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 impurities of VNX (III) viz., 1-[2-(amino)-1-(4-methoxyphenyl)

ethyl]cyclohexanol hydrochloride (I), 1-[2-(methylamino)-1-(4-methoxyphenyl)ethyl]

cyclohexanol hydrochloride (II), [2-cyclohex-1-enyl-2-(4-methoxy-phenyl)-ethyl]-dimethyl-

amine (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.

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.

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, 0.05-

0.5 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 r20.9999 for

VNX and 0.5-5.0 µg/ml with r20.9942 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), 4-[4-dimethyl-amino-1-(4-fluoro-

phenyl)-but-1-enyl]-3-hydroxymethyl-benzonitrile (IV), 1-(4-bromo-2-hydroxymethyl-

phenyl)-4-dimethylamino-1-(4-fluoro-phenyl)-butan-1-ol (VI), [3-[1-(4-fluoro-phenyl)-1, 3-

dihydro-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-bromo-

1-(4-fluoro-phenyl)-1,3-dihydro-isobenzofuran-1-yl]-propyl]-dimethyl-amine (IX) as shown

in Fig. 4 were separated and determined by HPLC.

Fig. 4 Chemical structures of CIT (V), degradation products (I, II, VIII) and its process- related 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 r20.999 for CIT

and 0.5-10 µg/ml with r20.9867 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

Fig. 6. ESI-MS/MS fragmentation patterns of A) I, B) II C) III, D) IV, E) V, F) VII and G) VIII.

CHAPTER 4

5

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 2-

methyl-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).

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 and well separated from MTZ under the present conditions. Different batches of MTZ

6

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, FT-IR, 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 r20.9999 for MTZ and 0.5-5.0 µg/ml with r20.9941 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-4,9-

diaza-benzo[f]azulene (I), 2-methyl-10-(4-methyl-4-oxy-piperazin-1-yl)-4H-3-thia-4,9-diaza-

benzo[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-methyl-

thiophene-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

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, FT-

IR, 1H NMR and ESI-MS-MS. The ESI-MS-MS fragmentation profiles have been discussed

(Fig. 11).

8

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 r20.9999 for OLZ and 0.5-10 µg/ml with r20.9867 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 process-

related 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-fluoro-

benzenesulfonyl)-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-trifluoromethyl-

phenyl)-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.

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 ESI-

MS-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 r20.9998 for BCT and 0.5-

5.0 µg/ml with r20.9838 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.

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: 2-

propanol (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.

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