Identification, isolation and characterization of potential degradation products in pioglitazone hydrochloride drug substance

Article abstract of DOI:10.1691/ph.2010.9239

During stress degradation studies of pioglitazone hydrochloride, one major unknown oxidative degradation impurity and two major unknown base degradation impurities were identified by LC-MS. These impurities were isolated using preparative liquid chromatography. Based on the spectral data (¹H NMR, ¹³C NMR, MS and IR), oxidative degradation impurity, base degradation impurity-1 and base degradation impurity-2 were characterized as pioglitazone N-oxide, 3-(4-(2-(5-ethylpyridine-2yl) ethoxy) phenyl)-2- mercaptopropanoic acid and 2-(1-carboxy-2-{4-[2-(5-ethylpyridine-2yl)-ethoxy] phenyl}-ethyl disulfanyl)-3-{4-[2-(5-ethylpyridine-2yl)-ethoxy] phenyl propanoicacid, respectively. The formation and mechanism of these impurities were discussed and presented.

Full text of DOI:10.1691/ph.2010.9239

ORIGINAL ARTICLES
Analytical Research1, Custom Pharmaceutical Services; Analytical Research Department2, Discovery Research,
Dr. Reddy’s Laboratories Ltd.; Andhra University3, Visakhapatnam, India
Identification, isolation and characterization of potential degradation
products in pioglitazone hydrochloride drug substance
K. Ramulu1, T. Thilak Kumar1, S. Radha Krishna1, R. Vasudev2, M. Kaviraj2, B. M. Rao3, N. Someswara Rao3
Received July 15, 2009, accepted August 3, 2009
Dr. B. M. Rao, Department of Analytical Chemistry, School of Chemistry, Andhra University, Visakhapatnam-530
003, India
drbmrao@hotmail.com
Pharmazie 65: 162–168 (2010)
doi: 10.1691/ph.2010.9239
During stress degradation studies of pioglitazone hydrochloride, one major unknown oxidative degra-
dation impurity and two major unknown base degradation impurities were identified by LC-MS. These
impurities were isolated using preparative liquid chromatography. Based on the spectral data (1H
NMR, 13C NMR, MS and IR), oxidative degradation impurity, base degradation impurity-1 and base
degradation impurity-2 were characterized as pioglitazone N-oxide, 3-(4-(2-(5-ethylpyridine-2yl) ethoxy)
phenyl)-2-mercaptopropanoic acid and 2-(1-carboxy-2-{4-[2-(5-ethylpyridine-2yl)-ethoxy] phenyl}-ethyl
disulfanyl)-3-{4-[2-(5-ethylpyridine-2yl)-ethoxy] phenyl propanoicacid, respectively. The formation and
mechanism of these impurities were discussed and presented.
1. Introduction
base stress degradation studies. The analytical HPLC chro-
matograms of pioglitazone hydrochloride, stressed samples in
base and oxidative degradations are shown in Fig. 1. Pioglita-
zone eluted with a retention time of about 7 min (Fig. 1a). In the
chromatogram of the base stressed sample two prominent degra-
dation impurities 1 and 2 were eluted at relative retention times
of 1.05 and 1.12 respectively with respect to the pioglitazone
peak (Fig. 1b). In the chromatogram of the oxidative stressed
sample one prominent degradation impurity eluted at 1.45 rela-
tive retention time with respect to pioglitazone peak (Fig. 1c).
To get further structural insight, LC-MS analysis was carried
out on the stressed samples. The mass spectra thus obtained
showed the protonated molecular ion of the oxidative degrada-
tion impurity at m/z 373, whereas the pioglitazone displayed
protonated molecular ion at m/z 357. The base degradation
mass spectra showed the protonated molecular ions of the
impurities −1 and 2 at m/z 332 and 661 respectively, whereas
the pioglitazone displayed protonated molecular ion at m/z 357.
Thus the oxidative degradation impurity and base degradation
impurity-2 has 16 amu and 304 amu more than the molecular
ion of pioglitazone and base degradation impurity-1 has
25 amu less than the molecular ion of pioglitazone.
