Alma Jed 2016

CHAPTER FIVE
Pioglitazone
A. Al-Majed, A.H.H. Bakheit, H.A. Abdel Aziz, H. Alharbi,
F.I. Al-Jenoobi
King Saud University, Riyadh, Kingdom of Saudi Arabia
Contents
1. Description 380
1.1 Nomenclature 380
1.2 Formulae 381
1.3 Elemental Analysis 382
1.4 Appearance 382
2. Uses and Applications 382
3. Methods of Preparation 382
4. Physical Characteristics 387
4.1 Color/Form 387
4.2 Melting Point 387
4.3 Dissociation Constant 387
4.4 Octanol/Water Partition Coefficient 387
4.5 Solubility 387
4.6 Vapor Pressure 387
4.7 X-Ray Powder Diffraction Pattern 388
4.8 Thermal Analysis 388
4.9 Spectroscopy 389
4.10 Mass Spectroscopy 390
4.11 NMR Spectrometry 390
5. Method of Analysis 391
5.1 Compendial Methods 391
5.2 Reported Method of Analysis 399
5.3 Electrochemical Method 406
5.4 Chromatography 409
6. Stability 427
7. Clinical Applications 428
7.1 Pharmcodynamics 428
7.2 Mechanism of Action 429
7.3 Pharmacokinetics 429
Profiles of Drug Substances, Excipients, and Related Methodology, Volume 41 # 2016 Elsevier Inc. 379
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/bs.podrm.2015.11.002
380 A. Al-Majed et al.
7.4 Absorption 430

7.5 Distribution 430
7.6 Metabolism 430
7.7 Excretion 431
7.8 Elimination Half-Life 431
References 431
1. DESCRIPTION
1.1 Nomenclature
1.1.1 Systemic Chemical Names
• 5-[[4-[2-(5-Ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,
3-thiazolidine-2,4-dione hydrochloride [1].
• (RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl)thiazolidine-2,
4-dione [2].
• ()-5-{p-[2-(5-Ethyl-2-pyridyl)-ethoxy] benzyl}-2,4-thiazolidinedione
hydrochloride [3].
• [()-5-[[4-[2-(5-ethyl-2-pyridinyl) ethoxy] phenyl] methyl]-2,4-]
thiazolidine-dione [4].
• 5-(4-(2-(5-ethylpyridin-2-yl)ethoxy)benzyl)thiazolidine-2,4-dione
hydrochloride [5].
• 5-[[4-[2-(5-ethylpyridin-2-yl) ethoxy] phenyl] methyl] thiazolidine-
2,4-dione.
• 5-[4-[2-(5-ethyl-2-pyridyl)ethoxy]benzyl]-2,4-thiazolidinedione.
• ()-5-[[4-[2-(5-Ethyl-2-pyridinyl)-ethoxy] phenyl] methyl]-2,
4-thiazolidinedione.
• 2,4-Thiazolidinedione, 5-[[4-[2-(5-ethyl-2-pyridinyl)ethoxy]phenyl]
methyl]-(9CI).
• 2,4-Thiazolidinedione, 5-((4-(2-(5-ethyl-2-pyridinyl)ethoxy)phenyl)
methyl)-.
• 5-({4-[2-(5-ethylpyridin-2-yl) ethoxy] phenyl}methyl)-1,
3-thiazolidine-2,4-dione.
• 5-[[4-[2-(5-ethyl-2-pyridinyl)ethoxy]phenyl]methyl]thiazolidine-2,
4-dione.
• 5-{4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl}-4-hydroxy-1,3-thiazol-2
(5H)-one.
• 2-Amino-5-{4-[2-(5-ethyl-pyridin-2-yl)-ethoxy]-benzyl}-thiazol-4-
one [6].
Pioglitazone 381
1.1.2 Nonpropriety Name

Generic: Pioglitazone Hydrochloride [1].
Synonyms: Pioglitazonum [INN-Latin]; Pioglitazona [INN-Spanish];
Duetact, AD-4833; Pioglitazone [INN:BAN]; U 72107, pioglitazone
(INN); Pioglitazone [BAN:INN].
1.1.3 Propriety Name
Brand names [3]:
Actos (Takeda Pharmaceuticals); Glustin (Eli Lilly and Company);
Glados (Tabuk Pharmaceuticals). Arg.: Actos; Cereluc; Higlucem; Pioglit;
Piotamax; Austral.: Actos; Austria: Actos; Belg.: Actos; Braz.: Actos;
Canad.: Actos; Chile: Actos†; Diabestat†; Tiazac; Cz.: Actos; Glustin;
Denm.: Actos; Fin.: Actos; Fr.: Actos; Ger.: Actos; Gr.: Actos; Hong
Kong: Actos; India: Diaglit; G-Tase; Glita; Glizone; Opam; P-Glitz;
Pepar; Piomed; Piosafe; Piozulin; Indon.: Actos; Deculin; Ital.: Actos;
Jpn: Actos; Malaysia: Actos; Mex.: Zactos; Neth.: Actos; Glustin; Norw.:
Actos; NZ: Actos; Philipp.: Actos; Prialta; Zypi; Port.: Actos; Glustin;
Rus.: Actos (Актос); S.Afr.: Actos; Spain: Actos; Swed.: Actos; Switz.:
Actos; Thai.: Actos; UK: Actos; USA: Actos; Venez.: Actos.
Multiingredient [3]:
Oseni (Alogliptin/pioglitazone systemic); Duetact (Glimepiride/
pioglitazone systemic); ActoPlus Met, ActoPlus Met XR (Metformin/
pioglitazone systemic); Cz.: Competact; Glubrava; Tandemact; Fr.:
Competact; Tandemact; India: Exermet P; P-Glitz M; Piomed M;
Piosafe MF; Port.: Competact; Tandemact; UK: Competact; USA:
Actoplus Met; Duetact.
1.2 Formulae
1.2.1 Empirical Formula, Molecular Weight, and CAS Number
Pioglitazone C19H20N2O3S 356.44 g/ [111025-46-8] [2]

mol
Pioglitazone C19H20N2O3SHCl 392.90 g/ [112529-15-4] [1,3]
Hydrochloride mol
1.2.2 Structural Formula

O
O
S
HN
N CH3
O
1.3 Elemental Analysis

Pioglitazone: C: 64.02%, H: 5.66%, N: 7.86%, O: 13.47%, S: 9.00%.
Pioglitazone Hydrochloride: C: 58.08%, H: 5.39%, N: 7.13%, O
12.22%, S: 8.16%, Cl: 9.02%.
1.4 Appearance
Colorless prisms from ethanol [1] and needles from dimethylformamide and
water [7].
2. USES AND APPLICATIONS

Pioglitazone is a thiazolidinedione antidiabetic with actions similar to
those of rosiglitazone. Pioglitazone depends on the presence of insulin for its
mechanism of action [8]. It is used to treat diabetes mellitus type 2. It may be
used alone or with other medicines such as insulin, metformin, or sulfonyl-
urea agents. Pioglitazone is used together with a proper diet and exercise to
help control blood sugar levels. It decreases insulin resistance in the periph-
ery and in the liver, resulting in increased insulin-dependent glucose disposal
and decreased hepatic glucose output. In animal models of diabetes,
pioglitazone reduces the hyperglycemia, hyperinsulinemia, and hyper-
triglyceridemia characteristic of insulin-resistant states such as type 2 diabe-
tes. The metabolic changes produced by pioglitazone result in increased
responsiveness of insulin-dependent tissues and are observed in many animal
models of insulin resistance. Pioglitazone is not intended for treating type 1
diabetes [3].
3. METHODS OF PREPARATION
A number of syntheses of pioglitazone have been disclosed. Fischer et al.
[9] and Les et al. [10] described two related syntheses of pioglitazone hydro-
chloride (Scheme 1). The tosylate of 2-(5-ethylpyridin-2-yl) ethanol 2, formed
in situ with tosyl chloride, was displaced by 4-hydroxybenzaldehyde 3 by
means of benzyltributylammonium chloride and NaOH to give 4-[2-(5-eth-
ylpyridin-2-yl)ethoxy]benzaldehyde 6. Alternatively, a nucleophilic aromatic
substitution reaction between alcohol 2 and 4-fluorobenzonitrile 4 using NaH
as the base provided 4-[2-(5-ethylpyridin-2-yl)ethoxy] benzonitrile 5, which
was reduced with Raney nickel and formic acid to aldehyde 6. Then
Pioglitazone 383
CN CN
H3C
H3C
NaH
+ F N O
N OH 4 5
2
Ra–Ni, HCOOH
TsCl, PhCH2NBu3Cl,
+ aq. NaOH
O
CHO S
CHO NH
H3C
HO O N
3 N O
6
, H
S
H3C O S
H3C O
O N
N O H2 or NaBH4
H N O N
O H
7 1
Scheme 1 Fischer and Les method for synthesis of pioglitazone 1.
condensation of 6 with thiazolidine-2,4-dione in basic medium afforded 5-[-

4-[2-(5-ethylpyridin-2-yl)ethoxy]benzylidene] thiazolidine-2,4-dione 7.
Finally, this compound was hydrogenated to provide pioglitazone 1.
Scientists from Takeda chemical industries Ltd. [11] synthesized
pioglitazone 1, by condensed 5-ethyl-2-pyridyl ethanol 2 with 4-fluoro
nitro benzene 3 to obtain nitro compound 4. Hydrogenation of nitro com-
pound 4 in methanol using 10% Pd/C afforded amine compound 5. Com-
pound 5 was treated with sodium nitrate in the presence of HBr to give diazo
compound 6, which when reacted with methyl acrylate in the presence of
cuprous oxide by applying Meerwein arylation conditions afforded α-
Bromo ester compound 7. Condensation of compound 7 with thio urea
in the presence of sodium acetate followed by acid catalyzed hydrolysis
resulted pioglitazone 1 (Scheme 2).
Meguro et al. [12] reported an alternative process for the synthesis of 1.
This involves protection of 2 with p-toluene sulfonyl chloride in the pres-
ence of phase transfer catalyst (benzyl tert-butyl ammonium chloride
(BTBAC)) resulted intermediate 3. Subsequently, intermediate 3 was sub-
jected to nucleophilic substitution. In particular, intermediate 3 was reacted
with 4-hydroxy benzaldehyde 4 in the presence of NaOH to give aldehyde
compound 5. The compound 5 was reacted with 2,4-thiazolidinedione 6 in
the presence of piperidine by employing knoevengal reaction conditions to
obtain benzylidene intermediate, which was hydrogenated using Pd/C in
dioxane to obtain (Scheme 3).
F NO2
Et NO2
OH Et Et NH2
3 10% Pd/C
N N O
2 NaH, DMF MeOH N O
4 5
O
1. NaNO2, HBr O
Et CH3
O Thio urea Et
Br NH
2. Methylacrylate, S
N O Sodium acetate
Cu2O N O
6 7 NH
O
2N HCl
Et
NH
S
N O
O
1
Scheme 2 Synthesis of pioglitazone 1 from (intermediate methyl 2-bromo-3-(4-(2-(5-

ethylpyridin-2-yl)ethoxy)phenyl)propanoate (6) from 1-fluoro-4-nitrobenzene through
SNAr and Meerwein arylation).
OH
Et Et CHO
OH OHC
TsCL Et 4
OTs
N BTBAC, DCM N O
2 NaOH, water
N 5
O NH O 3 O
O
Et
S 6 Et
NH 5% Pd/C
S NH
Piperidine N O S
O Dioxane N O
ethanol, water O
7 1
Scheme 3 Synthesis of pioglitazone 1 from the Knoevenagel condensation of

thiazolidine-2,4-dione 6 and 4-(2-(5-ethylpyridin-2-yl)ethoxy)benzaldehyde 5.
Huber [13] reported synthesis of pioglitazone 1 that involves a reduction

of 2 using sodiumborohydride in the presence of cobalt chloride/dimethyl-
glyoxime catalyst system (Scheme 4). An improved procedure by using sim-
ilar reagents was described by Andrzej Les [10] and coworkers for the
preparation of 1.
Scientists from Smithkline Beecham pharmaceuticals [14] reported a
reduction of benzylidene intermediate 2 using microbial reductase, derived
from suitable red yeast (Scheme 5).
Bipin Pandey et al. [15] described a process for the synthesis of 1 that
involves the usage of halohydrin compounds as intermediates. Reaction
of 5-ethyl-2-vinyl pyridine 2 with N-bromo succinamide provided bromo-
hydrin compound 3, which was reacted with 4 in the presence of base
(NaOH, K2CO3, or NaH) to afford compound 5. Condensation of 2,4-
thiazolidinedione 6 with compound 5 by employing Knoevenagel reaction
conditions resulted in benzylidene compound 7, which was hydrogenated
with sodium borohydride in the presence of cobalt chloride and
Pioglitazone 385
O
O
Et NaBH4
NH Et
S CoCl2, DMG NH
N O S
O THF, water N O
2 1 O
Scheme 4 Synthesis of pioglitazone from (Z)-5-(4-(2-(5-propylpyridin-2-yl)ethoxy)

benzylidene)thiazolidine-2,4-dione 2 using sodium borohydride in the presence of
cobalt chloride/dimethyl-glyoxime catalyst.
O
O
Et
NH Et
S Yeast NH
N O S
O Dioxane N O
2 1 O
Scheme 5 Synthesis of pioglitazone from (Z)-5-(4-(2-(5-propylpyridin-2-yl)ethoxy)

benzylidene)thiazolidine-2,4-dione 2 using microbial reductase.
OH
Et
Et
OHC Et CHO
CH2
N NBS Br 4
2 N
H NaOH, water N O
3 OH K2CO3 (or) NaH
O N O OH 5
O
S O
Et NaBH4
6 NH Et
S CoCl2, DMG NH
N O S
Piperidine O N O
OH DMF O
acetic acid 7 OH 8
etanol, water
O
O
PCl5 Et
NH Et
S Zn, acetic acid NH
CHCl3 N O S
O N O
Cl O
9 1
Scheme 6 Synthesis of pioglitazone 1 from intermediate halohydrin compounds.
dimethylglyoxime to furnish compound 8. Chlorination of 8 with PCl5,

