Development of new analytical methods for impurity profiling of

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12 Νοε 2013 (πριν από 3 χρόνια και 8 μήνες)

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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 p
rocess related impurities
.
Chapter
4
describes the

s
eparation

and determination of process
-
related substances of
an
antidepressant
Mirtazapine by reversed
-
phase HPLC.
Chapter
5

describes

the
d
evelopment of
a
RP
-
HPLC
method for impurity profile study of
an
antipsychotic drug Olanzapine and characterization of
process impurities by LC
-

ESI
-
MS
-
MS,
1
H
-
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 validat
ion

of
LC method
s for separation and
determination of
R
&
S

enantiomers
of Citalopram hydrobro
mide and
Bicalutamide using
polysaccharide chiral stationary phases.





















CHAPTER

1



Impact of impurities on quality and safety of psychiatric and cancer
D
rugs



1

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 an
d 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 N
orepinephrine 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 (
I
I), [2
-
cyclohex
-
1
-
enyl
-
2
-
(4
-
methoxy
-
phenyl)
-
ethyl]
-
dimethyl
-
amine (IV)

(1
-
h
ydroxy
-
cyclohexyl)
-
(4
-
methoxy
-
phenyl)
-
a
cetonit
r
ile

and
4
-
methoxy phenyl
acetonitrile

(
V
)

and
(1
-
h
ydroxy
-
cyclohexyl)
-
(4
-
methoxy
-
phenyl)
-
acetonit
r
ile (VI)

as shown in
Fig. 1 were separated and determined by HPLC.


H
3
C
O
N
O
H
C
H
3
C
H
3
H
3
C
O
C
N
H
3
C
O
O
H
C
N
H
3
C
O
N
O
H
C
H
3
H
H
3
C
O
N
O
H
H
H
H
3
C
O
N
C
H
3
C
H
3
(

I
I

)
(

I
I
I

)
(

I

)
(

I
V

)
(

V

)
(

V
I

)


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 pump
ed 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
-
5C
18

column,
temperature of
column

40
0
C±2
0
C
and detection at 225
nm (PDA).
The effects of organic modifier (i,e; acetonitrile and

2

methanol) and concentration (0.1% to 0.3%) and pH (3.0 to 6.0) of DEA buffer and
temperature of column (25
0
C to 40
0
C) on retention and resolution were studied to optimize
the chromatographi
c conditions.

H
3
C
O
N
O
H
H
C
H
3
H
3
C
H
3
C
O
N
O
H
C
H
3
H
3
C
H
H
H
C
l
H
3
C
O
N
C
H
3
H
3
C
-
H
2
O
+
+


H
C
l




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, 60
o
C temperature for 15 days and ii) extreme
conditions such as 0.2
-
1.0

N HCl, 0.05
-
0.5

N NaOH, and 3% H
2
O
2
.

Under acidic

conditions
one

degraded product (
IV
) w
as

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 ch
aracterized using modern spectroscopic techniques such as
UV, FT
-
IR,
1
H 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
r
2

0.9999 for
VNX
and 0.5
-
5.0 µg/ml with r
2

0.9942 for impurities), limit of detection (LOD) and limit of
quantitation (LOQ) and specificity. The developed method

was found to be selective,
sensitive, precise and stability indicating. The method was applied to determine VNX and its
process
-
related substances in bulk drugs and pharmaceutical formulations.

CHAPTER 3


3

Isolation and Characterization of Process Related
I
mpurities
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.,

i
ts 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
-
b
romo
-
2
-
hydroxymethyl
-
phenyl)
-
4
-
dimethylamino
-
1
-
(4
-
fluoro
-
phenyl)
-
butan
-
1
-
ol (VI), [3
-
[1
-
(4
-
f
luoro
-
phenyl)
-
1, 3
-
dihydro
-
isobenzofuran
-
1
-
yl]
-
propyl]
-
dimethyl
-
amine (VII),
1
-
(3
-
d
imethylamino
-
propyl)
-
1
-
(4
-
fluoro
-
phenyl)
-
1,3
-
dihydro
-
isobenzofuran
-
5
-
carbonitrile
-
N
-
oxide (VIII)

and

[
3
-
[5
-
b
romo
-
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.



