CHAPTER ONE

Alendronate Sodium
Gennady Ananchenko, Jasmina Novakovic, Anna Tikhomirova
Apotex Inc., Toronto, Ontario, Canada

Contents
1. General Information 2
1.1 Nomenclature 2
1.2 Formulae 2
1.3 Elemental analysis 2
1.4 Appearance 2
2. Physical Profile 2
2.1 Dissociation constants (Alendronic acid) 2
2.2 Solubility characteristics 2
2.3 Partition and distribution coefficients 5
2.4 Crystallographic properties and polymorphism 5
2.5 Hygroscopicity 8
2.6 Thermal methods of analysis 8
2.7 Spectroscopy 9
3. Stability 14
4. Methods of Chemical Synthesis 14
5. Analytical Profile 16
5.1 Impurities of Alendronate sodium 16
5.2 Compendial tests 17
5.3 Noncompendial analytical methods 17
5.4 Analysis in biological matrices 17
6. ADME 24
6.1 Absorption 28
6.2 Distribution 28
6.3 Metabolism 28
6.4 Elimination 28
Acknowledgments 28
References 29

Profiles of Drug Substances, Excipients, and Related Methodology, Volume 38 # 2013 Elsevier Inc. 1
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-407691-4.00001-0
2 Gennady Ananchenko et al.

1. GENERAL INFORMATION
This chapter deals with Alendronate sodium as a derivative of Alendronic
acid. The latter belongs to bisphosphonate (sometimes called diphosphonate)
group of drugs, which inhibit bone resorption by binding to bone surfaces and
slowing down the formation of hydroxyapatite crystals. A story about fascinat-
ing discovery and development of bisphosphonates has been published recently
[1]. Although this chapter is focused on the monosodium salt of Alendronic
acid, relevant information concerning free Alendronic acid is also presented.
1.1. Nomenclature
1.1.1 Systematic chemical name
4-Amino-1-hydroxybutane-1,1-diphosphonic acid sodium salt
P,P 0 -(4-amino-1-hydroxybutylidene)bisphosphonic acid sodium salt
(4-Amino-1-hydroxybutylidene)bisphosphonic acid monosodium salt
Sodium trihydrogen (4-amino-1-hydroxybutylidene)diphosphonate
1.1.2 Nonproprietary names
Alendronate sodium, Alendronic acid monosodium salt
1.1.3 Proprietary names
Adronat, Alendros, Bonalon, Dronal, Fosamac, Fosamax, Fosavance, Onclast,
MK-217, G-704650.
1.2. Formulae (Tables 1.1 and 1.2)
1.3. Elemental analysis
Alendronic acid: C 19.29%, H 5.26%, N 5.62%, O 44.96%, P 24.87%.
Alendronate sodium: C 14.78%, H 5.58%, N 4.31%, Na 7.07%,
O 49.21%, P 19.05%.

1.4. Appearance
White, crystalline, nonhygroscopic powder.

2. PHYSICAL PROFILE
2.1. Dissociation constants (Alendronic acid) (Figure 1.1
and Table 1.3)
2.2. Solubility characteristics
Alendronate sodium: highly soluble in water, very slightly soluble in etha-
nol, and practically insoluble in chloroform [11]. Solubility in water is
Alendronate Sodium 3

Table 1.1 Empirical formula, molecular weight, CAS number

Water
Formula content
Name Molecular formula weight (% w/w) CAS number
Alendronic C4H13NO7P2 249.10 – 66376-36-1
acid
Alendronate C4H12NNaO7P2 271.08 – 129318-43-0
sodium
Alendronate C4H12NNaO7P2H2O 289.09 6.2 260055-05-8
sodium
monohydrate
Alendronate C4H12NNaO7P23H2O 325.12 16.6 121268-17-5
sodium
trihydrate

Table 1.2 Structural formulae and abbreviations used in this chapter

O OH O OH H2O
P P
HO OH HO O−
H2N H2N Na+ H2O
OH OH
P P
OH OH H2O
O O
Alendronic acid, AA Alendronate sodium trihydrate, AS

H3L- H2L2-
100
H5L+ L4-

80

H4L HL3-
60
%

40

20

0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
pH
Figure 1.1 Calculated species distribution diagram for Alendronic acid based on pKa
values reported in Ref. [2].
Table 1.3 Dissociation constants of Alendronic acid
References HL3 H2L2 H3L H 4L H5Lþ Media
[3] 11.6  0.1 10.5  0.1 8.73  0.05 2.72  0.05 – 25  C, 0.1 M KCl
[4] 12 10.79 6.36 2.38 1 0.1 M KNO3
[5] 12.04 10.77 6.21 2.16 1 0.2 M KCl
[6] 12.68 11.07 6.36 2.19 <1.2 25  C, 0.1 M Me4NCl
[2] 11.82 10.96 6.39 2.22 1.33 0.1 M NaNO3 or NaCl or Me4NCl
[7] 11.4  0.2 10.68  0.06 6.38  0.03 2.24  0.01 – 0.1 M KCl
[7] 10.5  0.1 10.25  0.03 5.97  0.02 2.34  0.02 – 0.1 M cetylpyridinium chloride
[8] 8.66  0.04 6.75  0.05 5.79  0.05 – – 0.10 M Brij35; 0.10 M KCl
[8] 10.5  0.4 9.4  0.2 7.4  0.1 – – Water–ethanol, 1/1, v/v; 0.10 M KCl
[8] 10.45  0.07 9.58  0.04 7.4  0.02 – – Water–ethanol, 1/1, v/v; 0.10 M NaCl
[9] – 11.3 7.03 2.87 1.35 No details
[10] 12.44(3) 11.51(2) 6.73(1) 2.60(1) – 25  Ca
a
Thermodynamic pKa (i.e., extrapolated to ionic strength ¼ 0).
Alendronate Sodium 5

10 mg/mL [9]. Aqueous solubility (0.05 M phosphate buffer solutions) of

Alendronate sodium exceeds 20 mg/mL at pH range 3.1–6.4 and is between
10 and 20 mg/mL in 0.1 M HCl.

2.3. Partition and distribution coefficients

Alendronic acid: log P (octanol/water): 5.642 ([12], calculated).
Alendronate sodium: log P (octanol/water): 4.49 [13].

2.4. Crystallographic properties and polymorphism

2.4.1 Single-crystal structure
Alendronate sodium trihydrate has been characterized by single-crystal X-ray
diffraction [14]. The compound crystallizes in the monoclinic P21/n space
group with unit-cell parameters 7.3, 9.0, and 19.5 Å, b ¼ 100.6 . The unit
cell is represented by four Alendronate anions. The connection between
the Alendronate anions is provided by sodium cations and water molecules
in such a way that sodium is in distorted octahedral environment, which con-
sists of one bidentate and one monodentate phosphonate group of one
Alendronate unit, one monodentate phosphonate group and OH group of
the second Alendronate unit, and one water molecule (Figure 1.2). Amino
group of Alendronate is protonated.
The crystalline lattice of Alendronate sodium trihydrate consists of a lay-
ered structure where relatively hydrophobic layers of aminobutylidene

c
a
0

Figure 1.2 Unit cell of Alendronate sodium trihydrate. Sodium ions are shown as black
spheres, phosphonate groups, as well as oxygen in water coordinated to sodium are
shown as ball and stick.
6 Gennady Ananchenko et al.

Figure 1.3 Slice of the crystal structure of the dihydrate form of Alendronate sodium
trihydrate showing bilayers parallel to the (101) plane. Sodium ions are shown as black
spheres.

groups are separated by layers of water molecules H-bonded to phosphonate

and OH groups of Alendronate moieties (Figure 1.3).
In addition to the trihydrate form of Alendronate sodium [14], single-
crystal structures of anhydrous and hydrate forms of Alendronic acid have
also been reported [15,16].