Pioglitazone hydrochloride is an antidiabetic agent, which is
used to manage a certain type of diabetes like NIDDM (non-
insulin-dependent diabetes mellitus, type 2 diabetes). It is used
to decrease insulin resistance. Its chemical designation is 5-(4-
(2-(5-ethylpyridin-2-yl) ethoxy) benzyl) thiazolidine-2,4-dione
hydrochloride. Pioglitazone activates peroxisome proliferator-
activated receptor gamma (PPAR-gamma), a ligand-activated
transcription factor, thereby inducing cell differentiation and
inhibiting cell growth and angiogenesis. Pharmacological stud-
ies indicate that pioglitazone improves sensitivity to insulin in
muscle and adipose tissue and inhibits hepatic gluconeogenesis.
It improves glycemic control while reducing circulating insulin
levels (Sohda et al. 1990).
Several articles were published on analytical methods of piogli-
tazone hydrochloride and its metabolites for its biological stud-
ies (Ho et al. 2004; Berecka et al. 2005; Sripalakit et al. 2006).
Some papers were published on analytical methods for the bulk
drug and formulation of pioglitazone hydrochloride (Chandna
et al. 2005; Sane et al. 2004; Jyothi et al. 2006). Kumar et al.
(2008) reported a stability-indicating high-performance liquid
chromatographic method for pioglitazone hydrochloride with
stress degradation studies. Some other publications and patents
were published on process related impurities preparation and
characterization of pioglitazone (Richter et al. 2007; Balwant
et al. 2007; Radhakrishna et al. 2002; Dolitzky et al. 2006).
The present research work describes the identification, isolation
and characterization of three unknown impurities (major degra-
dation products) formed in the oxidative and base degradation
studies (ICH Q3BR2 2006).
As the molecular ion information is not just enough to arrive
at the structure, the drug substance was stressed and subjected
to purification by preparative HPLC to isolate more quantities
of the oxidative degradation impurity, the base degradation
impurity-1 and impurity-2 for spectroscopic studies. After
isolation oxidative and base degradation products (impurity-1
and impurity-2) were spiked to pioglitazone and the spiked
HPLC chromatogram was shown in Fig. 1d.
3. Experimental
3.1. Chemicals and reagents
2. Investigations, results and discussion
The objective of this work is to identify and characterize the
major unknown degradation products, formed in oxidative and
The investigated samples of pioglitazone hydrochloride were received
from Process Research Department of Custom Pharmaceutical Services of
162
Pharmazie 65 (2010) 3

ORIGINAL ARTICLES
V W D1 A, W avele ngth=22 5 n m (D1 224 10 6\CPIO000 0 06.D)
m AU
2 5 0
2 0 0
1 5 0
1 0 0
5 0
0
0
2
4
6
8
10
12
(a)
DAD1 A, Sig = 225, 4 Ref = off (K1113016\PIO00001.D)
mAU
250
200
150
100
50
0
0
2
4
6
8
10
(b)
VWD1 A, Wavelength = 225 nm (B1218016\PIO000001.D)
mAU
250
200
150
100
50
0
min
0
2
4
6
8
10
12
(c)
Fig. 1: Typical HPLC chromatograms of (a) pioglitazone drug substance, (b) stressed sample by base hydrolysis condition, (c) stressed sample by oxidative condition,
(d) pioglitazone spiked with N-oxide, impurity-1 and impurity-2
Pharmazie 65 (2010) 3
163

ORIGINAL ARTICLES
DAD1 A, Sig = 225,4 Ref = off (E1330016\CPIO000034.D)
mAU
250
200
150
100
50
0
0
4
6
8
10
12
2
(d)
Fig. 1: (Continued)
Dr. Reddy’s Laboratories Limited, Hyderabad, India. The chemicals used
in the present analysis are trifluoroaceticacid (Across), sodium hydrox-
ide (Rankem), hydrochloric acid (Rankem), hydrogenperoxide (S.D. Fine
Chemicals) and HPLC-grade acetonitrile (Rankem). Water used in the anal-
ysis and isolation was purified using Milli-Q plus purification system.
