutical fields are concerned with the analysis of cyto-
toxic drugs. The use of methods to analyze cytotoxic agents in various
matrices, such as pharmaceutical formulations and biological and environ-
mental samples, is discussed. Thus, an overview of reported analytical
methods for the determination of dacarbazine and the most commonly used
anticancer drugs is given.
Iradyan et al. [7] reviewed the physicochemical properties and antitumor
activity of dacarbazine, its analogs, and the new alkylating agent imidazene.
It is shown that the activity of dacarbazine is superior to most of its analogs.
Imidazene exhibits an advantage over dacarbazine with respect to both sta-
bility and activity and can be used for the treatment of malignant melanoma
and sarcoma of soft tissues and in combined chemotherapy.
Rooseboom et al. [52] reviewed the most important enzymes involved in
prodrug activation notably with respect to tissue distribution, upregulation in
tumor cells, and turnover rates. The following endogenous enzymes are dis-
cussed: aldehyde oxidase, amino acid oxidase, cytochrome P450 reductase,
374 Abdullah A. Al-Badr and Mansour M. Alodhaib

DT-diaphorase, cytochrome P450, tyrosinase, thymidylate synthase, thymi-

dine phosphorylase, glutathione S-transferase, deoxycytidine kinase, carbox-
ylesterase, alkaline phosphatase, beta-glucuronidase, and cysteine conjugate
beta-lyase. In relation to each of these enzymes, several prodrugs are discussed
regarding organ- or tumor-selective activation of clinically relevant prodrugs
of 5-fluorouracil, oxazaphosphorines (cyclophosphamide, ifosfamide, and
trofosfamide), paclitaxel, etoposide, anthracyclines (doxorubicin, daunorubi-
cin, epirubicin), mercaptopurine, thioguanine, cisplatin, melphalan, and
other important prodrugs such as menadione, mitomycin C, tirapazamine,
5-(aziridin-1-yl)-2,4-dinitrobenzamide, ganciclovir, irinotecan, dacarbazine,
and amifostine. In addition to endogenous enzymes, a number of non-
endogenous enzymes, used in antibody-, gene-, and virus-directed enzyme
prodrug therapies, are described. It is concluded that the development of pro-
drugs has been relatively successful; however, all prodrugs lack a complete
selectivity. Therefore, more work is needed to explore the differences
between tumor and nontumor cells and to develop optimal substrates in terms
of substrate affinity and enzyme turnover rates of prodrug-activating enzymes
resulting in more rapid and selective cleavage of the prodrug inside the
tumor cells.

ACKNOWLEDGMENTS
The authors wish to thank Mr. Tanvir A. Butt, Department of Pharmaceutical Chemistry,
College of Pharmacy, King Saud University for his secretarial assistance in typing this profile.

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CUMULATIVE INDEX

Bold numerals refer to volume numbers.

A Azathioprine, 10, 29
Acebutolol, 19, 1 Azintamide, 18, 1
Acetaminophen, 3, 1; 14, 551 Azithromycin, 39, 1
Acetazolamide, 22, 1 Aztreonam, 17, 1
Acetohexamide, 1, 1; 2, 573; 21, 1
Acetylcholine chloride, 31, 3, 21 B
Acyclovir, 30, 1 Bacitracin, 9, 1
Adenosine, 25, 1 Baclofen, 14, 527
Alendronate sodium, 38, 1 Benazepril hydrochloride, 31, 117
Allopurinol, 7, 1 Bendroflumethiazide, 5, 1; 6, 597
Amantadine, 12, 1 Benperidol, 14, 245
Amfebutamone, 41, 1 Benzocaine, 12, 73
Amikacin sulfate, 12, 37 Benzoic acid, 26, 1
Amiloride hydrochloride, 15, 1 Benzyl benzoate, 10, 55
Aminobenzoic acid, 22, 33 Betamethasone diproprionate, 6, 43
Aminoglutethimide, 15, 35 Bipolar disorder, 41, 133
Aminoketone, 41, 1 Bretylium tosylate, 9, 71
Aminophylline, 11, 1 Brinzolamide, 26, 47
Aminosalicylic acid, 10, 1 Bromazepam, 16, 1
Amiodarone, 20, 1 Bromcriptine methanesulfonate, 8, 47
Amitriptyline hydrochloride, 3, 127 Buclizine, 36, 1
Amlodipine besylate, 37, 31 Bumetanide, 22, 107
Ammonium carbonate, 41, 31 Bupivacaine, 19, 59
Amobarbital, 19, 27 Bupropion hydrochloride, 41, 1
Amodiaquine hydrochloride, 21, 43 Busulphan, 16, 53
Amoxicillin, 7, 19; 23, 1 Butyl methoxy dibenzoylmethane, 38, 87
Amphotericin B, 6, 1; 7, 502
Ampicillin, 2, 1; 4, 518 C
Antineoplastics, 41, 323 Caffeine, 15, 71
Apomorphine hydrochloride, Calcitriol, 8, 83
20, 121 Calcium acetate, 41, 31
Arginine, 27, 1 Calcium carbonate (CaCO3), 41, 31
Aripiprazole, 38, 35 Calcium chloride, 41, 31
Aripiprazole: polymorphs and Calcium hydroxide, 41, 31
solvatomorphs, 37, 1 Camphor, 13, 27
Ascorbic acid, 11, 45 Candesartan cilexetil, 37, 79
Aspartame, 29, 7 Captopril, 11, 79
Aspirin, 8, 1 Carbamazepine, 9, 87; 41, 133
Astemizole, 20, 173 Carbenoxolone sodium, 24, 1
Atenolol, 13, 1 Carbonate rocks, Dunhams classification of,
Atorvastatin calcium, 35, 1 41, 31
Atropine, 14, 325 Carbon dioxide, 41, 31

