143

Chapter – VI

Identification, separation and characterization

of impurities present in olanzapine drug substance

and the impurities enriched through oxidative

degradation #

6.1.0 Introduction

Olanzapine, chemically known as 2-Methyl-4-(4-methyl-1-piperazinyl)-10H-

thieno [2, 3-b] [1, 5] benzodiazepine, is a potential antipsychotic agent used in

chemotherapy. It has been approved by the food and drug administration (FDA) and it

is one of the most commonly used atypical anti psychotics. It is used in the treatment

of schizophrenia, acute mania in bipolar disorder, agitation associated with

schizophrenia and bipolar disorder. Chemical structure of olanzapine is:

# Part of this research work has been published in ACTA CHROMATOGRAPHICA, 20, 1 (2008) 81-93.

144

N

NH S

N

N

CH3

CH3

IUPAC name: 2-methyl-4-(4-methylpiperazin-1-yl)-10H-thieno[2,3-b][1,5] benzodiazepine. (Olanzapine)

In order to achieve a high level of safety and effectiveness of pharmacotherapy,

the requirements on quality of active pharmaceutical ingredients are growing [1, 2].

The investigation of the impurities present in the drug substance represents an

important issue in drug quality evaluation. The impurities present in the drug

substance may be formed because of the ageing or the drug is undergoing various

stresses during the manufacturing process. Many environmental conditions, for

example, heat, light, humidity as well as ability of drug substance for hydrolysis and

also oxidation can play an important role in formation of drug related impurities.

Stress testing of a drug substance can help to enrich the impurities present in drug

substance which can be used to isolate and characterize the impurities and provide

important information about the inherent stability of the drug substance under

hydrolytic, oxidative and photolytic conditions [3]. Several HPLC (high performance

liquid chromatography) procedures for the determination of the olanzapine in body

fluids as well as in pharmaceutical compounds have been reported in the literature.

HPLC analysis has been applied for the determination of the drug in human plasma

[4], serum [5] and in rat plasma [6, 7] with electrochemical detection. Monitoring the

olanzapine in serum by liquid chromatographic atmospheric pressure chemical

ionization mass spectrometry [8], and also for the pharmaceutical compounds [9, 10]

145

has been reported. To the best of our knowledge, the impurity profiling study and the

formation of impurities in oxidative condition has not been reported. In the present

research work, the author describes the identification, separation, isolation of

olanzapine impurities by preparative HPLC; and also after enriching the impurities by

exposing the drug substance to oxidative stress condition; and characterization of both

types of impurities using FT-IR (fourier transform infrared spectrometer), LC-

MS/MS (liquid chromatography-mass spectrometer) and FT-NMR (fourier transform

nuclear magnetic resonance spectrometer).

6.2.0 Experimental details

6.2.1 Reagents and samples

The sample of olanzapine bulk drug was received from Jubilient Organosys,

Mysore, India. HPLC grade methanol, acetonitrile and A.R.grade orthophosphoric

acid (85% v/v) were obtained from Spectrochem (India). A.R. grade disodium

hydrogen orthophosphate, A.R. grade sodium hydroxide, L.R.grade hydrochloric acid

and L.R.grade hydrogen peroxide (30% solution) were obtained from Rankem, India.

MilliQ water was obtained from Elix Millipore water purification system (Millipore,

India). GC grade, dichloromethane, was obtained from Merck, India.

6.2.2 High performance liquid chromatography (Analytical)

Chromatographic separation was performed on a Waters HPLC system

equipped with alliance 2695 low pressure quaternary gradient pump along with

degasser, photo diode array detector and auto sampler (Waters corporation, USA).

The data were collected and processed using Empower 2 version 6.00.00.00 software.