2.4.2 Powder X-ray diffraction (PXRD)

Experimental powder X-ray diffraction diagram of Alendronate sodium
trihydrate in comparison with the diagram calculated from single-crystal data
is shown on Figure 1.4.
A good quality PXRD diagram of the anhydrous Alendronate sodium
was also obtained [17] so that it allowed the authors to determine crystal
structure of the material using Rietveld refinement. Similar to the trihydrate
form, anhydrous Alendronate sodium crystallizes in the monoclinic P21/n
space group with unit-cell parameters 7.6, 9.1, and 14.5 Å, b ¼ 112.6 .
The unit cell is represented by four Alendronate moieties having zwitter-
ionic character, that is, with protonated amino group in analogy with the
trihydrate form. Sodium ion has distorted octahedral coordination
(Figure 1.5) with one bidentate and one monodentate phosphonate group
Alendronate Sodium 7

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

10 20 30 [ ⬚2q ] 40
Figure 1.4 X-ray (Cu Ka) powder diffractograms of Alendronate sodium trihydrate:
(A) calculated from single-crystal data and (B) experimental diffractogram.
8 Gennady Ananchenko et al.

Figure 1.5 Sodium ion in octahedral coordination in the crystal structure of anhydrous
Alendronate sodium. Sodium and phosphonate groups are shown as ball and stick.

of one Alendronate unit, one monodentate phosphonate group and OH

group of the second Alendronate unit, and, instead of water molecule in
the trihydrate form, one monodentate phosphonate group of the third
Alendronate unit.
In addition to the anhydrous and trihydrate forms, powder X-ray
diffractograms of various intermediate hydrates have been also published
[18,19].

2.5. Hygroscopicity
Alendronate sodium trihydrate is not hygroscopic. From dry state to 95%
relative humidity, the sample weight increase is about 0.3% on the absorp-
tion curve (Figure 1.6). The isotherm did not show plateaus corresponding
to the formation of hydrates.

2.6. Thermal methods of analysis

The trihydrate form of Alendronate sodium has a distinct endothermic peak
at about 126  C (Figure 1.7), which corresponds to loss of water and collapse
of the original trihydrate’s crystalline lattice so that the initial crystals do not
remain intact [17]. Heating beyond about 220  C leads to decomposition of
the material [17], which is accompanied by melting-like behavior around
250  C (e.g., 257–262.5  C [20]).
Alendronate Sodium 9

10.000

9.000
Adsorption
8.000 Desorption

7.000
Weight (% change)

6.000

5.000

4.000

3.000

2.000

1.000

0.000
0 10 20 30 40 50 60 70 80 90 100
−1.000
%RH
Figure 1.6 Dynamic vapor sorption isotherm of Alendronate sodium trihydrate.

−5
Heat flow (mW)

−10

−15

−20

−25
0 50 100 150 200 250 300
Exo Up Temperature (⬚C)
Figure 1.7 DSC thermogram of Alendronate sodium trihydrate.

2.7. Spectroscopy
2.7.1 UV spectroscopy
Alendronate sodium does not have distinct chromophores in the molecule,
so no significant UV absorption occurs beyond 200 nm.
10 Gennady Ananchenko et al.

2.7.2 Vibrational spectroscopy

The infrared spectrum of Alendronic acid is published in SDBS database
[21].
The FT-IR spectrum of Alendronate sodium trihydrate (Figure 1.8) was
obtained from potassium bromide pellets using a Perkin Elmer Spectrum
1000 Fourier Transform IR spectrometer. Strong bands in the region
1200–900 cm1 correspond to CdO and P]O stretches.

2.7.3 Nuclear magnetic resonance spectroscopy

For this chapter, the 1H, 13C NMR, and 31P spectra of Alendronate sodium
were obtained on a Bruker Avance 400 instrument using D2O as the solvent.
1
H and 13C NMR chemical shifts are reported in ppm relative to TMS (with
TSP as internal standard), and 31P NMR chemical shifts are relative to 85%
H3PO4. Refer to Figure 1.9 for the atom numbering used for assignment of

82.1
80
78
76
74
72
70
68
%T

66
64
62
60
58
56
54
52.5
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600.0
cm−1

Figure 1.8 FT-IR spectrum of Alendronate sodium trihydrate (KBr disk).

10
13
O OH
6 P
HO 8 O−14 Na+
H2N 3 1
5 4 2 7 OH
P 12
OH
O 11
9

Figure 1.9 Atom numbering in Alendronate sodium.

Alendronate Sodium 11

NMR resonances of Alendronate. Figures 1.10 and 1.11 demonstrate 1H and

13
C NMR spectra, respectively, and Table 1.4 contains 1H, 13C NMR, and
31
P NMR data.
1
H NMR chemical shifts for Alendronic acid have also also reported in
DMSO-d6 [23]: 2.95 t, 1.94 m.
2.0000

4.0372

ppm 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8

Figure 1.10 1H NMR spectrum (400 MHz, D2O) of Alendronate sodium.
76.8128
75.4800
74.1413

41.9319

32.5536

24.1218
ppm

ppm 70 60 50 40 30
13
Figure 1.11 C NMR spectrum (101 MHz, D2O) of Alendronate sodium.
12 Gennady Ananchenko et al.

Table 1.4 Assignment of the resonances in 1H, 13C, and 31P NMR (400 MHz, D2O) spectra
of Alendronate sodium (refer to Figure 1.9 for atom numbering)
Nuclei Assignment da (multiplicity, integration)
1
H H4 3.04 (t, 3JH–H ¼ 6.3 Hz, 2H)
H3, H2 2.07–1.96 (m, 4H)
13
C C1 75.48 (t, 1JC–P ¼ 134.4 Hz)
C4 41.93
C2 32.55
C3 24.12b
31
P P7, P8 18.47c
a
For 1H and 13C: relative to TMS with TSP as internal standard; for 31P: relative to 85% H3PO4 as internal
standard (in a capillary insert); t, triplet; m, multiplet.
b
Slightly broad signal (Figure 1.11). This signal is also reported as a triplet with 3JC–P ¼ 6.6 Hz [20].
c
This signal is reported as a triplet, when taken without 1H decoupling, d ¼ 18.46, 3JP–H ¼ 12.1 Hz [22].

Solid-state 31P NMR data for Alendronic acid (diso 15.9 and 16.6) and
Alendronate sodium trihydrate (diso 17.4 and 22.1) are reported in Refs.
[24,25], respectively. Both references also provide 31P chemical shift anisot-
ropy data. Also, Ref. [25] contains solid-state 13C NMR spectra for
Alendronate sodium trihydrate.
15
N NMR chemical shift of Alendronate sodium trihydrate (d 37.1 with
the reference to liquid NH3) in the solid state is reported in Ref. [26]. It is
noted that this chemical shift is essentially the same as for lysine (NH3 þ ) res-
idues in amino acids [26] and is also in good agreement with the chemical
shift of 38.0 ppm obtained by quantum mechanical calculation for proton-
ated nitrogen in Alendronate [26].
In addition to these “common” nuclei, a solid-state 2H NMR spectrum of
2
a H-enriched crystalline Alendronate sodium trihydrate is reported in the
study of interactions of the bisphosphonate side chains with human bone [27].

2.7.4 Mass spectrometry

The electrospray ionization mass spectroscopic study of Alendronate sodium
was carried out on an AB Sciex Q-Trap LC–MS/MS system with negative
ionization mode. Figure 1.12 demonstrates MS/MS spectrum of the parent
deprotonated molecular ion of Alendronate (m/z 248).
The fragmentation pathway via elimination of water and/or of H3PO3
from the parent deprotonated molecular ion (m/z 248) shown in
Scheme 1.1 generally agrees with that proposed in Ref. [28].
Alendronate Sodium 13

248.0
100

90

80

70

60
Rel. Int. (%)

148.0

50

40
194.0
30
230.0

20
212.0
166.0
142.9

0
80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260
m/z (Da)
Figure 1.12 Tandem mass spectrum of the deprotonated molecular ion (m/z 248) of
Alendronate.

O OH
P
HO O−
H2N
OH
P
OH
m/z 248 O

H2N O O
O O− OH OH
P P
P
HO O−
O−
H2N O H2N
OH O
P P
m/z 230 O OH OH m/z 166
m/z 230 O

H2N O O− O
O O− H
P N
P P
O OH
O
P
O O−
P
m/z 212 O OH NH m/z 148
m/z 194

Scheme 1.1 Proposed fragmentation pathways of the deprotonated molecular ion of

Alendronate.
14 Gennady Ananchenko et al.