439
440
Carbonic acid calcium salt, 41, 31
Carbonic anhydrase (CA), 41, 31
Carvedilol, 38, 113
Cefaclor, 9, 107
Cefamandole nafate, 9, 125; 10, 729
Cefazolin, 4, 1
Cefdinir, 39, 41
Cefixime, 25, 39
Cefotaxime, 11, 139
Cefoxitin sodium, 11, 169
Ceftazidime, 19, 95
Ceftriaxone sodium, 30, 21
Cefuroxime sodium, 20, 209
Celiprolol hydrochloride, 20, 237
Cephalexin, 4, 21
Cephalothin sodium, 1, 319
Cephradine, 5, 21
Chitin, 36, 35
Chloral hydrate, 2, 85
Chlorambucil, 16, 85
Chloramphenicol, 4, 47; 15, 701
Chlordiazepoxide, 1, 15
Chlordiazepoxide hydrochloride, 1, 39;
4, 518
Chloropheniramine maleate, 7, 43
Chloroquine, 13, 95
Chloroquine phosphate, 5, 61
Chlorothiazide, 18, 33
Chlorpromazine, 26, 97
Chlorprothixene, 2, 63
Chlortetracycline hydrochloride, 8, 101
Chlorthalidone, 14, 1
Chlorzoxazone, 16, 119
Cholecalciferol, 13, 655
Cimetidine, 13, 127; 17, 797
Cinnarizine, 40, 1
Ciprofloxacin, 31, 163, 179, 209
Cisplatin, 14, 77; 15, 796
Citric Acid, 28, 1
Clarithromycin, 24, 45
Clidinium bromide, 2, 145
Clindamycin hydrochloride, 10, 75
Clioquinol, 18, 57
Clofazimine, 18, 91; 21, 75
Clomiphene citrate, 25, 85
Clonazepam, 6, 61
Clonfibrate, 11, 197
Clonidine hydrochloride, 21, 109
Cumulative Index
Clopidogrel bisulfate, 35, 71
Clorazepate dipotassium, 4, 91
Clotrimazole, 11, 225
Cloxacillin sodium, 4, 113
Clozapine, 22, 145
Cocaine hydrochloride, 15, 151
Cocrystal Systems of Pharmaceutical
Interest: 20072008, 35, 373
Cocrystal Systems of Pharmaceutical
Interest: 2009, 36, 361
Codeine phosphate, 10, 93
Cockle shell, 41, 31
Colchicine, 10, 139
Cortisone acetate, 26, 167
Creatine monohydrate, 34, 1
Crospovidone, 24, 87
Curcumin, 39, 113
Cyanocobalamin, 10, 183
Cyclandelate, 21, 149
Cyclizine, 6, 83; 7, 502
Cyclobenzaprine hydrochloride, 17, 41
Cycloserine, 1, 53; 18, 567
Cyclosporine, 16, 145
Cyclothiazide, 1, 65
Cypropheptadine, 9, 155
Cytarabine, 34, 37
D
Dacarbazine, 41, 323
Dapsone, 5, 87
Dasatinib, 39, 205
Dexamethasone, 2, 163; 4, 519
Diatrizoic acid, 4, 137; 5, 556
Diazepam, 1, 79; 4, 518
Dibenzepin hydrochloride, 9, 181
Dibucaine, 12, 105
Dibucaine hydrochloride, 12, 105
Diclofenac sodium, 19, 123
Didanosine, 22, 185
Diethylstilbestrol, 19, 145
Diflunisal, 14, 491
Digitoxin, 3, 149; 9, 207
Dihydroergotoxine methanesulfonate, 7, 81
Diloxanide furoate, 26, 247
Diltiazem hydrochloride, 23, 53
Dioctyl sodium sulfosuccinate, 2, 199;
12, 713
Diosgenin, 23, 101
Cumulative Index 441