POCl3, or SOCl2, followed by reaction with zinc and acetic acid, resulted
pioglitazone 1 (Scheme 6). Reactions were also performed by replacing OH
group with other groups (Cl, Br. OMs, OTs, and OSO3H).
Takao et al. [16] reported an alternative synthesis of pioglitazone 1,
where 2,4-thiazolidinedione 3 was condensed with 4-hydroxy benzalde-
hyde 2 in the presence of sodium acetate, acetic anhydride, and
dimethylacetamide to obtain intermediate 4, which was hydrogenated with
Pd/C and H2 in acetic acid to furnish 5. The resultant compound 5 was sub-
jected to N-alkylation with triphenyl methyl chloride in methylene chloride
resulted 6. Hydrolysis of 6 using sodium methoxide in toluene afforded 7.
Condensation of 7 and tosylate intermediate 8 in basic medium (K2CO3)
H CH3COONa O O
OH N
O O (CH3CO)2O O 10% Pd/C O
NH NH
OHC + S
CH3CON(CH3)2 H3C O
S
H3C O
S
2
CH3COOH 5
3 O O
4 Et
O OTs
O
PPh3CCl O NaOMe N
NCPh3 NCPh3
S S
DCM, TEA H3C O Toluene HO K2CO3
O O
6 7
O O
Et Et
H+
NCPh3 NH
S S
N O N O
O O
8 1
Scheme 7 Synthesis of pioglitazone 1 from intermediate (Z)-4-((2,4-dioxothiazolidin-5-

ylidene)methyl)phenyl acetate 4 from condensation of 2,4-thiazolidinedione 3 with
4-hydroxybenzaldehyde 2.
O
CHO O O
Cl OtBu OtBu
OtBu
3 10% Pd/C
BnO O O
2 tBuOK, tBuOH BnO Ethyl acetate HO
5
4
Et
O
O
N OMs Et MsCl
6 OMe Et
TEA OCH3
OH
N O OMs
K2CO3, ACN N O
7
8
O
Et O
NH Et
2MHCl
Sodium acetate S NH
N O S
NH N O
Thiourea
9 O
ethanol
1
Scheme 8 Synthesis of pioglitazone 1 from intermediate (Z)-4-((2,4-dioxothiazolidin-5-

ylidene)methyl)phenyl acetate 4 from condensation of 4-benzylloxybenzaldehyde 2
with tert-butyl chloroacetate.
followed by deprotection of trityl group in the presence of hydrochloric acid

furnished compound 1 (Scheme 7).
Thijs and coworkers [17] have synthesized pioglitazone 1, where 4-
benzyloxybenzaldehyde 2 was condensed with tert-butyl chloroacetate 3,
employing Darzens condensation conditions, afforded α,β-epoxy ester 4,
which underwent for debenzylation using 10% Pd/C and hydrogen to obtain
intermediate 5. Intermediate 5 was reacted with 5-ethylpyridine-2-ethy
mesylate 6 in the presence of K2CO3 afforded α-hydroxy compound 7,
which was mesylated using methanesulfonyl chloride in the presence of
TEA to provide mesylate compound 8. Requisite compound 1 was prepared
by condensation of thio urea with mesylated intermediate 8 in the presence
of sodium acetate, followed by hydrolysis of imine compound 9 using
hydrochloric acid (Scheme 8).
Pioglitazone 387
4. PHYSICAL CHARACTERISTICS
4.1 Color/Form
Pioglitazone: Colorless needles from dimethylformamide and water.
Pioglitazone hydrochloride: Colorless prisms from ethanol [7], Odorless
white crystalline [18].
4.2 Melting Point

Pioglitazone: 183–184°C [7].
Pioglitazone hydrochloride: 193–194°C [7].
4.3 Dissociation Constant

pKa ¼ 5.19 [7].
4.4 Octanol/Water Partition Coefficient

Log Kow ¼ 3.96 [6].
4.5 Solubility
Soluble in DMF, DMSO (79 mg/mL); slightly soluble in ethanol (4 mg/
mL), acetone, or acetonitrile; practically insoluble in water; insoluble in
ether. Soluble in 25 mM of DMSO [18]; in water, 46.85 mg/L at 25°C [6].
Pioglitazone hydrochloride very soluble in dimethyl formamide; slightly
soluble in ethanol; very slightly soluble in acetone, acetonitrile. Practically
insoluble in water and ether [7,19].
Tao et al. [20] measured the solubility of pioglitazone hydrochloride
(form I) in methanol, ethanol, 1-propanol, acetic acid, and N,N-
dimethylacetamide between 278.15 and 323.15 K at atmospheric pressure.
The solubility of pioglitazone hydrochloride (form I) increases with increasing
temperature and the order is N,N-dimethylacetamide> methanol > acetic
acid > ethanol > 1-propanol.
4.6 Vapor Pressure

2.88 1014 mm Hg at 25°C [6].
4.7 X-Ray Powder Diffraction Pattern

The X-ray powder diffraction pattern of Pioglitazone HCl has been mea-
sured using a Scintag X’TRA X-ray powder Diffractometer, equipped with
a solid state Si(Li) detector thermoelectrically cooled and single channel ana-
lyzer and using a copper Kα (λ ¼ 1.5418 Å) radiation (tube operated at
40 kV, 40 mA). The data were collected over an angular range from 2 to
40 degree two theta continuous scan mode using a step size of 3 degree
two theta and a step time of 1 s. The peaks (reflections) of crystalline
pioglitazone hydrochloride are found at values of two theta of about 9.2,
10.4, 15.2, 16.4, 18.6, and 21.4 0.2 [21].
4.8 Thermal Analysis

4.8.1 Melting Behavior
Shirolkar et al. [22] determined the melting temperature of Pioglitazone by
melting point apparatus. It was found to be 194°C which is acceptable to the
values of the reported melting temperature [18].
4.8.2 Differential Scanning Calorimetry

The differential scanning calorimetry (DSC) thermogram of Pioglitazone
was recorded on microspheres using DSC. Samples were accurately weighed
and put into aluminum pans and then sealed with aluminum lids. The ther-
mograms of the samples were obtained at a scanning rate of 10°C/min. The
peak of pure drug was found at 192–193°C [22].
4.8.3 Thermogravimetric Analysis

Thermogravimetric analysis, derivative thermogravimetry, and differential
thermal analysis were carried out using simultaneous DTA-TGA thermal
analyzer apparatus (Shimadzu DTG-60H). The samples (4–7 mg) were
placed in platinum pan and heated up to 900°C at a rate of 10°C/min
under nitrogen purge (30 mL/min). Pioglitazone decomposed four stages
of decomposition: At the first stage (145–225.9°C), pioglitazone exhibits
a weight loss of 9.53% due to the loss of HCl molecule. A weight loss
of 57.09% observed between 225.9°C and 327.73°C which may be
attributed to the loss of C10H8NO3S. Beyond 389.34°C, the drug
decomposed in two stages due to the loss of C4H9 at 389.34–468°C
(weight loss of 14.71%) and the loss of C5H3N at 468–551.55°C (weight
loss of 19.47%) [23].
Pioglitazone 389
4.9 Spectroscopy
4.9.1 Ultraviolet Spectroscopy
The ultraviolet (UV) absorption spectra of 20 μg/mL pioglitazone HCl in
methanol is shown in Fig. 1. The figures were recorded using a Shimadzu
UV spectrophotometer; model no. UV-1800 with 1 cm matched quartz
cells was used for experiments. The absorption spectra of reference and
test solution were carried out in a 1 cm quartz cell over the range of
200–400 nm.
The molar absorptivity of pioglitazone HCl at 268 nm is
6561.43 L/mol cm.
4.9.2 Infrared Spectroscopy

Infrared (IR) spectrum of pioglitazone was recorded as KBr disk using
the Shimadzu FT-IR Spectrum BX apparatus. The IR absorption
spectrum of pioglitazone showed two carbonyl functions in the range of
1684–1743 cm1. The NH absorption band appeared at 3258 cm1 (Fig. 2).
2.000
4
1.000
3
Abs.
1
5
0.000
−0.572
200.00 250.00 300.00 350.00 400.00
nm.
Figure 1 Ultraviolet absorption spectrum of pioglitazoe HCl dissolved in methanol.
Figure 2 Infrared absorption spectrum of pure pioglitazone HCl.
4.10 Mass Spectroscopy

The mass spectrum of pioglitazone (C19H20N2O3S, 356.44) was obtained
using an Agilent 6320 Ion trap mass spectrometer (Agilent technologies,
USA) equipped with an electrospray ionization interface (ESI). A connector
was used instead of column. Mobile phase composed of a mixture of solvents
A and B (50:50), where A is high-performance liquid chromatography
(HPLC) grade water and B is acetonitrile. Compound was prepared by
weighing the solid substances to 1 mg/mL in DMSO and diluted with
mobile phase. Test solution was prepared by diluting the stock solutions
to 10–30 mg mL depending on the ion intensities—with mobile phase.
Flow rate was 0.4 mL/min and run time was 5 min. MS parameters were
optimized for each compound. The scan was ultra-scan mode. MS2 scans
were performed in the mass range of m/z 50–1000. The ESI was operated
in positive mode. The source temperature was set to 350°C nebulizer gas
pressure of 55.00 psi with dry gas flow rate of 12.00 L/min. Fig. 3 shows
the molecular ion peak of pioglitazone at m/z ¼ 357.1 [M]+.
4.11 NMR Spectrometry

4.11.1 1H NMR Spectrometry
1
H NMR spectrum of pioglitazone was scanned in DMSO-d6 on a Brucker
NMR spectrometer operating at 500 MHz. Chemical shifts are expressed in
Pioglitazone 391
×102 +Scan(0.297 min) pg00002.d

357.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2 320.1
0.1
0
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
Counts vs mass-to-charge (m/z)
Figure 3 Mass spectrum of pioglitazone.
δ-values (ppm) relative to TMS as an internal standard. Coupling constants

( J) are expressed in Hz (Table 1 and Fig. 4A–C).
4.11.2 13C NMR Spectrometry

13
C NMR spectrum of pioglitazone was scanned in DMSO-d6 on a
Brucker NMR spectrometer operating at 125 MHz. Chemical shifts are
expressed in δ-values (ppm) relative to TMS as an internal standard
(Table 2 and Fig. 5).
5. METHOD OF ANALYSIS
5.1 Compendial Methods
5.1.1 United States Pharmacopeia Methods [24]
Definition
Pioglitazone Hydrochloride contains NLT 98.0% and NMT 102.0% of
C19H20N2O3SHCl, calculated on the anhydrous basis.
Identification
A. IR absorption <197K >
B. Identification tests–General, Chloride <191 >: Dissolve 25 mg of
Pioglitazone Hydrochloride in 0.5 mL of nitric acid, and add 2 mL of
dilute nitric acid. It meets the requirements of the test for chloride.
C. The retention time of the pioglitazone peak of the sample solution cor-
responds to that of the standard solution, as obtained in the Assay.
Table 1 1H NMR of Pioglitazone (DMSO-d6)

6 8 11⬘ O
7 9 O 16
5 12⬘ S
10
N H
1 N
3 11 13 15 17
2 4 12 14
O
Pioglitazone
Signal Location (δ) Shape Integration Correspondences

1 1.21–1.26 m 3H dCH2dCH3 (Hs1)
2 2.76–2.82 m 2H dCH2dCH3 (Hs2)
3 3.03–3.09 m 1H dArdCH2dCH < (Hs14)
4 3.27–3.32 m 1H dArdCH2dCH < (Hs14´)
5 3.49–3.51 m 2H dCH2dCH2dOdArd
(sH8)
6 4.40–4.41 m 2H dCH2dCH2dOdArd
(Hs9)
7 4.85–4.89 m 1H ArdCH2dCH< (H15)
8 6.86–6.89 m 2H ArHs (11 + 11´)
9 7.13–7.17 m 2H ArHs (12 + 12´)
10 7.97–7.99 dd, J ¼ 3.5, 1H Pyridine H6
8.0 Hz
11 8.40–8.42 d, J ¼ 3.0, 8.0 Hz 1H Pyridine H5
12 8.72–8.73 d, J ¼ 6.0 Hz 1H Pyridine H4
13 12.05 s 1H NdH
Assay
Procedure:
• Mobile phase: Acetonitrile, 0.1 M ammonium acetate, and glacial
acetic acid (25:25:1)
• Standard solution: Prepare a 0.5 mg/mL solution of USP
Pioglitazone Hydrochloride RS in methanol and dilute with Mobile
phase to obtain a solution containing 50 μg/mL of pioglitazone
hydrochloride.
Pioglitazone 393
Figure 4 (A) 1H NMR spectrum of pioglitazone. (B) 1H NMR spectrum of pioglitazone

(aliphatic region).
(Continued)
Figure 4—Cont'd (C) 1H NMR spectrum of pioglitazone (aromatic region).
• System suitability stock solution: 0.5 mg/mL of USP Pioglitazone

Hydrochloride RS and 0.13 mg/mL of benzophenone in methanol.
• System suitability solution: Dilute system suitability stock solution
with mobile phase to obtain a solution containing 50 μg/mL of
pioglitazone hydrochloride and 13 μg/mL of benzophenone.
• Sample solution: Prepare a 0.5 mg/mL solution of pioglitazone
hydrochloride in methanol and dilute with mobile phase to obtain
a solution containing 50 μg/mL of pioglitazone hydrochloride.
• Mode: LC
• Detector: UV 269 nm
• Column: 4.6 mm 15 cm; 5 μm packing L1
• Column temperature: 25 2.5°C
• Flow rate: 0.7 mL/min
• (NOTE—Adjust the flow rate so that the retention time of the
pioglitazone peak is about 7 min.)
• Injection size: 20 μL
• System suitability
Pioglitazone 395
13
Table 2 C NMR of Pioglitazone (DMSO-d6)
6 8 11⬘ O
7 9 O 16
5 12⬘ S
10
N H
1
N
3 11 13 15 17
2 4 12 14
O
Pioglitazone
Signal Location (δ) Correspondences