3. STABILITY
Alendronate sodium is a remarkably stable substance. To the best of
our knowledge, no noticeable degradation with identified degradation
products has been mentioned in the literature. The European Medicine
Agency’s Scientific Discussion for Fosavance® [29], however, contains a
note that 2-phosphonopyrrolidine is a degradation product of Alendronate
sodium.

4. METHODS OF CHEMICAL SYNTHESIS

Alendronate sodium is normally prepared by converting Alendronic
acid into sodium salt using sodium hydroxide in appropriate media.
Alendronic acid (AA), in turn, can be prepared from g-aminobutyric acid
(1) by treatment with phosphorus trichloride and phosphoric acid in chlo-
robenzene followed by quenching with water, as described in the seminal
work of Kabachnik et al. [3] (Scheme 1.2).
While this method is reasonably suitable for laboratory scale, it does not
seem appropriate for commercial scale-up due to highly viscous reaction
mixture with increasing volume due to gases generated and formation of
glass-like solid upon cooling, which prevents effective quenching by water
[20]. Earlier improvements of the method included use of phosphorus acid
and PCl5 or phosphorus acid and POCl3 and some variation of the reaction
conditions [30].
Later, general modifications in the reaction conditions were undertaken,
for example, change of solvent to methanesulfonic acid [20], phenol [31], or
some others. Another modification was to use microwave irradiation with
sulfolane as the solvent [32].
Further modification of the synthesis was to use pyrrolidone (2) as a
starting material [33] (Scheme 1.3), which is hydrolyzed in situ by aqueous
methanesulfonic acid and is then treated with PCl3 and, finally, with water.

O OH
P
(1) H3PO4, PCl3 / C5H5Cl HO OH
H2N COOH H2N
(2) H2O OH
P
1 AA OH
O
Scheme 1.2 First synthesis of Alendronic acid [3].
Alendronate Sodium 15

H
N (1) CH3SO3H / H2O; (2) PCl3
O AA
(2) H2O
2
Scheme 1.3 Synthesis of Alendronic acid from pyrrolidone.

O O O
COOEt NaHCO3 SOCl2
N H2N COOH
+ N COOH N COCl

O O O
3 1 4 5
O O O OEt
P(OEt)3 O HPO(OEt)2 P HCl
5 N
OEt N
HO OEt AA
OEt
P OEt P
O O OEt
6 O 7 O

Scheme 1.4 Synthesis of Alendronic acid via phthalimido protection of amino group.

O O OR
(1) ROP(OSiMe3)2 P (1) N2H4
5 HO
N
OH AA
(2) MeOH OH (2) HCl
P
O OR
O
8a (R = Me)
8b (R = Ph)
Scheme 1.5 Synthesis of Alendronic acid by using alkyl bis(trimethylsilyl)phosphite.

Other modifications, which probably have mostly laboratory applications

for Alendronate, included several step synthetic schemes. In Ref. [34]
(Scheme 1.4), the phthalimido protection of amino group of g-aminobutyric
acid (1) was applied, followed by reaction with SOCl2 to prepare corresponding
acyl chloride (5, yield 92%), which was converted to acylphosphonate (6) in
the reaction with triethylphosphite (trimethylphosphite can also be used
[16]). The former without isolation reacted then with diethylphosphite (or
dimethylphosphite [16]) in the presence of triethylamine to yield the
corresponding tetraethyl bisphosphonate (7, yield 48%), which was further
hydrolyzed by 6N HCl to Alendronic acid (yield 90% after conversion to
monosodium salt).
As a modification of this scheme, phenyl or methyl bis(trimethylsilyl)
phosphite was used [35] to introduce the phosphonate group into 5. Further
methanolysis [35] (Scheme 1.5) afforded partial methyl or phenyl esters of
phthalimido-protected bisphosphonate (8), which after deprotection and
hydrolysis gave Alendronic acid (or its dimethyl ester).
16 Gennady Ananchenko et al.

It is worth noting that in addition to the phthalimido protection of the

amino moiety, other protecting groups, such as Boc or Cbz, were tried [35].
Also, instead of acyl chloride 5, the corresponding succinimido derivative
was employed. In all these attempts, the yield of the final product was sig-
nificantly lower [35].
As a method of synthesis without the need to use protection of the
amino group, a THF solution of catecholborane was employed for the
carboxyl group activation without the isolation of the intermediate
acyloxybenzodioxaborolane (9) [36] (Scheme 1.6). The reaction with n þ 1
equivalents of P(OSiMe3)3 followed by methanolysis afforded Alendronic acid
with 51% yield.

5. ANALYTICAL PROFILE
5.1. Impurities of Alendronate sodium
The current European Pharmacopeia (EP 7.0) monograph for Alendronate
sodium [37] and BP 2013 monograph for Alendronic acid tablets [38] list
three impurities (Table 1.5).
No impurities are specified in the USP 35 monographs for Alendronate
sodium drug substance [39] and Alendronate sodium tablets [40].

O
H B
O
O O (1) P(OSiMe3)3
1 AA
−H2 H2N B (2) MeOH
O O

9
Scheme 1.6 Synthesis of Alendronic acid via catecholborane activation of carboxy
group of g-aminobutyric acid (1).

Table 1.5 Related compounds of Alendronate sodium listed in EP and BP

Alendronate sodium drug substance,
Structure EP 7.0; Alendronic acid tablets, BP 2013
H2N COOH 4-Aminobutanoic acid
PO3
4 Phosphate ion
PO3
3 Phosphite ion
Alendronate Sodium 17

5.2. Compendial tests

Tests and limits listed in the USP 35 and EP 7.0 monographs for Alendronate
sodium drug substance and in USP 35 and BP 2013 monographs for
Alendronate drug product are listed in Tables 1.6 and 1.7, respectively.

5.3. Noncompendial analytical methods

Due to the lack of chromophore groups in the Alendronate moiety (see
Section 2.7.1), it is impossible to use UV detection in chromatographic
techniques and the analytical methods had to exploit alternative approaches
to detect Alendronate in bulk drug and formulations (single or in combina-
tion with other bisphosphonates) [41]. Tables 1.8–1.10 provide an overview
of the analytical methods, partially covered by the review [41], as well as of
the methods published after 2007.

5.4. Analysis in biological matrices

Analysis of a bisphosphonate drug Alendronate in biological samples presents
challenges for both sample purification and Alendronate detection.
Alendronate is a strongly polar and ionic compound, and therefore, its
extraction from biological fluids with organic solvents is restricted. Sample
pretreatment protocol involves typically deproteinization, precipitation of
the analyte, solid phase, and/or liquid–liquid extraction [41]. Due to chela-
tion properties, Alendronate interacts with metals in HPLC system resulting
in poor peak shape and irreproducible chromatography. Alendronate has no
chromophore or fluorophore in its structure, and therefore, derivatization
prior to UV or fluorescence detection is necessary. For GC analysis, deriv-
atization to volatile derivatives is a prerequisite [70–72]. Analytical/
bioanalytical methods for determination of bisphosphonates have been
reviewed by Zacharis and Tzanavaras [41]. The review is covering relevant
techniques for determination of bisphosphonates and sample preparation
techniques published until 2007. Since then, several articles have been pub-
lished describing different techniques for determination of Alendronate in
biological matrices, such as column-switching HPLC [73], LC–MS/MS
[74], and capillary electrophoresis with fluorescence detection [71]. The
methods, relevant for determination of Alendronate in biological matrices,
along with sample preparation and derivatization techniques, are summa-
rized in Table 1.11.
18 Gennady Ananchenko et al.

