J Therm Anal Calorim (2015) 120:795–805

DOI 10.1007/s10973-014-4142-3

Polymorphic characterization and compatibility study

of clozapine: implications on its stability and some
biopharmaceutics properties
Simone Buarque Tavares Dias • Ticiano Gomes Nascimento •
Ana Flávia Oliveira Santos • Iana Mayane M. Nicácio Viana •
Rusiene Monteiro Almeida • Irinaldo Diniz Bası́lio Júnior •
Rui Oliveira Macêdo • João Xavier de Araújo-Júnior

Received: 31 December 2013 / Accepted: 27 August 2014 / Published online: 21 September 2014
Ó Akadémiai Kiadó, Budapest, Hungary 2014

Abstract Clozapine is an antipsychotic drug used for
refractory schizophrenia and severe psychiatric disorders
associated with several side effects. Studies on standardi-
zation of raw material and bulk products are necessary to
ensure reproducibility batch to batch during all stages of the
industrial pharmaceutical process. The aim of this study was
to conduct studies of polymorphic characterization and
compatibility study of clozapine. Different solvatomorphic
forms of clozapine were obtained by recrystallization tech-
nique. Polymorphic characterization was performed using
optical microscopy, SEM, intrinsic dissolution, and thermal
analysis. Compatibility studies of clozapine:excipients were
performed by TG and DSC techniques. The polymorphic
characterization obtained by analytical and thermal
S. B. T. Dias
Laboratório Industrial Farmacêutico do Estado de Alagoas, Polo
Multissetorial Dr. Luiz Cavalcanti, s/n Tabuleiro dos Martins,
Maceió, AL 57082-000, Brazil
e-mail: simone.dias@lifal.al.gov.br
T. G. Nascimento (&)
R. M. Almeida
I. D. B. Júnior
Laboratory of Quality Control of Drugs and Medicines,
Postgradute Program in Pharmaceutical Sciences, School of
Nursing and Pharmacy, Federal University of Alagoas, Campus
A. C. Simões, University City, Maceió, AL 57072-970, Brazil
e-mail: ticianogn@yahoo.com.br
techniques showed the formation of a solvatomorphic form
of clozapine (clozapine monohydrate) when recrystallized
in aqueous solvents and in alkaline medium. The polymor-
phic form (clozapine anhydrate) showed higher intrinsic
dissolution rate compared to solvatomorphic form (cloza-
pine monohydrate). All industrial batches of clozapine
presented in anhydrate form. The DSC/TG data demon-
strated similar melting peaks for 2 polymorphic forms, but
desolvation peaks characteristic of monohydrate form was
observed in clozapine monohydrate. Studies of binary
mixtures showed no incompatibilities between clozapine
and excipients, except for clozapine:lactose which can
reduce the stability of bulk and tablets of clozapine. Tablets
of clozapine presented the same thermal analysis profile of
clozapine:lactose but did not contribute in decreasing shelf
life of clozapine tablets before 24 months of storage. Dis-
solution studies of the tablets did not show variability
between batches of clozapine during 24 months but pre-
sented decreasing on stability for 36 months of storage.
Keywords Clozapine
Polymorphism
Compatibility
study
Thermal analysis
Intrinsic dissolution
SEM
Introduction

A. F. O. Santos
R. O. Macêdo Clozapine is chemically defined as 8-chloro-11(4 methyl-

Unified Laboratories of Pharmaceutical Development and
Assays, Department of Pharmaceutical Sciences, Federal
University of Paraı́ba, Campus I, University City, João Pessoa,
PB 58059-970, Brazil
I. M. M. Nicácio Viana
J. X. de Araújo-Júnior
Postgraduate Program in Biological and Health Sciences,
Institute of Biological and Health Sciences, Federal University
of Alagoas, Campus A. C. Simões, University City, Maceió,
AL 57072-970, Brazil
1-piperazinyl)-5H-dibenzo(b,e) (1,4) diazepine, and the
molecular structure is shown in Fig. 1. Clozapine is char-
acterized as a yellow crystalline powder, almost insoluble
in water, freely soluble in methylene chloride, and soluble
in ethanol and diluted acetic acid [1].
Clozapine (CZP) is an antipsychotic drug used for refractory
schizophrenia and severe psychiatric disorders associated with
several side effects including agranulocytosis, neutropenia,