An Inertsil ODS 3V (150 * 4.6 mm, 5µ particle size, 100 Å pore size) (GL Sciences,

146

Japan) column was employed for the separation of the impurities from olanzapine. A

linear gradient program was optimized for the separation of impurities from

olanzapine bulk drug, where the initial mobile phase consisted of mobile phase A (10

mM disodium hydrogen phosphate, pH adjusted to 7.4 with orthophosphoric acid) and

mobile phase B (acetonitrile) in a ratio of 70:30 (v/v) for 15 min. Subsequently, the

percentage of mobile phase B was increased from 30 to 80 up to 10 min. The same

ratio was held for 5 min, and brought back to initial condition within 5 min. The

column was allowed to get equilibrated for 10 min. before performing the next

injection. Chromatography was performed at room temperature (25° ± 2°C) using a

flow rate of 1.0 ml min-1. The column eluent was monitored at 271 nm. Sample

concentration was about 1.0 mg ml-1 prepared in acetonitrile.

6.2.3 High performance liquid chromatography (preparative)

An Agilent preparative HPLC system equipped with 1200 series

pump(DE55055052), photo diode array detector(DE60555523), auto sampler(DE60555110)

and 1200 series preparative fraction collector(DE60555117) (Agilent technologies,

USA) were used. The data were collected and processed using Agilent “Chemstation”

1200 series software. A 150 * 20 mm i.d. column packed with 5µ particle size Inertsil

ODS 3 (GL Sciences, Japan) was employed for loading the sample. An analytical

method was modified to resolve these impurities followed by scaling up the same

method for preparative HPLC to collect the required fractions. A linear gradient

program was optimized for the separation of impurities from olanzapine bulk drug,

where the initial mobile phase was a mixture of mobile phase A (water) and mobile

phase B (acetonitrile) in the ratio of 80:20 (v/v) for about 5 min. Subsequently, the

percentage of mobile phase B in the mobile phase ratio was increased from 20 to 60

147

in 10 min. duration. The same ratio was held for 4 min, and brought back to initial

condition within 4 min. The flow rate was set at 20 ml min -1. The UV detection

wavelength was 271 nm. Approximately 200 mg ml-1 of sample solution was

prepared in 10% hydrogen peroxide (v/v) solution and kept at room temperature for

24 hours. From the above sample solution, 900µl was injected into the preparative

HPLC system.

6.2.4 Mass Spectroscopy (LC-MS/MS)

LC-MS/MS analysis was performed on API 2000 (Applied Biosystems)

coupled with HPLC system consisting of Agilent 1100 series low pressure quaternary

gradient pump along with degasser, auto sampler and the column oven (Agilent

Technologies, USA). The analysis was done in a positive electrospray ionization

mode with turbo ion spray interface under the following conditions. Ion source

voltage (IS) = 5500 V; declustering potential (DP) = 70 V; focusing potential (FP) =

400V; capillary temperature = 350°C; entrance potential (EP) = 10 V with nitrogen as

nebuliser gas at 40 psi and nitrogen curtain gas at 25 psi. An Inertsil C18 (150 * 4.6

mm, 5µ particle size, 100 Å pore size) (GL Sciences, Japan) column was used for the

separation. The separation of impurities for mass analysis was done in a manner

similar to linear gradient programme mentioned in section 6.2.3. The flow rate was set

at 1.0 ml min -1. The UV detection wavelength was set as 271 nm. The column eluent

was introduced into the electron spray ionization (ESI) chamber of the mass

spectrometer with the split ratio of 3:7. Mass fragmentation studies were carried out

by maintaining normalized collision energy at 35 eV with the range of m/z 50-1000

amu. The representative mass spectra for olanzapine, impurity I, II and III are given

below (Figure 6.1, 6.2 , 6.3 and 6.4).

148

Figure 6.1: Mass spectrum of olanzapine drug substance.

Figure 6.2: Mass spectrum of impurity I.

Figure 6.3: Mass spectrum of impurity II.

Figure 6.4: Mass spectrum of impurity III

149

6.2.5 NMR Spectroscopy

The 1H NMR, 13C NMR and DEPT (distortionless enhancement by polarization

transfer) NMR studies of the impurities were carried out at precessional frequencies

of 399.939 MHz and 100.574 MHz, respectively. The solvent dimethylsulfoxide-d6

was used at 25°C on a Varian AS-400 FT NMR spectrometer. The 1H and 13C, DEPT

chemical shifts are reported on the δ scale in ppm, relative to tetramethyl silane

(TMS) (δ 0.00) present in dimethylsulfoxide-D6 solvent. Deuterium exchange

experiment was performed to confirm the exchangeable protons. The recorded 1H

NMR and 13C NMR spectra for olanzapine, impurity I, II and III are given below

(Figure 6.5, 6.6, 6.7, 6.8, 6.9, 6.10, 6.11 and 6.12).