1 14.08 CH3 (C1)
2 25.07 CH2 (C2)
3 32.79 CH2 (C8)
4 36.67 CH2 (C14)
5 53.42 CH (C15)
6 65.91 CH2 (C9)
7 114.90 (2C) CH (C11 + C11´)
8 127.53 CH (C6)
9 129.56 C (C13)
10 130.88 (2C) CH (C12 + CC12´)
11 140.67 CH (C4)
12 141.71 C (C10)
13 145.56 CH (C5)
14 151.74 C (C3)
15 157.49 C (C7)
16 172.12 CdO (C16)
17 176.15 CdO (C17)
• Samples: System suitability solution and standard solution

• (NOTE—The approximate relative retention times for pioglitazone
and benzophenone are 1.0 and 2.6, respectively.)
Suitability requirements:
• Tailing factor: NMT 1.5 for pioglitazone and benzophenone, system
suitability solution
13
Figure 5 C NMR spectrum of pioglitazone.
• Resolution: NLT 15 between pioglitazone and benzophenone,

system suitability solution
• Relative standard deviation: NMT 2.0% for six replicate injections,
standard solution.
Analysis:
• Samples: Standard solution and Sample solution
• Calculate the percentage of C19H20N2O3SHCl in the portion of
Pioglitazone Hydrochloride taken:

rU CS
Result ¼ 100
rS CU
rU ¼ peak response from the Sample solution

rS ¼ peak response from the Standard solution
CS ¼ Concentration of USP PioglitazoneHydrochloride RS in
the Standard solution (μg/mL).
Pioglitazone 397
CU ¼ Concentration of Pioglitazone Hydrochloride in the Sam-

ple solution (μg/mL).
• Acceptance criteria: 98.0–102.0% on the anhydrous basis
Impurities
Inorganic impurities:
• Residue on ignition <281 >: NMT 0.1%
• Heavy metals
Sodium sulfide solution: 5 g of sodium sulfide in 10 mL of water
and 30 mL of glycerin
Magnesium nitrate solution: 100 mg/mL of magnesium nitrate in
alcohol
Standard solution: Place 10 mL of magnesium nitrate solution in a
platinum or porcelain crucible. Ignite the alcohol to burn. Cool,
add 1 mL of sulfuric acid, heat carefully, and ignite at 550 50°C.
Cool and add 3 mL of hydrobromic acid. Proceed as directed from
this point under Sample solution, adding 1.0 mL of Standard Lead
Solution (see Heavy Metals <231 >, Special Reagents) before
adding water to make 50 mL.
Sample solution: Place 1.0 g of pioglitazone hydrochloride in a
platinum or porcelain crucible. Mix with 10 mL of magnesium
nitrate solution. Ignite the alcohol to burn and carbonize by gradual
heating. Cool, add 1 mL of sulfuric acid, heat carefully, and incin-
erate by ignition at 550 50°C. If carbonized substances remain,
moisten with a small amount of sulfuric acid and incinerate by
ignition. Cool, dissolve the residue in 3 mL of hydrobromic acid,
and evaporate on a water bath to dryness. Wet the residue with three
drops of hydrochloric acid, add 10 mL of water, and dissolve by
warming. Add one drop of phenolphthalein TS and add ammonia
TS dropwise until a pale red color develops. Add 2 mL of 1 N acetic
acid, filter if necessary, wash with 10 mL of water, transfer the filtrate
and washings to a Nessler tube, and add water to make 50 mL.
Analysis: Add one drop of Sodium sulfide solution to each of the
tubes containing the Standard solution and Sample solution. Mix
thoroughly and allow to stand for 5 min. Compare the colors of
both solutions by viewing the tubes downward or transversely
against a white background. The Sample solution has no more
color than the Standard solution.
• Acceptance criteria: NMT 10 ppm
Organic impurities
• Procedure
Mobile phase and System suitability stock solution: Proceed as
directed in the Assay.
System suitability solution: Dilute the System suitability stock
solution with Mobile phase to obtain a solution containing
25 μg/mL of pioglitazone hydrochloride and 6.5 μg/mL of
benzophenone.
Sample solution: 0.2 mg/mL of pioglitazone hydrochloride dis-
solved in 20% of the final volume with methanol, then diluted
with Mobile phase to final volume.
Standard solution: 1 μg/mL of pioglitazone hydrochloride pre-
pared by diluting the Sample solution with Mobile phase.
Mode: LC
Detector: UV 269 nm
Column: 4.6 mm 15 cm; 5 μm packing L1
Column temperature: 25 2.5°C
Flow rate: 0.7 mL/min
(NOTE—Adjust the flow rate so that the retention time of the
pioglitazone peak is about 7 min.)
Injection size: 40 μL
Run time: At least 4 the retention time of pioglitazone.
• System suitability
Samples: System suitability solution and Standard solution
Suitability requirements
A. Tailing factor: NMT 1.5 for pioglitazone and benzophenone,
System suitability solution
B. Resolution: NLT 15 between pioglitazone and benzophe-
none, System suitability solution
C. Relative standard deviation: NMT 3.0%, Standard solution
• Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of
Pioglitazone Hydrochloride taken:

rU
Result ¼ D 100
rS
Pioglitazone 399
rU ¼ peak response of each individual impurity from the

Sample solution
rS ¼ peak response of pioglitazone from the Standard solution
D ¼ dilution factor used to prepare the Standard solution,
0.005
Acceptance criteria
Individual impurities: See Table 3.
Total impurities: NMT 0.5%.
5.2 Reported Method of Analysis
5.2.1 Spectroscopy Methods
5.2.1.1 UV Spectrometry, Colorimetry, and Thermal Analysis
Ulu et al. [25] developed three rapid, sensitive, and simple spectrophoto-
metric methods for the determination of pioglitazone in pure and pharma-
ceutical preparations. For the first method, UV spectrophotometry,
standard solutions were measured at 270.2 nm. It was linear from 5.0 to
20.0 μg/mL. For the second method, the distances between two extre-
mum values (peak-to-peak amplitudes), 272.0 and 287.4 nm, were mea-
sured in the second-order derivative spectra of standard solutions. The
linearity was found to be 2.0–12.0 μg/mL for pioglitazone standards in
acetonitrile. The third method was based on the formation of an ion asso-
ciation complex with bromocresol green (BCG), bromocresol purple
(BCP), bromophenol blue (BPB), and bromothymol blue (BTB). The
assay was linear over the concentration range of 20.0–100.0 μg/mL for
BCG, 10.0–100.0 μg/mL for BCP, 20.0–120.0 μg/mL for BPB, and
10.0–100.0 μg/mL for BTB. The three proposed methods have been suc-
cessfully applied to the assay of pioglitazone in pure and in pharmaceutical
preparations.
Table 3 Impurity
Relative Retention Acceptance Criteria,
Name Time NMT (%)
Hydroxypioglitazonea 0.7 0.15
Pioglitazone 1.0 —
Didehydropioglitazoneb 1.4 0.15
N-alkylpioglitazonec 3.0 0.15
Any other individual impurity — 0.10
Adhikari et al. [26] used a spectroscopic method to quantify three anti-

diabetics in multicomponent formulations. In the present study, three wave-
length spectroscopic method and multiwavelength method was carried out
for determination of metformin hydrochloride, glipizide, pioglitazone
hydrochloride in their bulk and preparations using acetonitrile:methanol:
water in the proportion of 5:4:1. The λmax was found at 236.5, 226.4,
and 227.3 nm, respectively. The isobestic point was found to be 254 nm.
Method II is based on multiwavelength spectroscopy. All the three drugs
obey the Beer–Lambert limit within the concentration range of 5–50 μg/
mL. The method can be used for routine quantitative analysis of metformin,
glipizide, and pioglitazone in pure and tablet dosage forms.
Amanlou et al. [27] developed an extractive spectrophotometric method
for the determination of pioglitazone hydrochloride in pure and pharmaceu-
tical formulations. This method is based on the formation of yellow ion-pair
complex between the basic nitrogen of the drug and bromocresol green
(BCG) in phthalate buffer of pH 2.4. The formed complexes were extracted
with chloroform and measured at 419 nm. Beer’s law was obeyed in the
range 2.5–14 μg/mL. The proposed method has been applied for the deter-
mination of drug in commercial tablets dosage forms.
Deepa et al. [28] developed a method of analysis to determine gimepride
(GLM), pioglitazone hydrochloride, and metformin hydrochloride (MET)
in combined dosage forms using second-derivative spectrophotometry. The
combined preparations were quantified using the second-derivative
responses at 233.4 nm for gimepride, 265.4 nm for pioglitazone hydrochlo-
ride, and 252.6 nm for MET in spectra of their solution in methanol. The
calibration curves were linear in the concentration range 5–25 μg/mL for
glimepiride, 5–25 μg/mL for pioglitazone hydrochloride, and 2–12 μg/
mL for metformin hydrochloride. The method was applied for estimation
of glimepiride, pioglitazone hydrochloride, and metformin hydrochloride
in combined tablet formulation.
Dhole et al. [29] described a UV spectrophotometric method for the
simultaneous determination of pioglitazone HCl, metformin HCl, and
glibenclamide in combined tablet dosage form using ethanol (95%) as sol-
vent. The wavelengths selected for the analysis were 237, 268, and 300 nm
for estimation of metformin HCl, pioglitazone HCl, and glibenclamide,
respectively. Beer’s law was obeyed in the concentration ranges of 3–
30, 10–100, and 1–10 μg/mL for pioglitazone HCl, metformin HCl,
and glibenclamide, respectively. The mean percentage drug content for
Pioglitazone 401
pioglitazone HCl, metformin HCl, and glibenclamide were found to be

99.48%, 99.77%, and 99.35%, respectively. The method was found to
be suitable for the routine quality control analysis of pioglitazone HCl,
metformin HCl, and glibenclamide in pure and pharmaceutical dosage
forms.
Game [30] developed a spectrophotometric method for simultaneous
estimation of glimepiride and pioglitazone HCl in capsules by employing
first order derivative zero crossing method. The wavelengths selected for
quantitation were 230.0 nm (zero cross point of glimepiride) for
pioglitazone HCl and 250.0 nm (zero cross point of pioglitazone HCl)
for glimepiride. Linearity was maintained within a wide concentration range
from 4.0 to 30.0 μg/mL for glimepiride and 6 to 30 μg/mL for pioglitazone
HCl. The limit of detection and limit of quantification for glimepiride were
found to be 2.0 and 4.0 μg/mL, respectively, and for pioglitazone HCl 4.0
and 6.0 μg/mL, respectively. The method was applied in the analysis of
commercial capsules.
Havele et al. [31] developed a spectrophotometric method for simulta-
neous estimation of atorvastatin and pioglitazone in bulk and tablet. The
method showed maximum absorbance at 210 nm for atorvastatin while
showed maximum absorbance for pioglitazone at 225 nm. The overlain
spectra showed maximum absorbance at 242 nm. The method was applied
for simultaneous determination for both drugs in tablet dosage form.
Kishore et al. [32] developed spectrophotometric methods for the simul-
taneous estimation of pioglitazone and glimepiride. First method used was
the simultaneous equation method, in which two wavelengths (216 and
225 nm) were selected for the measurement of absorbance. Second method
was the absorption ratio method in which measurements are made based on
the absorptivity at the isosbestic point (228 nm) and absorption maxima of
pioglitazone (216 nm). The absorption maximum wavelengths of
pioglitazone and glimepiride were observed at 216 and 225 nm, respec-
tively, and the isosbestic point at 228 nm. Linearity ranges were 5–25 μg/
mL for both drugs. The proposed methods were recommended for routine
analysis of pioglitazone and glimepiride as they are rapid, precise, accurate,
and reproducible.
Ali et al. [33] developed a UV spectrophotometric method for the quan-
titative estimation of pioglitazone in bulk and pharmaceutical dosage forms.
Pioglitazone hydrochloride has absorption maxima at 224.4 nm in ethanol
and obeyed Beer’s law in a concentration range of 5–25 μg/mL.
Mohd et al. [34] developed another UV spectroscopic method for the

estimation of pioglitazone hydrochloride in bulk and tablet dosage form.
Pioglitazone hydrochloride shows maximum absorption at 269 nm with
molar absorptivity of 9.6013 104 L/mol cm. Beer’s law was obeyed in
the concentration range of 10–70 μg/mL. The proposed method was found
to be accurate and precise for estimation of pioglitazone hydrochloride in
bulk and tablet dosage form.
Patil Pallavi et al. [35] developed UV derivative spectrophotometric
methods for the simultaneous determination of glimepiride, metformin
HCL, and pioglitazone HCL in tablets. The first derivative UV spectropho-
tometric method was performed at 227, 233, and 265.5 nm for glimepiride,
metformin HCL, and pioglitazone HCL, respectively, in 0.1 N NaOH
solution and distilled water (50:50).
Patil et al. [36] developed two visible spectrophotometric methods (A
and B) for the quantitative estimation of pioglitazone, in bulk drug and phar-
maceutical dosage forms. Methods were based on the formation of pale yel-
low colored and green colored chromo gens, which were measured 267 and
297 nm, respectively. For the first method, UV spectrophotometry, stan-
dard solutions were measured at 267 nm. The first method was linear from
2.5 to 20 mg/mL. The second method was based on the formation of an ion
association complex with Methyl orange (MO) and Bromocresol Green
(BCG). The assay was found to be linear over the concentration range of
2.5–20 μg/mL. The two methods have been successfully applied to the assay
of pioglitazone.
Rathod et al. [37] developed and validated simple spectrophotometric
method for simultaneous quantitation of metformine hydrochloride and
pioglitazone hydrochloride in tablet dosage form without previous sepa-
ration. In simultaneous equation method, metformine hydrochloride
and pioglitazone hydrochloride were quantified using their absorptivity
values at selected wavelengths, 233 and 265.5 nm, respectively. The simul-
taneous equation method permits simple, rapid, and direct determination
of metformine hydrochloride and pioglitazone hydrochloride in commer-
cially available tablet dosage form without previous separations and can
therefore be used for routine analysis of both drugs in quality control
laboratories.
Shakya et al. [38] developed and validated a UV spectrophotometric
method for the analysis of pioglitazone in tablets. The proposed method
was performed in phosphate buffer (pH 7.4). Beer’s law was valid in a con-
centration range of 10–50 μg/mL and UV detection was done at 238 nm.
Pioglitazone 403
The proposed method was applied to the determination of pioglitazone in