O
H
2
N
O
C
N
C
H
3
C
H
3
F
N
C
N
C
H
3
C
H
3
F
O
H
O
O
C
N
C
H
3
C
H
3
F
O
N
C
N
C
H
3
C
H
3
F
O
N
C
H
3
C
H
3
F
O
B
r
N
C
H
3
C
H
3
F
O
H
O
H
B
r
N
C
H
3
C
H
3
F
O
H
O
H
O
H
N
C
N
C
H
3
C
H
3
F
O
N
C
N
C
H
3
C
H
3
F
O
[
I
]
[
I
I
]
[
I
I
I
]
[
V
]
[
V
I
]
[
V
I
I
]
[
V
I
I
I
]
[
I
V
]
[
I
X
]
H
B
r

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 acco
rding 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 50
0
C±1
0
C and
detection at 225 nm (PDA).
The effects of organic modifi
er (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 (35
0
C to 50
0
C) on retention and resolution were studied to optimize the
chromatographic conditions (Fig. 5).


4



Fig.
5

Typica
l 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, 60
o
C temperature for 15 days and ii) extreme conditions such
as 0.2
-
1.0 N HCl, 0.05
-
0.5
N NaOH, and 3% H
2
O
2
.

Under alkaline conditions two degraded products (I and II) and
under peroxide conditions one degraded impurity (VI
II) 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,
1
H
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 impuri
ties)

linearity (range 10
-
300 µg/ml with r
2

0.999 for CIT
and 0.5
-
10 µg/ml with r
2

0.9867 for impurities), limit of detection and limit of quantitation.
The developed method was found to be selective, sensitive, precise and stability indicating.
The method

was applied to determine CIT (V) and its process
-
related substances in bulk
drugs and pharmaceutical formulations.