123
796
CH3
N
N processes (compaction, compression, powder density,
N hardness, and dissolution) and bioavailability of the drug
Cl
NH
Fig. 1 Chemical structure of clozapine
myocarditis, hepatitis, and lupus erythematosus [2]. CZP is
considered as class II drug with low water solubility and high
permeability by the Biopharmaceutical Classification System
(BCS) [3]. Its bioavailability is between 27 and 50 % due to
extensive first pass metabolism [4] by CYP3A4 and CYP1A2.
Clozapine has present high variation in plasma concentration
due to genetic variants of CYP1A2 and may affect clozapine
plasma concentrations (reduce or increase) and consequently
can be observed in the clinical effects and adverse drug reac-
tions [5].
Several techniques have been used to characterize and
monitor pharmaceutical in solid state. Studies on stan-
dardization of this raw material and bulk products and
pharmaceutical solids are necessary to ensure reproduc-
ibility batch to batch during all stages of the industrial
pharmaceutical process [6]. The combination of the
methods and use of supplementary methods, which is not
described in pharmacopeia assay, is usually recommended.
The set of official and unofficial pharmacopeia tests are
being a powerful tools to ensure quality of pharmaceutical
products that are on the market with generic medicine
name.
Characterization studies of polymorphic forms for
drugs have been extensively explored in order to solve
trouble qualifying of manufacturers and suppliers of raw
materials [6, 7], as well as establish new traceability
assays as indicative of quality pharmaceutical raw mate-
rials. The intrinsic dissolution studies can be decisive in
the process of purchase of pharmaceutical raw material,
and to establish the criteria for standardization of raw
materials during a specific industrial pharmaceutical pro-
cess, so this study is considered essential to prove the
quality of the raw material and some steps of pharma-
ceutical manufacturing process. The thermal techniques
(thermogravimetry and differential scanning calorimetry)
have significant use in various kinds of studies including
polymorphism and pseudo-polymorphism investigation,
[6, 8–10] characterization of pharmaceutical [7, 11–13],
123
S. B. T. Dias et al.
compatibility studies [14–16], and assessment of drug
stability [17, 18]. The characterization of the size and
shape of the pharmaceutical solids using SEM and
microscopy has been widely applied in the pharmaceutical
industry to assess changes related to the pharmaceutical
in bodily fluids [19–21].
The aim of this work was to conduct studies of poly-
morphic characterization and compatibility study of clo-
zapine using different analytical techniques as intrinsic
dissolution and dissolution, microscopy, SEM, and thermal
analysis relating with the stability and some biopharma-
ceutical properties of clozapine.
Experimental
Chemicals
Samples of Clozapine (lots: 4182, 4292, 4303, and 4336)
were donated by Pharmaceutical Industrial Laboratory of
Alagoas-Brazil (LIFAL), which were acquired by only
China supplier. Clozapine Reference Standard (CRS), Lot
GOD 315, was purchased from United States Pharmaco-
peia. The excipients used in the compatibility study were
starch (Colorcon, Inc.), magnesium stearate (Mallinckrodt
Inc.), colloidal silicon dioxide (aerosil) (Cabot Creating
What Matters), lactose monohydrate supertab SD (DMV-
Fonterra Excipients), and talc (Magnesite SA). Tablets A,
B, C, and D were manufactured by LIFAL using clozapine
Lot 4303. Tablets E, F, and G were manufactured by LI-
FAL using clozapine Lot 4292 and tablet H also manu-
factured by LIFAL using clozapine Lot 4182. All
excipients and drug cited were used in the process of
manufacture of 25 mg clozapine tablets like a generic
medicine name. All clozapine tablets were donated by
LIFAL for this research.
Preparation of the clozapine solvatomorphic form
Clozapine, Lot 4292, was tested to evaluate the formation
of different polymorphous using four different processes of
recrystallization at low temperature (-20 °C). The cloza-
pine (1.00 g) was dissolved in ethanol, followed by addi-
tion of an anti-solvent: (i) distilled water, (ii) 0.1 M NaOH,
(iii) phosphate buffer pH 8.0, and (iv) borate buffer pH 8.6.
The recrystallization time varied between 5–8 days at room
temperature. After the recrystallization process, clozapine
were subjected to filtration and drying process in a desic-
cator with silica at room temperature. Clozapine recrys-
tallized and non-recrystallized have been submitted to
polymorphic characterization.
Polymorphic characterization and compatibility
Compatibility study of the clozapine binary mixtures
The compatibility study was performed using binary mix-
tures of clozapine and excipients starch, lactose, colloidal
silicon dioxide (aerosil), magnesium stearate, and talc in
proportions previously defined. Binary mixtures were pre-
pared in 30-mL plastic bags, followed by physical mixture
for 1 min in the following proportions: Clozapine:starch
(1:0.3), clozapine:lactose (1:2.3), clozapine:colloidal sili-
con dioxide (aerosil) (1:0.06), clozapine: magnesium
stearate (1:0.06), and clozapine:talc (1:0.096) (w/w).
Polymorphic characterization
Optic microscopy (OM) and scanning electron microscopy
(SEM)
Clozapine Lot 4303 non-recrystallized and clozapine Lot
4292 recrystallized in phosphate buffer pH 8.0
(1.0 ± 0.1 mg) were accurately weighed and placed on
slides, dispersed in mineral oil, and then analyzed under a
microscope Nikon, model Eclipse 50i using a objective of
20X. The images were captured on camera DS-Ri1. The
data were analyzed to evaluate the crystal form and the
particle size using Ferret diameter template. A total of 150
particles were measured for each (08) classes of size, using
a program DP2—BSW version Olympus Soft Imaging
solution GmbH with 50 lm strip.
Clozapine non-recrystallized (Lot 4182) and clozapine
recrystallized in water (Lot. 4292) were analyzed by
scanning electron microscopy (SEM) from Shimadzu,
model SSX-550 (Tokyo, Japan). The metallization of the
samples was performed in Sanyu electron equipment,
model QuickCoater SC 701. The formation of plasma gold
film was necessary to capture images in increments of 100,
200, 500-fold.
Intrinsic dissolution and dissolution test
Clozapine recrystallized and non-recrystallized were sub-
jected to sample preparation for intrinsic dissolution test-
ing. Masses of 0.1530 grams of clozapine were subjected
to compression force of 20 kN for 1 min to form pellets of
1.0 cm2 in area using rotating disk apparatus in a die of
8 mm of diameter [6]. The dissolution profile for tablets
(A, B, C, and D storage for 24 months) and (E, F, G, and H
storage for 36 months) of clozapine (25 mg) was per-
formed using paddle apparatus.
The intrinsic dissolution and dissolution tests were
performed in a Nova Ética (São Paulo, Brazil), model
299. A volume of 900 mL of dissolution medium (ace-
797
tate buffer pH 4.0), temperature of 37 ± 0.5 8C, and
rotation speed of 100 rpm (Brazilian Pharmacopoeia 5th
edition, 2010). Aliquots (10 mL) of dissolution medium
were collected (with replacement) at the times of 10, 20,
30, 45, 60, and 90 min. Aliquots were filtered, diluted at
the ratio (2:10, v/v), and analyzed by UV spectropho-
tometry at 290 nm in Jasco apparatus, model 7800
(Tokyo, Japan).
Clozapine (CRS, Lot: GOD315) was used to obtain the
calibration curve. A stock solution (1.00 mg mL-1) was
solubilized in ethanol and then diluted to obtain standard
curve of absorbance versus concentration from 28.00 to
1.80 lg mL-1. The sink condition was maintained, and the
intrinsic dissolution rate was assessed in six replicate,
except to clozapine recrystallized (triplicate).
Thermal analysis (polymorphic characterization
and compatibility study)
Clozapine (Lot: 4182, 4292, 4303, 4336) and clozapine
recrystallized (in water and 0.1 M NaOH) were subjected
to Differential Scanning Calorimetry (DSC) and Thermo-
gravimetry (TG). Binary Mixtures and tablets also were
subjected to thermal analysis to observe the compatibility
of the bulk preparation clozapine excipients contained in
tablets manufactured like a generic medicine name. The
excipients: starch, lactose, aerosil, magnesium stearate,
talc, binary mixtures, and clozapine tablets A, B, C, and D
followed the same conditions.
Differential scanning calorimetry (DSC)
The calorimetric curves were obtained from Shimadzu
(Tokyo, Japan) Model DSC 50, using a mass of
(2.0 ± 10% mg), which were packed in aluminum cap-
sules hermetically sealed. The heating rate was 10 °C
min-1 in the temperature range 30–400 °C under an
atmosphere of nitrogen and flow rate of 50 mL min-1. The
DSC was calibrated using indium and zinc standards
according to the Shimadzu procedures.
Thermogravimetric analysis (TG)
TG curves were acquired in Shimadzu equipment (Tokyo,
Japan), model TGA 50H. TG curves were performed using
a mass of (5.0 mg ± 10 %), packed in an alumina crucible,
heating rate of 10 °C min-1, and temperature range of
10–900 °C under atmosphere of synthetic air and nitrogen
with a flow rate of 20 and 50 mL min-1, respectively. The
equipment was calibrated under the same conditions with
standard calcium oxalate monohydrate.
123
798 S. B. T. Dias et al.

