The thesis entitled “Development of new analytical methods for impurity profiling of
psychiatric and cancer drugs” has been divided into seven chapters. Chapter 1 deals with a
brief introduction of psychiatric and cancer drugs and importance of analytical methods in
studying the impurity profiles of drugs. Chapter 2 describes development and validation of
a liquid chromatographic method for monitoring of reactions involved in synthesis of
antidepressant Venlafaxine hydrochloride and characterization of degradation product and
process impurities. Chapter 3 deals with the impurity profiling of Citalopram hydrobromide
and characterization of degradation products and process related impurities. Chapter 4
describes the separation and determination of process-related substances of an antidepressant
Mirtazapine by reversed-phase HPLC. Chapter 5 describes the development of a RP-HPLC
method for impurity profile study of an antipsychotic drug Olanzapine and characterization of
process impurities by LC- ESI-MS-MS, 1H-NMR and FT-IR spectroscopy. Chapter 6 deals
with impurity profiling of an anticancer drug bicalutamide by RP-HPLC and characterization
of degradation products and unknown process impurities by spectroscopic techniques.
Chapter 7 describes the development and validation of LC methods for separation and
determination of R&S enantiomers of Citalopram hydrobromide and Bicalutamide using
polysaccharide chiral stationary phases.
CHAPTER 1
Impact of impurities on quality and safety of psychiatric and cancer Drugs
Chapter 1 gives a brief introduction to quality, safety and efficacy of drugs and
pharmaceuticals with some examples of L-tryptophan, thalidomide, and aspirin. The origin
of impurities, types of different impurities in drugs and pharmaceuticals, impurity profiling of
drugs, identification of impurities by analytical techniques such as HPLC, LC-MS, GC-MS,
LC-NMR and MS were discussed. The pharmacopoeial status, regulatory aspects and
analytical methodologies were presented. Statement of the problem, aims and objectives of
the present investigation were given at the end of the chapter. All the experimental details
were given in the respective chapters.
CHAPTER 2
Liquid Chromatographic Studies on Impurity Profiles of Venlafaxine Hydrochloride, a
Serotonin Norepinephrine Reuptake Inhibitor
This chapter describes reversed phase liquid chromatographic studies for monitoring
of process related substances of venlafaxine hydrochloride (VNX) an antidepressant. The
process
related
ethyl]cyclohexanol
impurities
of
hydrochloride
VNX
(III)
(I),
viz.,
1-[2-(amino)-1-(4-methoxyphenyl)
1-[2-(methylamino)-1-(4-methoxyphenyl)ethyl]
cyclohexanol hydrochloride (II), [2-cyclohex-1-enyl-2-(4-methoxy-phenyl)-ethyl]-dimethylamine (IV) (1-hydroxy-cyclohexyl)-(4-methoxy-phenyl)-acetonitrile and 4-methoxy phenyl
acetonitrile (V) and (1-hydroxy-cyclohexyl)-(4-methoxy-phenyl)-acetonitrile (VI) as shown in
Fig. 1 were separated and determined by HPLC.
CH 3
H
N
N
H
H
CH 3
N
N
OH
OH
OH
CH 3
CN
OH
CH 3
CH 3
CN
H3CO
(I)
( II )
H3CO
H3CO
H3CO
H3CO
H3CO
( IV )
( III )
(V)
( VI )
Fig. 1 Process-related impurities and degradation products of venlafaxine (III).
The HPLC conditions developed were as follows; mobile phase: (A: 0.3%
diethylamine, pH adjusted to 3.0 with ortho-phosphoric acid and B: acetonitrile: methanol
(90: 10 v/v) was pumped at a flow rate of 1.0 ml/min according to the gradient elution
program: 0 min. 33% B, 0-5 min. 33% B, 5-14 min. 85% B, 14-18 min. 85% B; 18-22 min.