Table 1.6 Compendial tests and limits for Alendronate sodium drug substance
Test USP 35a EP 7.0a
Identification A. IR (in mineral oil) A. IR (KBr disk)
B. Flame test for sodium: B. Reaction of sodium:
h191i (2.3.1—test a)
Loss on drying 16.1–17.1% (140  C in 16.1–17.1% (1.000 g,
vacuum NMT 5 mm Hg) 140–145  C)
Appearance No requirements (2.2.2, Method II)
of solution
pH No requirements 4.0–5.0
Heavy metals NMT 0.001% (h231i NMT 20 ppm (2.4.8—test F,
Method III) for 1.0 g)
Assay Isocratic RP-HPLC with Ion chromatography
precolumn derivatization Detector: refractometer.
with FMOCb Column: 4.6 mm  15 cm,
Detector: 266 nm. Column: packing: anion-exchange resin
4.1 mm  25 cm, packing with [CH2Nþ(CH3)3]-groups
L21. Flow rate: 1.2 mL/min. attached to methacrylate
Column temperature: 35  C. lattice (7 mm). Flow rate:
Mobile phase: pH 8 buffer, 1.2 mL/min. Column
ACN, MeOH, 70/25/5. temperature: 35  C. Mobile
Limit: 98.0–102.0% (dried phase: pH 3.5 formate buffer.
basis) Limit: 98.0–102.0% (dried
basis)
Chromatographic Gradient RP-HPLC with 4-Aminobutanoic acid (TLC
purity/related precolumn derivatization test)
substances with FMOC Plate: TLC silica gel.
Chromatographic system Developing solution: water,
Detector: 266 nm. Column: glacial acetic acid, butanol,
4.1 mm  25 cm, packing 20/20/60. Detection: with
L21. Flow rate: 1.8 mL/min. ninhydrin solution.
Column temperature: 45  C. Limit: NMT 0.5%.
Mobile phase: pH 8 buffer, Phosphate and phosphite (using
acetonitrile, gradient program. Assay method)
Limits Limits
Any individual impurity: Phosphate: NMT 0.5%
NMT 0.1% Phosphite: NMT 0.5%
Total: NMT 0.5%
a
Here and in Table 1.7, numbers in hi and () refer to method conditions specified in general chapters of
the USP and EP, respectively.
b
FMOC, 9-fluorenylmethyl chloroformate; ACN, acetonitrile.
Table 1.7 Compendial tests and limits for Alendronate sodium (USP) and Alendronic acid (BP) tablets
Test USP 35 BP 2012
Identification HPLC: by retention time A. TLC
using the Assay test B. HPLC: by retention time using the Assay test
Uniformity of dosage units Meet the requirements No requirements
Related substances No requirements Ion chromatography
Detector: 250 nm. Column: 4.1 mm  25 cm, packing:
styrene-divinylbenzene copolymer with a chemically
bonded, strongly basic quaternary ammonium anion-
exchange coating (10 mm). Column temperature: ambient.
Flow rate: 1.6 mL/min. Mobile phase: 0.0456% (v/v) nitric
acid.
Limits
Phosphate: NMT 0.5%
Phosphite: NMT 0.5%
Any other impurity: NMT 0.2%
Total (excluding phosphate and phosphite): NMT 1.5%
Disregard threshold: 0.05%
4-Aminobutanoic acid No requirements RP-HPLC: As per the Chromatographic Purity Method in
the USP monograph for Alendronate sodium drug substance
Limit: 4-Aminobutanoic acid: NMT 0.5%
Assay Isocratic RP-HPLC with Ion chromatography
precolumn derivatization with FMOC Detector: 240 nm. Column: same as in related substances
Detector: 266 nm, Column: method. Column temperature: 30  C. Flow rate:
4.1 mm  25 cm, packing L21.
Continued
Table 1.7 Compendial tests and limits for Alendronate sodium (USP) and Alendronic acid (BP) tablets—cont'd
Test USP 35 BP 2012
Column temperature: 35  C. Flow rate: 1.6 mL/min, 0.0456% (v/v) nitric acid
1 mL/min. Mobile phase: pH 8 buffer, Limit: 95.0–105.0%
acetonitrile, methanol, 75/20/5
Limit: 90.0–110.0%
Dissolution One of the following tests: Medium: water, 900 mL. Apparatus 2: 50 rpm.
Test 1 Time: 45 min.
Medium: water, 900 mL. Apparatus Tolerance: Q: NLT 75% (general acceptance criteria as
2: 50 rpm. Time: 15 min specified in BP 2013, Appendix XII B1)
Tolerance Q: NLT 80%
Q: NLT 75% (for tablets labeled for
weekly dosing)
Test 2
Medium: water, 900 mL. Apparatus
2: 50 rpm. Time: 30 min
Tolerance Q: NLT 80%
Table 1.8 HPLC methodsa
Type of Linearity, LOD,
chromatography Derivatizing agent Column Mobile phase Detection LOQ Refs.
RP-HPLC Precolumn: FMOC PRP-1 0.05 M sodium citrate, UV, 266 nm [42]: Linearity: [42,43]
0.05 M potassium 1–100 mg/mL
phosphate, pH 8/ACN/ [43]: Linearity:
MeOH (75/20/5, v/v/v) 0.5–20 mg/mL
LOD: 0.3 mg/mL
RP-HPLC Postcolumn: OPA/ IONPAC NS1 0.1 M pH 8.0 phosphate Fluorescence, Linearity: [44]
2-mercaptoethanol with a neutral buffer/ACN (60/40, v/v) lex ¼ 230 nm/ 0.02–0.08 mg/mL
macroporous lem ¼ 455 nm LOD:
resin 0.02 mg/mL
Ion N/A IC-Pak HR (for 1.6 mM nitric acid (for Conductivity, Linearity: [45]
chromatography intravenous intravenous solution) negative 0.1–10 mg/mL
solution) 1.76 mM nitric acid þ 20% polarity LOD: 100 ng
Omnipac PAX- ACN (for tablets)
100 (for tablets)
Ion N/A pSt–DVB base 1 mM 1,3,5- Indirect UV Linearity: [46]
chromatography benzenetricarboxylic acid detection, 0.1–2 mg/mL
(trimesic acid), pH 5.5 254 nm LOD: 250 ng
Ion N/A IC-Pak HR 1.6 mM nitric acid Indirect UV Linearity: [47]
chromatography detection, 0.02–0.08 mg/mL
235 nm LOD:
0.001 mg/mL
Continued
Table 1.8 HPLC methods—cont'd
Type of Linearity, LOD,
chromatography Derivatizing agent Column Mobile phase Detection LOQ Refs.
Ion N/A AX-300 0.1% aqueous HCOOH/ ESI-MS [48]
chromatography ACN, 95/5, v/v
Ion N/A IC-Pak HR 6 mM nitric acid, pH 2.3 Refractive Linearity: [49]
chromatography 1.5 mM nitric acid, pH 2.9 index 0.2–0.6 mg/mL
LOD:
4  104 mg/mL
Ion Postcolumn: Al3þ– pSt–DVB base 30–10 mM of NaNO3, Fluorescence, LOD: 10 ng [50]
chromatography morin complex (Hamilton pH 9.5 lex ¼ 420 nm/
PRP-X100) lem ¼ 505 nm
Ion N/A AS7 analytical 1.5 mmol/L nitric acid ICP-MS, m/z Linearity: [51]
chromatography column with (1.4 mL/min) 31 0.6–20 mg/L
AG7 guard LOD: 0.20 mg/L
column
RP-HPLC Precolumn: OPA/2- pSt–DVB ACN/phosphate buffer pH UV, 333 nm Linearity: [52]
mercaptoethanol 9.6 (15/85) containing 10–60 mg/mL
3 mg% of LOQ: 0.3 mg/mL
tetrabutylammonium LOD: 90 ng/mL
perchlorate
RP-HPLC N/A C18 18 mM n-amylamine Evaporative Linearity: [53]
aqueous solution, pH 7.0/ light- 32–505 mg/mL
ACN (95/5, v/v) scattering LOD: 16 mg/mL
Ion N/A Sphereclone 20 mM sodium citrate, Indirect UV Linearity: [54]
chromatography SAX pH 4.6 detection, 100–500 mg/mL
222 nm LOD: 10.0 mg/
mL
RP-HPLC Precolumn: OPA C18 0.005 M sodium hexane UV, 325 nm Linearity: [55]
sulfonate with 0.1% 10–120 mg/mL
triethanolamine, pH 2.8/ LOQ: 10 mg/mL
ACN (70/30, v/v) LOD: 2.5 mg/mL
RP-HPLC Postcolumn: C18 50 mmol/L borate buffer Fluorescence, Linearity: [56]
peroxydisulfate-assisted pH 9.0 containing lex ¼ 375 nm/ 0.10–100 nmol/
photolysis followed by 0.25 mmol/L lem ¼ 440 nm mL
reaction with molybdate tetrabutylammonium LOD: 23 nmol/L
and then with thiamine chloride and 0.5 mmol/L
to form thiochrome EDTA/ACN (97/3)
RP-HPLC N/A C18 50 mM aqueous tricationic ESI-MS LOD: [57]
ion-pairing reagent/ 0.40–1.3 ng
MeOH, 80/20 (depends on
reagent used)
a
pSt, polystyrene; DVB, divinylbenzene; OPA, o-phthalaldehyde.
24 Gennady Ananchenko et al.