(a) (b)
60

Relative frequency/%

Relative frequency/%
60
50 50
40 40
30 30
20 20
10 10
0 0
9

47

51

03

80

58

36

14

10 10

80

1– 1

1– 1

2– 2

3– 3

3
.6

.6

.3

.0
5.

2.

9.

5.

2.

9.

6.

3.

19 19.

5.

0.
–8

–8

36

48
–1

–2

–2

–3

–4

–4

–5

–1

4
91

39

0–

2–
69

6
1.

.4

.5

.0

.8

.5

.3

2.

.8

.5

.2

.9

.6

.3
8.

8.
15

22

29

35

42

49

13

25

30

36

42
Diameter/µm Diameter/µm
(c) (d)
80

Relative frequency/%
Relative frequency/%

50
70
40 60
50
30
40
20 30
20
10 10
0 0
6

46

85

5
7

0
.3

.7

.6

.5

.4

.3

.2

.1
.5

.6

.7

.8

.9

.1
4.

1.
–8

20

29

38

47

56

65

74
20

26

32

38

44

51
–1

–1
55

5–

5–

5–

5–

5–

5–

5–
6–

7–

7–

8–

8–

9–
36

95
2.

.8

.7

.6

.5

.4

.3

.2
.4

.5

.6

.7

.8

.9
8.

2.