The mass spectrometer was equipped with a Waters Acquity system.
Pioglitazone and impurities were dissolved in water: acetonitrile (50:50 v/v)
diluent at a concentration level of 0.25 mg/mL. The sample solutions were
introduced to the mass spectrometer via a HPLC column. The chromato-
graphic conditions used were: A: water: trifluoroacetic acid in the ratio
of 100:0.05 (v/v); B: acetonitrile and trifluoroacetic acid in the ratio of
100:0.05 (v/v); using the following gradient elution (T/%B: 0/10, 12/62,
16/65, 17/10) with a flow rate of 1.0 mL/min and monitored at 225 nm. A
Zorbas Bonus RP18 column with 150 mm length, 4.6 mm internal diameter
and 3.5 ␮m particle size was used.
3.2. Instrumentation
3.2.1. High-performance liquid chromatography
An Agilent 1100 series HPLC with photodiode array detector with chemsta-
tion data handling system was used. The analysis was carried out on Zorbax
Bonus RP18 column with 150 mm length, 4.6 mm internal diameter and
3.5 ␮m particle size. Mobile phase A was water and trifluoroacetic acid in
the ratio of 100:0.05 (v/v) and mobile phase B was acetonitrile and triflu-
oroacetic acid in the ratio of 100:0.05 (v/v). UV detection was carried out
at 225 nm and flow rate was kept at 1.0 mL/min. Column oven temperature
was maintained at 30 ◦C. The gradient program: time/% of MP-B was 0/10,
12/62, 16/65, 17/10 with post run 5 min.
3.2.5. NMR spectroscopy
The 1H and 13C NMR experiments for pioglitazone and impurities were
performed on Mercury plus 400 MHz FT NMR spectrometer using DMSO-
d6 as solvent. The 1H chemical shift values were reported on the ␦ scale in
ppm, relative to TMS (␦ = 0.00 ppm), while 13C chemical shifts relative to
DMSO-d6 (␦ = 39.50 ppm) as internal standards.
3.2.6. ESI-MS/MS
3.2.2. High-performance liquid chromatography (preparative)
The MS/MS experiments were performed on a PESCIEX API 3000. The
sample was introduced through a turbo ion spray interface in positive ion-
ization mode using infusion pump. The nebulizer and curtain gases used
were zero air and nitrogen respectively, ion spray voltage was maintained at
4500 V. Focussing potential, declustering potential and entrance potential
were kept at 180 V, 70 V and 10 V respectively.
An Agilent 1100 series preparative liquid chromatography equipped with
a photodiode array detector system was used. Data was processed through
chemstation software. Zorbax SB C18 (250 mm long × 9.4 mm i.d.) prepara-
tive column packed with 5 ␮m particle size was employed for isolation of the
impurities. Mobile phase A was water and trifluoroacetic acid in the ratio of
100:0.05 (v/v) and mobile phase B was acetonitrile and trifluoroacetic acid
in the ratio of 100:0.05 (v/v). The flow rate was kept at 10 mL/min and the
UV detection was carried out at 225 nm. The gradient program employed
with T (min)/% mobile phase-B (v/v) as 0/20, 5/25, 7/70, 10/70, 11/20 with
post run time of 5 min.
3.2.7. FT-IR spectroscopy
FT-IR spectra were recorded as KBr pellet on Perkin-Elmer instrument
model—spectrum one.
3.2.3. LC-MS
3.3. Preparation, analysis and isolation of degradation samples
LC-MS was carried out on the degraded drug substance of pioglitazone.