33% B; 22-30 min. 33% B; Kromasil KR100-5C18 column, temperature of column 400C±20C
and detection at 225 nm (PDA). The effects of organic modifier (i,e; acetonitrile and
1
methanol) and concentration (0.1% to 0.3%) and pH
(3.0 to 6.0) of DEA buffer and
temperature of column (250C to 400C) on retention and resolution were studied to optimize
the chromatographic conditions.
CH3
CH3
N
N
H3C
H3C
H
CH3
H
N
H
H3C
H
O+
OH
-H2O
HCl
+ HCl
H3CO
H3CO
H3 CO
Fig. 2. The degradation of venlafaxine by acid hydrolysis.
Forced degradation studies were carried out by stressing VNX under i) UV light at
254 nm, 60oC temperature for 15 days and ii) extreme conditions such as 0.2-1.0 N HCl, 0.050.5 N NaOH, and 3% H2O2. Under acidic conditions one degraded product (IV) was formed
and well separated from VNX under the present conditions (Fig. 2). Different batches of VNX
were analyzed by developed HPLC method and one impurity having >0.1% area at retention
time 2.45 min (0.32 RRT) (i.e., marked as II) did not match with any of the process
intermediates (Fig. 3). The unknown impurity (II) and degradation product (IV) were isolated
by semi-preparative HPLC and characterized using modern spectroscopic techniques such as
UV, FT-IR, 1H NMR and ESI-MS-MS.
Fig. 3 Typical chromatograms of A) VNX (III) spiked with 10% (w/w) of each of
impurities; B), C) & D) Different process samples of VNX (III).
The method was validated with respect to precision (inter and intra day assay of VNX,
R.S.D<2%), accuracy (99.08-100.21% with R.S.D 0.28-0.68% for VNX and 96.19-101.14%
with R.S.D 0.39-1.15% for impurities), linearity (range 25-300 µg/ml with r20.9999 for
VNX and 0.5-5.0 µg/ml with r20.9942 for impurities), limit of detection (LOD) and limit of
quantitation (LOQ) and specificity. The developed method was found to be selective,
sensitive, precise and stability indicating. The method was applied to determine VNX and its
process-related substances in bulk drugs and pharmaceutical formulations.
CHAPTER 3
2
Isolation and Characterization of Process Related Impurities Including the
Degradation Products of Citalopram Hydrobromide, a Selective Serotonin Reuptake
Inhibitor
This chapter describes a gradient reversed phase liquid chromatographic method for
monitoring of process related substances and degradation products of a SSRI antidepressant,
citalopram hydrobromide (CIT). The process related impurities of CIT (V) viz., its process
related
substances
viz.,
1-(3-dimethylamino-propyl)-1-(4-fluoro-phenyl)-1,3-dihydro-
isobenzofuran-5-carboxylic acid amide (I), 1-(3-dimethylamino-propyl)-1-(4-fluoro-phenyl)1,3-dihydro-isobenzofuran-5-carboxylic acid (II), 4-[4-dimethylamino-1-(4-fluoro-phenyl)1-hydroxy-butyl]-3-hydroxymethyl-benzonitrile-(III),
phenyl)-but-1-enyl]-3-hydroxymethyl-benzonitrile
4-[4-dimethyl-amino-1-(4-fluoro(IV),
1-(4-bromo-2-hydroxymethyl-
phenyl)-4-dimethylamino-1-(4-fluoro-phenyl)-butan-1-ol (VI), [3-[1-(4-fluoro-phenyl)-1, 3dihydro-isobenzofuran-1-yl]-propyl]-dimethyl-amine (VII), 1-(3-dimethylamino-propyl)-1(4-fluoro-phenyl)-1,3-dihydro-isobenzofuran-5-carbonitrile-N-oxide (VIII) and [3-[5-bromo1-(4-fluoro-phenyl)-1,3-dihydro-isobenzofuran-1-yl]-propyl]-dimethyl-amine (IX) as shown
in Fig. 4 were separated and determined by HPLC.