Table 1.9 Spectrophotometrya

Detection Derivatizing agent Linearity, LOD, LOQ Refs.
UV, 290, 300, FeCl3 Linearity: 8.1–162.5 mg/mL [58]
310 nm LOD: 2 mg/mL
LOQ: 7 mg/mL
UV, 568 nm Ninhydrin in MeOH in Linearity: 3.75–45 mg/mL [59]
the presence of LOD: 1.2 mg/mL
NaHCO3 LOQ: 4.1 mg/mL
UV, 320 nm Ceric (IV) sulfate in the Linearity: 2–24 mg/mL [59]
presence of sulphuric LOD: 0.66 mg/mL
acid LOQ: 2 mg/mL
UV, 333 nm OPA/2- Linearity: 14–60 mg/mL [52]
mercaptoethanol LOQ: 14 mg/mL
UV, 240 nm CuCl2 Linearity: 1.0–60.0 mg/L [60]
LOD: 0.3 mg/L
Stopped-flow OPA/2- Linearity: 0.13–10.0 mg/L [60]
fluorimetry mercaptoethanol LOD: 0.04 mg/L
(lex ¼ 340 nm/
lem ¼ 455 nm)
UV, 233, 245, CuSO4, HNO3, pH 2.8 Linearity: 25–600 mmol/L [61]
and 254 nm LOD: 2.13 mg/mL
LOQ: 7.11 mg/mL
UV, 472 nm NBD-Cl Linearity: 1.0–20.0 mg/mL [62]
LOD: 0.09 mg/mL
UV, 378 nm DNFB Linearity: 4.0–40.0 mg/mL [62]
LOD: 1.06 mg/mL
UV, 374 nm DNFB Linearity: 1.5–30.0 mg/mL [62]
LOD: 0.06 mg/mL
Fluorimetry, Al3þ–morin complex Linearity: 250–1250 mg/L [63]
lex ¼ 340 nm, LOQ: 62.5 mg/L
lem ¼ 455 nm
a
NBD-Cl, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole; DNFB, 2,4-dinitrofluorobenzene.

6. ADME
A pharmacokinetic model for Alendronate, indicating rapid distribu-
tion throughout the body followed by quick redistribution into target tissue
(i.e., bone) and elimination via urine, no evidence of metabolism and a long
terminal half-life, was established based on the preclinical data in animals [81].
Table 1.10 Other techniques
Technique
ICP
Capillary electrophoresis
Capillary electrophoresis
Voltammetry
Potentiometric titration
Capillary electrophoresis
Experimental conditions Linearity, LOD, LOQ Refs.
The wavelength used to detect phosphorus was Linearity: 0.0017–0.115 mg/mL [64]
178.3 nm. A fixed quartz torch was used with 1.2 kW
power. Argon gas was used for purging. The sample
flow rate was 2 mL/min with an integration time of
3 s on the peak used
Fused, uncoated silica capillary of 75 mm ID and Linearity: 0.025–0.225 mg/mL [65]
70 cm length, 25 kV applied voltage, 1.6 mM of
HNO3, 2 mM CuSO4 as electrolyte
Multidimensional approach. A preseparation Linearity: 10–100 mg/mL [66]
fluorinated ethylene–propylene copolymer capillary LOD: 1.2 mg/mL
and a fused silica analytical capillary were employed in
the first and second dimensions, respectively. HCl
(5 mM), Tris (5 mM), HEC (0.2%, w/v) as
electrolyte. Conductivity detection
The procedure is based on the formation of Linearity: 0.096–0.288 mg/mL [67]
Alendronate/copper(II) complex when shaking with LOD: 0.0086 mg/mL
copper(II) phosphate suspension. The voltammetric
peaks, which correspond to the reduction of the
copper(II) moiety of the formed complexes, are
obtained at 153 mV
Titrant: 0.001 mol/L NaOH LOD: 20.0 mg/mL [68]
Electrolyte: 0.05 M sodium chromate (pH 7.8–10.5), LOD: 1.6 mg/L [69]
1 mM 3,6-ionen, 0.1% ACN. UV detection at
254 nm
Table 1.11 Alendronate determination in biological matrices
Repeatability
Technique Pretreatment Derivatization Detection Matrix LOD LOQ RSD (%) Accuracy (%) Refs.
a
RP-HPLC Calcium NDA Fluorescence Urine N/A N/A N/A N/A [75]
precipitation
RP-HPLC Calcium NDA Fluorescence, Urine N/A 1 ng/mL 3–8 (urine) 94–109 [76]
precipitation electrochemical and (urine) 3–7 (plasma) (urine)
detection plasma 5 ng/mL 94–105
(plasma) (plasma)
RP-HPLC Calcium FMOC Fluorescence Urine N/A 3.5 ng/mL 8 99.1–105.4 [77]
precipitation
RP-HPLC Calcium FMOC Fluorescence Plasma N/A 1 ng/mL <15 N/A [78]
precipitation
RP-HPLC Sodium OPA Fluorescence Urine 0.2 ng/mL 2 ng/mL 0.4–3.3 92.9–99.3 [52]
hydroxide
and calcium
precipitation
RP-HPLC Automated FMOC Fluorescence Urine N/A N/A <15 N/A [79]
SPE using a
96-well
cartridge
plate
RP-HPLC N/A Dual (isobutyl MS/MS, Urine N/A 6.667 ng/mL <15 N/A [80]
chloroformate þ positive ion
trimethyl mode
orthoacetate)
Column- Sodium FMOC Fluorescence Plasma 0.5 ng/mL 2 ng/mL 3.97–12.07 98.36–103.93 [73]
switching hydroxide
HPLC and calcium
precipitation
RP-HPLC N/A Trimethylsilyldiazo- MS/MS, Plasma N/A 1 ng/mL 5.0–8.2 N/A [74]
methane positive ion
mode
Capillary Solid-phase NDA Fluorescence Urine 1.5 ng/mL 5 ng/mL 4.1–10.3 99–115 [71]
electrophoresis extraction and (urine)
plasma 96–112
(plasma)
a
NDA, 2,3-naphtalene dicarboxyaldehyde.
28 Gennady Ananchenko et al.

Since most of the studies suggesting the model could not be conducted in
human subjects, a similar model is assumed in humans.
6.1. Absorption
Alendronate is highly polar and charged compound at physiological pH
(Figure 1.1), and therefore, it is proposed that its absorption across gastro-
intestinal tract occurs primarily paracellulary [82]. Oral bioavailability of
Alendronate under fasting conditions is similar in postmenopausal women
(0.76%) and in male subjects (0.6%). Dosing after a meal reduces absorption
dramatically (80–90%) [83]. The effect of gastric pH on absorption was eval-
uated in postmenopausal women, and the results showed that elevation of
gastric pH by ranitidine increased the absorption of Alendronate about two-
fold comparing with native stomach pH [84].
6.2. Distribution
Studies in rats, dogs, and monkeys showed that Alendronate is approximately
80%, 73%, and 70% plasma protein (predominantly albumin) bound, respec-
tively [85,86]. Radiolabeled Alendronate, administered to rats intravenously, is
initially widely distributed in soft tissues, followed by rapid redistribution into
bone [87]. By analogy with the results in animals and humans, Alendronate is
probably bound to the mineral phase of the skeleton, from which it is released at
a rate that is proportional to the rate of bone turnover. The apparent steady-
state volume of distribution was estimated at more than 28 L [88].
6.3. Metabolism
There is no evidence of Alendronate metabolism in animals and humans [88].