The mobile phase used was water: trifluoroacetic acid in the ratio of
100:0.05 (v/v) as a mobile phase - A and acetonitrile: trifluoroacetic acid
in the ratio of 100:0.05 (v/v) as a mobile phase - B using the following gra-
dient/elution (T/%B: 0/10, 12/62,16/65,17/10) flow rate of 1.0 mL/min and
monitored at 225 nm. A Zorbax Bonus RP18 column (150 mm × 4.6 mm
i.d., with a particle size of 3.5 ␮m) was used. The injected volume was
10 ␮L. Zero air was used in nebulizer and curtain gas and high pure nitro-
gen was used as a collision assisted dissociation gas. The following MS
parameters were used for data acquisition: Nebulizer: 8.00 psi, curtain gas
8.00 psi, ion spray voltage 4500 V, Temperature 0 ◦C, declustering potential
70 V, focusing potential 180 V, entrance potential 10 V.
3.3.1. Preparation of degradation samples
3.3.1.1. Base degradation. A solution of pioglitazone hydrochloride
(250 mg) in 25 mL of 0.5 N sodium hydroxide was kept for 48 h.
3.3.1.2. Oxidative degradation. A solution of pioglitazone hydrochloride
(250 mg) in 25 mL of 5% w/v hydrogen peroxide was kept at 60 ◦C for 24 h.
3.3.2. Analysis of degradation samples by analytical HPLC
The degradation samples were diluted to required concentration and ana-
lyzed with analytical LC.3.2.1. In base degradation two major unknown
degradation impurities were found (one is with about 9.0% and another
was about 4.5% by area normalization). At oxidative degradation one major
unknown degradation impurity was found about 3.0% by area normalization.
3.2.4. High resolution mass
All samples were analyzed on the Micromass LCT Premier mass spectrome-
terequippedwithanESILockspraysourceforaccuratemassvalues. Leucine
enkephalin was used as internal reference compound which was introduced
via the Lockspray channel using the Waters reagent manager. The mass
range was calibrated with the cluster ions of sodium formate using a fifth
order polynomial fit. Data were acquired using the W mode.
3.3.3. Isolation of degradation products by preparative HPLC
3.3.3.1. Isolation of base degradation products (impurity-1 and impurity-
2). Pioglitazone hydrochloride (2.0 g) was treated with sodium hydroxide
(0.5N, 25 mL) and kept at 80 ◦C for 24 h. Degraded solution was subjected
164
Pharmazie 65 (2010) 3

ORIGINAL ARTICLES
Table 1: NMR assignments of pioglitazone and N-oxide (oxidative degradation impurity)
1
Position
Pioglitazone
N-oxide impurity
1
2
13
1
2
13
H
␦ (ppm)
J
C
H
␦ (ppm)
J
C
2
3
4
5
6
7
8
10
1H
–
1H
1H
–
2H
2H
–
2H
2H
–
Ha
Hb
1H
–
N-H
–
8.36/d
–
7.57/m
7.27/d
–
3.13/t
4.29/t
–
6.86/d
7.13/d
–
3.05/m
3.02
4.84/m
–
11.98/s
–
2.0
–
–
8.1
–
6.6
6.6
–
8.6
8.6
–
–
–
–
–
–
–
7.6
7.6
148.40
136.61
135.66
122.98
155.32
36.27
1H
–
1H
1H
–
2H
2H
–
2H
2H
–
Ha
Hb
1H
–
N-H
–
8.19/s
–
7.40/d
7.19/m
–
3.25/t
4.28/t
–
6.89/d
7.14/d
–
3.07/m
3.03
4.86/m
–
12.0/s
–
2.55/q
1.23/t
–
–
8.0
–
–
6.3
6.3
–
8.7
8.7
–
140.70
126.36
137.96
124.95
144.81
30.00
66.66
63.49
157.42
114.27
130.26
128.53
36.72
157.23
114.29
130.29
128.66
36.27
11, 15
12, 14
13
16
–
17
18
19
20
22
23
52.98
175.58
–
171.55
24.94
15.36
–
–
–
–
7.5
7.5
52.96
175.58
–
171.55
24.67
14.67
2H
3H
2.59/q
1.20/t
2H
3H
1
1
1
Refer Fig. 2 for numbering; 2: This column gives H - H coupling constant; (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet
O
O
O
NH
S
NH
NH
N
O
S
S
O
N
O
O
N
O
O
O
+
O
O
O
H
H
–
OH
H
(a)
O
O
O
NH
N
H
S
OH
S
N
O
N
O
O
_
CO2
O
OH
O
NH2
OH
S
S
N
O
N
O
OH
H
O
OH
O
S
N
O
N
O
OH
S
SH
OH
N
O
O
IMP-1
IMP-2
(b)
Scheme: Proposed degradation pathways for the (a) Oxidative degradation product, (b) base degradation products (impurity-1 and impurity-2)
to preparative LC as described in section 3.2.2. Fractions collected were ana-
lyzed by analytical HPLC as per the conditions mentioned in Section 3.2.1.