11

20

29

38

47

56

65
14

20

26

32

38

44

Diameter/µm Diameter/µm

Fig. 2 Particle size distribution for clozapine raw materials during class interval experiment obtained by optic microscopy. a Lot. 4182, b Lot
4292, c Lot 4303, d Lot 4336

Results and discussion Optic microscopy and SEM

Polymorphic characterization During morphologic analysis clozapine non-recrystallized

(Lot 4303 and Lot 4182) presented a shape of hexagonal
Particle size distribution crystals with brown coloration on the boarder and clear on
the center. These are observed when analyzed by optic
Clozapine non-recrystallized (raw materials) showed a microscopy (Fig. 3a, b) and SEM (Fig. 3e, f). Clozapine
particle size distribution divided into class interval Lot 4292 after processes of recrystallization in different
according to each clozapine sample. Clozapine lot 4182 anti-solvents (distilled H2O, 0.1 M NaOH, phosphate buf-
showed a size distribution in 8 class intervals fer pH 8.0, and borate buffer pH 8.6) has observed a plate-
(1.91–56.14 lm), clozapine lot 4292 presented in 6 class type crystal which was formed during the recrystallization
intervals (2.39–48.03 lm), clozapine lot 4303 presented in process in alkaline medium (Fig. 3c, d) and distilled water
7 class intervals (2.25–51.10 lm), and clozapine lot 4336 (Fig. 3g, h). This morphology was completely distinct from
showed only 4 class intervals (2.95–74.15 lm) (Fig. 2). original shape on clozapine non-recrystallized, which pre-
The higher frequency, 67.50 %, was observed in the range sented it like as hexagonal-type crystals.
of 2.95–11.85 microns for the clozapine lot 4336. The other The TG and DSC data also showed that the planar
lots also showed high frequency at intervals of small par- crystals are presented as clozapine hydrates form, while the
ticles with the results of 53.75 % (Lot 4182), 50.62 % (lot hexagonal presented themselves as clozapine anhydrate
4292), and 46.00 % (Lot 4303), and the smaller diameter crystals (Figs. 4, 5). The planar crystalline form when
found was 1.91 lm for the lot 4182 (Fig. 2), probably due analyzed by intrinsic dissolution rate showed lower values
to the milling process described by the manufacturer [22]. in relation to the hexagonal crystalline form (Fig. 6). It is
High frequency ([85 %) of small particles (\30 lm) important to identify the crystal form after recrystallization
observed in all clozapine batches can justify the similar because in the most cases the crystalline structures are
values of IDR for clozapine, and it has demonstrated the dependent of the solvents used. The anhydrate crystal
standardization of particle size in clozapine lots [23]. presented in intact form with particle size of 23.65 lm and

123
Polymorphic characterization and compatibility
Fig. 3 Optic photomicroscopy of the clozapine anhydrate (Lot 4303)
(a and b) in 50 lm scale. Optic photomicroscopy of the clozapine
monohydrate (Lot 4292) after process of anti-solvent crystallization
in phosphate buffer pH 8.0 in 50 lm scale (c and d). SEM of the
clozapine anhydrate (Lot 4182) (e and f). SEM of the clozapine
monohydrate (Lot 4292) after process of anti-solvent crystallization
in water (g and h)
clozapine hydrate crystals presented particle size greater
than 100 lm when observed by SEM (Fig. 3f, g).
DSC and TG
Four samples of clozapine (raw materials) presented calori-
metric behavior quite similar (Fig. 4a), evidenced by three
events: three endothermic peaks: the first due to loss of mois-
ture, the second related to the melting point of clozapine
(185 °C), and the third related to the decomposition of the drug
in temperatures above 260 °C (Table 1), which were confirmed
by TG curves (Table 2). Clozapine presents melting point
between 182–186 °C according to data from drug monograph
described in the Brazilian Pharmacopoeia [1]. The melting
point detected in the exact peak of fusion for drug discards any
other phase transition related to processes of polymorphic
impurities or melting of drug-related impurities demonstrating
the crystalline purity of the crystals of clozapine [6, 7].
Clozapine (Lot 4192) after recrystallization process in
water and 0.1 M NaOH solution presented four endothermic
events: two desolvation endotherms were observed: first one
799
at 100.8 °C (water), 101.5 °C (0.1 M NaOH solution) and the
second one at 121.2 °C (water), 123.6 °C (0.1 M NaOH
solution) corresponding to the desolvation of water in cloza-
pine molecule; the third peak remained on the clozapine
melting point at 185 °C (Fig. 4b; Table 1); and fourth peak
representing the clozapine decomposition. The endothermic
desolvation energies for recrystallized clozapine were much
higher compared to endothermic processes of loss of moisture
found in 4 lots of non-recrystallized clozapine (raw materials).
TG curves of clozapine (non-recrystallized) showed no
mass loss in the temperature range of 100 °C; however,
recrystallized clozapine in H2O and 0.1 M NaOH solution
showed mass loss of 5.50 and 5.76 %, respectively, in
temperature range between 80–150 °C (Fig. 5a, b). Stoi-
chiometrically, one mole of water corresponds to 5.22 % of
mass loss in relation to a clozapine molecule. Thus, calo-
rimetric and TG and SEM/optic microscopy data demon-
strated the presence clozapine anhydrate in all raw
materials and a solvatomorphic form of clozapine (cloza-
pine monohydrate) after recrystallization processes.
Intrinsic dissolution
The intrinsic dissolution rate (IDR) obtained in clozapine
raw materials has met the sink condition (suitable condition
of dissolution medium, drug mass, temperature, etc.) and
presented a model of linear velocity with coefficient of
correlation between 0.9969 and 0.9994 (Table 3). It was
necessary to standardize the experiment, and main
parameters were established for these raw materials. The
variables such as mass of the solid, compression force, and
time were exhaustively performed to get these results. The
clozapine Lot 4292 was used to standardized the experi-
ment, and we observed the necessity to add 20 lL of dis-
tilled water in the die before to insert the 0.1530 grams of
clozapine in rotate disk apparatus and to promote the
compression of these highly anhydrate raw materials.
The comparison between batches of clozapine has
shown similar release rates (IDR) of drug from dissolution
medium (Fig. 6a, b), regardless of the variations found in
the particle size distribution (Fig. 2). The intrinsic disso-
lution values between 0.0899–0.1050 mg/cm2 min-1
(Table 3) were below and very closed from minimum
specification of 0.1 mg/cm2 min-1 (limit value for poorly
soluble drug in water), indicating that the dissolution of
this drug may be a limiting factor for its absorption [6, 24],
which is in agreement with drug class (Class II: low sol-
ubility and low permeability) in the biopharmaceutical
classification system.
We observed a expressive reduction of clozapine IDR
after the recrystallization processes. When the recrystal-
lization was performed in water and 0.1 M NaOH, there
123
800 S. B. T. Dias et al.