6.4. Elimination
Upon administration, Alendronate is cleared from plasma by deposition into
bone and excretion via urine. The renal clearance was found to average
4.26 L/h. Terminal elimination half-life was estimated to be on average
10.5 years [81]. A very long elimination half-life is a reflection of the rate
of bone turnover and as such related to Alendronate’s mechanism of action.

ACKNOWLEDGMENTS
The authors are indebted to Dr. Yan Alsmeyer for encouragement and management support.
We also wish to express our sincere appreciation to Dr. Yuri Goldberg for his guidance, to
Ms. Janet Mensah for her assistance in retrieving literature cited, to Dr. Konstantin Udachin
for fruitful discussion on X-ray data, and lastly to many colleagues in our laboratories who
contributed material needed for the preparation of this chapter.
Alendronate Sodium 29

REFERENCES
[1] R.G.G. Russell, Bisphosphonates: the first 40 years, Bone 49 (2011) 2–19.
[2] G. Hägele, Z. Szakács, J. Ollig, S. Hermens, C. Pfaff, NMR-controlled titrations: char-
acterizing aminophosphonates and related structures, Heteroatom Chem. 11 (2000)
562–582.
[3] M.I. Kabachnik, T.J. Medved, N.M. Djatlova, J.M. Polikarpov, B.K. Sherbakov,
F.I. Belskij, Synthesis and acid–base and complexing properties of amino-
substituteda-hydroxyalkylidenediphosphonic acids, Bull. Acad. Sci USSR Div. Chem.
Sci. (Eng. Ed.) 27 (1978) 374–377 Izv. Akad. Nauk., Ser. Khim. 1978, 27, 433–437
(Russian edition).
[4] M. Dyba, H. Kozlowski, A. Tlalka, Y. Leroux, D.E. Manouni, Oxovanadium(IV)
complexes of 1-hydroxyalkane-1,1-diyldiphosphonic acids, Pol. J. Chem. 72 (1998)
1148–1153.
[5] M. Dyba, M. Jezowska-Bojczuk, E. Kiss, T. Kiss, H. Kozlowski, Y. Leroux,
D.E. Manouni, 1-Hydroxyalkane-1,1-diyldiphosphonates as potent chelating agents
for metal ions. Potentiometric and spectroscopic studies of copper (II) coordination,
J. Chem. Soc. Dalton Trans. (1996) 1119–1123.
[6] V. Kubicek, J. Kotek, P. Hermann, I. Lukes, Aminoalkylbis(phosphonates): their com-
plexation properties in solution and in the solid state, Eur. J. Inorg. Chem. 2007 (2007)
333–344.
[7] A.P. Boichenko, V.V. Markov, H.L. Kong, A.G. Matveeva, L.P. Loginova, Re-
evaluated data of dissociation constants of alendronic, pamidronic and olpadronic acids,
Cent. Eur. J. Chem. 7 (2009) 8–13.
[8] N.N. Kamneva, A.P. Boichenko, V.V. Ivanov, V.V. Markov, L.P. Loginova,
Acid–base properties and complexation of alendronic acid in water-ethanol and
ultramicroheterogenous micellar media of Brij 35, Ukr. Khim. Zh. 78 (2012) 74–80.
[9] B. Palazzo, M. Iafisco, M. Laforgia, N. Margiotta, G. Natile, C.L. Bianchi, D. Walsh,
S. Mann, N. Roveri, Biomimetic hydroxyapatite–drug nanocrystals as potential bone sub-
stitutes with antitumor drug delivery properties, Adv. Funct. Mater. 17 (2007) 2180–2188.
[10] M. Meloun, Z. Ferenčı́ková, L. Netolická, T. Pekárek, Thermodynamic dissociation
constants of alendronate and ibandronate by regression analysis of potentiometric data,
J. Chem. Eng. Data 56 (2011) 3848–3854.
[11] The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 14th ed.,
Merck & Co., Inc., Whitehouse Station, NJ, USA, monograph 229, 2006, p. 42.
[12] J. Li, J. Sun, Z. He, Quantitative structure–retention relationship studies with
immobilized artificial membrane chromatography II: partial least squares regression,
J. Chromatogr. A 1140 (2007) 174–179.
[13] H. Sanderson, M. Thomsen, Comparative analysis of pharmaceuticals versus industrial
chemicals acute aquatic toxicity classification according to the United Nations classifi-
cation system for chemicals. Assessment of the (Q)SAR predictability of pharmaceuticals
acute aquatic toxicity and their predominant acute toxic mode-of-action, Toxicol. Lett.
187 (2009) 84–93.
[14] D. Vega, R. Baggio, M.T. Garland, Monosodium 4-amino-1-hydroxy-
1,1-butanediyldiphosphonate trihydrate (alendronate), Acta Cryst. C52 (1996) 2198–2201.
[15] J. Ohanessian, D. Avenel, D.E. Manouni, M. Benramdane, The molecular structure of
4-amino-1-hydroxy butylidene-1 bisphosphonic acid (AHBBPA); an uncommon
anhydrous hydroxybisphosphonic acid, Phosphorus Sulfur Silicon Relat. Elem. 129
(1997) 99–110.
[16] Y. Leroux, D. El Manouni, A. Safsaf, A. Neuman, H. Gillier, Etude structurale de
l’acide butane hydroxy-1 amino-4 diphosphonique-1,10 , Phosphorus Sulfur Silicon
Relat. Elem. 63 (1991) 181–191.
30 Gennady Ananchenko et al.

[17] M. Asnani, K. Vyas, A. Bhattacharya, S. Devarakonda, S. Chakraborty,

A.K. Mukherjee, Ab initio structure determination of anhydrous sodium alendronate
from laboratory powder X-ray diffraction data, J. Pharm. Sci. 98 (2009) 2113–2121.
[18] US2003/0065214 (2003), Novel hydrate forms of alendronate sodium, processes for
manufacture thereof, and pharmaceutical compositions thereof.
[19] US2005/0113343 (2005), Solid-state form of alendronate sodium and preparation thereof.
[20] G.R. Kieczykowski, R.B. Jobson, D.G. Melillo, D.F. Reinhold, V.J. Grenda,
I. Shinkai, Preparation of (4-amino-1-hydroxybutylidene)bisphosphonic acid sodium
salt, MK-217 (alendronate sodium). An improved procedure for the preparation of
1-hydroxy-1,1-bisphosphonic acids, J. Org. Chem. 60 (1995) 8310–8312.
[21] http://riodb01.ibase.aist.go.jp/sdbs/cgi-bin/cre_frame_disp.cgi?spectrum_type¼ir&
sdbsno¼52123.
[22] M.T. Rubino, M. Agamennone, C. Campestre, P. Campiglia, V. Cremasco, R. Faccio,
A. Laghezza, F. Loiodice, D. Maggi, E. Panza, A. Rossello, P. Tortorella, Biphenyl
sulfonylamino methyl bisphosphonic acids as inhibitors of matrix metalloproteinases
and bone resorption, ChemMedChem 6 (2011) 1258–1268.
[23] WO2005/047282 (2005), Antiresorptive mutual salt of raloxifene and biphosphonic
acid 2005.
[24] G. Grossmann, A. Grossmann, G. Ohms, E. Breuer, R. Chen, G. Golomb, H. Cohen,
G. Hägele, R. Classen, Solid-state NMR of bisphosphonates adsorbed on hydroxyap-
atite, Magn. Res. Chem. 38 (2000) 11–16.
[25] M.S. Ironside, M.J. Duer, D.G. Reid, S. Byard, Bisphosphonate protonation states,
conformations, and dynamics on bone mineral probed by solid-state NMR without iso-
tope enrichment, Eur. J. Pharm. Biopharm. 76 (2010) 120–126.
[26] J. Mao, S. Mukherjee, Y. Zhang, R. Cao, J.M. Sanders, Y. Song, Y. Zhang, G.A. Meints,
Y.G. Gao, D. Mukkamala, M.P. Hudock, E. Oldfield, Solid-state NMR, crystallo-
graphic, and computational investigation of bisphosphonates and farnesyl diphosphate
synthase-bisphosphonate complexes, J. Am. Chem. Soc. 128 (2006) 14485–14497.
[27] S. Mukherjee, Y. Song, E. Oldfield, NMR investigations of the static and dynamic
structures of bisphosphonates on human bone: a molecular model, J. Am. Chem.
Soc. 130 (2008) 1264–1273.
[28] Z. Qu, X. Chen, C. Qu, L. Qu, J. Yuan, D. Wei, H. Li, X. Huang, Y. Jiang, Y. Zhao,
Fragmentation pathways of eight nitrogen-containing bisphosphonates (BPs) investi-
gated by ESI-MSn in negative ion mode, Int. J. Mass Spectr. 295 (2010) 85–93.
[29] http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Scientific_
Discussion/human/000619/WC500024249.pdf, 2012 (accessed 20.08.12).
[30] US4407761A, Process for the production of o-amino-1-hydroxyalkylidene-
1,1-bisphosphonic acid, 1983.
[31] D.V.N. Srinivasa Rao, R. Dandala, G.K.A.S.S. Narayanan, R. Lenin, M. Sivakumaran,
A. Naidu, Novel procedure for the synthesis of 1-hydroxy-1,1-bisphosphonic acids
using phenols as medium, Synth. Commun. 37 (2007) 4359–4365.
[32] D.A. Mustafa, B.A. Kashemirov, C.E. McKenna, Microwave-assisted synthesis of
nitrogen-containing 1-hydroxymethylenebisphosphonate drugs, Tetrahedron Lett.
52 (2011) 2285–2287.
[33] G. Xu, Y. Xie, X. Wu, A facile and direct synthesis of alendronate from pyrollidone,
Org. Prep. Proc. Int. 36 (2004) 185–187.
[34] D.M. Mizrahi, T. Waner, Y. Segall, a-Amino acid derived bisphosphonates. Synthesis
and anti-resorptive activity, Phosphorus Sulfur Silicon Relat. Elem. 173 (2001) 1–25.
[35] E. Guénin, M. Monteil, N. Bouchemal, T. Prangé, M. Lecouvey, Syntheses of phos-
phonic esters of alendronate, pamidronate and neridronate, Eur. J. Org. Chem. 2007
(2007) 3380–3391.
[36] M. Egorov, S. Aoun, M. Padrines, F. Redini, D. Heymann, J. Lebreton,
M. Mathé-Allainmat, A one-pot synthesis of 1-hydroxy-1,1-bis(phosphonic acid)s
Alendronate Sodium 31