H2NOC
CH 3
N
CH3
O
F
[I]
NC
O
F
[V]
OH
OH
CH3
N
CH3
F
[IV]
[III]
Br
NC
Br
CH3
N HBr
CH 3
OH
OH
O
CH 3
N
CH3
CH3
N
CH 3
F
[VI]
O
F
[VIII]
[VII]
CH 3
N
CH3
OH
CH3
N
CH 3
F
[II]
F
F
NC
NC
HOOC
O
O
CH3
N
CH3
O
CH 3
N
CH3
F
[IX]
Fig. 4 Chemical structures of CIT (V), degradation products (I, II, VIII) and its processrelated impurities (III, IV, VI, VII and IX).
The HPLC conditions developed were as follows; mobile phase: A: 0.3%
diethylamine, pH adjusted to 3.0 with ortho-phosphoric acid and B: acetonitrile- methanol
(55:45 v/v) was pumped at a flow rate of 1.0 ml/min according to the gradient elution
program: 0 min. 40% B, 0-13 min. 40% B, 13-25 min. 65% B, 25-28 min. 65% B; 28-29 min.
40% B; 29-40min. 40% B; Inertsil ODS 3V column, temperature of column 500C±10C and
detection at 225 nm (PDA). The effects of organic modifier (i,e; acetonitrile and methanol)
and concentration (0.1% to 0.4%) and pH (3.0 to 6.0) of DEA buffer and temperature of
column (350C to 500C) on retention and resolution were studied to optimize the
chromatographic conditions (Fig. 5).
3
Fig. 5 Typical HPLC chromatograms of A) CIT (V) (200 μg/ml) spiked with 5% (w/w)
of each of the related substances (I-IV and VI- IX); B), C), D) & E) Different process
samples of CIT (V).
Forced degradation studies were carried out by stressing CIT under i) UV light at 254
nm, 60oC temperature for 15 days and ii) extreme conditions such as 0.2-1.0 N HCl, 0.05-0.5
N NaOH, and 3% H2O2. Under alkaline conditions two degraded products (I and II) and
under peroxide conditions one degraded impurity (VIII) were formed. Different batches of
CIT were analyzed by developed HPLC and four impurities having >0.1% area at retention
times 5.82 min (0.46 RRT) (III), 10.41 min (0.83 RRT) (IV) and 15.59 (1.24 RRT) (VII)
were detected (Fig. 5). These impurities did not match with any of the process intermediates.
The unknown impurities (III, IV and VII) and degradation products (I, II and VIII) were
isolated and characterized using modern spectroscopic techniques such as UV, FT-IR, 1H
NMR and ESI-MS-MS. The ESI-MS-MS fragmentation profiles have been discussed (Fig.
6). The method was validated with respect to precision (inter and intra day assay of CIT,
R.S.D<1%), accuracy (99.83-100.15% with R.S.D 0.19-0.41% for CIT and 95.73-104.75%
with R.S.D. 1.37-3.44% for impurities) linearity (range 10-300 µg/ml with r20.999 for CIT
and 0.5-10 µg/ml with r20.9867 for impurities), limit of detection and limit of quantitation.
The developed method was found to be selective, sensitive, precise and stability indicating.
The method was applied to determine CIT (V) and its process-related substances in bulk
drugs and pharmaceutical formulations.