Degradation impurity-1 and impurity-2 fractions were collected separately.
Fractions of >95% were pooled together for impurity-1 and impurity-2 sep-
arately and concentrated on rotavapour to remove solvent mixture for both
impurities. The concentrated fractions of impurity-1 and impurity-2 were
again subjected to preparative HPLC using mobile phase consisting of a mix-
ture of water and acetonitrile in the ratio of 50:50 (v/v) with UV detection
at 225 nm and a flow rate of 10 mL/min. Again the eluate was concentrated
using rotavapour for both impurities 1 and 2. To confirm the retention time
of the isolated impurities the isolated fractions were analyzed by analytical
HPLC as mentioned in section 3.2.1. The impurity-1 is obtained as off-white
solid and the chromatographic purity is 95.0%.The impurity-2 is obtained
as off-white solid and the chromatographic purity is 96.0%.
3.3.3.2. Isolation of oxidative degradation product. Pioglitazone hydro-
chloride (2.0 g) was treated with hydrogen peroxide (5%, 25 mL) and
kept at 80 ◦C for 24 h. Degraded solution was subjected to preparative
LC as described in section 3.2.2. Fractions collected were analyzed by
analytical HPLC as per the conditions mentioned in Section 3.2.1. Frac-
tions of >95% were pooled, concentrated on rotavapour to remove solvent
Pharmazie 65 (2010) 3
165

ORIGINAL ARTICLES
O
O
22
12
4
16
17
22
12
4
18
16
17
13
18
23
3
5
6
11
13
23
3
5
6
11
NH
NH.HCl
8
8
19
19
2
S
2
S
14
14
10
10
N
1
O
9
21
N
O
21
20
20
15
7
1
15
7
9
O
O
Pioglitazone
Pioglitazone hydrochloride
O
22
12
4
16
17
18
O
13
23
3
5
11
21
12
4
16
17
18
13
NH
22
3
5
6
11
8
6
19
2
S
OH
14
8
10
N
O
O
21
1
20
19
15
7
2
9
SH
14
10
O
N
O
20
15
7
1
9
N-Oxide
Impurity-1
O
21
12
4
16
18
13
17
22
3
5
11
OH
19
8
20
S
6
2
14
10
9
N
1
O
7
15
15
9
7
N1
O
10
14
20S
2
6
8
19
OH
5
11
22
3
13
17
18
16
4
21
12
O
Impurity-2
Fig. 2: Structures of Pioglitazone and degradation products
mixture. The concentrated fractions were again subjected to preparative
HPLC using mobile phase consisting of a mixture of water and acetoni-
trile in the ratio of 50:50 (v/v) with UV detection at 225 nm and a flow rate
of 10 mL/min. Again the eluate was concentrated on rotavapour. To con-
firm the retention time of the isolated impurity the isolated fraction was
analyzed by analytical HPLC as mentioned in section 3.2.1. The impurity
was obtained as off-white solid and the chromatographic purity is 97.0% by
area normalization.