two pharmaceutical formulations.
Singhvi et al. [39] developed one simple, accurate, economical, and
reproducible UV spectrophotometric method for simultaneous estimation
of pioglitazone and glimepiride in combined tablet dosage form. The devel-
oped method employs multiwavelength spectroscopy using 280 and 238 nm
as two wavelengths for estimation of pioglitazone and glimepiride,
respectively.
Sujana et al. [40] developed difference spectrophotometric methods for
the estimation of pioglitazone and metformin in bulk drug and in pharma-
ceutical formulations. Difference spectrum obtained by keeping
pioglitazone and metformin separately in 0.1 M NaOH in the sample cell
and 0.1 M HCl as blank, showed characteristic peaks (λmax) at 228.1 nm
pioglitazone and 228.2 nm metformin and the characteristics peaks for phar-
maceutical formulations were also found. The proposed method can be used
for routine estimation of pioglitazone and metformin in pharmaceutical dos-
age form.
Sujana et al. [41] developed two new UV spectrophotometric methods
for simultaneous estimation of pioglitazone hydrochloride and metformin
hydrochloride in tablets. The first method was based on application of
Vierordt’s method which involves the formation and solving of simulta-
neous equations at 225 and 237 nm, as absorbance maxima of pioglitazone
hydrochloride and metformin hydrochloride, respectively. The second
method employed was absorption correction method which involves direct
estimation of pioglitazone hydrochloride at 267 nm, as at this wavelength
metformin hydrochloride has zero absorbance and shows no interference.
For estimation of metformin hydrochloride, corrected absorbance was cal-
culated at 237.0 nm due to the interference of pioglitazone hydrochloride at
this wavelength. Calibration curves were linear with a correlation coefficient
of 0.999 over the concentration ranges of 6–14 and 1–5 μg/mL for both the
drugs. The proposed methods can be used in the quality control of pharma-
ceutical formulations and routine laboratory analysis.
Jani et al. [42] developed another analytical method for simultaneous esti-
mation of valsartan and pioglitazone hydrochloride. This method involves
solving of simultaneous equations based on the measurement of absorbance
at two wavelengths, 248 and 268 nm, from 0.1 N HCl and phosphate buffer
solutions. Both drugs obey the Beer’s law in the concentration ranges
employed for this method. The method can be used to estimate the amount
of valsartan and pioglitazone hydrochloride in pharmaceutical formulations.
Abdelmonem et al. [43] represented simple atomic absorption spectro-

scopic and spectrophotometric methods for determination of pioglitazone
hydrochloride and carvedilol based on formation of ion-pair associates
between drugs and inorganic complex, bismuth(III) tetraiodide (Method A)
and between drugs and organic acidic dyes, fast green and orange G (Method
B). Method A is based on formation of ion-pair associate between drugs and
bismuth (III) tetraiodide in acidic medium to form orange-red ion-pair asso-
ciates, which can be quantitatively determined by two different procedures.
The formed ion-pair associate is extracted by methylene chloride, dissolved
in acetone, and quantified spectrophotometrically at 490 nm. Method B is
based on formation of ion-pair associate between drugs and either fast green
dye or orange G dye in acidic medium to form ion-pair associates. The
formed ion-pair associate is extracted by methylene chloride and quantified
spectrophotometrically at 630 nm (for fast green dye method) or 498 nm
(for orange G dye method).
Okdeh et al. [44] developed simple extractive spectrophotometric
method for the rapid determination of pioglitazone in pure form and phar-
maceutical formulations. The method was based on the formation of binary
complex (ion-pair complex) between the Pioglitazone and Chromotrope
2R in an acidic buffer, giving purple color, and the absorbance of dic-
hloromethane extracted complex was measured at 514 nm. The complexa-
tion reaction was extremely rapid at room temperature and the absorption
values remains unchanged up to 24 h. Beer’s law was obeyed in the concen-
tration range of 1.0–65.0 μg/mL, detection limit was 0.16 μg/mL, and the
molar absorptivity coefficients were 9.934 103 L/mol/cm. Recoveries
were between 99.13% and 102.17%. Interferences of the other ingredients
and excipients were not observed.
Dubey et al. [45] developed a UV spectroscopy method for the estima-
tion of Pioglitazone hydrochloride tablet dosage form and validated by
ICH guidelines. The standard (10 μg/mL) was scanned between 200 and
400 nm and maximum absorption was recorded at 231.5 nm. The assay
results were found to be 100.52%. The linearity range of 15–65 μg/mL
proved that it obeyed Beer’s Law, and the correlation coefficient (r2) was
found to be 0.9983 at 270 nm with an intercept of 0.0008 and a slope
of 0.0018 with RSD less than 2% complied ICH. The pH degradation
study of API was found to be less at pH 7–12. The force degradation
study of Pioglitazone were done on Stress degradation by hydrolysis
under alkaline condition by using 0.1 N NaOH and was found to be
5.76% for 60 min, 9.61% for 90 min. Stress degradation by hydrolysis under
Pioglitazone 405
acidic condition by using 3 N HCl and product degradation was found to

be 11.53% for 60 min and 21.15% for 90 min for API. Dry heat-induced
degradation was done by using 70°C temperature and was found to
be 1.93% for API for 48 h. Oxidative degradation was done by using
hydrogen peroxide and product degradation was found to be 19.23% at
15 min. Photolytic degradation was found to be 9.61% for 3 h and
15.38% for 5 h for API.
Kashyap et al. [46] developed a UV spectroscopic method for the simul-
taneous estimation of Alogliptin and Pioglitazone bulk and pharmaceutical
dosage forms. First order derivative and dual wavelength methods were
developed and validated using solvent methanol. Both methods show line-
arity at 5–30 μg/mL. The first order derivative spectra of each solution were
obtained. ZCP of Alogliptin was found to be 275.60 nm and ZCP of
Pioglitazone was found to be 268.20 nm. Pioglitazone was measured at
the zero crossing point (ZCP) of Alogliptin and Alogliptin. In dual wave-
length method, spectra two wavelengths 270.20 and 265 nm were selected
as λ1 and λ2 for the estimation of Alogliptin. Pioglitazone shows the same
absorbance at these wavelengths. Similarly, wavelengths 280 and 271 nm
were selected as λ3 and λ4 for estimation of Pioglitazone. Alogliptin shows
the same absorbance at these wavelengths.
Dubey et al. [47] developed a simple procedure for the estimation of
Pioglitazone by first order derivative spectroscopy. The method is based
upon determination of D1 value of Pioglitazone at 231.5 nm, in 0.1 N
NaOH. Pioglitazone at its λmax shows linearity in the concentration range
of 15–65 μg/mL.
5.2.1.2 Atomic Absorption Spectroscopic

Sarat et al. [48] developed a method of determination of Cobalt (Co) in the
Pioglitazone hydrochloride sample by the atomic absorption spectroscopy
(AAS). The linearity with a correlation coefficient value of 0.9993 and accu-
racy recoveries at LOQ are ranging from 93.2% to 105.2%. The limit of
detection (LOD) obtained under the optimum condition was 0.3 μg/g
and relative standard deviation for six replicate determinations of 10 μg/L
was 2.66%.
Abdelmonem et al. [43] represented simple atomic absorption spectro-
scopic and spectrophotometric methods for determination of pioglitazone
hydrochloride and carvedilol based on the formation of ion-pair associates
between drugs and inorganic complex, bismuth(III) tetraiodide (Method
A). Method A is based on the formation of ion-pair associate between drugs
and bismuth (III) tetraiodide in acidic medium to form orange-red ion-pair

associates, which can be quantitatively determined by two different proce-
dures. The formed ion-pair associate is extracted by methylene chloride, dis-
solved in acetone, dried, and then decomposed by hydrochloric acid, and
bismuth content is determined by direct atomic absorption spectrometric
technique.
5.2.1.3 Spectrofluorimetric Method

Alarfaj et al. [49] established a convenient and sensitive spectrofluorimetric
method for the determination of two antidiabetic drugs, ie, pioglitazone
HCl and glimepiride, in pharmaceutical formulations and biological fluids.
The method is based on the native fluorescence of the studied drugs in meth-
anol. The fluorescence intensity was measured in methanol at 512 and at
522 nm for pioglitazone HCl excitation and glimepiride, respectively.
The range of 0.005–1.3 μg/mL for pioglitazone HCl with lower limit of
detection (LOD) of 1.61 103 μg/mL.
5.3 Electrochemical Method

5.3.1 Potentiometric Measurement
Saber et al. [50] developed simple poly(vinyl chloride) membrane sensors for
the determination of pioglitazone in biological samples (urine) and pharma-
ceutical preparations. Potentiometric measurements were based on
iodobismuthite-drug ion-pair as novel electroactive materials incorporating
a plasticized PVC membrane with o-nitrophenyl octyl ether or dioctyl
phthalate. Each sensor was conditioned for at least 2 days in 0.1 M drug solu-
tion before use. It exhibited fast and stable Nernstian response for
pioglitazone over the concentration range of 1.0 107–1.0 102 M,
pH range of 3.0–7.0 pioglitazone sensors. Results with an average recovery
not more than 100.4% and a mean standard deviation less than 1.0% of the
nominal were obtained for the two sensors.
Mandil et al. [51] described a method for the determination of
pioglitazone HCl as antidiabetic drug in its pure form and pharmaceutical
formulations. The proposed methods depend on the polarographic activity
of pioglitazone HCl in Britton–Robinson buffer over the pH range 2–12
using direct current (DC) and differential pulse polarography (DPP), and
it showed well-defined two cathodic peaks with high selectivity. Its electro-
chemical behavior at a dropping mercury electrode (DME) and stating mer-
cury drop electrode (SMDE) has been investigated. Polarograms of the drug
at DME & SMDE in B–R buffer at pH 6.0 exhibited two two-electron
Pioglitazone 407
irreversible cathodic peaks, the first peak (Ep1) is in the range of potential
at 0.05 to 0.10 V, while the second peak (Ep2) is in the potential ranges
at 0.975 to 1.10 V vs Ag/AgCl. The first and second peaks may be attrib-
uted to the reduction of oxy group (peak 1) and C]N group (peak 2),
respectively.
The diffusion current–concentration relationship was found to be recti-
linear over the range 1.6–224 and 1.6–28 μg/mL for Ep1 and over the
ranges 1.6–256 and 1.6–32 μg/mL for Ep2 using DME & SMDE, respec-
tively, with limit of quantifying pioglitazone HCl as 1.6 μg/mL, and relative
standard deviation (RSD) 4.0% and 4.3% for Ep1 and Ep2 using DME
and 3.6% and 3.8% for Ep1 and Ep2 using SMDE. The peaks were char-
acterized as being irreversible and diffusion-controlled although adsorption
phenomenon played a limited role in the electrode process.
5.3.2 Potentiometric Titration

Mostafa et al. [52] described the construction and electrochemical response
characteristics of poly (vinyl chloride) membrane sensors for determination
of pioglitazone HCl in which the authors used polyvinyl chloride (PVC)
membrane sensors. These membrane sensors incorporate ion association
complexes of pioglitazone cation and sodium tetraphenylborate (NaTPB)
(sensor 1) or phosphomolybdic acid (PMA) (sensor 2) or phosphotungstic
acid (PTA) (sensor 3) as electroactive materials. The sensors display a fast,
stable, and near-Nernstian response over a relative wide pioglitazone con-
centration range (1 102 to 106 M). The direct determination of 2.5–
3900.0 μg/mL of pioglitazone show an average recovery a mean relative
standard deviation of 98.5 1.6, 99.0 1.5, and 98.4% 1.7% and at
100.0 μg/mL for sensors 1, 2, and 3, respectively. These sensors were
applied for direct determination of pioglitazone in some pharmaceutical
preparations and have been used as indicator electrodes for potentiometric
titration.
El-Ghobashy et al. [53] applied Polyvinyl chloride (PVC) membrane
sensors for the determination of pioglitazone hydrochloride pioglitazone
and metformin hydrochloride (MET) by using the ion association com-
plexes between these drugs with either sodium tetraphenyl-borate (TPB)
or ammonium reineckate (RNC) counter ions. The performance character-
istics of the sensors evaluated according to IUPAC recommendations reveal
a fast, stable, and linear response over the concentration range 3.162 105–
1 102 M for PIO and 1 103–1 101 M for MET.
Faridbod et al. [54] selected pioglitazone–tetraphenyl borate as a suitable

ion-pair reagent in making pioglitazone potentiometric PVC membrane
sensor. The proposed method showed a wide linear range of 105–
102 mol/L and detection limit of 6.0 106 mol/L.
Badawy et al. [55] applied carbon paste and polyvinyl chloride as
membrane electrodes for the determination of anti-diabetic drugs for
type 2 diabetic patients. The authors used these electrodes for the
potentiometric determination of rosiglitazonne, pioglitazone, glime-
piride, and glyburide in their standard forms and also as pharmaceutical
preparation. The prepared ion-selective electrodes showed a Nernstian
response with the limit of detection amounting to 106 M in a pH range
of 3–5.
5.3.3 Voltammetric Method