5

A
)
B
)
C
)
O
N
F
N
F
F
O
F
O
F
-
H
2
O
m
/
z

3
0
0
m
/
z

2
8
2
m
/
z

2
5
5
m
/
z

2
3
7
m
/
z

1
4
1
m
/
z

9
1
m
/
z

1
0
9
+
H
+
H
+
H
+
H
+
H
+
H
-
H
N
-
(
C
H
3
)
2
-
C
2
H
2
-
H
N
-
(
C
H
3
)
2
m
/
z

1
5
9
m
/
z

2
1
1
D
)
E
)
F
)
O
N
C
N
F
N
C
F
O
N
C
F
N
C
O
N
C
F
N
C
m
/
z

3
4
1
m
/
z

2
8
0
m
/
z

2
6
2
m
/
z

1
6
6
m
/
z

1
1
6
m
/
z

1
0
9
+
H
+
H
+
H
+
H
+
H
-
H
N
-
(
C
H
3
)
2
-
H
O
N
-
(
C
H
3
)
2
m
/
z

1
8
4
O
N
C
N
F
N
C
N
F
N
C
F
O
H
N
C
F
N
C
O
N
C
F
N
C
-
H
2
O
m
/
z

3
2
5
m
/
z

3
0
7
m
/
z

2
8
0
m
/
z

2
6
2
m
/
z

1
6
6
m
/
z

1
1
6
m
/
z

1
0
9
+
H
+
H
+
H
+
H
+
H
+
H
-
H
N
-
(
C
H
3
)
2
-
H
N
-
(
C
H
3
)
2
m
/
z

1
8
4
O
H
O
N
C
N
F
N
C
N
F
N
C
F
O
N
C
F
N
C
O
N
C
F
N
C
-
H
2
O
m
/
z

3
2
5
m
/
z

3
0
7
m
/
z

2
8
0
m
/
z

2
6
2
m
/
z

1
6
6
m
/
z

1
1
6
m
/
z

1
0
9
+
H
+
H
+
H
+
H
+
H
+
H
-
H
N
-
(
C
H
3
)
2
-
H
N
-
(
C
H
3
)
2
m
/
z

1
8
4
O
H
2
N
O
C
N
F
H
2
N
O
C
N
F
H
2
N
O
C
F
O
H
2
N
O
C
F
F
O
H
2
N
O
C
O
H
2
N
O
C
F
H
2
N
O
C
-
H
2
O
m
/
z

3
4
3
m
/
z

3
2
5
m
/
z

2
9
8
m
/
z

2
0
2
m
/
z

2
8
0
m
/
z

2
6
3
m
/
z

1
8
4
m
/
z

1
3
4
m
/
z

1
0
9
+
H
+
H
+
H
+
H
+
H
+
H
+
H
-
H
N
-
(
C
H
3
)
2
-
N
H
3
-
H
N
-
(
C
H
3
)
2
N
C
N
F
-
H
2
O
O
N
C
N
F
O
H
O
H
m
/
z

3
4
3
+
H
m
/
z

3
2
5
+
H
G
)
O
H
O
O
C
N
F
H
O
O
C
N
F
H
O
O
C
F
O
H
O
O
C
F
F
O
H
O
O
C
O
H
O
O
C
F
H
O
O
C
-
H
2
O
m
/
z

3
4
4
m
/
z

3
2
6
m
/
z

2
9
9
m
/
z

2
0
3
m
/
z

2
8
1
m
/
z

2
6
3
m
/
z

1
8
5
m
/
z

1
3
5
m
/
z

1
0
9
+
H
+
H
+
H
+
H
+
H
+
H
+
H
-
H
N
-
(
C
H
3
)
2
-
H
N
-
(
C
H
3
)
2
-
C
6
H
5
F
-
C
2
H
2
-
C
6
H
5
F
-
C
6
H
5
F
-
C
6
H
5
F
-
C
6
H
5
F
m
/
z

2
3
7
-
C
2
H
2
-
C
6
H
5
F
-
C
6
H
5
F
-
C
6
H
5
F
-
C
6
H
5
F
-
C
6
H
5
F
-
C
6
H
5
F
-
C
6
H
5
F
F
O
+
H
m
/
z

2
3
7
F
O
+
H
-
H
2
O
F
+
H


Fig. 6.

ESI
-
MS/MS fragmentation patterns of A) I,
B)

II C) III, D) IV, E) V,
F) VII and

G) VIII.




6

CHAPTER 4

Reversed Phase HPLC Separation

and Determination of Process Related Substances of
Mirtazapine, a Noradrenergic and Specific Serotonergic Antidepressant



This chapter describes an isocratic reversed phase liquid chromatographic method for
monitoring of process related substances of mirtazapine (MTZ). The process related
impurities of

MTZ (V
)

viz., 1
-
methyl
-
3
-
phenyl
-
piperazine (I), 2
-
(4
-
m
ethyl
-
2
-
phenyl
-
pipe
razin
-
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
-
oxi
de

(VI)

as
shown in Fig. 7 were separated and determined by HPLC on a
BDS Hypersil
C
18

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 a
t 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
(25
0
C to 40
0
C) on retention and resolution we
re studied to optimize chromatographic
conditions (Fig. 8).


N
N
N
O
O
H
N
N
N
H
O
N
N
N
H
C
H
3
N
N
N
H
H
N
N
N
H
C
H
3
O
H
N
N
(

I

)
(

I
I

)
(

I
I
I

)
(

I
V

)
(

V

)
(

V
I

)

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 s
piked 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, 60
o
C temperature for 15 days and i
i) extreme conditions such as 0.2
-
1.0

N HCl, 0.05
-
0.5

N NaOH, and 3% H
2
O
2
.