Diperodon, 6, 99 Flecainide, 21, 169

Diphenhydramine hydrochloride, 3, 173
Diphenoxylate hydrochloride, 7, 149
Dipivefrin hydrochloride, 22, 229
Dipyridamole, 31, 215
Disopyramide phosphate, 13, 183
Direct Crystallization of Enantiomers and
Dissociable Diastereomers, 36, 331
Disulfiram, 4, 168
Dodecyl dimethyl betaine, 41, 31
Dobutamine hydrochloride, 8, 139
Dolomite, 41, 31
Donepezil, 35, 117
Dopamine hydrochloride, 11, 257
Dorzolamide hydrochloride, 26, 283; 27, 377
Doxorubicine, 9, 245
Droperidol, 7, 171
Fluconazole, 27, 67
Flucytosine, 5, 115
Fludrocortisone acetate, 3, 281
Flufenamic acid, 11, 313
Fluorouracil, 2, 221; 18, 599
Fluoxetine, 19, 193
Fluoxymesterone, 7, 251
Fluphenazine decanoate, 9, 275; 10, 730
Fluphenazine enanthate, 2, 245; 4, 524
Fluphenazine hydrochloride, 2, 263; 4, 519
Flurazepam hydrochloride, 3, 307
Flurbiprofen, 37, 113
Flutamide, 27, 115
Fluvoxamine maleate, 24, 165
Folic acid, 19, 221
Furosemide, 18, 153

E G
Echothiophate iodide, 3, 233 Gadoteridol, 24, 209
Econazole nitrate, 23, 127 Gatifloxacin, 37, 183
Edetic Acid (EDTA), 29, 57 Gefitinib, 39, 239
Emetine hydrochloride, 10, 289 Gemifloxacin, 36, 151
Enalapril maleate, 16, 207 Gentamicin sulfate, 9, 295; 10, 731
Ephedrine hydrochloride, 15, 233 Glafenine, 21, 197
Epinephrine, 7, 193 Glibenclamide, 10, 337
Ergonovine maleate, 11, 273 Glimepiride, 36, 169
Ergotamine tartrate, 6, 113 Glutathione, 40, 43
Erthromycin, 8, 159 Gluthethimide, 5, 139
Erthromycin estolate, 1, 101; 2, 573 Gramicidin, 8, 179
Estradiol, 15, 283 Griseofulvin, 8, 219; 9, 583
Estradiol valerate, 4, 192 Guaifenesin, 25, 121
Estrone, 12, 135 Guanabenz acetate, 15, 319
Ethambutol hydrochloride, 7, 231 Guar gum, 24, 243
Ethynodiol diacetate, 3, 253
Etodolac, 29, 105
H
Halcinonide, 8, 251
Etomidate, 12, 191
Haloperidol, 9, 341
Etopside, 18, 121
Halothane, 1, 119; 2, 573; 14, 597
Eugenol, 29, 149
Heparin sodium, 12, 215
Ezetimibe, 36, 103
Heroin, 10, 357
Hexestrol, 11, 347
F Hexetidine, 7, 277
Famotidine, 34, 115 Histamine, 27, 159
Fenoprofen calcium, 6, 161 Homatropine hydrobromide,
Fenoterol hydrobromide, 27, 33 16, 245
Flavoxoate hydrochloride, 28, 77 Hydralazine hydrochloride, 8, 283
Fexofenadine hydrochloride, 34, 153 Hydrochlorothiazide, 10, 405
442 Cumulative Index