Al-Arfaj et al. [56] used square-wave adsorptive cathodic stripping
voltammetry for the determination of pioglitazone HCl in Britton Robin-
son buffer of pH 5. The adsorptive cathodic peak was observed at 1.5 V vs
Ag/AgCl. Under optimal conditions, the peak current is proportional to the
concentration of pioglitazone HCl, and linear calibration graphs were
obtained within the concentration levels of 108 and 104 M following dif-
ferent accumulation time periods (0–300 s). The detection limit is
8.08 109 M (3.17 ng/mL) using 300 s preconcentration time, whereas
the quantitative limit is 2.45 108 M (9.63 ng/mL).
Al-Arfaj et al. [57] developed a flow injection chemiluminescent (FI-CL)
method for the determination of pioglitazone HCl. It is based on the sen-
sitizing effect of the drug on the oxidation reaction of sulfite with cerium
(IV). The method permits the determination of 0.05–3.0 mg/mL of
pioglitazone HCl with correlation coefficient r ¼ 0.9999. The lower limit
of detection (LOD) is 0.01 mg/mL (S/N ¼ 2) and the lower limit of quan-
titation (LOQ) is 0.05 mg/mL.
Wang et al. [58] developed a method that uses electrochemical imped-
ance spectroscopy (EIS) to quantitatively determine pioglitazone, a
thiazolidine-2,4-diones derivative. The method uses a silver electrode.
An increase in the pioglitazone concentration results in an increase
in the Faradaic electron-transfer resistance (Ret) obtained from the EIS
measurements. Pioglitazone is quantified from the linear variation of
the sensor response (Ret) as a function of the pioglitazone concentration
in solution.
Pioglitazone 409
Wang et al. [59] developed a method that uses flow-through

voltammetric sensor to quantitatively determine thiazolidine-2,4-diones
(TZDs) derivatives which are pioglitazone, rosiglitazone, and troglitazone.
The method uses a gold electrode. The dynamic range for determining
thiazolidine-2,4-diones (TZDs) is extended by more than two orders of
magnitude. The quantification limits are 0.10, 23, 15, and 0.005 ng for
thiazolidine-2,4-dione, pioglitazone, rosiglitazone, and troglitazone,
respectively. The method can be applied to the quantitatively determining
pioglitazone and rosiglitazone in anti-diabetic drugs. Findings using HPLC
with a flow-through voltammetric detector and UV detector are
comparable.
5.4 Chromatography
5.4.1 Thin-Layer Chromatography
Kucher et al. [60] established a thin layer chromatography method for sep-
aration and quantitative analysis of pioglitazone hydrochloride tablet. Meth-
anol was selected as solvent for pioglitazone according to physical–chemical
properties. Behavior of pioglitazone was investigated in different chromato-
graphic conditions. Chromatographic plate Sorbfi l, Armsorb, and Merck
were used as stationary phase. General systems of TLC screening of acid
and neutral agents and others were used as mobile phase. When using the
mobile phase chloroform–methanol (90:10) RF value of pioglitazone was
0.82, 0.7, 0.79; chloroform–acetone (80:20): 0.55, 0.42, 0.42; toluene–
acetone–methanol-25% solution of ammonia (50:20:10:0.02): 0.57, 0.57,
0.57; and chloroform–toluene–acetate acid conc.–ethanol (4.5:4.5:1:1):
0.45, 0.45, 0.51, respectively. The most acceptable defined system is
chloroform–toluene–acetic acid conc.–ethanol (4.5:4.5:1:1). Detection of
spots (areas of absorbance) of substances on the chromatogram was carried
out in two ways: irradiation by UV light, and action by general and
specific detecting reagents. The most optimal reagent was Dragendorff spray
modified on Munje (limit of detection 0.01 mg).
5.4.2 Capillary Electrophoresis

Calixto et al. [61] established an alternative electrophoretic method for
determination Pioglitazone and its main metabolites in rat liver microsomal
fraction. The electrophoretic analyses were performed using an uncoated
fused-silica capillary of 50 μm i.d., 48 cm in total length and 40 cm in effec-
tive length, and 50 mmol/L sodium phosphate buffer solution (pH 2.5).
All experiments were carried out under the normal mode. The capillary
temperature was set at 35°C and a constant voltage of +30 kV was applied
during the analyses. Samples were introduced into the capillary by hydrody-
namic injection (50 mbar, 15 s) and detection was performed at 190 nm.
The sample preparation procedure, based on hollow-fiber liquid-phase
microextraction, was optimized using multifactorial experiments. Next,
the following optimal condition was established: sample agitation at
1500 rpm, extraction for 15 min, 0.01 mol/L hydrochloric acid as acceptor
phase, 1-octanol as organic phase, and donor phase pH adjustment to 6.0.
The method demonstrated LOQs of 200 ng/mL. Additionally, it was linear
over the concentration range of 200–25,000 ng/mL for Pioglitazone and
200–2000 ng/mL for the metabolites.
Yin et al. [62] established a capillary zone electrophoresis method for chi-
ral separation of pioglitazone hydrochloride. Methods by optimizing factors
which affect the chiral separation, the kinds and concentration of cyclodex-
trin, the pH value and concentration of buffer, the voltage and temperature,
and the optimum conditions for chiral separation were selected. The results
of the optimal separation were conditioned as a phosphate buffer 40 mmol/
L containing hydroxypropyl-γ-cyclodextrin (6 mmol/L), detection wave-
length at 200 nm, voltage at 18 kV and separation temperature at 20°C,
by which the separation of pioglitazone hydrochloride enantiomers was
achieved. Conclusion The established method is convenient, which can
be applied for chiral separation of pioglitazone hydrochloride enantiomers.
5.4.3 HPLC Methods

Tahmasebi et al. [63] evaluated the applicability of hollow fiber-liquid phase
microextraction (HF-LPME) for extraction and preconcentration of trace
amounts of pioglitazone as an anti-diabetic drug in biological fluids, prior
to the determination by HPLC. In this technique, the target drug was
extracted into di-n-hexyl ether immobilized in the wall pores of a porous
hollow fiber from 10 mL of the aqueous sample (source phase, SP) with
pH 8.0, and then back extracted into the receiving phase (RP) with pH
2.2 located in the lumen of the hollow fiber. The extraction occurred
due to a pH gradient between the two sides of the hollow fiber. After
extracting for a prescribed time, 24 μL of the RP solution was taken back
into the syringe and injected directly into an HPLC instrument for quanti-
fication. The Taguchi orthogonal array (OAD) experimental design with an
OA16 (45) matrix was employed to optimize the HF-LPME conditions.
Under the optimum conditions (di-n-hexyl ether as membrane impregna-
tion solvent, pHs of the SP and RP equal to 8.0 and 2.2, respectively,
Pioglitazone 411
extraction time of 30 min, stirring speed of 500 rpm and 10% (w/v) NaCl
for adjusting the ionic strength), preconcentration factor of 180, linear
dynamic range (LDR) of 2.5–250 μg/L with good correlation of determi-
nation(r2 > 0.998) and limit of detection (LOD) of 1.0 μg/L were obtained
for the target drug.
Ravikanth et al. [64] developed a rapid high-performance liquid chro-
matography with UV–visible (HPLC-UV) detection method for the deter-
mination of Pioglitazone in rat serum. Rosiglitazone was used as internal
standard. Pioglitazone and Rosiglitazone are extracted from serum using a
liquid–liquid extraction procedure using ethyl acetate. Isocratic separation
of Pioglitazone and Rosiglitazone is carried out using a reversed-phase
phenomenex C18 (250 mm 4.6 mm, 5 μm) column with mobile phase
consisting of methanol and 30 mM ammonium acetate buffer (pH adjusted
to 5 with ortho-phosphoric acid) in the ratio of 60:40 (v/v) and quantified
by UV detection at 269 nm. Analytical run time was less than 10 min. Mean
recovery was 97.12% for 0.1–10 μg/mL concentrations. The assay exhibited
good linear relationship. Quantification limit was at 50 ng/mL of
Pioglitazone and accuracy and precision were over the concentration range
of 0.1–10 μg/mL.
Lakshmi et al. [65] developed reverse-phase HPLC method for the deter-
mination of Pioglitazone and Glimepiride on a Shimadzu Class vp series
HPLC system with a phenomenex C18 column (150 4.6 mm, 5 μm) using
a mobile phase mixture containing methanol and ammonium acetate buffer
(pH 3.5) in the ratio of 55:45 with the flow rate was 0.5 mL/min. The efflu-
ents were monitored at 252 nm and eluted at 5.63 min Pioglitazone and
7.18 min glimepiride. Calibration curve was plotted with a range from 25
to 25,000 ng/mL for Pioglitazone and 10 to 10,000 ng/mL for glimepiride.
The drugs were extracted from rat plasma by simple liquid–liquid extraction
using diethyl ether as an extraction solvent.
Islambulchilar et al. [58] developed a simple and rapid HPLC method
with UV detection for the determination of pioglitazone in human plasma.
The method was based on protein precipitation using perchloric acid on an
ODS column. The mobile phase consisted of a mixture of phosphate buffer,
methanol, acetonitrile, and 12 M perchloric acid (54:33:12:1, v/v/v/v).
The UV detector was set at 269 nm. Under these conditions, the retention
time of pioglitazone was 5.2 min. The standard curve was linear over the
range of 50–2000 ng/mL pioglitazone in human plasma. The within-day
and between-day precision studies showed high reproducibility, with CV
less than 5. The LOQ was 44.2 ng/mL. The method has been applied to
a bioequivalence study after administration of pioglitazone as 30 mg tablets

to 12 healthy volunteers.
Arayne et al. [66] developed, validated, and applied a reversed-phase
high-performance liquid chromatographic (RP-HPLC) method for the
simultaneous determination of gliquidone, pioglitazone hydrochloride,
and verapamil in tablets and human serum. Chromatographic separation
was achieved on a C18 column (5 μm, 25 0.46 cm) with a mobile phase
consisting of methanol–water–acetonitrile (80:10:10, v/v/v) with a flow
rate of 0.7 mL/min and pH adjusted to 3.50 with phosphoric acid at
230 nm. Glibenclamide was used as internal standard. The experimentally
derived limit of detection and limit of quantitation were determined to
be 0.24, 0.93, 0.40, and 0.80, 3.11, 1.36 μg/mL for gliquidone,
pioglitazone, and verapamil, respectively.
Saber [67] developed a rapid and accurate HPLC method for the deter-
mination of pioglitazone hydrochloride in tablets. Chromatographic analysis
was performed on a Nova-Pak® C18 column (3.9 mm 150 mm, 5 μm)
with a mixture of ammonium formate buffer adjusted with formic acid to
pH 3 and acetonitrile (75:25, v/v) as mobile phase, at flow rate of
1.0 mL/min, and UV detection at 225 nm. The determination was com-
pleted in less than 12 min. Linearity 0.5 μg/mL, accuracy 99.14%,
and precision 0.6% were found to be acceptable over the range 0.5–
20 μg/mL.
Jedlička et al. [68] developed a reversed-phase gradient HPLC method
for the evaluation of pioglitazone hydrochloride (PG-HCl) in tablets.
The limit of detection for PG-HCl was found to be 42 ng/mL. Analyses
were performed on a C18 column (Symmetry C18, 5 μm, 2504.6 mm)
and mobile phase was a mixture of ammonium formate buffer adjusted with
formic acid to pH 4.1 and acetonitrile. Shortened purity method was used as
the assay method.
Sane et al. [69] developed a rapid HPLC method for simultaneous
determination of pioglitazone and glimepiride. Chromatographic separa-
tion of the two pharmaceuticals was performed on a Cosmosil C18 column
(150 mm 4.6 mm, 5 mm) with a 45:35:20 (v/v) mixture of 0.01 m
triammonium citrate (pH adjusted to 6.95 with orthophosphoric acid),
acetonitrile, and methanol as mobile phase, at a flow rate of 1.0 mL/
min, and detection at 228 nm. Separation was complete in less than
10 min. The method showed a linear response for concentrations over
the ranges of 2.50–30.00 μg/mL for pioglitazone and 0.10–10.00 μg/
mL for glimepiride.
Pioglitazone 413
Swapna et al. [70] developed an RP-HPLC method for the estimation of

Metformin HCl (MET) and Pioglitazone (PIO) in pure and in pharmaceu-
tical dosage forms. A BDS Hypersil C18 column (250 4.6 mm, 5 μm) was
used with a mobile phase containing a mixture of Acetonitrile and Potassium
dihydrogen ortho phosphate buffer (pH 3) in the ratio of 50:50. The flow
rate was 1 mL/min and effluents were monitored at 238 nm and eluted at
2.81 min (MET) and 4.57 min (PIO). Calibration curve was plotted with
ranges of 40-240 μg/mL for MET and 12–72 μg/mL for pioglitazone.
Karthik et al. [71] developed a reverse-phase isocratic HPLC method for
the separation and quantification of pioglitazone and glimepiride in bulk
drug and pharmaceutical dosage form. The quantification was carried out
using Inertsil ODS (250 4.6 mm, 5 μm) column and mobile phase com-
prised of acetonitrile and ammonium acetate (pH 4.5, 20 mM) in proportion
of 60:40 (v/v). The flow rate was 1.0 mL/min and the effluent was moni-
tored at 230 nm. The retention time of pioglitazone and glimepiride were
7.0 0.1 and 10.2 0.1 min, respectively. Linearity of pioglitazone and
glimepiride were in the range of 2.0–200.0 and 0.5–50 μg/mL, respectively.
The percentage recoveries of both the drugs were 99.85% and 102.06% for
pioglitazone and glimepiride, respectively from the tablet formulation.
Jain et al. [72] developed an RP-HPLC method for the simultaneous
estimation of metformin hydrochloride (MET), pioglitazone hydrochloride
(PIO), and glimepiride (GLP) present in multicomponent dosage forms.
Chromatography is carried out isocratically at 25°C 0.5°C on an Inertsil
ODS-3 (C18) column (250 4.60 mm, 5 μm) with a mobile phase com-
posed of methanol–phosphate buffer (pH 4.3) in the ratio of 75:25 (v/v)
at a flow rate of 1 mL/min. Detection is carried out using a UV-PDA detec-
tor at 258 nm. The retention times for MET, PIO, and GLP are 2.66 + 0.5,
7.12 + 0.5, and 10.17 + 0.5 min, respectively. The linearity range and per-
centage recoveries for MET, PIO, and GLP are 10–5000, 10–150, and
1–10 μg/mL and 100.4%, 100.06%, and 100.2%, respectively.
Sarat et al. [73] developed and validated HPLC method for the analysis of
Sexaliptin and Pioglitazone. Chromatographic separation achieved
isocratically on a C18 column (Use Inertsil C18, 5 m, 150 mm 4.6 mm)
utilizing a mobile phase of acetonitrile/phosphate buffer (60:40, v/v, pH
7.0) at a flow rate of 0.8 mL/min with UV detection at 260 nm. Aceclofenac
was used as an internal standard. The retention time of Sexagliptin,
pioglitazone, and aceclofenac was 2.48, 4.45, and 6.34 min, respectively.
Kalyankar et al. [74] developed a reversed-phase liquid chromatographic
method for simultaneous determination of pioglitazone HCl in combination
with glimepiride. This method uses a Eurosphere-100 C18 (250 Ã—