Under peroxide
conditions
one

degraded product (
V
I
) w
as

formed

7

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 prod
uct (VI) were matched. The two impurities were isolated by
column
chromatography
and characterized using modern spectroscopic techniques such as UV, FT
-
IR,
1
H 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
-
2
00 µg/ml
with r
2

0.9999 for
MTZ

and 0.5
-
5.0 µg/ml with r
2

0.994
1

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,
1
H
-
NMR and FT
-
IR Spectroscopy




This chapter describes a gradient rever
sed 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
-
m
ethyl
-
10
-
(
4
-
methyl
-
4
-
oxy
-
piperazin
-
1
-
yl)
-
4H
-
3
-
thia
-
4,9
-
diaza
-
benzo[f]azulene

(II),
2
-
m
ethyl
-
4H
-
3
-
thia
-
4,9
-
diaza
-
benzo[f]azulen
-
10
-
ylamine hydrochloride

(IV),

2
-
amino
-
5
-
methyl
-
thiophene
-
3
-
carbonitrile (V),
2
-
(2
-
a
mino
-
phenylamino)
-
5
-
methyl
-
thiophene
-
3
-
carbonitrile (VII)

and

5
-
methyl
-
2
-
(2
-
nitro
-
phenylamino)
-
thiophene
-
3
-
carbonitrile (VIII)

as shown in Fig.

9

wer
e separated and determined by HPLC.
The HPLC
conditions developed were as follows; mobile phase:
A:
ammoniu
m acetate (0.2 M in H
2
O)
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 mi
n. 20% B; Inertsil ODS 3V column,
temperature of column 25
0
C±2
0
C 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).


8


N
H
N
S
N
H
2
.

H
C
l
S
C
H
3
N
C
H
2
N
S
N
H
N
O
2
N
C
N
H
N
S
N
N
N
O
2
F
N
H
N
+
+
K
O
H
,

E
t
h
a
n
o
l
S
n
C
l
2
,

H
C
l
,
E
t
h
a
n
o
l
T
o
l
u
e
n
e
/
D
M
S
O
S
N
H
N
H
2
N
C
N
H
N
S
N
N
H
N
H
N
S
N
N
N
H
H
N
O
H
N
N
S
N
N
N
H
N
S
O
x
i
d
a
t
i
o
n
S
i
d
e

p
r
o
d
u
c
t
S
i
d
e

p
r
o
d
u
c
t
S
i
d
e

p
r
o
d
u
c
t
N
H
H
N
I
I
I
V
I
V
I
I
I
I
I
I
V
V
V
I
I
I


Fig
. 9
. The scheme of reactions involved in the synthesis of OLZ (III) and formation
of


impurities I, II, VI and VII.




Fig.
10
. Typical

HPLC chromatograms of a) OLZ (III) (200 μg/ml) spiked with 2.5%



(w/w) of each of the impurities (I, II and IV
-
VIII); b), c), d) & e) Different process



samples of OLZ (III).



Different batches of OLZ were anal
yzed by developed HPLC and four impurities
having >0.1% area at retention
times 8.53 min (0.6
9

RRT)

(I)
, 10.12 (0.79 RRT)

(II),

22.22
(1.74

RRT)

(VI)

and 26.61 (2.0
9
RRT)
(VII)
were
did not match with any of the process
intermediates (Fig. 10). The unknow
n impurities (I, II, VI and VII) were isolated by column
chromatography and characterized using modern spectroscopic techniques such as UV, FT
-
IR,
1
H NMR and ESI
-
MS
-
MS. The ESI
-
MS
-
MS fragmentation profiles have been discussed
(Fig. 11).