4
B)
A)
F
H2NOC
H2NOC
+H
O
+H
N
+H
F
m/z 344
+H
-HN-(CH3)2
+H
+H
HOOC
O
m/z 135
+H
HOOC
O
-NH3
O
-H2O
m/z 280
m/z 263
F
-C 2H2
F
-C 6H5F
m/z 281
m/z 298
F
F
-C 2H2
-C 6H5F
HOOC
+H
-C 6H5F
O
+H
H2NOC
+H
HOOC
O
m/z 184
m/z 299
F
+H
O
+H
m/z 263
F
-C 6H5F
+H
H2NOC
HOOC
-HN-(CH3)2
m/z 134
H2NOC
O
m/z 109
m/z 326
F
-HN-(CH3)2
+H
N
-H2O
N
H2NOC
F
m/z 343
F
-HN-(CH3)2
H2NOC
+H
m/z 109
m/z 325
+H
O
N
-H2O
F
HOOC
HOOC
O
m/z 237
m/z 185
m/z 237
m/z 202
F
m/z 203
F
OH
C)
NC
NC
+H
OH
+H
O
N
-H2O
N
m/z 343
m/z 325
F
F
D)
E)
F
NC
NC
+H
NC
-HN-(CH3)2
-HN-(CH3)2
-HN-(CH3)2
+H
m/z 109
F
m/z 325
F
-HN-(CH3)2
N
-H2O
m/z 307
NC
F
m/z 325
NC
N
m/z 109
m/z 307
F
+H
O
+H
N
-H2O
N
F
NC
NC
+H
OH
m/z 116
m/z 116
+H
NC
+H
NC
+H
NC
O
OH
m/z 262
m/z 262
m/z 280
F
F
-C 6H5F
NC
-C 6H5F
-C 6H5F
NC
+H
NC
+H
m/z 280
F
F
-C 6H5F
+H
NC
+H
O
O
F)
G)
F
+H
O
+H
F
NC
N
-H2O
N
m/z 184
m/z 166
m/z 184
m/z 166
m/z 109
N
-HN-(CH3)2
m/z 282
+H
O
+H
NC
m/z 109
O
F
m/z 300
F
-HN-(CH3)2
-HN-(CH3)2
-HON-(CH3)2
F
+H
+H
NC
F
m/z 341
m/z 262
m/z 91
m/z 116
+H
O
-C 6H5F
+H
NC
-C 2H2
O
m/z 237
F
-C 6H5F
m/z 211
F
F
NC
m/z 255
-C 6H5F
+H
NC
m/z 166
F
m/z 280
O
+H
+H
O
m/z 141
-C6H5F
+H
m/z 184
m/z 159
Fig. 6. ESI-MS/MS fragmentation patterns of A) I, B) II C) III, D) IV, E) V, F) VII and G) VIII.
5
CHAPTER 4
Reversed Phase HPLC Separation and Determination of Process Related Substances of
Mirtazapine, a Noradrenergic and Specific Serotonergic Antidepressant
This chapter describes an isocratic reversed phase liquid chromatographic method for
monitoring of process related substances of mirtazapine (MTZ). The process related
impurities of
MTZ (V) viz., 1-methyl-3-phenyl-piperazine (I), 2-(4-methyl-2-phenyl-
piperazin-1-yl)-nicotinic
acid
(II),
[2-(4-methyl-2-phenyl-piperazin-1-yl)-pyridin-3-yl]-
methanol(III), 1,2,3,4,9,13b-hexahydro-2,4a,5-triaza-tribenzo[a,c,e]cyclohep-tene (IV) and 2methyl-3,4,9,13b-tetrahydro-1H-2,4a,5-triaza-tribenzo[a,c,e]cycloheptene 2-oxide (VI) as
shown in Fig. 7 were separated and determined by HPLC on a BDS Hypersil C18 column with
0.3% triethylamine, pH adjusted to 3.0 with ortho-phosphoric acid and acetonitrile (78:22 v/v)
as a mobile phase at a flow rate of 1.0 ml/min and detection at 215 nm using photo diode
array detector (PDA). The effects of organic modifier (i,e; acetonitrile from 20% to 25%) and
concentration (0.1% to 0.3%) and pH (3.0 to 6.0) of TEA buffer and temperature of column
(250C to 400C) on retention and resolution were studied to optimize chromatographic
conditions (Fig. 8).
OH
HO
O
N
N
HN
(I)
N
N
N
H
N
H
N
H
N
N
N
( II )
N
N
N
( III )
NH
( IV )
N
N
(V)
CH3
O
CH3
( VI )
Fig. 7. Process-related impurities (I, II, III), side products (IV and VI) and degradation
product (VI) of Mirtazapine (V).