The 1H and 13C NMR spectral data of oxidative degradation impurity
were compared with those of pioglitazone in Table 1. The numbering
Scheme for the NMR assignments is shown in Fig. 2. The number of pro-
tons and carbon signals obtained in NMR spectra of degradation impurity
are same as that of signals present in pioglitazone. However, the 1H and
the 13C chemical shifts of the CH groups attached to the nitrogen atom
in the pyridine ring are shifted to down-field when compared to those
of pioglitazone. The NMR experiments were done using DMSO-d6 sol-
vent. This observation lends support to the hypothesis of the formation of
N-Oxide in the pyridine ring. The electrospray ionization (ESI) mass spec-
trum of this impurity exhibited a molecular ion peak at m/z, 373 amu (MH+)
in positive ion mode which is more than 16 amu to the molecular ion of
pioglitazone. The IR (KBr) spectral data of impurity was compared with
those of pioglitazone in Table 2. In IR absorption spectrum of degradation
impurity, the absorption bands corresponding to major functional groups
3.4. Structure elucidation of degradation products
3.4.1. Oxidative degradation impurity
The HR MS data of this oxidative degradation impurity showed exact mass
of the protonated molecular ion at m/z 373.1204 (Calcd. 373.1222 for
C19H21N2O4S) which corresponds to the molecular formula C19H20N2O4S.
are similar to pioglitazone. IR absorption bands for this impurity (cm−1
)
Table 2: FT-IR spectral data of pioglitazone and impurities
a
Compound
IR
Pioglitazone
N-Oxide
3416 (N-H stretching), 3084 (aromatic C-H stretching), 1692 (C = O stretching), 1509 (aromatic C = C stretching),
1461 (C-H bending), 1243 (C-O stretching).
3422 (N-H stretching), 3090 (aromatic C-H stretching), 1701 (C = O stretching), 1513 (aromatic C = C stretching),
1421 (C-H bending), 1246 (C-O stretching).
Impurity-I
Impurity-II
3446 (O-H stretching, broad), 3030 (aromatic C-H stretching), 2567 (S-H weak, sharp), 1685 (C = O stretching),
1512 (aromatic C = C stretching), 1404 (C-H bending), 1211 (C-O stretching), 1136 (C-O stretching).
3440 (O-H stretching, broad), 3070 (aromatic C-H stretching), 1692 (C = O stretching), 1512 (aromatic C = C
stretching), 1404 (C-H bending), 1200 (C-O stretching), 1028 (C-O stretching).
a
KBr
166
Pharmazie 65 (2010) 3

ORIGINAL ARTICLES
Table 3: NMR assignments of impurity-1 and impurity-2 (base degradation impurities)
1
Position
Impurity-1
Impurity-2
1
2
13
1
2
13
H
␦ (ppm)
J
C
H
␦ (ppm)
J
C
2
3
4
5
6
7
1H
–
1H
1H
–
Ha
Hb
2H
–
2H
2H
–
Ha
Hb
1H
–
2H
3H
8.36/d
–
7.56/m
7.26/d
–
3.19/m
3.15
4.39/t
–
6.79/d
7.09/d
–
3.13/m
3.11
3.33/m
–
2.58/q
1.18/t
1.7
–
–
7.8
–
–
–
6.3
–
8.8
8.8
–
–
–
–
–
7.3
7.5
148.47
136.58
135.61
122.97
155.48
36.85
1H
–
1H
1H
–
8.71/s
–
8.36/d
7.93/d
–
–
–
7.3
8.0
–
144.93
141.08
140.58
126.88
151.51
32.66
2H
3.43/t
5.9
8
10
11, 15
12, 14
13
66.68
2H
–
2H
2H
–
Ha
Hb
1H
–
2H
3H
4.36/t
–
6.84/d
7.11/d
–
2.99/m
2.95/m
3.75/m
–
2.77/q
1.23/t
6.1
–
8.3
8.3
–
–
–
–
–
65.49
156.65
113.95
129.98
132.51
42.00
156.69
114.35
130.12
130.03
35.86
16
17
18
21
22
47.21
174.98
24.97
15.38
53.76
171.86
24.65
14.61
7.5
7.5
1
1
1
Refer Fig. 2 for numbering; 2: This column gives H - H coupling constant; (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet
are 3422 (N-H stretching), 3090 (aromatic C-H stretching), 1701 (C = O
stretching), 1513(aromatic C = C stretching), 1421 (C-H stretching), 1246
(C-O stretching). From the spectral data, the structure of this degradation
impurity is characterized as pioglitazone N-Oxide with molecular formula
C19H20N2O4S and molecular weight 372.1.