9


N
H
N
S
N
N
H
N
H
N
S
N
N
H
N
S
N
H
2
N
H
N
S
N
N
H
N
S
N
H
N
S
N
H
N
S
N
N
O
N
H
N
S
N
N
H
N
S
N
N
N
H
N
S
N
N
H
H
N
H
N
S
N
H
2
S
N
N
H
N
N
S
N
N
H
N
H
2
S
N
N
H
N
S
N
N
H
N
H
N
N
S
N
H
N
N
N
N
H
N
S
S
N
N
H
N
S
N
N
H
N
S
N
N
H
H
N
N
S
N
H
N
S
H
N
N
S
N
H
N
S
H
N
N
S
N
H
N
S
N
H
N
N
S
N
H
N
S
N
H
S
N
N
H
N
H
2
S
N
N
H
N
N
m
/
z

2
9
9
m
/
z

2
8
2
m
/
z

2
3
9
m
/
z

2
5
6
m
/
z

2
1
3
+
H
+
H
+
H
+
H
+
H
-
N
H
3
-
C
2
H
2
-
C
2
H
2
-
2
C
2
H
2
-
N
H
2
-
C
4
H
7
N
-
C
2
H
5
N
-
C
2
H
5
N
-
C
4
H
9
N
2
+
m
/
z

2
3
0
m
/
z

2
8
5
-
C
2
H
2
-
C
2
H
4
O
+
H
+
H
+
H
+
H
+
H
m
/
z

2
3
0
m
/
z

3
1
1
m
/
z

2
5
4
-
C
2
H
9
N
O
-
C
5
H
9
N
O
-
C
5
H
7
N
-
H
2
O
A
)
m
/
z

3
1
3
m
/
z

2
8
2
m
/
z

2
5
6
m
/
z

2
3
0
-
C
2
H
2
-
C
2
H
2
-
C
H
3
N
H
2
-
-
C
3
H
5
N
H
2
-
-
C
5
H
7
N
H
2
+
H
+
H
+
H
+
H
C
)
B
)
m
/
z

3
2
9
-
2
C
2
H
2
m
/
z

9
9
+
H
+
H
+
H
+
H
m
/
z

4
5
1
m
/
z

4
4
2
m
/
z

2
5
6
m
/
z

2
3
9
m
/
z

4
7
7
m
/
z

4
6
8
+
H
+
H
+
H
+
H
+
H
m
/
z

5
1
1
-
2
N
H
3
-
N
H
3
-
C
2
H
2
-
C
2
H
2
-
C
2
H
2
-
C
4
H
7
N
-
N
H
3
-
C
2
H
5
N
m
/
z

2
3
0
+
H
-
2
C
2
H
2
-
C
1
6
H
1
5
N
S
-
C
1
4
H
1
0
N
2
S
-
C
1
2
H
1
1
N
3
S
-
C
1
2
H
8
N
2
S
-
C
2
H
2
-
C
1
2
H
1
0
N
2
S
-
C
1
2
H
8
N
2
S
+
H
m
/
z

2
9
7
m
/
z

2
8
2
S
N
C
N
S
N
H
N
H
2
C
N
D
)
m
/
z

2
1
3
m
/
z

2
3
0
+
H
+
H
-
N
H
3
E
)

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
-
0.4
8% for
OLZ

and 95.18
-
104.32% with R.S.D 0.87
-
3.85% for impurities) linearity (range 100
-
300 µg/ml
with r
2

0.9999 for
OLZ

and 0.5
-
10 µg/ml with r
2

0.9867 for impurities), limit of detection
(LOD) and limit of quantitation

(LOQ) and specificity. The developed method was found to
be selective, sensitive and precise. The method was applied to determine OLZ and its process
-
related substances in bulk drugs and pharmaceutical formulations.






10

CHAPTER 6


Isolation and Characteri
zation of Process Related Impurities and Degradation Products
of Bicalutamide, an Antiandrogen
-
Development of Impurity Profile
s 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
-
f
luoro
-
benzenesulfonyl)
-
2
-
hydroxy
-
2
-
methyl
-
propionic acid

(I),
N
-
(4
-
cyano
-
3
-
trifluoromethyl
-
phenyl)
-
2,3
-
dihydroxy
-
2
-
methyl
-
propionamide (II),
4
-
a
mino
-
2
-
fluoromethyl
-
benzenonitrile

(III),
N
-
(4
-
cyano
-
3
-
trifluoromethyl
-
phenyl)
-
3
-
(4
-
fluoro
-
benzene sulfinyl)
-
2
-
hydrox
y
-
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
-
phenylsul
fa
-
nyl)
-
2
-
hydroxy
-
2
-
methyl
-
propionamide (VIII)

as shown in Fig.
12

were separated and determined
by HPLC on a symmetry C
18

column with

p
otassium dihydrogen ortho
-
phosphate (10 mM in
H
2
O) 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 p
otassium dihydrogen ortho
-
phosphate
buffer o
n retention and resolution were studied
to optimize the chromatographic conditions.