Figure 6.5: 1H NMR spectrum of olanzapine drug substance recorded in d6-DMSO

Figure 6.6: 13C NMR spectrum of olanzapine drug substance recorded in d6-DMSO

150

Figure 6.7: 1H NMR spectrum of impurity I recorded in d6-DMSO

Figure 6. 8: 13C NMR spectrum of impurity I recorded in d6-DMSO

Figure 6.9: 1H NMR spectrum of impurity II recorded in d6-DMSO

151

Figure 6.10: 13C NMR spectrum of impurity II recorded in d6-DMSO

Figure 6.11: 1H NMR spectrum of impurity III recorded in d6-DMSO

Figure 6.12: 13C NMR spectrum of impurity III recorded in d6-DMSO

152

6.2.6 FT-IR

IR spectra of the olanzapine and its impurities were recorded in the solid state

as KBr dispersion (Figure 6.13, 6.14, 6.15 and 6.16) using Perkin Elmer spectrum 100

series FT-IR spectrophotometer with DRS (Diffuse Reflectance Sampler) technique.

Figure 6. 13: Infrared spectrum of olanzapine drug substance recorded in the solid

state as KBr dispersion

Figure 6.14: Infrared spectrum of impurity I recorded in the solid state as KBr

dispersion.

153

Figure 6.15: Infrared spectrum of impurity II recorded in the solid state as KBr

dispersion.

Figure 6.16: Infrared spectrum of impurity III recorded in the solid state as KBr

dispersion.

154

6.2.7 Trials to enrich impurities present in the drug substance

All stress test trials were carried out on a single batch olanzapine. The drug

concentration in solutions was about 10 mg mlֿ¹. The photo stability test was carried

out in chemically inert and UV-VIS transparent quartz containers using xenon lamp.

The conditions of the stress studies are presented in the Table-6.1.

Table 6.1: The different stress test trials conducted to enrich the impurities present in the

drug substances by hydrolysis (under acidic and alkaline conditions), oxidation and

photolysis of olanzapine drug substance .

Stress test

condition

solvent Time(h) Temperature

Acidic 0.1M HCl 6 Reflux

0.1M HCl 12 Reflux

Basic 0.1M NaOH 6 Reflux

0.1M NaOH 12 Reflux

Oxidation 3 % (v/v) H2O2 6 Room temperature (25°C ± 2°C)

3 % (v/v) H2O2 24 Room temperature (25°C ± 2°C)

10 % (v/v) H2O2 Room temperature (25°C ± 2°C)

Photolysis UV light 24 Room temperature (25°C ± 2°C)

UV light 72 Room temperature (25°C ± 2°C)

155

6.3.0 Results and discussion

6.3.1 Detection of impurities by HPLC

Olanzapine drug substance sample solution was prepared in the acetonitrile

solvent to get the resultant concentration of about 1 mg mL-1. The sample was

analyzed using the smobile phase system as described under the section 6.2.2. On the

chromatograms, along with the peak due to olanzapine, three other peaks were

observed and these peaks were marked as impurity I, II and III. The retention times of

the impurities I, II, III and olanzapine were approximately 12.5, 15.5, 25.7 and 22.5

min., respectively. The typical chromatogram (Figure 6.17) showing all the peaks

appeared distinctly. Percent area on the chromatogram for the three peaks (other than

drug) ranged from 0.13%-0.4%. Resolution for each peak was more than 2.0 from any

other nearest peak. Further, the peaks were checked for purity to ascertain any co-

eluting peaks, and it was found that all the peaks were pure. This indicated that there

are three potential impurities that may be present in olanzapine.

Figure 6.17: Analytical HPLC chromatogram of olanzapine and its impurities I, II & III.

