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 3deals with the impurity profiling of Citalopramhydrobromide 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 5describesthe 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 6deals with impurity profiling of an anticancer drug bicalutamide by RP-HPLC and characterization of degradation products and unknown process impurities by spectroscopic techniques. Chapter 7describes the development and validationof LC methods for separation and determination of RSenantiomersof Citalopramhydrobromide and Bicalutamide using polysaccharide chiral stationary phases.
CHAPTER1
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.
CHAPTER2
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)-acetonitrileand 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 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 0.2-1.0N HCl, 0.05-0.5N 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. 3Typical 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
Isolation and Characterization of Process Related Impurities Including the Degradation Products of CitalopramHydrobromide, 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, citalopramhydrobromide (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).
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.
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
Reversed Phase HPLC Separationand 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]cycloheptene2-oxide (VI)as shown in Fig. 7 were separated and determined by HPLC on a BDS HypersilC18column 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 0.2-1.0N HCl, 0.05-0.5N 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 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) and5-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).
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 (1.74RRT) (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).
Fig. 11. ESI-MS/MS fragmentation patterns for A) I, B) II C) III D) VIand 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.
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 dihydrogenortho-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 dihydrogenortho-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 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).
CHAPTER 7
Enantiospecific Resolution of CitalopramHydrobromide and Bicalutamide by HPLC on Polysaccharide Based Stationary Phases Connected with Ultraviolet and Polarimetric Detectors in series
Chiral liquid chromatographic separation of citalopramhydrobromide (CIT) and bicalutamide (BCT) (Fig. 16) have been described on Chiralpak AD-H and Chiralcel OD-H columns. Chiralcel OD-H column containing amylosetris-(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 amylosetris-(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 enantiomersof citalopram and S-clopidogrel (Internal
standard) and enantiomers of bicalutamide.
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.
1