cating that it can be the dimmer impurity of impurity-1. To confirm that
1H and 13C NMR spectral data of impurity-2 was compared with those of
impurity-1 (Table 3). The 1H and the 13C chemical shifts of the CH group at
the17th positionofimpurity-2areshiftedtoup-fieldwhencomparedwiththe
impurity-1. This is in well agreement with the IR pattern of impurity-2. The
IR (KBr) spectral data of impurity-2 was compared with those of impurity-1
inTable2. IR(KBr)absorptionbandsforthisimpurity(cm−1)are3440(O-H
stretching, broad), 3070 (aromatic C-H stretching), 1692 (C = O stretching),
1512 (aromatic C = C stretching), 1404 (C-H bending), 1200 (C-O stretch-
ing), 1028 (C-O stretching). From the spectral data, the structure of this
impurity is characterized as 2-(1-carboxy-2-{4-[2-(5-ethylpyridine-2yl)-
ethoxy] phenyl}-ethyl disulfanyl)-3-{4-[2-(5-ethylpyridine-2yl)-ethoxy]
phenyl propanoicacid with molecular formula C36H40N2O6S2 and molec-
ular weight 660.2.
3.4.2. Structure elucidation of base degradation impurity-1
The HR MS data of impurity-1 showed exact mass of the protonated
molecular ion at m/z 332.1332 (Calcd. 332.1320 for C18H22NO3S) which
corresponds to the molecular formula C18H21NO3S.
The 1H and 13C NMR spectral data of impurity-1 (Table 3) was compared
with those of pioglitazone (Table 1). The numbering scheme for the NMR
assignments is shown in Fig. 2. The 1H and the 13C chemical shifts of the CH
group at the 17th position of impurity-1 are shifted to down-field when com-
pared with the pioglitazone. In 13C NMR, the presence of carbonyl group at
20th position in pioglitazone, was missing in impurity-1. Thus the impurity-1
structure can be rationalized in terms of opening of thiazolidine ring. The
electrospray ionization (ESI) mass spectrum of this impurity exhibited a
molecular ion peak at m/z, 332 amu (MH+) in positive ion mode which is
less than 25 amu to the molecular ion of pioglitazone, indicating that loss of
one carbon, one nitrozen and addition of one hydrogen atom in the molecule.
IR absorption spectrum also supporting that loss of one carbonyl func-
tional group and formation of the thiol functional group in the impurity-1.
The IR (KBr) spectral data of impurity-1 was compared with those of
pioglitazone in Table 2. IR absorption bands for this impurity (cm−1) are
3446 (O-H stretching, broad), 3030 (aromatic C-H stretching), 2567 (S-
H weak, sharp) 1685 (C = O stretching), 1512 (aromatic C = C stretching),
1404 (C-H bending), 1211 (C-O stretching) and1136 (C-O stretching). From
the spectral data, the structure of this impurity-1 is characterized as 3-
(4-(2-(5-ethylpyridine-2yl) ethoxy) phenyl)-2-mercaptopropanoic acid with
molecular formula C18H21NO3S and molecular weight 331.1.
3.5. Formation of impurities
Oxidative degradation impurity formed in the prescence of perox-
ide stress degradation, to the corresponding to pyridine ring. Both
impurity-1 and impurity-2 were formed in the prescence of base stress
degradation.Impurity-1 is formed due to cleavage of thiazolidine ring and
the impurity-2 is obtained as the dimmer impurity of impurity-1 by forming
the disulfide bond formation. The probable degradation pathway is shown
in the Scheme 1.
Acknowledgements: The authors wish to thank the management of, Dr.
Reddy’s Laboratories Ltd. for supporting this work. Authors would also
like to thank colleagues of Analytical Research of Custom Pharmaceutical
services and Analytical Research Department of Discovery Research for
their co-operation in carrying out this work.