156

Several trials were done to enrich the impurities in the olanzapine drug samples

since the quantity of olanzapine drug substance obtained was very small (about 900

mg olanzapine drug substance obtained from the manufacturer). As mentioned in

section 6.2.7, the olanzapine drug substance was exposed to different stress conditions

and these stressed samples were diluted to the required concentration and injected to

analytical HPLC using the linear gradient system mentioned in section 6.2.2. Impurity

enrichment was not observed in olanzapine sample when it was subjected to light,

acid as well as base hydrolysis. Olanzapine impurities I, II & III were enriched under

oxidative condition with hydrogen peroxide (Figure 6.18). Peak purity test result

confirmed that olanzapine peak is pure in all the analyzed stress samples. As

expected, major impurities were obtained from olanzapine under oxidative stress

condition.

Figure 6.18: Analytical HPLC chromatogram of enriched olanzapine impurities I, II & III

under oxidative stress condition.

157

6.3.2 LC-MS/MS analysis

LC-MS/MS analysis of olanzapine bulk drug sample was performed using the

linear gradient system as described in section 6.2.3. Results of LC-MS/MS analysis

revealed that the impurities I, II and III exhibited the molecular ion peak at m/z

(M+1): 247.4, 231.4 and 459.3 amu (Figure 6.2, 6.3 and 6.4) (Table 6.2). The

fragmentation pattern of these impurities (Figure 6.19, 6.20 and 6.21) indicated that

the oxidation occurred at the diazepine ring in the olanzapine molecule.

Figure 6.19 : Fragmentation pattern of the impurity I

Figure 6.20 : Fragmentation pattern of the impurity II

158

Figure 6.21 : Fragmentation pattern of the impurity III

6.3.3 Isolation of the impurities by preparative HPLC

A simple reverse phase solvent system mentioned in section 6.2.3 was used for

the isolation of impurities. In this solvent system, olanzapine eluted at about 7.9 min.

whereas the impurities I, II & III were eluted at about 5.6, 12.0. and 15.2 min.,

respectively. Approximately, 0.6 g of oxidized olanzapine bulk drug sample was

loaded onto the preparative HPLC and the impurity fractions were collected

separately and concentrated at room temperature under high vacuum on a Buchii

Rotavapour Model R124. The remaining aqueous layer was subjected to liquid-liquid

extraction using dichloromethane. The organic layer was again concentrated under

high vaccum to obtain the impurities in solid form. The solids thus obtained were re-

analyzed to check the purity of individual isolated impurities (impurity I, II and III)

on analytical HPLC (Figure 6.22, 6.23 and 6.24). The purity of isolated impurities

was found to be in the range 98.0 - 99.0 % (area percent), which was relatively good

enough for carrying out spectroscopic experiments.

159

Figure 6.22: Analytical HPLC chromatogram of isolated impurity I

Figure 6.23: Analytical HPLC chromatogram of isolated impurity II

Figure 6. 24: Analytical HPLC chromatogram of isolated impurity III

160

6.3.4 Structural elucidation of impurity I

The mass, IR and NMR spectral analysis data of the impurities were compared

with that of olanzapine. Major mass fragments are given in Table 6.2, which were

obtained from LC-MS/MS analysis.

Table 6.2: Mass spectral data and major fragments of Olanzapine and its impurities

Olanzapine (M+1) 313.2, 282.1, 256.1, 213, 198.2, 186.2, and 84.1

Impurity I (M+1) 247.4, 213.8, 134.2, 113.0 and 85.0

Impurity II (M+1) 231.4, 214.0, 198.2, 186.1 and 169.1

Impurity III (M+1)459.3, 326.3, 293.1 and 229.2

The FT-IR, 1H, 13C and DEPT NMR spectral data are given in Table-6.3 &

6.4. Based on the above data, it is inferred that the impurities exhibited the chemical

structures without methyl piperazine ring compared to that of olanzapine. Impurity I

shows oxidation at diazepine ring in olanzapine and formed a ketooxime compound.

The LC-MS/MS analysis data of this product exhibited the molecular ion peak (M+1)

at m/z 247.4 amu and the fragmentation pattern also confirmed to the structure

(Figure 6.25). The IR spectrum values shown at 3196,1695,1592,1481 and 1320 cm-1

reveal that the compound contains NH, C=O, C=C, C=N and C-N groups,

respectively.