starting from the corresponding carboxylic acids, Eur. J. Org. Chem. 2011 (2011)
7148–7154.
[37] Sodium Alendronate, European Pharmacopoeia 7.0, Council of Europe, 2010,
pp. 2908–2909.
[38] Alendronic Acid Tablets, British Pharmacopoeia 2013, Medicines and Healthcare Prod-
ucts Regulatory Agency, vol. III, 2012, pp. 2487–2489.
[39] Alendronate Sodium, U.S. Pharmacopeia—National Formularly, USP35/NF30, The
United States Pharmacopoeial Convention, 2011, pp. 2093–2094.
[40] Alendronate sodium Tablets, U.S. Pharmacopeia—National Formularly, USP35/
NF30, The United States Pharmacopoeial Convention, 2011, pp. 2094–2096.
[41] C.K. Zacharis, P.D. Tzanavaras, Determination of bisphosphonate active pharmaceu-
tical ingredients in pharmaceuticals and biological material: a review of analytical
methods, J. Pharm. Biomed. Anal. 48 (2008) 483–496.
[42] J.D. De Marco, S.E. Biffar, D.G. Reed, M.A. Brooks, The determination of 4-amino-
1-hydroxybutane-1,1-diphosphonic acid monosodium salt trihydrate in pharmaceutical
dosage forms by high-performance liquid chromatography, J. Pharm. Biomed. Anal. 7
(1989) 1719–1727.
[43] S. Samdancioglu, S. Calis, S. Kir, M. Sumnu, The determination of alendronate sodium
in microparticular systems by high performance liquid chromatography, FABAD
J. Pharm. Sci. 28 (2003) 183–192.
[44] E. Kwong, A.M. Chiu, S.A. McClintock, M.L. Cotton, HPLC analysis of an amino
bisphosphonate in pharmaceutical formulations using postcolumn derivatization and
fluorescence detection, J. Chromatogr. Sci. 28 (1990) 563–566.
[45] E.W. Tsai, D.P. Ip, M.A. Brooks, Determination of alendronate in pharmaceutical dos-
age formulations by ion chromatography with conductivity detection, J. Chromatogr.
596 (1992) 217–224.
[46] R. Thompson, N. Grinberg, H. Perpall, G. Bicker, P. Tway, Separation of
organophosphonates by ion chromatography with indirect photometric detection,
J. Liq. Chromatogr. 17 (1994) 2511–2531.
[47] E.W. Tsai, S.D. Chamberlin, R.J. Forsyth, C. Bell, D.P. Ip, M.A. Brooks, Determina-
tion of bisphosphonate drugs in pharmaceutical dosage formulations by ion chromatog-
raphy with indirect UV detection, J. Pharmaceut. Biomed. Anal. 12 (1994) 983–991.
[48] X.-Z. Qin, E.W. Tsai, T. Sakuma, D.P. Ip, Pharmaceutical application of liquid
chromatography-mass spectrometry: II.1 ion chromatography-ion spray mass spectro-
metric characterization of Alendronate, J. Chromatogr. A 686 (1994) 205–212.
[49] Y.-H.R. Han, X.-Z. Qin, Determination of alendronate sodium by ion chromatogra-
phy with refractive index detection, J. Chromatogr. A 719 (1996) 345–352.
[50] M.J. Lovdahl, D.J. Pietrzyk, Anion-exchange separation and determination of
bisphosphonates and related analytes by post-column indirect fluorescence detection,
J. Chromatogr. A 850 (1999) 143–152.
[51] M. Kovacevic, A. Gartner, M. Novic, Determination of bisphosphonates by ion
chromatography-inductively coupled plasma mass spectrometry, J. Chromatogr. A
1039 (2004) 77–82.
[52] S.K. Al Deeb, I.I. Hamdan, S.M. Al Najjar, Spectroscopic and HPLC methods for the
determination of alendronate in tablets and urine, Talanta 64 (2004) 695–702.
[53] Z. Xie, Y. Jiang, D.Q. Zhang, Simple analysis of four bisphosphonates simultaneously
by reverse phase liquid chromatography using n-amylamine as volatile ion-pairing
agent, J. Chromatogr. A 1104 (2006) 173–178.
[54] C. Fernandes, R.S. Leite, F.M. Lanças, Rapid determination of bisphosphonates by
ion chromatography with indirect UV detection, J. Chromatogr. Sci. 45 (2007) 236–241.
[55] O.I. Abd El-Sattar, A.A. Ahmad, T. ElKady, Validated and stability indicating HPLC-
method for determination of alendronate sodium using phthaldialdehyde as a pre-
column derivatizing agent, Egypt. J. Biomed. Sci. 11 (2003) 35–45.
32 Gennady Ananchenko et al.