(a) (b)
10.00
1 - Clozapine Lot 4182

Exo
Exo

10.00
1 - Clozapine Lot 4182

2 - Clozapine recrystalized H2O

Heat flow/mW Endo

Heat flow/mW Endo

2 - Clozapine Lot 4292 0.00

0.00

3 - Clozapine Lot 4303

Tmf
–10.00 Tmf 3 - Clozapine recrystalized 0.1M NaOH
4 - Clozapine Lot 4336
–10.00

–20.00 Vh = 10 °C min–1 Tmp Vh = 10 °C min–1 Tmp

50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00
Temperature/°C Temperature/°C

(c) (d) Tmf

20.00
1 - Lactose Tmf 1 - Clozapine + lactose

Exo
Exo

10.00 Tmp
Tmp 2 - Clozapine + starch
2 - Starch 10.00

Heat flow/mW Endo

Heat flow/mW Endo

3 - Clozapine + talc
0.00
3 - Talc
0.00 4 - Clozapine + aerosil

4 - Aerosil
–10.00 5 - Clozapine + Mg estearate Tmf
5 - Mg estearate –10.00

–20.00 Vh = 10 °C min–1 Vh = 10 °C min–1 Tmp

50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00
Temperature/°C Temperature/°C

Fig. 4 DSC curves of clozapine anhydrate, (a) clozapine monohydrate, (b) excipients, (c) and binary mixtures of clozapine (d) at heating rate of
10 °C min-1

(a) (b)
1
100.00 100.00 2
1 - Clozapine Lot 4182 1 - Clozapine Lot 4303
3
2 - Clozapine Lot 4336 2 - Clozapine recrystalized in H2O
80.00 80.00
3 - Clozapine Lot 4292 3 - Clozapine recrystalized in 0.1 NaOH
4 - Clozapine Lot 4303
Mass/%
Mass/%

60.00 60.00

40.00 40.00

20.00 1 20.00
2
4
–0.00 Vh = 10 °C min
–1
–0.00 Vh = 10 °C min–1 3
–0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 –0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00
Temperature/°C Temperature/°C

(c) 6 (d)
100.00 5 100.00
1 - Clozapine + lactose
1 - Clozapine Lot 4303
80.00 2 - Clozapine lot 4303
2 - Starch 80.00 1 3 - Clozapine + starch
3 - Lactose
4 - Clozapine + aerosil
3
Mass/%

60.00 4 4 - Mg estearate
Mass/%

60.00
5 - Clozapine + Mg estearate
5 - Talc
6 - Aerosil
40.00 40.00
4
1 2
20.00 2 5
20.00
3

–0.00 Vh = 10 °C min–1 –0.00 Vh = 10 °C min–1

–0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 –0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00
Temperature/°C Temperature/°C

Fig. 5 TG curves of clozapine anhydrate, (a) clozapine monohydrate, (b) clozapine (Lot 4303) and excipients, (c) and binary mixtures of
clozapine (d) at heating rate of 10 °C min-1

123
Polymorphic characterization and compatibility 801

Fig. 6 Intrinsic dissolution of (a) (c)

clozapine anhydrate (a) and Clozapine Lot 4292 100
14
clozapine monohydrate (b) in Clozapine Lot 4336