References
3.4.3. Structure elucidation of base degradation impurity-2
Balwant DP, Mohansinh RD, Onkarsingh TS (Alembic Limited, India).
Indian Pat. Appl. (2007) 12pp. CODEN: INXXBQ IN 2005MU01255
A 20070817.
Berecka A, Gumieniczek A, Hopkala H (2005) Retention behavior of new
oral antidiabetic drugs in reversed-phase chromatography. J Planar Chro-
matogr modern TLC 18: 61–66.
Chandna S, Kasture AV, Yeole PG (2005) Simultaneous spectrophotometric
determination of pioglitazone hydrochloride and glimepiride in tablets.
Indian J Pharm Sci 67: 627–629.
Dolitzky, Ben-Zion; Pesachovich, Michael. (Israel). U.S. Pat. Appl. Publ.
(2006), 9pp, Cont.-in-part of U.S. Ser. No. 324, 928.
The HR MS data of impurity-2 showed exact mass of the protonated molec-
ular ion at m/z 661.2383 (Calcd. 661.2406 for C36H41N2O6S2) which
corresponds to the molecular formula C36H40N2O6S2.
The 1H and 13C NMR spectral data of impurity-2 (Table 3) was com-
pared with those of pioglitazone (Table 1). The numbering scheme for
the NMR assignments is shown in Fig. 2. The proton chemical shifts of
the CH group at the 17th position of impurity-2 are shifted to down-field
when compared with the pioglitazone. In 13C NMR, the presence of car-
bonyl group at 20th position in pioglitazone, was missing in impurity-2.
Thus the impurity-2 structure can be rationalized in terms of opening of
thiazolidine ring. The electrospray ionization (ESI) mass spectrum of this
impurity exhibited a molecular ion peak at m/z, 661 amu (MH+) in positive
ion mode which is more than 304 amu to the molecular ion of pioglitazone
and the 329 amu to the molecular ion of impurity-1. Thus spectral data indi-
Ho ENM, Yiu KCH, Wan, TSM, Stewart, BD, Watkins K L (2004) Detection
of anti-diabetics in equine plasma and urine by liquid chromatography-
tandem mass spectrometry. J Chromatogr B 811: 65–73.
Pharmazie 65 (2010) 3
167

ORIGINAL ARTICLES
International Conference on Harmonization tripartite guideline Q3B R2,
“Impurities in New Drug Products”, June 2006.
Jyothi N, Sujan DP, Kumar PR, Rao JVLNS, RaoVJ (2006) Int J Chem Sci
4: 279–284.
Kumar TT, Rao BM, Abdul Nasser AJ, Nallasivam A, Balakumaran K
(2008) Trade Science Inc - Anal Chem 7: 255–262.
Radhakrishna T, Rao S, Reddy (2002) Determination of pioglitazone
hydrochloride in bulk and pharmaceutical formulations by HPLC and
MEKC methods. J Pharm Biomed Anal 29: 593–607.
Richter J, Jirman J, Havlicek J, Hrdina R (2007) Pioglitazone impurities.
Pharmazie 62: 580–584.
Sane RT, Menon SN, Inamdar S, Mote M, Gundi G (2004) Simultaneous
determinationofpioglitazoneandglimepiridebyhigh-performanceliquid
chromatography. Chromatographia 59: 451–453.
Sohda T, Momose Y, Meguro K, Kawamatsu Y, Sugiyama Y, Ikeda H (1990)
Studies on antidiabetic agents – Synthesis and hypoglycemic activity of
5-[4-(pyridylalkoxy)benzyl]-2,4-thiazolidinediones. Arzneim-Forsch 40:
37–42.
Sripalakit P, Neamhom P, Saraphanchotiwitthaya
A (2006) High-
performance liquid chromatographic method for the determination of
pioglitazone in human plasma using ultraviolet detection and its applica-
tion to a pharmacokinetic study. J Chromatogr B 843: 164–169.
168
Pharmazie 65 (2010) 3