Fig. 8 Typical chromatograms of (a) MTZ spiked with 5% (w/w) each of impurities; (b)
(c) & (d) Different process samples of MTZ (V).
Forced degradation studies were carried out by stressing MTZ under i) UV light at 254
nm, 60oC temperature for 15 days and ii) extreme conditions such as 0.2-1.0 N HCl, 0.05-0.5
N NaOH, and 3% H2O2. Under peroxide conditions one degraded product (VI) was formed
6
and well separated from MTZ under the present conditions. Different batches of MTZ were
analyzed by developed HPLC and two impurity having >0.05% area at retention times 8.53
min (0.91 RRT) (IV) and 10.79 min (1.15 RRT) (VI) did not match with any of the process
intermediates (Fig. 8). The retention time and absorption spectra of unknown impurity (VI)
and degradation product (VI) were matched. The two impurities were isolated by column
chromatography and characterized using modern spectroscopic techniques such as UV, FTIR, 1H NMR and ESI-MS-MS. The method was validated with respect to precision (inter and
intra day assay of MTZ, R.S.D<1%), accuracy (99.42 -100.32 with R.S.D. 0.28-0.61% for
MTZ and 95.54-102.22 with R.S.D. 0.58-2.52% for impurities), linearity (range 25-200 µg/ml
with r20.9999 for MTZ and 0.5-5.0 µg/ml with r20.9941 for impurities), limit of detection
(LOD) and limit of quantitation (LOQ) and specificity. The developed method was found to
be selective, sensitive, precise and stability indicating. The method was applied to determine
MTZ and its process-related substances in bulk drugs and pharmaceutical formulations.
CHAPTER-5
Isolation and Characterization of Process Impurities of Olanzapine, an Atypical
Antipsychotic by LC, ESI-MS-MS, 1H-NMR and FT-IR Spectroscopy
This chapter describes a gradient reversed phase liquid chromatographic method for
monitoring of process related substances of an atypical antipsychotic drug, olanzapine (OLZ).
The process related impurities of OLZ (III) viz., 2-methyl-10-piperazin-1-yl-4H-3-thia-4,9diaza-benzo[f]azulene (I), 2-methyl-10-(4-methyl-4-oxy-piperazin-1-yl)-4H-3-thia-4,9-diazabenzo[f]azulene (II), 2-methyl-4H-3-thia-4,9-diaza-benzo[f]azulen-10-ylamine hydrochloride
(IV), 2-amino-5-methyl-thiophene-3-carbonitrile (V), 2-(2-amino-phenylamino)-5-methylthiophene-3-carbonitrile
(VII)
and
5-methyl-2-(2-nitro-phenylamino)-thiophene-3-
carbonitrile (VIII) as shown in Fig. 9 were separated and determined by HPLC. The HPLC
conditions developed were as follows; mobile phase: A: ammonium acetate (0.2 M in H2O)
pH adjusted to 4.50 with acetic acid and B: acetonitrile was pumped at a flow rate of 1.0
ml/min according to the gradient elution program: 0 min. 20% B, 0-5 min. 20% B, 5-30 min.
85% B, 30-34 min. 85% B; 34-35 min. 20% B; 35-45 min. 20% B; Inertsil ODS 3V column,
temperature of column 250C±20C and detection at 254 nm (PDA). The effects of organic
modifier (i,e; acetonitrile and methanol) and concentration (0.05 M to 0.3M) and pH (4.0 to
6.5) of ammonium acetate buffer on retention and resolution were studied to optimize the
chromatographic conditions (Fig. 9).