Hydrocortisone, 12, 277 Levothyroxine sodium, 5, 225

Hydroflumethaizide, 7, 297 Lidocaine, 14, 207; 15, 761
Hydroxyprogesterone caproate, 4, 209 Lidocaine hydrochloride, 14, 207; 15, 761
Hydroxyzine dihydrochloride, 7, 319 Lincomycin, 23, 275
Hyoscyamine, 23, 155 Lisinopril, 21, 233
Lithium carbonate, 15, 367
I Lobeline hydrochloride, 19, 261
Ibuprofen, 27, 265 Lomefloxacin, 23, 327
Imatinib mesylate, 39, 265 Lomustine, 19, 315
Imipramine hydrochloride, 14, 37 Loperamide hydrochloride, 19, 341
Impenem, 17, 73 Lorazepam, 9, 397
Indapamide, 23, 233 Lornoxicam, 36, 205
Indinivar sulfate, 26, 319 Losartan, 40, 159
Indomethacin, 13, 211 Lovastatin, 21, 277
Iodamide, 15, 337
Iodipamide, 2, 333
Iodoxamic acid, 20, 303 M
Iopamidol, 17, 115 Mafenide acetate, 24, 277
Iopanoic acid, 14, 181 Malic Acid, 28, 153
Ipratropium bromide, 30, 59 Magnesium Silicate, 36, 241
Iproniazid phosphate, 20, 337 Maltodextrin, 24, 307
Isocarboxazid, 2, 295 Mandelic Acid, 29, 179
Isoniazide, 6, 183 Maprotiline hydrochloride, 15, 393
Isopropamide, 2, 315; 12, 721 Mebendazole, 16, 291
Isoproterenol, 14, 391 Mebeverine hydrochloride, 25, 165
Isosorbide dinitrate, 4, 225; 5, 556 Mefenamic acid, 31, 281
Isosuprine hydrochloride, 26, 359 Mefloquine hydrochloride, 14, 157
Itraconazole, 34, 193 Melatonin: comprehensive profile, 38, 159
Ivermectin, 17, 155 Melphalan, 13, 265
Menadione, 38, 227
Meperidine hydrochloride, 1, 175
K Meprobamate, 1, 207; 4, 520; 11, 587
Kanamycin sulfate, 6, 259 Mercaptopurine, 7, 343
Ketamine, 6, 297 Mesalamine, 25, 209; 27, 379
Ketoprofen, 10, 443 Mestranol, 11, 375
Ketotifen, 13, 239 Metformin hydrochloride, 25, 243
Khellin, 9, 371 Methadone hydrochloride, 3, 365; 4, 520;
9, 601
L Methaqualone, 4, 245
Lactic acid, 22, 263 Methimazole, 8, 351
Lactose, anhydrous, 20, 369 Methixen hydrochloride, 22, 317
Lamotrigine, 37, 245 Methocarbamol, 23, 377
Lansoprazole, 28, 117 Methotrexate, 5, 283
Leucovorin calcium, 8, 315 Methoxamine hydrochloride, 20, 399
Levallorphan tartrate, 2, 339 Methoxsalen, 9, 427
Levarterenol bitartrate, 1, 149; 2, 573; Methylclothiazide, 5, 307
11, 555 Methylphenidate hydrochloride, 10, 473
Levodopa, 5, 189 Methyprylon, 2, 363
Cumulative Index 443