161

Table-6.3: IR spectral data of Olanzapine and its impurities

The 1H NMR spectrum of the impurity I (Figure 6.7) showed two broad

singlets at δ 9.67 and 10.97 ppm, which corresponds to the proton at amide (HN-

C=O) and oxime (N-OH), respectively. The signals corresponding to the aromatic

protons appeared as multiplet from δ 6.51 to 7.02 ppm. Furthermore, the amidic and

oxime protons exchanged with deuterium by D2O exchange experiment. In olanzapine

1H NMR (Figure 6.5)there were two multiplets at δ 2.38 and 3.32 ppm corresponding

to piperazine ring attached with the olanzapine main ring but in impurity I, piperazine

ring was not seen, so it was confirmed that oxidation occurs at diazepine ring and a

stable impurity I formed by keto-enol tautomerism. Similarly, the 13C NMR spectrum

(Figure 6.8) of the impurity I showed signal corresponding to the carbonyl

carbon(C=O) at about δ 155.1 ppm, and N-OH attached quaternary aromatic carbons

of benzene and thiophene ring shifted to δ 137.4 and 151.0 ppm, respectively, where

as in olanzapine these quaternary carbons appeared at δ 144.6 and 154.0 ppm,

respectively. In olanzapine bulk drug piperazine ring carbons exhibited two peaks at δ

47.1 and 55.1 ppm (Figure 6.6), where as these two peaks were absent in the impurity

I. Based on the above data it is concluded that the structure of the impurity I has been

characterized as 10-Hydroxy-2-methyl5,10-dihydro-4H-benzo[b]thieno[2,3-

e][1,4]diazepine-4-one (Figure 6.25).

Olanzapine Impurity -I Impurity -II Impurity -III Group assignment

3239 3196 3282 3195 NH and OH Stretching

2929 3064 3034 3063 C-H Stretching

- 1695 1637 1702 C=O Stretching

1587 1592 1595 1581 C=C Stretching

1421 - - 1433 C=N Stretching

1287 1320 1351 1393 C-N Stretching

162

Table 6.4: 1H NMR and 13C NMR assignments for Olanzapine and impurities-I,II and III

Position Olanzapine Impurity I Impurity II Impurity III

ppm/ 1H / multiplicity 13C DEPT

ppm/1H / multiplicity 13C DEPT

ppm/ 1H / multiplicity 13C DEPT

ppm/ 1H / multiplicity 13C DEPT

1 2.20 / 3H / s 15.7 15.7 2.38 / 3H / s 16.9 16.9 2.20 / 3H / s 15.01 15.01 2.23 / 3H / s 15.9 15.9

2 2.26/ 3H/ s 46.4 46.4

3 2.38 / 4H / m 55.1 55.1

4 3.32 / 4H / m 47.1 47.1

5 6.63 / 1H / s 123.2 123.2 6.51 / 1H / s 109.7 109.7 6.58 / 1H / s 124.3 124.3 6.62 / 1H / s 111.3 111.3

6 7.58 / 1H / brs 8.84 / 1H /brs 6.90 / 1H / brs

7 128.7 129.2 130.4 126.2

8 154.0 151.0 155.5 153.1

9 118.8 105.6 115.9 116.1

10 158.1 155.3

11 141.3 132.4 125.3 132.3

12 144.6 137.4 130.4 133.3

Aromatic

6.67-6.84/

4H/m

119.5,123.2

123.9,124.1

119.5,123.2

1123.9,124.1 6.51-7.02/ 4H/m

110.3,119.0

122.2,123.0

110.3,119.0

122.2,123.0 6.76-6.89/ 4H/m

119.4,123.1

123.9,124.6

119.4,123.1

123.9,124.6 6.37-6.95/ 8H/m

108.4,119.1

122.2,123.0

123.2,124.7

125.5,127.4

108.4,119.1

122.2,123.0

123.2,124.7

125.5,127.4

14 9.67 / 1H /brs 9.06 / 1H /brs 119.4 9.58 / 1H / brs

15 155.1 165.1 165.1

16 10.97 / 1H /brs

17 2.49 / 3H / s 16.9 16.9

18 6.72 / 1H / s 125.7 125.7

19,20 140.4,135.3

21,22 137.0,124.4

Note:. s=singlet; m=multiplet; brs=broad singlet; for numbering refer figure 6..25.