4.6 mm, 5 μm) analytical column, a mobile phase of acetonitrile and buffer
containing 0.01 M potassium dihydrogen orthophosphate in the ratio 50:50
(v/v), and pH adjusted to 6.2 with orthophosphoric acid. The instrumental
settings are at the flow rate of 1.4 mL/min and a detector wavelength of
225 nm. The retention times for pioglitazone HCl and glimepiride are
4.34 and 6.33 min, respectively. The linearity range for pioglitazone HCl
and glimepiride were in the range of 0.9–2.4 and 0.12–0.32 μg/mL,
respectively.
Havaldar et al. [75] developed a gradient RP-HPLC method and subse-
quently validated for the determination of glimipiride, rosiglitazone, and
pioglitazone hydrochloride. Separation was achieved with a nucleodur
C18 column having 250 4.6 mm i.d. with 5 μm particle size and water
HPLC grade adjusted to pH 3.0 using diluted orthophosphoric acid and ace-
tonitrile (80:20, v/v) with gradient program as eluent at a constant flow rate
of 0.8 mL/min. UV detection was performed at 215 nm. The retention time
of glimipiride, rosiglitazone, and pioglitazone hydrochloride were about
17.9, 6.31, and 8.24 min, respectively.
Shankar et al. [76] developed two methods of analysis to determine
pioglitazone hydrochloride pioglitazone and metformin hydrochloride in
combined dosage forms using second-derivative spectrophotometry and
reversed-phase liquid chromatography (LC). In the LC method, analysis
was performed on a Hypersil ODS C18 column with 5 μm particle size using
the mobile phase acetonitrile–water–acetic acid (75:25:0.3), adjusted to pH
5.5 with liquor ammonia, at a flow rate of 0.5 mL/min. Measurement was
made at a wavelength of 230 nm. Both the drugs were well resolved on the
stationary phase, and the retention times were 8.5 min for pioglitazone and
16.0 min for metformin. The calibration curves were linear in the concen-
tration range of 4–20 μg/mL for pioglitazone and metformin.
Nirupa et al. [77] developed a reverse-phase HPLC method for the
separation and estimation of three drugs glimepiride, pioglitazone, and
metformin in bulk drug mix and pharmaceutical dosage forms. The
estimation was carried out using Inertsil ODS-3V (250 mm 4.6 mm,
5 μm) column; mobile phase consisting of acetonitrile, tetrahydrofuran,
and buffer at pH 5; the flow rate of 1.7 mL/min and UV detection at
228 nm. All the three drugs were properly resolved having run time of
5, 3.9, and 1.3 min for glimepiride, pioglitazone, and metformin, respec-
tively. The validated method applied to the commercially available
pharmaceutical dosage form.
Pioglitazone 415
Sakuntala et al. [78] developed an RP-HPLC method for the determi-

nation of Glimepiride (GLM) and Pioglitazone HCl (PIO) on shimadzu
HPLC systems with Inertsil ODS C18 column (150 4.6 mm, 5 μm) and
using a mobile phase mixture containing mixed phosphate buffer and ace-
tonitrile in the ratio of 40:60. The flow rat e was 1.5 mL/min and the efflu-
ent was monitored at 225 nm. The retention time of Glimepiride and
Pioglitazone HCl were 3.06 and 1.97 min, respectively. Linearity of
Glimepiride and Pioglitazone HCl were in the range of 1.32–7.92 and
10–60 μg/mL. The percentage recoveries of both the drugs were 99.78%
and 100.10% for GLM and PIO, respectively from the tablet formulations.
The proposed method is suitable for simultaneous determination of
Glimepiride and Pioglitazone HCl for routine quality control of drugs in
bulk drug and formulation.
Havele et al. [79] developed and validated an RP-HPLC method for
simultaneous analysis of metformin hydrochloride, gliclazide, and
pioglitazone hydrochloride in a tablet dosage form. Chromatography was
performed on a 25 cm 4.6 mm i.d., 5 μm particle, C18 column with
85:15 (v/v) methanol:20 mM potassium dihydrogen phosphate buffer as
mobile phase at a flow rate of 1.2 mL/min. UV detection at 227 nm; met-
formin hydrochloride, gliclazide, and pioglitazone hydrochloride were
eluted with retention times of 2.15, 3.787, and 4.57 min, respectively. Cal-
ibration plots were linear over the concentration ranges 50–250 μg/mL for
metformin hydrochloride, 3.0–15.0 μg/mL for gliclazide, and 2–10 μg/mL
for pioglitazone hydrochloride. Limits of detection were 0.20, 0.04, and
0.10 μg/mL and limits of quantification were 0.75, 0.18, and 0.30 μg/mL
for metformin hydrochloride, gliclazide, and pioglitazone hydrochloride,
respectively.
Souri et al. [80] developed a new, simple, and reproducible HPLC
method for the determination of pioglitazone in human plasma. After
liquid–liquid extraction with diethyl ether, samples were quantitated on a
Nova-Pak C8 column using a mixture of acetonitrile–140 mM K2HPO4
(40:60, v/v, pH 4.45) as mobile phase with UV detection at 269 nm.
The flow rate was set at 1.4 mL/min. Ethylparaben was used as internal stan-
dard and the total run time of analysis was approximately 7 min. The method
was linear over the range of 25–1500 ng/mL of pioglitazone in plasma
(r2 > 0.999). The within- and between-day precision values were in the
range of 2.4–6.8%. The limit of quantitation of the method was 25 ng/mL.
Radhakrishna et al. [81] developed HPLC and Micellar Electrokinetic
Chromatographic (MEKC) methods for the determination of pioglitazone,
a new englycemic antidiabetic agent. Pioglitazone and its unsaturated impu-

rity were separated by MEKC in less than 7 min using a 43 cm 50 μm i.d.
uncoated fused-silica capillary with extended light path for better sensitivity
(25 kV at 30°C) and a background electrolyte (BGE) consisting of 20% ace-
tonitrile (v/v) in 20 mM sodium borate buffer pH 9.3 containing 50 mM
sodium dodecyl sulfate (SDS). The HPLC method is capable of detecting
all process-related compounds, which may be present at trace levels in fin-
ished products.
Yamashita et al. [82] developed an HPLC method for the simultaneous
determination of pioglitazone and its metabolites (M-I to M-V) in human
serum and urine. The method for serum involved the solid-phase and
liquid–liquid extraction. Urine with and without enzymatic hydrolysis using
β-glucuronidase was treated with liquid–liquid extraction. The compounds
in the extract were analyzed using HPLC with UV detection at 269 nm. The
detection limits of pioglitazone, M-I, M-II, M-III, M-IV, and M-V in
serum were 0.01–0.05 μg/mL; those in urine were 0.1–0.5 μg/mL; and
those in urine after enzymatic hydrolysis were 0.3–0.5 μg/mL, respectively.
Sripalakit et al. [83] developed an analytical method based on HPLC-UV
detection (269 nm) for the determination of pioglitazone in human plasma.
Rosiglitazone was used as an internal standard. Chromatographic separation
was achieved with a reversed-phase Apollo C18 column and a mobile phase
of methanol–acetonitrile mixed phosphate buffer (pH 2.6, 10 mM)
(40:12:48, v/v/v) with a flow rate of 1.2 mL/min. The calibration curve
was linear over the range of 50–2000 ng/mL and the lower limit of quan-
tification was 50 ng/mL.
Vinod et al. [84] developed a method for the simultaneous estimation of
Pioglitazone and glimepiride in a combined tablet dosage form by using RP-
HPLC method and UV spectrophotometric method. Both methods were
validated and compared for sensitivity and linearity. The RP-HPLC method
utilized Gliburide (Glibenclamide) as an internal standard and the mobile
phase composition was methanol and water that gave a retention time of
4.34 and 5.19 min for Glimepiride and Pioglitazone, respectively. The lin-
earity range was between 1–15 and 3–45 μg/mL for glimepiride and
pioglitazone, respectively. The accuracy of the method was found to be
in the range of 98–102%. Both these methods applied to pharmaceutical
dosage formulation and were validated according to ICH guidelines.
PremAnand et al. [85] developed and validated simple, rapid, fast, and
precise RP-HPLC method for the simultaneous estimation of Telmisartan
and Pioglitazone in tablet dosage form. The quantification was carried out
Pioglitazone 417
using Phenomenex C8 (250 4.6 mm, 5 μm) column and mobile phase
comprised of acetonitrile and ammonium dihydrogen phosphate (pH 4.5,
20 mM) in proportion of 65:35 (v/v). The flow rate was 1.0 mL/min
and the effluent was monitored at 210 nm. The retention time of
Telmisartan and Pioglitazone were found to be 2.38 and 3.16 min, respec-
tively. Linearity of Telmisartan and Pioglitazone were in the range of 10–50
and 7.5–37.5 μg/mL, respectively. The percentage recoveries of both the
drugs were 99.85% and 102.06% for telmisartan and pioglitazone, respec-
tively from the tablet formulation. The proposed method is suitable for
simultaneous determination of Telmisartan and pioglitazone in pharmaceu-
tical dosage form and bulk drug.
Lakshmi et al. [86] developed an HPLC method and a UV derivative
spectrophotometric method for the simultaneous determination of metfor-
min (MFN), pioglitazone (PLZ) and glimepiride (GLM), in tablets. HPLC
was carried out by using the reversed-phase technique on an phenomenex
RP-18 column (150 4.6 mm, 5 μm) with a mobile phase consisting of a
cetonitrile and phosphate buffer (pH 3) in the ratio of 65:35. The flow rate
was fixed at 0.5 mL/min and the drugs were monitored at 245 nm with UV
dual absorbance detector and the elution time was found less than 10 min,
indicating shorter analysis time.
Lakshmi et al. [87] developed a rapid RP-HPLC method for the
estimation of Metformin HCl and Pioglitazone in pure and in pharma-
ceutical dosage forms. A Gemini C18 column (150 4.6 mm, 5 μm) was
used with a mobile phase containing a mixture of acetonitrile and ammo-
nium acetate buffer (pH 3) in the ratio of 42:58. The flow rate was
0.3 mL/min and effluents were monitored at 255 nm and eluted at
5.17 min Metformin and 8.1 min Pioglitazone. Calibration curve was plot-
ted with a range from 0.5 to 50 μg/mL for metformin and 0.3 to 30 μg/mL
for Pioglitazone.
Sharma et al. [88] reported a liquid chromatographic procedure that uses
micellar mobile phase containing only Tween-20 and n-butanol, for the
simultaneous determination of Atorvastatin Calcium (ATV) and
Pioglitazone (PIO) in tablet dosage form. The estimation was carried out
on Luna C18column (5 μm 25 cm 4.6 mm) with a mixture of Tween-
20 and n-butanol phosphate buffer, pH 4.2 (50:25:25, v/v) at flow rate
of 1.5 mL/min at 25°C temperature. Quantitation was achieved by
UV detection at 322 nm over lain spectra and the concentration range
was 5–210 μg/mL for both the drugs with mean recoveries of 99.01%
±0.12 and 100.64% ±0.20 for ATV and PIO, respectively.
Rashmithaa et al. [89] developed an RP-HPLC method for Pioglitazone

hydrochloride in the presence of its impurities and degradation products,
generated from forced degradation studies. The drug substance was sub-
jected to stress conditions of hydrolysis, oxidation, photolysis and thermal
degradation. The separation of the drug from the process-related impurities
and degradation products formed under stress conditions was achieved on an
Inertsil ODS-3V (150 4.6 mm), 5 μm column. The gradient LC method
employs solution A and solution B as mobile phase. The solution A contains
phosphate buffer pH 3.1 and Solution B contains acetonitrile.
Madhukar et al. [90] developed an isocratic RP-HPLC method and sub-
sequently validated the determination of Pioglitazone Hydrochloride. Sep-
aration was achieved with a Symmetry-Extend-C18 HPLC column
(150 mm 4.6 mm). A mobile phase comprising 0.01 M buffer: Methanol
in the volume ratio of 40:60 was developed. The detection was carried out
using a UV detector set at a wavelength of 240 nm. The method was linear
over the concentration range of 1–200 μg/mL and can be used for quality
control assay of Pioglitazone Hydrochloride.
Abro et al. [91] developed a rapid and reliable analytical method based on
HPLC with UV detection (221 nm) for the determination of the
antihyperglycemic agent Pioglitazone in pharmaceutical formulations and
biological fluids (serum and urine) after clean up with solid-phase extraction.
Chromatographic separation was achieved with a Chromolith® Perfor-
mance RP-18e (100 4.6 mm) column using mobile phase composition
of acetonitrile: mixed phosphate buffer (pH 2.5, 10 mM) (30:70, v/v) with
a flow rate of 2.0 mL/min. The total run time was only 2 min under opti-
mized conditions. The calibration curve was found to be linear in the range
of 1–10 μg/mL with regression coefficient of 0.9996, and the lower limit of
detection was 72 ng/20 μL.
Kumar et al. [92] developed and validated an HPLC-UV detection for
simultaneous determination of Pioglitazone and Clopidogrel. Separation
was performed on a C18 column by isocratic elution with a mobile phase
of methanol:acetonitrile:water (80:10:10) at pH 4.6. The UV detection
was set at 230 nm. The method proved to be specific, accurate, precise,
and linear over the concentration ranges of 20–120 ppm for both
Pioglitazone and Clopidogrel with correlation coefficients always >0.999
for both drugs. The intra- and inter-day precision and accuracy were less
than 2 for both analytes.
Srinivasulu et al. [93] developed and validated specific and stability-indi-
cating reversed-phase gradient liquid chromatographic method for
Pioglitazone 419
determination of Pioglitazone Hydrochloride along with its impurities

