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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).