Metipranolol, 19, 367 Ondansetron hydrochloride, 27, 301

Metoclopramide hydrochloride, 16, 327 Ornidazole, 30, 123
Metoprolol tartrate, 12, 325 Oxamniquine, 20, 601
Metronidazole, 5, 327 Oxazepam, 3, 441
Mexiletine hydrochloride, 20, 433 Oxyphenbutazone, 13, 333
Miconazole nitrate, 32, 3 Oxytetracycline, 32, 97
Minocycline, 6, 323 Oxytocin, 10, 563
Minoxidil, 17, 185
Mitomycin C, 16, 361 P
Mitoxanthrone hydrochloride, 17, 221 Paclitaxel, 34, 299
Morphine, 17, 259 Pantoprazole, 29, 213
Moxalactam disodium, 13, 305 Papaverine hydrochloride, 17, 367
Moxidectin, analytical profile, 38, 315 Parbendazole, 35, 263
Moxifloxacin hydrochloride, 39, 299 Particle Size Distribution, 31, 379
Paroxetine hydrochloride, 38, 367
N Paroxetine hydrochloride: polymorphs
Nabilone, 10, 499 and solvatomorphs, 38, 407
Nadolol, 9, 455; 10, 732 Penicillamine, 10, 601; 32, 119, 131, 149
Nalidixic acid, 8, 371 Penicillin-G, benzothine, 11, 463
Nalmefene hydrochloride, 24, 351 Penicillin-G, potassium, 15, 427
Nalorphine hydrobromide, 18, 195 Penicillin-V, 1, 249; 17, 677
Naloxone hydrochloride, 14, 453 Pentazocine, 13, 361
Naphazoline hydrochloride, 21, 307 Pentoxifylline, 25, 295
Naproxen, 21, 345 Pergolide Mesylate, 21, 375
Natamycin, 10, 513; 23, 405 Phenazopyridine hydrochloride, 3, 465
Neomycin, 8, 399 Phenelzine sulfate, 2, 383
Neostigmine, 16, 403 Phenformin hydrochloride, 4, 319; 5, 429
Niclosamide, 32, 67 Phenobarbital, 7, 359
Nicotinamide, 20, 475 Phenolphthalein, 20, 627
Nifedipine, 18, 221 Phenoxymethyl penicillin potassium, 1, 249
Nimesulide, 28, 197 Phenylbutazone, 11, 483
Nimodipine, 31, 337, 355, 371 Phenylephrine hydrochloride, 3, 483
Nitrazepam, 9, 487 Phenylpropanolamine hydrochloride, 12,
Nitrofurantoin, 5, 345 357; 13, 767
Nitroglycerin, 9, 519 Phenytoin, 13, 417
Nizatidine, 19, 397 Phosgene, 41, 133
Norepinephrinedopamine disinhibitor Physostigmine salicylate, 18, 289
(NDDI), 41, 1 Phytonadione, 17, 449
Norethindrone, 4, 268 Pilocarpine, 12, 385
Norfloxacin, 20, 557 Pimozide, 37, 287
Norgestrel, 4, 294 Pioglitazona, 41, 379
Nortriptyline hydrochloride, 1, 233; 2, 573 Pioglitazone, 41, 379
Noscapine, 11, 407 Pioglitazonum, 41, 379
Nystatin, 6, 341 Piperazine estrone sulfate, 5, 375
Pirenzepine dihydrochloride, 16, 445
O Piroxicam, 15, 509
Ofloxacin, 34, 265 Polymorphism 2004, 32, 263
Omeprazole, 35, 151 Polythiazide, 20, 665
444 Cumulative Index

Polyvinyl alcohol, 24, 397 Salmeterol xinafoate, 40, 321

Polyvinylpyrollidone, 22, 555 Scopolamine hydrobromide, 19, 477
Povidone, 22, 555 Secobarbital sodium, 1, 343
Povidone-Iodine, 25, 341 Second-generation antidepressant, 41, 1
Pralidoxine chloride, 17, 533 Seizure disorders, 41, 133
Prasugrel hydrochloride, 40, 195 Sertraline hydrochloride, 24, 443
Pravastatin sodium, 39, 433 Sertraline lactate, 30, 185
Praziquantel, 25, 463 Sildenafil citrate, 27, 339
Prazosin hydrochloride, 18, 351 Silver sulfadiazine, 13, 553
Prednisolone, 21, 415 Simvastatin, 22, 359
Primaquine diphosphate, 32, 153 Sodium bicarbonate, 41, 31
Primidone, 2, 409; 17, 749 Sodium nitroprusside, 6, 487; 15, 781
Probenecid, 10, 639 Sodium valproate, 32, 209
Procainamide hydrochloride, 4, 333; 28, 251 Solasodine, 24, 487
Procaine hydrochloride, 26, 395 Sorbitol, 26, 459
Procarbazine hydrochloride, 5, 403 Sotalol, 21, 501
Promethazine hydrochloride, 5, 429 Spironolactone, 4, 431; 29, 261
Proparacaine hydrochloride, 6, 423 Starch, 24, 523
Propiomazine hydrochloride, 2, 439 Streptomycin, 16, 507
Propoxyphene hydrochloride, 1, 301; Strychnine, 15, 563
4, 520; 6, 598 Succinycholine chloride, 10, 691
Propyl paraben, 30, 235 Sucralose, 38, 423
Propylthiouracil, 6, 457 Sucrose, 41, 31
Pseudoephedrine hydrochloride, 8, 489 Sulfacetamide, 23, 477
Pyrazinamide, 12, 433 Sulfadiazine, 11, 523
Pyridoxine hydrochloride, 13, 447 Sulfadoxine, 17, 571
Pyrimethamine, 12, 463 Sulfamethazine, 7, 401
Sulfamethoxazole, 2, 467; 4, 521
Sulfasalazine, 5, 515
Q Sulfathiazole, 22, 389
Quicklime, 41, 31
Sulfisoxazole, 2, 487
Quinidine sulfate, 12, 483
Sulfoxone sodium, 19, 553
Quinine hydrochloride, 12, 547
Sulindac, 13, 573
Sulphamerazine, 6, 515
R Sulpiride, 17, 607
Ranitidine, 15, 533 Sunitinib malate, 37, 363
Reserpine, 4, 384; 5, 557; 13, 737
Riboflavin, 19, 429 T
Rifampin, 5, 467 Tadalafil, 36, 287
Risperidone, 37, 313 Talc, 23, 517
Rocuronium bromide, 35, 285 Telmisartan, 40, 371
Rutin, 12, 623 Teniposide, 19, 575
Tenoxicam, 22, 431
S Terazosin, 20, 693
Saccharin, 13, 487 Terbutaline sulfate, 19, 601
Salbutamol, 10, 665 Terfenadine, 19, 627
Salicylamide, 13, 521 Terpin hydrate, 14, 273
Salicylic acid, 23, 427 Testolactone, 5, 533
Cumulative Index 445