in bulk samples. Drug substance was subjected to stress conditions of
hydrolysis (acid and base), oxidation, photolytic, humidity, and thermal
degradation as per International Conference on Harmonization (ICH) to
show the stability-indicating power of the method. Significant degrada-
tion was observed with alkali and hydrogen peroxide. The impurities
were characterized using spectral techniques like IR, 1H NMR, and
MS. Successful separation of impurities was achieved on C18 ODS
(150 4.6 mm) 3.5 μm column using mobile phase consisting of Solvent
A: Ammonium acetate buffer and Acetonitrile in the ratio (57:43, v/v)
for 0–7 min and Solvent B: Ammonium acetate buffer and Acetonitrile
in the ratio (20:80, v/v) at a flow rate of 1.0 mL/min from 7 to 20 min
followed by Solvent A from 20 to 21 min. The retention times of impurity
A, impurity B, impurity C, and Pioglitazone were 3.44, 10.65, 17.95, and
8.32 min, respectively. The detection wavelength was set at 254 nm with
column temperature at 45°C.
Pallapolu et al. [94] described a simple, economic, selective, accurate,
precise RP-HPLC method for the simultaneous estimation of metformin
and pioglitazone in pure and pharmaceutical dosage forms. Metformin
and pioglitazone were well separated using a Hypersil BDS C18 column
of dimension 250 4.6, 5 μm and Mobile phase consisting of Phosphate
buffer:Methanol (Adjusted with Ortho phosphoric acid to pH 4.5) in the
ratio of 70:30 (v/v) at the flow rate 1 mL/min and detection was carried
out at 240 nm with PDA detector. The retention time for Metformin
and Pioglitazone were found to be 1.945 and 3.595 min, respectively.
Prava et al [95] developed and validated an RP-HPLC method for the
determination of process-related impurities in pioglitazone hydrochloride.
High-quality separation was achieved on a Luna C18 column (150 mm
4.6 mm, 3 μm) using gradient elution at a flow rate of 1 mL/min and a
column temperature of 45°C. UV detection was performed at 254 nm.
The method gives satisfactory separation of impurities of pioglitazone
hydrochloride and so it is suitable for quantification of the process-related
impurities as well as for the assay of the active compound.
Madhukar et al. [96] developed a precise reverse-phase isocratic HPLC
method for the separation and quantification of pioglitazone and glimepiride
in bulk drug and pharmaceutical dosage form. The quantification was car-
ried out using X-Bridge ODS (150 4.6 mm, 5 μm) column and mobile
phase comprised of Acetonitrile and Ammonium Acetate (pH 4.3,
20 mM) in proportion of 40:60 (v/v). The flow rate was 1.0 mL/min
and the effluent was monitored at 235 nm. The retention time of
Pioglitazone and Glimepiride were found to be 2.61 and 3.50 min, respec-
tively. Linearity of pioglitazone and glimepiride were in the range of 1.5–
225 and 0.20–30 μg/mL, respectively. The percentage recoveries of both
the drugs were 98.95–101.22% and 98.46–100.98% for Pioglitazone and
Glimepiride, respectively, from the tablet formulation.
Shaik et al. [97] developed and validated an HPLC-UV method for the
determination of pioglitazone hydrochloride. It is an oral antidiabetic agent
belonging to the class of thiazolidinediones. Isocratic separation of
Pioglitazone is carried out using a reversed-phase Intersil ODS C18 column
(150 mm 4.6 mm, 5 μm) with mobile phase consisting of Ammonium
acetate buffer with Acetonitrile and Glacial acetic acid in the ratio of
50:50:1 (v/v) and quantified by UV detection at 269 nm with flow rate
of 0.7 mL/min.
Najma et al. [98] achieved a method for the simultaneous quantification
of four NIDDM drugs (metformin, glimepride, glibenclamide, and
pioglitazone) on a Purospher Start C18 (5 μm, 25 0.46 cm) and Supelco
C18 column in 2, 3, 7, 9 min, respectively. The optimized method involves
a C18 column thermostated at 30°C, UV detection at 235 nm, at a flow rate
of 1 mL/min. Good separation of the analytes was achieved by gradient
HPLC-UV/visible detector in API, pharmaceutical dosages, and serum;
The mobile phase was a mixture of methanol:water (70:30, v/v) and the
pH of which was adjusted to 3.0 by phosphoric acid.
The method exhibited consistent, high-quality recoveries of the four
analytes which ranged from 93.8 2.1 to 99.8 1.5 (mean RSD) with
a high precision for the drug and impurities. Validation under Food
and Drug Administration (FDA) guideline of the analytical parameters
includes: linearity (r2 > 0.9996), LLODs (0.315, 2.3, 0.2,0.1 ng/mL),
LLOQs (0.95, 0.7, 0.59,0.32 ng1), intra-day precision (0.001), and
inter-day precision 0.9 expressed as relative standard deviation (RSD),
and robustness parameters (less than 1.98%) with accuracies between
98% and 102%.
Sharma et al. [99] developed the method of RP-HPLC coupled with
a diode array detector (DAD) for the pharmacokinetic interaction study
of atorvastatin with pioglitazone and cholestyramine, respectively, in
Wistar rats. Atorvastatin and pioglitazone were resolved on a C18 column
with a mobile phase composed of 48% methanol, 19% acetonitrile, and
33% 10 mM ammonium formate (v/v/v; pH 3.5 0.3, by formic acid)
and a 260 nm detection wavelength on the diode array detector. The
Pioglitazone 421
method was validated according to international standards with good

reproducibility and linear response; mean (r) 0.9987 and 0.9972 to Ator-
vastatin and pioglitazone, respectively. The coefficients of variation of intra-
and inter-assay precision ranged between 4.95–8.12 and 7.29–9.67,
respectively.
Neelima et al. [100] developed a new stability-indicating RP-HPLC
method for estimation of Alogliptin and Pioglitazone in bulk and pharma-
ceutical dosage form. To optimize the mobile phase, various combinations
of buffer and organic solvents were used on Hypersil BDS C18 column.
Then the mobile phase containing a mixture of phosphate buffer:Acetoni-
trile in the ratio of 45%:55% (v/v) was selected at a flow rate of 1.0 mL/min
for developing the method and the peaks with good shape and resolution
were found resulting in short retention time, baseline stability, and mini-
mum noise. The retention times of Alogliptine and Pioglitazone were found
to be 3.42 and 5.24 min, respectively. Quantitative linearity was obeyed in
the concentration range of 31–187 and 75–450 μg/mL of Alogliptin and
Pioglitazone, respectively. The limit of detection and limit of quantitation
were found to be 0.399 and 1.21 μg/mL for Alogliptine and 0.516 and
1.565 μg/mL Pioglitazone, respectively, which indicates the sensitivity of
the method.
Mohamed et al. [101] developed HPLC method for determination of
both metformin hydrochloride and pioglitazone hydrochloride in tablet
dosage form. The chromatographic separation was conducted on Shimadzu
(Prominence LC 20 UFLC XR) connected with PDA detector, using
mixed column ODS/Cyano; ACE (100 4.6 mm, 5 μm). The mobile
phase was isocratic and consisted of Acetonitrile:Phosphate buffer in the
ratio of (50:50, v/v) (buffer was composed of 3.55 gm disodium hydrogen
phosphate per liter, adjusted by 85% phosphoric acid to pH 5) and was deliv-
ered to the system at a flow rate of 1.2 mL/min. An injection volume of
20 μL was used for pioglitazone hydrochloride and 5 μL for metformin
hydrochloride. The detection wavelength (λmax) was 235 nm for metformin
HCl and 266 nm for pioglitazone hydrochloride. All assays were performed
at ambient conditions. The calibration curve of metformin hydrochloride in
mobile phase was linear with correlation coefficient (r2) ¼ 0.99995; over a
concentration range of 30–750 mg/L; with a retention time of 1.07 min.
While the calibration curve of pioglitazone HCl in mobile phase was
linear with correlation coefficient (r2) ¼ 0.99859; over a concentration
range of 1–25 mg/L; with a retention time of 1.85 min. The percentage
recoveries of metformin hydrochloride and pioglitazone hydrochloride
were 100.13% and 100.22%, respectively. The relative standard deviation

(RSD) was found to be <2.
Du et al. [102] developed and validated a selective chiral HPLC method to
separate and quantify the pioglitazone enantiomers in rat plasma. After extrac-
tion of the plasma samples with ethyl acetate, the separation of pioglitazone
enantiomers and internal standard (IS, dexamethasone acetate) was achieved
on a cellulose tris(3,5-dichlorophenylcarbamate) column known as Chiralpak
IC with a mobile phase of hexane–isopropanol (70:30, v/v) at a flow rate
of 1.0 mL/min. The UV detection wavelength was set at 225 nm. Baseline
separation of pioglitazone enantiomers and IS, free from endogenous inter-
ferences, was achieved in less than 25 min. Ratio of peak area of each enan-
tiomer to IS was used for quantification of plasma samples. Linear calibration
curves were obtained over the range of 0.25–50 μg/mL in plasma for both
enantiomers (r2 > 0.9990) with quantitation limit of 0.25 μg/mL. The mean
extraction recoveries were 82.37–91.38% for pioglitazone enantiomers and
95.76% for IS from rat plasma. The mean relative error (RE %) of accuracy
and the mean relative standard deviation (RSD %) of intra- and inter-day
precision for both enantiomers were <10%. The method was validated
with accuracy, precision, recovery, and stability and used to determine the
pharmacokinetics of pioglitazone enantiomers, after a single oral administra-
tion of racemic pioglitazone (30 mg/kg). The differences between the phar-
macokinetic parameters Cmax, AUC0–24, AUC0–1, CL/F of (+)-pioglitazone
and ()-pioglitazone were significant, suggesting that the disposition of
pioglitazone in rats may be enantio-selective. Moreover, the plasma levels
of (+)- and ()-pioglitazone in female rats were apparently higher than that
in male rats, respectively.
Malichetti et al. [103] developed a method for the determination of Met-
formin and Pioglitazone by using RP-HPLC in pharmaceutical dosage
form. Chromatographic separation was carried out by using mobile phase
0.02 M potassium dihydrogen ortho phosphate:acetonitrile (55:45, v/v,
pH 5.64 adjusted with Orthophosphoric acid) on Agilent Thermo Scientific
Hypersil, C18 column (250 4.6 mm, 5 μm) at a flow rate 1.0 mL/min with
UV detection at 228 nm. The retention times for Metformin and
Pioglitazone were 2.579 and 5.633 min, respectively, and both drugs
showed good linearity in the range of 500–2000 and 30–120 μg/mL. The
percentage recovery for Metformin and Pioglitazone was found between
99.48–100.85% and 99.48–100.89%, respectively, indicating the proposed
method was accurate and precise.
Sachin et al. [104] developed an isocratic RP-HPLC method and
validation for the determination of Pioglitazone and Glimepiride.
Pioglitazone 423
The separation was achieved using a Inertsil, Phenyl C18 column

(25 cm 4.6 mm i.d. and 5 μm) with a mobile phase—Composition of
Buffer (1.15 g of diammonium hydrogen orthophosphate, 1.36 g of
potassium dihydrogen orthophosphate, and 1 mL of triethylamine in
1000 mL distilled water adjust pH 5.0 with orthophosphoric acid) and
Acetonitrile in the ratio of 50:50. Detection was carried out at
230.0 nm and the flow rate 1.0 mL/min. The method was capable of
resolving Pioglitazone and Glimepiride having resolution 6.76 between
Pioglitazone and Glimepiride.
5.4.4 HPLC–Tandem Mass Spectrometry Methods

Xue et al. [105] developed and validated a direct-injection HPLC–tandem
mass spectrometry method (LC–MS/MS) for the quantitation of
pioglitazone in human serum. After mixing the internal standard with a sam-
ple, a 10 μL portion of the mixture was directly injected into a high-flow
LC–MS/MS system, which included an extraction column, an analytical
column and a six-port switching valve. The online extraction was achieved
on an Oasis® HLB column (1 mm 50 mm, 30 μm) with a 100% aqueous
loading mobile phase containing 5 mM ammonium acetate (pH 4.0) at a
flow rate of 4 mL/min. The extracted analyte was eluted by a mobile phase
which contained 5 mM ammonium acetate and acetonitrile. The analytical
column was a Luna C18 column (4.6 mm 50 mm, 5 μm). Detection was
achieved by positive ion electrospray tandem mass spectrometry. The lower
limit of quantitation of the method was 9 ng/mL. The standard curve,
which ranged from 9 to 1350 ng/mL, was fitted by a weighted (1/x2) qua-
dratic regression model. This method was used to analyze pioglitazone con-
centrations in human serum samples from a bioequivalence study of a
blinded Actos® formulation (encapsulated 15 mg tablet) and an Actos®
15 mg tablet. The blinded formulation was shown to be bioequivalent to
an Actos® 15 mg tablet.
Surendra et al. [106] developed ultra-performance liquid chromatogra-
phy (UPLC) method and validated for the simultaneous determination of
Metformin and pioglitazone in bulk and pharmaceutical dosage forms.
The chromatographic separation of the drug components were achieved
on Waters Acquity BEH C18, 50 2.1 mm, 1.7 μm UPLC column using
0.2% triethylamine in water:acetonitrile(70:30); pH adjusted with 3.8 with
o-phosphoric acid as mobile phase 0.5 mL/min and detection wavelength
was 243 nm. Within a short runtime of 5.0 min the linearity range of met-
formin and pioglitazone were found to be 300–700 ppm and 10–50 ppm
respectively, with correlation coefficient is 0.999.
Lin et al. [107] developed and validated a liquid chromatography–

tandem mass spectrometry (LC–MS/MS) method for the simultaneous
determination of pioglitazone (PIO) and its two metabolites: M-III
(keto-derivative) and M-IV (hydroxyl derivative) in human plasma. Human
plasma samples of 0.2 mL were extracted by a single step liquid/liquid
extraction procedure and analyzed using an HPLC electrospray tandem mass
spectrometer system. The compounds were eluted isocratically on a C18
column, ionized using a positive ion atmospheric pressure electrospray
ionization source and analyzed using multiple reaction monitoring mode.
The ion transitions monitored were m/z 357 ! 134 for PIO, m/z
371 ! 148 for M-III, m/z 373 ! 150 for M-IV, and m/z 413 ! 178 for
the internal standard. The chromatographic run time was 2.5 min per injec-
tion, with retention times of 1.45, 1.02, and 0.95 min for PIO, M-III, and
M-IV, respectively. The calibration curves of pioglitazone, M-III and M-IV
were well fit over the range of 0.5–2000 ng/mL by using a weighted (1/x2)
quadratic regression. The proposed method applied to sample analysis for
clinical studies.
Kumari et al. [108] developed and validated an LC–MS/MS assay
method for simultaneous quantification of pioglitazone and candesartan in
human plasma. Irbesartan was used as an internal standard. The analytes were
extracted from human plasma samples by solid-phase extraction technique
using a Strata-X 33 mm polymeric sorbent. The reconstituted samples
were chromatographed on a C18 column by using a 80:20 (v/v) mixture
of acetonitrile and 0.1% formic acid as the mobile phase at a flow rate of
0.8 mL/min. The calibration curves obtained were linear over the concen-
tration range of 15–3000 ng/mL for pioglitazone and 5–608 ng/mL for
candesartan. A run time of 2.7 min for each sample made it possible to
analyze more than 300 plasma samples per day. The proposed method
was found to be applicable to clinical studies.
Kawaguchi-Suzuki et al. [109] developed a simple and rapid HPLC–
tandem mass spectrometry (LC–MS/MS) method for the simultaneous
determination of pioglitazone and its active metabolites hydroxypiogli-
tazone and ketopioglitazone in human plasma. Samples were processed
by protein precipitation with acetonitrile and selective phospholipid deple-
tion in a 96-well plate format. The method used deuterated internal stan-
dards for each analyte. Chromatographic separation was achieved with
gradient elution on a Hypersil GOLD C18 column. The mass spectrometer
was operated in electrospray positive ion mode with detection by selected
reaction monitoring using the transitions m/z 357.1 > 134.0 for
Pioglitazone 425
pioglitazone, m/z 373.1 > 150.0 for hydroxypioglitazone, and m/z