Clozapine/µg mL–1
different anti-solvents. 12 80

Dissolution/%
Clozapine Lot 4182
Dissolution profile of different 10
lots of clozapine tablets (c) on Clozapine Lot 4303 60 Tablet A
8
the shelf life of drug (storage
6 40 Tablet B
during 24 months) and d after
the shelf life of drug (storage 4 Tablet C
20
during 36 months) 2 Tablet D
0 0
0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 35 40 45 50
Time/min Time/min
(b) (d)
5
pH 8.0 Phosphate buffer 100
Clozapine/µg mL–1

4 pH 8.6 Borate buffer 80

Dissolution/%
3 0.1M NaOH
60 Tablet E
H2O
2 40 Tablet F
Tablet G
1 20
Tablet H
0 0
0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 35 40 45 50
Time/min Time/min

Table 1 Calorimetric data of the clozapine (raw materials), clozapine hydrates, and binary mixtures at the heating rate of 10 °C min-1
Sample Desolvation peak Melting point peak Decomposition range
-1 -1
Ton set/°C DH/J g Ton set/°C DH/J g T/°C DH/J g-1

Lot 4182 97.9 -3.9 185.7 -88.7 259–353 -220.3

Lot 4292 100.5 -5.2 185.5 -67.4 273–354 -123.3
Lot 4303 100.8 -5.1 185.3 -92.7 261–374 -274.9
Lot 4336 101.4 -5.4 185.6 -92.4 293–366 -136.4
Clozapine hydrate in H2O 100.8 -46.8 185.1 -72.9 260–363 -189.1
121.2 -21.3
Clozapine hydrate in 0.1 M NaOH 101.5 -66.3 185.0 -96.1 318–360 -68.6
123.6 -32.7
Cloz-Lactose 101.4 -5.1 187.7 -24.2 212–218 -9.73
151.4 -69.6 212.2 -14.2
Cloz-Starch 100.3 -4.3 185.9 -70.1 250–310 -100.0
Cloz-Aerosil 96.4 -4.7 185.7 -82.6 260–378 -284.8
Cloz-Talc 100.7 -4.2 185.6 -89.7 260–378 -255.4
Cloz-Mg Estearate 99.5 -4.6 185.6 -74.2 300–378 -98.2

was a delay in the release close to 53 % of IDR and a
decrease of approximately 64 % when the solvents were
phosphate buffer and borate buffer (Table 3). This
decrease in clozapine IDR can be related to the changing
of morphology in solid crystalline state and a higher
particle size observed in clozapine monohydrate form.
Optic microscopy and SEM have demonstrated differ-
ences on the morphology between clozapine recrystal-
lized (monohydrate) and non-recrystallized (anhydrate)
(Fig. 3f, g). It demonstrated a higher intrinsic dissolution
rate (IDR) for the anhydrate form of clozapine and
showed that it is more soluble in aqueous medium than
its corresponding monohydrate form. Generally, solvato-
morphic forms with higher grade of hydration can pres-
ent lower IDR as showed by some researchers [25, 26].
The intrinsic dissolution rate is a characteristic of the
drug on the kinetic study that depends on the solubility,
wettability, and diffusion of this compound in the

123
802 S. B. T. Dias et al.

Table 2 Thermogravimetric data of clozapine, clozapine recrystallized, and binary mixtures obtained by TG dynamical at heating rate of 10 °C
min-1
Sample 1st step of decomposition 2nd step of decomposition Residue
Temperature range/°C Mass/% Temperature range/°C Mass/% Temperature range/°C Mass/%

Lot 4182 180–322 94.2 338–604 5.3 [604 0.5

Lot 4292 180–338 92.6 338–628 6.4 [628 0.9
Lot 4303 180–338 93.1 338–628 6.5 [628 0.4
Lot 4336 180–338 91.5 338–628 7.7 [628 0.8
Clozapine hydrate in H2O 80–150 5.5 350–638 4.8 260–363 0.6
150–350 89.1
Clozapine hydrate in 0.1 M NaOH 80–150 5.7 348–640 6.7 318–360 0.1
150–348 87.5
Cloz-Lactose 40–160 3.3 160–348 51.7 348–660 43.8
Cloz-Starch 40–160 1.4 160–348 82.5 348–660 14.2
Cloz-Aerosil 40–160 0.1 160–348 67.3 348–660 26.1
Cloz-Talc 40–160 0.3 160–348 85.7 348–660 2.7
Cloz-Mg Estearate 40–160 0.02 160–348 84.0 348–660 11.5