7
+
F
NO 2
NC
NO 2
H2N
KOH, Ethanol
S
NC
N
S
H
VIII
CH 3
V
Side product
NH 2
SnCl2, HCl,
Ethanol
NC
N
N
N
N
Toluene/DMSO
N
H
III
+
N
Side product
H
N
N
H
NH
N
N
H
N
N
N
N
N
HN
II
S
N
H
NH
S
S
N
H
S
S
Side product
H
N
N O
N
N
H
IV
S
Oxidation
N
N
H
VII
NH 2. HCl
H
N
S
VI
I
Fig. 9. The scheme of reactions involved in the synthesis of OLZ (III) and formation of
impurities I, II, VI and VII.
Fig. 10. Typical HPLC chromatograms of a) OLZ (III) (200 μg/ml) spiked with 2.5%
(w/w) of each of the impurities (I, II and IV-VIII); b), c), d) & e) Different process
samples of OLZ (III).
Different batches of OLZ were analyzed by developed HPLC and four impurities
having >0.1% area at retention times 8.53 min (0.69 RRT) (I), 10.12 (0.79 RRT) (II), 22.22
(1.74 RRT) (VI) and 26.61 (2.09 RRT) (VII) were did not match with any of the process
intermediates (Fig. 10). The unknown impurities (I, II, VI and VII) were isolated by column
chromatography and characterized using modern spectroscopic techniques such as UV, FTIR, 1H NMR and ESI-MS-MS. The ESI-MS-MS fragmentation profiles have been discussed
(Fig. 11).
8
A)
-C4H9N2
N
D)
NH
+H
N
N
N
-C4H7N
-C2H5N
N
H S
m/z 299
+H
N
NH2
+H
-NH2
N
+H
N
N
N
S
H
m/z 239
N
S
H
m/z 256
-C2H2
-C12H11N3S
S H
N
S
N
NH
N
HN
S
B)
+H
+H
-C2H4O
+H
N
-H2O
N
H S
m/z 329
-C2H9NO
+H
N
N
N
H S
m/z 311
-C5H7N
+H
NH2
N
-C5H9NO
-C4H7N
N N
HN
m/z 511
-C12H8N2S
-C12H10N2S
S
-C2H2
H
N
-2NH3
+H
+H
N
N
H S
m/z 285
HN
N
N
S
N
S
N
N
NH
NH
HN
S
m/z 468
m/z 477
+H
N
S
N
+H
N
N S
H
m/z 297
-C2H2
-NH3
HN
N
H S
m/z 230
N
H S
m/z 254
N
-C2H2
+H
N N
NH
m/z 442
-C14H10N2S
H N
N
N
N
N S
H
m/z 239
+H
-C2H5N
N
-NH3
-C12H8N2S
m/z 282
N O
+H
N
N S
H
m/z 256
N S
H
m/z 282
-NH3
-C2H2
-C2H5N
N
N
N
N
-C16H15NS
+H
+H
+H
N
N S
H
m/z 230
-C2H2
-2C2H2
-C2H2
-2C2H2
S
+
N
H S
m/z 213
N
H S
m/z 230
N
N
H
+H
NH2
m/z 99
+H
N
N
C)
+H
N
N
-CH3NH2
N
S
H
m/z 313
--C3H5NH2
N
NH2
m/z 451
N
NH2 CN
E)
N
S
H
m/z 282
--C5H7NH2
-NH3
+H
NC
N
+H
N
-C2H2
N
S
H
m/z 230
+H
N
S
H
m/z 230
-C2H2
+H
-2C2H2
S
S
N
N
NH
HN
+H
N
N
S
H
m/z 256
S
m/z 213
Fig. 11. ESI-MS/MS fragmentation patterns for A) I, B) II C) III D) VI and E) VII
The developed HPLC method was validated with respect to precision (inter and intra
day assay of OLZ, R.S.D<1%), accuracy (99.79-100.35% with R.S.D 0.29-0.48% for OLZ
and 95.18-104.32% with R.S.D 0.87-3.85% for impurities) linearity (range 100-300 µg/ml
with r20.9999 for OLZ and 0.5-10 µg/ml with r20.9867 for impurities), limit of detection
(LOD) and limit of quantitation (LOQ) and specificity. The developed method was found to
be selective, sensitive and precise. The method was applied to determine OLZ and its processrelated substances in bulk drugs and pharmaceutical formulations.