163

N

NH

S CH3

N

N

CH3

1

2

5

6 78

9

1011

12

3

34

4

HN

NS CH3

HO

O

5

78

911

12

14

15

16

1

Olanzapine Impurity I

HN

NH

S CH3

O

5

6 78

911

12

14

15

1

HN

NS

CH3N

NH S

CH3

O

O

5

67

8

9

1011

12

14

1516

1720

1

21 19

22

Impurity II Impurity III

N

NH

S CH3

1

5

6

78

9

1011

12

Intermediate (Loss of piperazine ring)

Figure 6.25: Chemical structures of olanzapine, impurity I, impurity II, and impurity III and

Intermediate (Loss of piperazine ring)

164

6.3.5 Structural elucidation of impurity II

Impurity II showed oxidation at diazepine ring in olanzapine and formed a

stable keto compound by keto-enol tautomerism. The LC-MS/MS analysis data

(Table 6.2) of this product exhibited the molecular ion peak (M+1) at m/z 230.4 amu

and the fragmentation pattern also confirmed the structure (Figure 6.25). The Peaks in

IR spectrum (Figure 6.15) at 3282,1637,1595,1351 cm-1 accounted for the NH, C=O,

C=C and C-N groups, respectively. The 1H NMR spectrum of impurity II ((Figure

6.9) showed two broad singlets at δ 8.84 and 9.06 ppm, which correspond to the

protons at 2°amine(N-H) and amide(HN-C=O), respectively. The signals

corresponding to the aromatic protons appeared as multiplet from δ 6.76 to 6.89 ppm.

The protons at 2º amine and amide exchanged with deuterium by D2O exchange

experiment. In olanzapine 1H NMR spectrum ((Figure 6.5), there were two multiplets

at δ 2.38 and 3.32 ppm which correspond to the piperazine ring attached with the

olanzapine main ring but, in impurity II piperazine ring was not seen. So it was

confirmed that oxidation occured at diazepine ring and a stable impurity II formed by

keto-enol tautomerism. Similarly, the 13C NMR spectrum of the impurity II (Figure

6.10) showed signal corresponding to the carbonyl carbon (C=O) at about δ 165.1

ppm. In olanzapine bulk drug piperazine ring exhibited two peaks at δ 47.1 and 55.1

ppm, where as these two peaks were absent in impurity II. Based on the above data it

is concluded that the structure of the impurity II can be characterized as 2-Methyl-5,

10-dihydro-4H-benzo[b]thieno[2,3-e][1,4]diazepin-4-one (Figure 6.25).

6.3.6 Structural elucidation of impurity III

Impurity III again showed oxidation at diazepine ring in olanzapine and formed

a dimeric compound. The LC-MS/MS analysis data (Table 6.2) of this product

165

exhibited the molecular ion peak (M+1)at m/z 459.4 amu and the fragmentation

pattern also confirmed the structure(Figure 6.25). The IR peaks (Figure 6.16) at 3195,

1702, 1581, 1433 and 1393 cm-1 revealed that the compound contained NH, C=O,

C=C and C-N groups, respectively. The 1H NMR spectrum of impurity III (Figure

6.11) showed two broad singlets at δ 9.58 and 6.90 ppm, which correspond to the

protons at amide(HN-C=O) and 2°amine(NH), respectively. The signals

corresponding to the aromatic protons appeared as multiplet from δ 6.37 to 6.95 ppm.

There were two singlets at δ 2.23 and 2.26 ppm which indicate the two methyl groups

in the impurity are same as in olanzapine (methyl protons at δ 2.20 and 2.26 ppm).

The protons at amide and 2° amine groups exchanged with deuterium by D2O

exchange experiment. Similarly, the 13C NMR spectrum of the impurity III (Figure

6.12) showed signal at δ 165.1 ppm which correspond to the carbonyl carbon (C=O).