Table 3 Intrinsic Dissolution parameters of clozapine raw materials crystal growth in different polymorphic forms, and conse-
and recrystallized quently decrease the solubilization and absorption of clo-
Clozapine Intrinsic dissolution Coefficient of zapine in biological medium [28, 29]. This fact could be
rate/J s-1 correlation/R2 harmful for patients with genetic variations causing thera-
peutic failure or increasing adverse drug reactions [5].
4182 0.0917** 0.9994
Dissolution profile of the clozapine tablets was com-
4292 0.0983** 0.9969
pared from different lots. No variation was observed on
4303 0.1050** 0.9981
dissolution profiles of clozapine during the shelf life of
4336 0.0899** 0.9978
drug (24 months), but a great variation was observed in the
Recrystallized clozapine
dissolution profile after the clozapine shelf life (36 months
H2O 0.0464* 0.9964
of storage) (Fig. 6c, d). A delay in drug release was
0.1 M NaOH 0.0460* 0.9075
observed in the first 10 min, and decrease of 9 % in the
pH 8.0 phosphate buffer 0.0355* 0.9586 concentration of clozapine was also observed at 36 months
pH 8.6 borate buffer 0.0365* 0.9806 of storage, which demonstrates maximum storage time for
There was no significance difference between these values using One- this product. It did not observe polymorphic change in
way ANOVA (F = 0.0709 and P value = 0.0252) clozapine tablets. The solid dosage form (tablets) contrib-
** Six replicate uted to the preservation and integrity of the polymorphic
* Triplicate purity of clozapine (Fig. 6c).
solvents and provides indications about changes in bio-
availability in vitro and in vivo depending on the par- Compatibility study
ticularities of the dissolution test [24, 27].
The recrystallization study of clozapine using anti-sol- Differential scanning calorimetry (DSC)
vent process is also important because they can simulate
the conditions that the biopharmaceutical raw material may Lactose showed a loss of moisture process 100.6 °C
be subjected to biological fluids, and then recrystallization (-4.66 J g-1), endothermic dehydration peak 149.9 °C
studies were performed not only to indicate the polymor- (-88.87 J g-1), exothermic peak corresponding to muto-
phic purity for industrial pharmaceutical process but also to rotation between a and b forms of lactose [30] 180.3 °C
evaluate certain biopharmaceutical conditions that the drug (8.48 J g-1), endothermic peak of melting at 221.5 °C
will suffer in gastrointestinal fluids. These conditions tested (-57.85 J g-1), and endothermic peaks of decomposition
(precipitation in water and alkaline medium) can simulate at temperatures higher than 235 °C. Starch showed two
some biological conditions that clozapine is subject, such phase transitions at temperatures between 70 and 110 °C
as intestinal fluids causing problems of precipitation and representing the loss of moisture and peak of

123
Polymorphic characterization and compatibility 803

Fig. 7 DSC curves of clozapine (a)

(Lot 4303) and tablets A, B, C, 20.00 Vh = 10 °C min–1
and D (a) and TG curves of

Exo
clozapine (Lot 4303) and tablets Tmf
A, B, C, and D (b) at heating 1 - Clozapine Lot 4303
rate of 10 °C min-1 10.00
Tmp

Endo
2 - Clozapine Tablet A

Heat flow/mW
0.0 3 - Clozapine Tablet B

4 - Clozapine Tablet C
–10.00

5 - Clozapine Tablet D

50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00

Temperature/°C

(b)
100.00 1 - Clozapine Lot 4303
1
2 - Clozapine Tablet C
80.00 3 - Clozapine Tablet A
5 4 - Clozapine Tablet B
Mass/%

60.00 5 - Clozapine Tablet D

40.00 2 3

20.00

4
–0.00 Vh = 10 °C min–1
–0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00
Temperature/°C

decomposition at temperatures above 288 °C. Magnesium
stearate showed three principal phase transitions: the first
peak at 109 °C (dehydration), the second peak at
128.90 °C related to the melting, and decomposition at
temperatures above 300 °C. Talc and aerosil (colloidal
silicon dioxide) showed no thermal events due to the
characteristics of mineral substances (Fig. 4c).
Binary mixtures showed three thermal events: (1)
characterized by the loss of moisture in temperature close
to 100.0 °C; (2) On set temperature of melting for cloza-
pine at 185 °C, and (3) thermal decomposition at temper-
atures above 235 °C were observed (Table 1). The
superposition of DSC curves in binary mixtures did not
show any disappearance of peak or displacement of the
melting temperature of clozapine suggesting compatibility
between the drug and excipients tested. The binary mix-
ture, clozapine:lactose, presented the same Tonset
(187.7 °C) for drug and binary mixtures, but it showed a
slight shift in peak of fusion of lactose (212.2 °C;
-14.2 J g-1) with reducing in the enthalpy of fusion
(Fig. 4d). The shift in melting peak of lactose (in mixture
clozapine:lactose) can be related to modification of the
solid state to a solid–liquid transition state and then to a
liquid state after the process of the lactose dehydration
(151.4 °C) and melting point of clozapine (187.7 °C). This
solid–liquid transition state can be favoring the chemical
equilibrium between the liquid phase (liquid state) and the
solid crystal state (solid state) promoting a chemical shift
of a temperature of 221.5 °C to temperature of 212.3 °C,
but it was favorable to liquid phase in temperatures higher
than 212.2 °C.
It was observed decrease in the enthalpy of fusion for
both clozapine (-24.2 J g-1) and lactose (-14.2 J g-1) in
this binary mixture (Table 1), and these events can be
associated with a high proportion of the excipient (1:2.3) in
this composition. However, the calorimetric profile of this
binary mixture (clozapine:lactose) showed a sum of the
phase transition peaks from the isolated DSC curves of
pure drug and pure lactose, occurring a maintenance of the
all thermal events (calorimetric profile) (Fig. 4c, d).