9
CHAPTER 6
Isolation and Characterization of Process Related Impurities and Degradation Products
of Bicalutamide, an Antiandrogen-Development of Impurity Profiles by RP-HPLC
This chapter describes an isocratic reversed phase liquid chromatographic method for
monitoring of process related substances and degradation products of an anticancer drug,
bicalutamide (BCT). The process related impurities of BCT (VII) viz., 3-(4-fluorobenzenesulfonyl)-2-hydroxy-2-methyl-propionic
acid
(I), N-(4-cyano-3-trifluoromethyl-
phenyl)-2,3-dihydroxy-2-methyl-propionamide (II), 4-amino-2-fluoromethyl-benzenonitrile
(III),
N-(4-cyano-3-trifluoromethyl-phenyl)-3-(4-fluoro-benzene sulfinyl)-2-hydroxy-2-
methyl-propionamide
(IV),
3-chloro-N-(4-cyano-3-trifluoromethyl-phenyl)-2-hydroxy-2-
methyl-propionamide (V), 2-methyl-oxirane-2-carboxylic acid (4-cyano-3-trifluoromethylphenyl)-amide (VI) and N-(4-cyano-3-trifluoromethyl-phenyl)-3-(4-fluoro-phenylsulfa-nyl)2-hydroxy-2-methyl-propionamide (VIII) as shown in Fig. 12 were separated and determined
by HPLC on a symmetry C18 column with potassium dihydrogen ortho-phosphate (10 mM in
H2O) pH adjusted to 3.0 with diluted ortho-phosphoric acid- acetonitrile (50:50 v/v) as a
mobile phase at a flow rate of 1.0 ml/min and detection at 215 nm using a photo diode array
detector (PDA). The effects of organic modifier (i,e; acetonitrile 45% to 55%) and pH (3.0 to
6.0) of potassium dihydrogen ortho-phosphate buffer on retention and resolution were studied
to optimize the chromatographic conditions.
O
HO
HO
CH3
S
OH
O
N
O
O
CF3
O
F
F
N
CN
(V)
CN
HO
N
O
O
F
( VII )
( VI )
CH3 H
N
CF3
O
F
CN
( IV )
CH3 H
S
O
HO
S
( III )
F
H
N
CF3
CN
( II )
CH3 H
Cl
H2N
CF3
O
(I)
HO
N
HOH2C
O
F
O
CH3 H
HO
CN
CH3 H
S
CF3
F
N
O
CF3
CN
( VIII )
Fig. 12. The chemical structures of bicalutamide (VII) its degradation products (I and III)
and process-related impurities (II, IV, V, VI and VIII).
Forced degradation studies were carried out by stressing BCT under i) UV light at 254
nm, 60oC temperature for 15 days and ii) extreme conditions such as 0.2-1.0 N HCl, 0.05-0.5
N NaOH, and 3% H2O2.Under alkaline conditions two degraded products (I and III) were
formed (Figs. 13 & 14). The kinetics of degradation of BCT was studied by developed HPLC
method. Different batches of BCT were analyzed by developed HPLC and two impurities
having >0.1% area at retention times 4.28 min (0.38 RRT) (II) and 7.95 (0.71 RRT) (IV) did
10
not match with any of the process intermediates (Fig. 15). The unknown impurities (II and
IV) and degradation products (I and III) were isolated by semi-preparative HPLC and
characterized using modern spectroscopic techniques such as UV, FT-IR, 1H NMR and ESIMS-MS. The ESI-MS-MS fragmentation profiles have been discussed. The method was
validated with respect to specificity, precision (inter and intra day assay of BCT, R.S.D<1%),
accuracy (99.75-100.29% with R.S.D 0.21-0.51% for BCT and 96.31-103.54% with R.S.D
0.61-2.87% for impurities) linearity (range 10-300 µg/ml with r20.9998 for BCT and 0.5-5.0
µg/ml with r20.9838 for impurities), limit of detection (LOD) and limit of quantitation
(LOQ). The developed method was found to be selective, sensitive, precise and stability
indicating. The method was applied to determine BCT and its process-related substances in
bulk drugs and pharmaceutical formulations.