Olanzapine bulk drug showed two signals at δ 15.7 and 46.4 ppm (Figure 6.5)

correspond to two methyl groups in olanzapine attached with thiophene ring and

nitrogen in piperazine ring, respectively, whereas in impurity III the two signals

appered at δ 15.9 and 16.9 ppm which reveals that two methyl groups are present at

thiophene ring only, but not attached to any nitrogen. Olanzapine bulk drug exhibited

two peaks at δ 47.1 and 55.7 ppm (Figure 6.6), whereas these two peaks were absent

in impurity III which further substantiated the absence of piperazine ring in the

molecule. Based on the above data it is concluded that the structure of the impurity III

can be characterized as 2-Methyl-10-(2-methyl-10H-benzo[b]thieno[2,3-

e][1,4]diazepin-4-yloxy)-5,10-dihydro-4H-benzo[b]thieno[2,3-e]diazepin-4-one

(Figure 6.25).

166

6.3.7 Formation of the impurities

The three impurities I, II &III were formed from olanzapine after the loss of

methyl piperazine from the parent drug. Probably, the impurities I, II and III were

formed by radical mechanism. Impurity I may be formed by the addition of oxygen

radical to the C10, and N6 of the intermediate product (Figure 6.25). However,

impurity II may be formed by the addition of oxygen radical at C10 of the

intermediate and followed by the distribution of electron towards the formation of

stable keto compound II. But, the impurity III may be formed by the dimerization of

impurities I and II by radical mechanism. The chemical pathway depicting the

formation of impurities I, II and III is depicted below (Figure 6.26).

NH

N

S

H

NH

N

S

OH

H+

NH

N

S

OH

N

NH

S

O

H

N

N

S

OH

OH

OH2

O' O'

Impurity I

NH

N

S

H

NH

N

S

OH

H+

NH

N

S

OH

NH

NH

S

OO'

Impurity II

167

O

NH

NS

N

NH S

ONH

N

S

OH N

NH

S

O

H

Impurity III

Figure 6.26: The chemical pathway showing the formation of impurities I, II &III.

6.4.0 Conclusion

Besides the impurity profiling of olanzapine drug substance, the present

research work details a HPLC method for separation of impurities from olanzapine,

preparative LC method for isolation of the impurities from the olanzapine drug

substance and also discusses the formation of impurities under oxidative stress

conditions. The identification, isolation, characterization and formation of the

impurities have been discussed in detail.

168

6.5.0 References

[1] Stability Testing of New Drug Substances and Products QIA (R2), International

Conference on Harmonization of Technical Requirements for Registration of

Pharmaceuticals for Human Use, August (2003)

[2] Stability Testing: Photo stability Testing of New Drug Substances and Products.

QIB, International Conference on Harmonization of Technical Requirements for

the Registration of Pharmaceuticals for Human Use, January (1998)

[3] S. Singh, M. Bakshi, Pharmaceutical Technology on-line, April (2000)

[4] Leon J. Dusei, L. Peter Hackett, Linda. M. Fellows, Kenneth F. Ilett, Journal of

Chromatography B, 774, 191 (2002)

[5] O.V. olesen and K. Linnet, Journal of chromatography B, 714 , 309 (1998)

[6] J.A.Chiu and R.B. franklin, Journal of pharma & Biomed.Anal., 14 , 609 (1996)

[7] J.T. catlow, R.D. Barton, M. Clemens, T.A. Gillespic, M. Goodwin, S.P.Swanson,

Journal of Chromatography B, 668 , 85 (1999)

[8] M.J. Bogusz, K.D. Kruger, R.D. Maier, R. Erkwoh, F. Juchtenhagen, Journal of

Chromatography B, 732 , 257 (1999)

[9] L.A. Larew, B.A. Olsen, J.D. Stafford, M.V. Wilhelm, Journal of

Chromatography A, 692 , 183 (1995)

[10] M.A. Raggi, G. Lasamenti, R. Mandroli, G.Izzo, & E. Kenndler, Journal of

Pharma. and Biomed. Anal., 23 , 973 (2000).