123
804
Tablets of clozapine presented the same calorimetric pro-
file of clozapine:lactose (Fig. 7a).
Modifications in melting point of the lactose (peak of
fusion) and the enthalpy of fusion for both clozapine and
lactose in the binary mixture were observed and suggest
only physical interactions between drug and excipient
during thermal analysis assays (accelerate tests) [31], but it
demonstrated no chemical (expressive decrease in the
concentration of drug) or physical signals (changing in the
coloration of tablets) after 24 months. Dissolution test was
performed and presented in Fig. 6c to confirm this fact.
Thermogravimetric analysis (TG)
The four lots of clozapine showed similar thermogravimetric
profile featuring two main steps of thermal decomposition.
The first starting close to the melting temperature with rapid
and expressive percentage of mass loss representing a per-
centage between 91.5–94.2 %; followed by the second
decomposition step representing a percentage of mass loss of
5.3–7.7 % and the third stage represents the amount of
mineral residue (\1.0 %) (Table 2).
All samples showed three events of thermal decompo-
sition in binary mixtures (Fig. 5c; Table 2). The binary
mixtures (clozapine:starch, clozapine:talc, clozapine:aero-
sil, clozapine:Mg stearate) presented decomposition profile
similar to clozapine drug. Only one binary mixtures pre-
sented difference in decomposition process. The binary
mixture clozapine:lactose showed a shift in the thermal
decomposition process to lower temperatures similar to TG
profile of the lactose (Fig. 5c, d) [31].
The TG data showed that high proportion of lactose
(70 %) in binary mixture (clozapine:lactose; 1:2.3 w/w) as
well as the presence of water in the excipient lactose
(lactose monohydrate) favors the reduction of the initial
decomposition temperature of clozapine. TG curves of the
tablets of clozapine showed TG profile similar to the clo-
zapine:lactose suggesting by means accelerated assay some
decrease on the stability of clozapine bulk and tablets
containing lactose in this proportion used (Fig. 7b). TG and
DSC data are coherent results and can predict this reduc-
tion on clozapine stability in a accelerate way. But this
decrease on clozapine stability did not affect during
24 months but can affect after 36 months. This fact can be
seen in the dissolution test for tablets after 36 months of
storage (Fig. 6c, d).
Additional experiments using other analytical methods
like as DSC-Photovisual, FTIR or NIR, LC-DAD-MS in
long-term studies should be performed to confirm the
probable chemical interaction between clozapine and lac-
tose. Our experiments were not enough to prove chemical
interaction for this composition but detected any physical
interaction which is promoting any reduction on stability of
123
S. B. T. Dias et al.
the clozapine tablets. Because the stability studies gener-
ally presented the focus on drug aging and forget the focus
on excipients aging. These new strategies should be used to
detect chemical or physical interaction and reduction on
stability of preformulation and formulation of medicines.
Conclusions
The techniques of intrinsic dissolution, SEM, optical
microscopy, and thermal analysis were useful to distin-
guish polymorphic forms of clozapine. The polymorphic
characterization studies using optical microscopy/SEM,
intrinsic dissolution, and thermal analysis (TG and DSC)
showed the presence of anhydrous crystalline form (clo-
zapine anhydrate) in industrial batches of clozapine, but it
was also possible to characterize the clozapine monohy-
drate when subjected to experimental procedures of
recrystallization. Industrial batches of clozapine did not
present variations in IDR and can avoid high variations in
biological fluids due to presence of only one crystalline
form (clozapine anhydrate).
Compatibility study of clozapine:excipients using TG
and DSC did not show chemical interaction of clozapine
with the excipients studied, only evidence of the decrease
on stability of clozapine bulk and tablets containing lactose
monohydrate. Dissolution test did not detect the decrease in
shelf life of clozapine tablets during 24 months of storage,
but at 36 months of storage, it was detected. Thermal
analysis has demonstrated to be a rapid and reproducible
technique to detect possible decrease in stability of cloza-
pine in accelerated preformulation studies.
Acknowledgements The authors thank to the CAPES and CNPq for
its financial support with Grant Number 478390/2010-06 of the
funding of research no 14/2010—Universal/MS/CNPq. The authors
thank to FINEP for its financial support (CT-INFRA) in acquisition of
some equipments and laboratorial facilities. The authors also want to
thank to the Pharmaceutical Industrial Laboratory of Alagoas (LI-
FAL) in the persons of Vânia do Nascimento Rocha, manager of the
department of quality assurance, Solange S. Moura and Denise M. de
Melo França pharmacists and Maria Cı́cera C. Santos for the technical
support in the selection of the raw materials and Intrinsic Dissolution
and dissolution profile and Ana Rúbia Ribeiro for the support in the
SEM analysis at the Institute of Physics from Federal University of
Alagoas.
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