Fig. 13.Typical HPLC chromatograms of a) BCT (VII) (200 μg/ml); b) Degradation of
BCT at 0.1N NaOH.
O
HO
S
O
F
(VII)
CH3H
N
O -OH
O
CF3
CN
HO
CH3H
N
S
0.1N NaOH
O NaO
O
F
O
CF3
HO
S
H
O
CN
F
CH3
H2N
ONa
O
(I)
CF3
+
CN
(III)
Fig. 14.The degradation of BCT by alkaline hydrolysis.
Fig. 15.Typical chromatograms of BCT (VII) A) Spiked with 2.5% (w/w) of each of
impurities; B), C) & D) Different process samples of BCT (VII).
11
CHAPTER 7
Enantiospecific Resolution of Citalopram Hydrobromide and Bicalutamide by HPLC on
Polysaccharide Based Stationary Phases Connected with Ultraviolet and Polarimetric
Detectors in series
Chiral liquid chromatographic separation of citalopram hydrobromide (CIT) and
bicalutamide (BCT) (Fig. 16) have been described on Chiralpak AD-H and Chiralcel OD-H
columns. Chiralcel OD-H column containing amylose tris-(3, 5-dimethylphenylcarbamate)
as a stationary phase was found to be suitable for the determination of enantiomers of CIT
while Chiralpak AD-H column containing amylose tris-(3, 5-dimethylphenylcarbamate) as a
stationary phase was found to be suitable for the determination of enantiomers of BCT. The
effects of organic modifiers viz., ethanol and 2-propanol and temperature on selectivity and
resolution were studied. The optimum separation was obtained on Chiralcel OD-H column
for CIT and chromatographic conditions were: n-hexane:2-propanol:triethylamine (95:05:0.1
v/v/v) as mobile phase and UV detector at 240 nm and the column temperature was at 25oC
(Fig. 17). For BCT optimized conditions were: Chiralpak AD-H column, n-hexane: 2propanol (65: 35 v/v) as a mobile phase and UV detector at 270 nm (Fig. 18). Polarimetric
detector connected in series to UV was used for the identification of the two enantiomers.
Both the separations were found to be enthalpy driven processes. These chromatographic
methods are suitable not only for qualifying optical purity but also isolation of individual
enanatiomers. The proposed methods were validated and applied to determine the
enantiomeric purity of CIT and BCT in bulk drugs and pharmaceutical formulations.
N
N
C
C
H3 C
O
N
O
H3 C
N
CH 3
O
CH 3
CH 3
O
H
N
Cl
F
F
(I.S)
(R)-(-)-CIT
(S)-(+)-CIT
O
S
H
HO
S
N
O
CF3
OH
S
O
H
N
CF3
O
O
F
O
F
CN
(S)-(+)-BCT
CN
(R)-(-)-BCT
Fig.16. Structural representation of enantiomers of citalopram and S-clopidogrel (Internal
standard) and enantiomers of bicalutamide.
12
Fig.17 Typical chromatograms showing the separation of CIT enantiomers and the
internal standard (I.S) on Chiralcel OD-H column with n-hexane:2-propanol:
TEA (95:05:0.1 v/v/v) as mobile phase at 25˚C using UV detector
A) (RS)-Citalopram, B) (S)-Citalopram and C) using polarimetric detector.
Fig.18. Typical chromatograms showing the separation of BCT enantiomers on
Chiralpak AD-H column with n-hexane:2-propanol (65:35 v/v) as a mobile
phase at 25˚C A) (RS)-BCT and B) (R)-(-)-BCT using UV detector at 270 nm
and C) (RS)-BCT using polarimetric detector.
13