[56] T. Pérez-Ruiz, C. Martı́nez-Lozano, M.D. Garcı́a-Martı́nez, A sensitive post-column

photochemical derivatization/fluorimetric detection system for HPLC determination of
bisphosphonates, J. Chromatogr. A 1216 (2009) 1312–1318.
[57] M.M. Warnke, Z.S. Breitbach, E. Dodbiba, J.A. Crank, T. Payagala, P. Sharma,
E. Wanigasekara, X. Zhang, D.W. Armstrong, Positive mode electrospray ionization
mass spectrometry of bisphosphonates using dicationic and tricationic ion-pairing
agents, Anal. Chim. Acta 633 (2009) 232–237.
[58] J. Kuljanin, I. Janković, J. Nedeljković, D. Prstojević, V. Marinković, Spectrophoto-
metric determination of alendronate in pharmaceutical formulations via complex for-
mation with Fe(III) ions, J. Pharm. Biomed. Anal. 28 (2002) 1215–1220.
[59] E.A. Taha, N.F. Youssef, Spectrophotometric determination of some drugs for osteo-
porosis, Chem. Pharm. Bull. 51 (2003) 1444–1447.
[60] P.D. Tzanavaras, C.K. Zacharis, G.A. Theodoridis, E.A. Kalaitzantonakis,
A.N. Voulgaropoulos, Normal spectrophotometric and stopped-flow spectrofluorimetric
sequential injection methods for the determination of alendronic acid, an anti-osteoporosis
amino-bisphosphonate drug, in pharmaceuticals, Anal. Chim. Acta 547 (2005) 98–103.
[61] M. Koba, K. Koba, L. Przyborowski, Application of UV-derivative spectrophotometry
for determination of some bisphosphonates drugs in pharmaceutical formulations, Acta
Pol. Pharm. 65 (2008) 289–294.
[62] M.I. Walash, M.E. Metwally, M. Eid, R.N. El-Shaheny, Validated spectrophotometric
methods for determination of Alendronate sodium in tablets through nucleophilic aro-
matic substitution reactions, Chem. Cent. J. 6 (2012) 25–40.
[63] W. Ulbrich, A. Lamprecht, Fluorimetric quantification of clodronate and Alendronate
in aqueous samples and in serum, Talanta 84 (2011) 437–442.
[64] D.G. Reed, G.P. Martin, J.M. Konieczny, M.A. Brooks, The determination of
alendronate sodium in tablets by inductively coupled plasma (ICP), J. Pharm. Biomed.
Anal. 13 (1995) 1055–1058.
[65] E.W. Tsai, M.M. Singh, H.H. Lu, D.P. Ip, M.A. Brooks, Application of capillary elec-
trophoresis to pharmaceutical analysis. Determination of alendronate in dosage forms,
J. Chromatogr. A 626 (1992) 245–250.
[66] D. Bexheti, E.I. Anderson, A.J. Hutt, M. Hanna-Brown, Evaluation of multidimensional
capillary electrophoretic methodologies for determination of amino bisphosphonate phar-
maceuticals, J. Chromatogr. A 1130 (2006) 137–144.
[67] O.A. Razak, S.F. Belal, M.M. Bedair, R.S. Haggag, The utilization of copper(II) phos-
phate for the anodic stripping voltammetric assay of alendronate sodium, des-
ferrioxamine mesylate and lisinopril, Talanta 59 (2003) 1061–1069.
[68] A. de Haro Moreno, H.R. Pezza, L. Pezza, Potentiometric determination of Alendronate
in pharmaceutical formulations, Chem. Anal. (Warsaw) 49 (2004) 351–358.
[69] P. Svidritskii, M.S. Jiang, V.I. Il’in, D.I. Dyn’kov, A.V. Pirogov, O.A. Shpigun, The
determination of alendronate ion and certain inorganic ions using capillary electropho-
resis, Moscow Univ. Chem. Bull. (Eng. Ed.) 65 (2010) 42–48. Vestnik Moskovskogo
Universiteta, Seriya 2: Khimiya 2010, 51, 53–61 (Russian Edition).
[70] L.S. Zhu, V.N. Lapko, J.W. Leey, Y.J. Basir, C. Kafonek, R. Olsen, C. Briscoe, A gen-
eral approach for the quantitative analysis of bisphosphonates in human serum and urine
by high-performance liquid chromatography/tandem mass spectrometry, Rapid
Commun. Mass Spectrom. 20 (2006) 3421–3426.
[71] S.-W. Su, Y.-C. Liao, C.-W. Whang, Analysis of alendronate in human urine and
plasma by magnetic solid-phase extraction and capillary electrophoresis with fluores-
cence detection, J. Sep. Sci. 35 (2012) 681–687.
[72] R.W. Sparidans, J. den Hartigh, Chromatographic analysis of bisphorphonates, Pharm.
World Sci. 21 (1999) 1–10.
Alendronate Sodium 33

[73] E. Ban, J.-Y. Park, H.T. Kim, C.-K. Kim, Determination of alendronate in low vol-
umes of plasma by column-switching high-performance liquid chromatography
method and its application to pharmacokinetic studies in human plasma, Arch. Pharm.
Res. 34 (2011) 2079–2086.
[74] M. Chen, K. Liu, D. Zhong, X. Chen, Trimethylsilyldiazomethane derivatization
coupled with solid-phase extraction for the determination of alendronate in human
plasma by LC-MS/MS, Anal. Bioanal. Chem. 402 (2012) 791–798.
[75] W.F. Kline, B.K. Matuszewski, W.F. Bayne, Determination of 4-amino-1-
hydroxybutane-1,1-bisphosphonic acid in urine by automated pre-column derivatization
with 2,3-naphthalenedicarboxyaldehyde and high-performance liquid chromatography
with fluorescence detection, J. Chromatogr. 534 (1990) 139–149.
[76] W.F. Kline, B.K. Matuszewski, Improved determination of the bisphosphonate
alendronate in human plasma and urine by automated precolumn derivatization and
high-performance liquid chromatography with fluorescence and electrochemical detec-
tion, J. Chromatogr. 583 (1992) 183–193.
[77] P. Ptáček, J. Klı́ma, J. Macek, Determination of alendronate in human urine as
9-fluorenylmethyl derivative by high-performance liquid chromatography, J. Chromat-
ogr. B 767 (2002) 111–116.
[78] M.-H. Yun, K. Kwon, High-performance liquid chromatography method for
determining alendronate sodium in human plasma by detecting fluorescence: applica-
tion to a pharmacokinetic study in humans, J. Pharm. Biomed. Anal. 40 (2006)
168–172.
[79] C. Apostolou, Y. Dotsikas, C. Kousoulos, G. Tsatsou, F. Colocouri, G.-S. Soumelas,
Y.L. Loukas, Application of a semi-automated 96-well format solid-phase extraction,
column-switching, fluorescence detection protocol for the determination of
alendronate in human urine samples obtained from a bioequivalence study, J. Pharm.
Biomed. Anal. 43 (2007) 1151–1155.
[80] I. Tarcomnicu, L. Silvestro, S.R. Savu, A. Gherase, C. Dulea, Development and appli-
cation of a high-performance liquid chromatography–mass spectrometry method to
determine alendronate in human urine, J. Chromatogr. A 1160 (2007) 21–33.
[81] A.G. Porras, S.D. Holland, B.J. Gertz, Pharmacokinetics of alendronate, Clin. Pharma-
cokinet. 36 (1999) 315–328.
[82] J.H. Lin, I.-W. Chen, F.A. Deluna, On the absorption of alendronate in rats, J. Pharm.
Sci. 83 (1994) 1741–1746.
[83] B.J. Gertz, S.D. Holland, W.F. Kline, B.K. Matuszewski, A.G. Porras, Clinical phar-
macology of alendronate sodium, Osteoporos. Int. 3 (Suppl. 3) (1993) S13–S16.
[84] B.J. Gertz, S.D. Holland, W.F. Kline, B.K. Matuszewski, A. Freeman, H. Quan,
K.C. Lasseter, J.C. Mucklow, A.G. Porras, Studies of the oral bioavailability of
alendronate, Clin. Pharm. Ther. 58 (1995) 288–298.
[85] J.H. Lin, Bisphosphonates: a review of their pharmacokinetic properties, Bone 18
(1996) 75–85.
[86] H. Lin, I.W. Chen, F.A. deLuna, M. Hichens, Role of calcium in plasma protein bind-
ing and renal handling of alendronate in hypo- and hypercalcemic rats, J. Pharmacol.
Exp. Ther. 267 (1993) 670–675.
[87] H. Lin, I.W. Chen, D.E. Duggan, Effects of dose, sex, and age on the disposition of
alendronate, a potent antiosteolytic bisphosphonate, in rats, Drug Metab. Dispos. 20
(1992) 473–478.
[88] V. Cocquyt, W.F. Kline, B.J. Gertz, S.J. Van Belle, S.D. Holland, M. DeSmet,
H. Quan, K.P. Vyas, K.E. Zhang, J. De Grève, A.G. Porras, Pharmacokinetics of intra-
venous alendronate, J. Clin. Pharmacol. 39 (1999) 385–393.