Testosterone enanthate, 4, 452 Tropicamide, 3, 565

Tetracaine hydrochloride, 18, 379 Tubocurarine chloride, 7, 477
Tetracycline hydrochloride, 13, 597 Tybamate, 4, 494
Theophylline, 4, 466
Thiabendazole, 16, 611 U
Thiamine hydrochloride, 18, 413 Urea, 41, 31
Thiamphenicol, 22, 461
Thiopental sodium, 21, 535 V
Thioridazine, 18, 459 Validation, Analytical Methods,
Thioridazine hydrochloride, 18, 459 37, 439
Thiostrepton, 7, 423 Validation, Chromatographic Methods,
Thiothixene, 18, 527 32, 243
Ticlopidine hydrochloride, 21, 573 Valproate sodium, 8, 529
Timolol maleate, 16, 641 Valproic acid, 8, 529; 32, 209
Titanium dioxide, 21, 659 Valsartan, 40, 431
Tobramycin, 24, 579 Vardenafil dihydrochloride, 39, 515
a-Tocopheryl acetate, 3, 111 Varenicline, 37, 389
Tolazamide, 22, 489 Verapamil, 17, 643
Tolbutamide, 3, 513; 5, 557; 13, 719 Vidarabine, 15, 647
Tolnaftate, 23, 549 Vigabatrin, 35, 309
Tramadol hydrochloride, 38, 463 Vinblastine sulfate, 1, 443; 21, 611
Tranylcypromine sulfate, 25, 501 Vincristine sulfate, 1, 463; 22, 517
Trazodone hydrochloride, 16, 693 Vitamin D3, 13, 655
Triamcinolone, 1, 367; 2, 571; 4, 521;
11, 593 W
Triamcinolone acetonide, 1, 397; 2, 571; Warfarin, 14, 423
4, 521; 7, 501; 11, 615 Wollastonite, 41, 31
Triamcinolone diacetate, 1, 423;
11, 651 X
Triamcinolone hexacetonide, 6, 579 X-Ray Diffraction, 30, 271
Triamterene, 23, 579 Xylometazoline hydrochloride, 14, 135
Triclobisonium chloride, 2, 507
Trifluoperazine hydrochloride, 9, 543
Triflupromazine hydrochloride, 2, 523; Y
4, 521; 5, 557 Yohimbine, 16, 731
Trimethaphan camsylate, 3, 545
Trimethobenzamide hydrochloride, 2, 551 Z
Trimethoprim, 7, 445 Zaleplon, 35, 347
Trimipramine maleate, 12, 683 Zidovudine, 20, 729
Trioxsalen, 10, 705 Zileuton, 25, 535
Tripelennamine hydrochloride, 14, 107 Zolpidem tartrate, 37, 413
Triprolidine hydrochloride, 8, 509 Zomepirac sodium, 15, 673