S
O
O
N
O
H
C
F
3
C
N
F
C
H
3
H
O
H
N
F
F
F
O
N
O
S
N
O
H
C
F
3
C
N
F
C
H
3
H
O
S
O
O
O
F
C
H
3
H
O
H
2
N
C
F
3
C
N
O
H
H
O
H
2
C
N
O
H
C
F
3
C
N
C
H
3
H
O
N
O
H
C
F
3
C
N
C
H
3
H
O
C
l
S
N
O
H
C
F
3
C
N
F
C
H
3
H
O
O
(

I

)
(

I
I

)
(

I
I
I

)
(

I
V

)
(

V

)
(

V
I

)
(

V
I
I

)
(

V
I
I
I

)

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, 60
o
C temperature for 15 days and ii) extreme conditions such as 0.2
-
1.0 N HCl, 0.05
-
0.5
N NaOH, and 3% H
2
O
2
.Un
der 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

11

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
characteri
zed using modern spectroscopic techniques such as UV, FT
-
IR,
1
H 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%),
accur
acy (
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 r
2

0.9998 for
BCT

and 0.5
-
5.0
µg/ml with r
2

0.9838 for impurities),

limit of detection (LOD) and limit of
quantitation
(LOQ). The developed method was found to be selective, sensitive, precise and stability
indicating. The method was applied to determine BCT and its process
-
related substances in
bulk drugs and pharmaceutical formulations.




Fig. 1
3
.
Typical
HPLC chromatograms

of a) BCT (VII) (200 μg/ml); b) Degradation of


BCT at 0.1N NaOH
.


S
H
N
O
F
O
O
C
F
3
C
N
H
O
C
H
3
S
H
N
F
O
O
C
F
3
C
N
H
O
C
H
3
-
O
H
N
a
O
O
H
S
F
O
O
H
O
C
H
3
H
2
N
C
F
3
C
N
O
N
a
O
0
.
1
N

N
a
O
H
+
(
I
)
(
I
I
I
)
(
V
I
I
)

Fig
.

14
.The degradation of BCT by alkaline hydrolysis.




Fig
.

15
.
Typical chromatograms of BCT (VII) A) Spiked wit
h 2.5% (w/w) of each of


impurities; B), C) &

D) Different process samples of BCT (VII).





12

CHAPTER 7


Enantiospecific Resolution of Citalopram Hydrobromide and Bicalutamide

by HPLC on
Polysaccharide Based Stationary Phases Connected with Ultraviolet and
P
olarimetric
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:triethyl
amine

(95:05:0.1
v/v/v) as mobile phase
and UV detector at 240 nm and the column temperature was at 25
o
C
(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 t
he separations w
ere 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.


O
C
N
F
N
C
H
3
H
3
C
O
C
N
F
N
C
H
3
H
3
C
C
l
H
O
O
C
H
3
N
S
(
S
)
-
(
+
)
-
C
I
T
(
R
)
-
(
-
)
-
C
I
T
(
I
.
S
)

F
S
O
O
N
O
H
C
F
3
C
N
O
H
F
S
O
O
N
O
H
C
F
3
C
N
H
O
(
S
)
-
(
+
)
-
B
C
T
(
R
)
-
(
-
)
-
B
C
T

Fig.16.

Structural representation of enantiomers
of
citalopram and S
-
clopidogrel (Internal



s
tandard
)

and
enantiomers of bicalutamide.




13



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 m
obile 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.