371.0 > 148.0 for ketopioglitazone. A linear standard curve was established
for the range of 10–1800 ng/mL for all three analytes. Intra- and inter-run
precision and accuracy (relative error) were less than 15%, and the mean
extraction recoveries of all analytes were more than 87.8%. The validated
method is sensitive and selective and was successfully applied to analyze
clinical samples obtained from patients with nonalcoholic fatty liver disease
taking pioglitazone.
Tengli et al. [110] developed and validated an UPLC–tandem mass
spectrometry method for simultaneous estimation and validation of tablet
dosage form containing glimepiride, metformin, and pioglitazone using
Tolzamide as an internal standard (IS). The chromatography separation
was achieved with Waters ACQUITY HSS C18 column, 1.8 μm,
2.1 50 mm, with mobile phase containing acetonitrile (A) and 1% ammo-
nium acetate buffer (B) (pH 2.5 adjusted with trifluoro acetic acid) with
gradient mode Gradient mode [2 min: 20 A: 80% B, 2–4 min: 70% A:
30% B, 4–5 min: 80% A: 20% B, 8–10 min: 90% A: 10% B]. The flow rate
was 0.4 mL/min column maintained at 25°C and the injection volume was
2 μL. The selected chromatographic condition was found to effectively
separate glimepiride, metformin, and pioglitazone with retention time of
3.17, 0.425, 2.3 min, respectively. The proposed method was found to
be rectilinear over the range of 2–12, 500–3000, and 15–90 ng/mL for
glimepiride, metformin, and pioglitazone, respectively. The signal intensi-
ties obtained in both positive and negative ion mode for all drugs including
internal standard found to be much higher in positive ion mode (M+H)+
parent ions at m/z 491.11, m/z 129.87, m/z 357.04, and m/z 312.04,
respectively, in QUATTROZQ full scan mass spectra.
Gananadhamu et al. [111] developed an LC–MS/MS-based method for
the simultaneous monitoring plasma levels of Sitagliptin and Pioglitazone for
applicability to pharmacokinetic studies. The method was based on HPLC
separation on the reversed-phase Phenomenex Synergy C18 column
(30 mm length, 4.6 mm internal diameter, and 4.0 μm particle size) at a
temperature of 40°C using a binary gradient mobile phase consisting of
methanol and 2 mM ammonium acetate buffer pH adjusted to 4.5 with
acetic acid, at a flow rate of 1 mL/min. Tolbutamide was used as an internal
standard. Detection of analytes was achieved with LC–MS/MS system in
Multiple Reaction Monitoring (MRM) mode. The method was validated
over concentration range of 10.98–2091.77 ng/mL for SIT and
8.25–1571.63 ng/mL for PIO, and lower limit of quantification was
10.98 and 8.25 ng/mL for Sitagliptin and Pioglitazone, respectively. Recov-
eries from spiked controls were within acceptance criteria for all the analytes
and internal standard at all QC levels. Within-batch and between-batch
accuracy for Sitagliptin was found within 96.9–100.3% and for Pioglitazone
was found within 100.0–104.3%. Within-batch and between-batch preci-
sion for Sitagliptin was less than 3.1% CV (coefficient of variation) and
for Pioglitazone was less than 5.3% CV at all concentrations levels.
Jafari et al. [112] described a method based on ESI ion mobility spectrom-
etry as a detection technique after treatment with a molecularly imprinted
polymer for the analysis of pioglitazone. In addition to the molecularly
imprinted polymer separation methodology, the positive ion mobility spec-
trum and the reduced mobility values for pioglitazone are reported for the
first time. The method was exhaustively validated in terms of sensitivity,
imprinting factor, enrichment factor, and sorption capacity. A linear
dynamic range of 0.10–20.00 μg/mL and an RSD below 6% were obtained
for the analysis of this compound. The average recovery for the analysis of
spiked samples was calculated to be about 91%.
5.4.5 High-Performance Thin-Layer Chromatography Methods

Singh et al. [113] developed and validated a simple high-performance thin-
layer chromatographic (HPTLC) method for simultaneous determination of
pioglitazone and glimepiride in bulk and tablet dosage form. The method
employed TLC aluminum plates precoated with silica gel 60 F254 as the sta-
tionary phase. The mobile phase used was a mixture of Benzene:Ethyl ace-
tate:Diethyl ether 6:3:1 (v/v). The detection of spot was carried out at
254 nm. The calibration curve of pioglitazone was found to be linear
between responses of 600–3600 ng/mL with regression coefficient 0.9984
and calibration curve of glimepiride was found to be linear between 200
and 1200 ng/mL for glimepiride with regression coefficient of 0.9991.
The limit of detection was 57.22 and 16.67 ng/mL and the quantification
limit was 190.73 and 55.58 ng/mL for pioglitazone and glimepiride, respec-
tively. The proposed method is applicable to routine analysis of Pioglitazone
in bulk and pharmaceutical formulations.
Sharma et al. [114] developed an HPTLC method for the simultaneous
estimation of Atorvastatin Calcium (ATV) and Pioglitazone (PIO) in tablet
dosage formulation. In this method, standard and sample solutions of Ator-
vastatin Calcium and Pioglitazone in tablet dosage form were separated on
the stationary phase used was precoated silica gel 60 F254. The mobile phase
used was a mixture of chloroform:methanol:toluene (6:3:4, v/v). The
Pioglitazone 427
detection of spot was carried out at 259 nm. The calibration curve was found
to be linear between 100 and 400 ng/spot for Atorvastatin Calcium and
Pioglitazone. The proposed method can be used to determine the drug
content of marketed formulations.
Kale et al. [115] developed and validated an HPTLC method for the
simultaneous determination of pioglitazone, metformin, and glimepiride
in multicomponent pharmaceutical preparations. Pioglitazone, metformin,
and glimepiride from the formulations were separated on silica gel 60 F254
HPTLC plates with acetonitrile, methanol, propyl alcohol, and ammonium
acetate solutions in the proportion of 7:2:1:1 (v/v) as mobile phase.
Densitometric quantification was performed at 240 nm. Well-resolved
bands were obtained with RF values 0.83, 0.21, and 0.89 for pioglitazone,
metformin, and glimepiride, respectively. The calibration curve was found
to be linear in the concentration range of 0.3–1.2, 10–40, and 0.04–0.16 μg
per band by area for pioglitazone, metformin, and glimepiride, respectively.
The method is selective and specific, with potential application in pharma-
ceutical analysis of these drugs in bulk and formulations.
6. STABILITY
Reddy et al. [116] developed and validated a stability indicating RP-
HPLC method for the determination of pioglitazone drug substance. Chro-
matographic separation was achieved on a Prontosil C8 SH colunm
(250 4.6 mm, 5 μm), using a mobile phase consisting of 550 mL of pH
4.0 phosphate buffer, 300 mL acetonitrile, and 150 mL methanol at a flow
rate of 1.5 mL/min. The detection was made at 254 nm. The retention time
of pioglitazone peak was 5.9 min. The method was found linear over the
range of 50–150%. The proposed method was validated as per the ICH
and USP guidelines.
Sharma et al. [117] developed an RP-HPLC method for the deter-
mination and stability indicating of pioglitazone hydrochloride in pure
and tablet forms. The recovery of the drug was calculated to be in the range
of 100.45–100.53% from a mixture of degradation products. The method
was specific to drug and also selective to degradation products. The method
showed a linear response for concentrations in the range of 10–65 μg/mL
using 0.01 M potassium dihydrogen phosphate buffer (pH 3.5):methanol
[55:45] as the mobile phase with detection at 241.0 nm and a flow rate of
1.5 mL/min resulting in a retention time of 6.15 min.
Wanjari et al. [118] developed a stability indicating simple, rapid, and

precise RP-HPLC method for the quantitation of pioglitazone in tablet
on a Hypersil C8 column (250 4.6 mm) using a mobile phase consisting
of acetonitrile:0.15% (v/v) triethylamine (40:60, v/v) adjusted to pH 4.6
with orthophosphoric acid at a flow rate of 1.5 mL/min and detection at
220 nm. The retention time of pioglitazone has been found to be
7.6 min and recoveries were between 99% and 101%.
Sriram et al. [119] described an RP-HPLC method for the quantitation
of pioglitazone hydrochloride in the presence of its impurities. The active
pharmaceutical ingredient (API) of pioglitazone hydrochloride was sub-
jected to stress conditions viz., hydrolysis, oxidation, photolysis, and thermal
degradation. The drug was found to be sensitive under basic and oxidation
environment. Successful separation of the drug from the degradation prod-
ucts were achieved on Gemini C18 column (250 4.6 mm, 5 μm) using a
mobile phase consisting of 50:50 (v/v) acetonitrile:0.05 M potassium
dihydrogen orthophosphate buffer of pH 3.0 and a flow rate of 1.0 mL/
min. Column oven temperature was kept at 40°C and quantitation was
achieved with UV detection at 225 nm. The proposed method can be
employed as a stability-indicating method for studying stability of
pioglitazone hydrochloride.
Navaneethan et al. [120] developed and validated an RP-HPLC method
for the simultaneous estimation and stability indicating of pioglitazone,
glimepiride, and glimepiride impurities, ie, related compound B and related
compound C from combination drug product containing pioglitazone,
glimepiride, and metformin HCl. The chromatographic separation was
achieved on a cyano stationary phase (250 4.6 mm, 5.0 μm particles) with
simple mobile phase combination delivered in gradient mode at a flow rate
of 0.8 mL/min at 230 nm.
7. CLINICAL APPLICATIONS
7.1 Pharmcodynamics
Clinical studies demonstrated that pioglitazone improves insulin sensitivity
in insulin resistant. Pioglitazone enhances cellular responsiveness to insulin,
increases insulin-dependent glucose disposal, and improves dysfunctional
glucose homeostasis. In patients with type II diabetes, the decreased insulin
resistance produced by pioglitazone has resulted in lowering plasma glucose
concentration, plasma insulin levels, and HbA1c values. Based on the results
from an open-label extension clinical, the glucose lowering effect of
Pioglitazone 429
pioglitazone appear to persist for at least 1 year. In controlled clinical trials,

pioglitazone in combination with sulfonylurea, metformin, or insulin had an
additive effect on glycemic control [121,122].
Patients with lipid abnormalities were included in clinical trials with
pioglitazone. Overall, patients treated with pioglitazone had mean decreases
in triglycerides, mean increases in HDL cholesterol, and no consistent mean
changes in LDL and total cholesterol [121,122].
7.2 Mechanism of Action

Insulin resistance may occur in type II diabetes mellitus in which the normal
levels of insulin do not activate the signal for glucose absorption.
Thiazolidinediones such as pioglitazone are potent synthetic peroxisome
proliferator-activated receptor (PPARγ) ligands that have been shown effec-
tive in the treatment of diabetes [121]. Their glucose lowering effect is medi-
ated mainly through improved insulin sensitivity and therefore, facilitating
glucose uptake and utilization. Thiazolidinediones can enter the nucleus
where they bind to PPARγ. PPARγ is expressed most abundantly in adipose
tissue but is also found in pancreatic beta cells, vascular endothelium, and
macrophages [122]. Its discovery as the target for thiazolidinediones was
followed by large number of clinical trials of several agents [123].
Pioglitazone is a potent and highly selective agonist for PPARγ. This recep-
tor regulates the expression of more than 100 genes, which cluster together
but are not identical. In addition, insulin secretory responses have been
reported to increase in subjects with impaired glucose tolerance and type
II diabetes, even after an improvement in insulin sensitivity. Other possible
mechanisms of thiazolidinediones may involve a reduction in adipocyte
cytokines and hormones that are involved in the pathogenesis of insulin
resistance [123].
7.3 Pharmacokinetics
Serum concentrations of total pioglitazone (pioglitazone plus its active
metabolites) remain elevated 24 h after once daily dosing. Steady-state
serum concentrations of both pioglitazone and total pioglitazone are
achieved within 7 days. At steady-state, two of the pharmacologically active
metabolites of pioglitazone, metabolites III (M-III) and IV (M-IV), reach
serum concentrations equal to or greater than pioglitazone. In both healthy
volunteers and patients with type II diabetes, pioglitazone comprises
approximately 30–50% of the total pioglitazone serum peak concentrations
and 20–25% of the total area under the serum concentration–time curve
(AUC). Maximum serum concentration (Cmax), AUC, and trough
serum concentration for both pioglitazone and total pioglitazone increase
proportionally at doses of 15 mg and 30 mg/d. There is a slightly less than
proportional increase for pioglitazone and total pioglitazone at a dose of
60 mg/d [123].
7.4 Absorption
Following oral administration, in the fasting state, Eckland et al. [124] found
that the first measurable pioglitazone serum concentration was within
30 min, with peak concentrations observed within 2 h. Food slightly delays
the time to peak serum concentration to 3–4 h, but does not alter the extent
of absorption [125].
7.5 Distribution
The mean apparent volume of distribution (Vd/F) of pioglitazone following
single-dose administration is 0.63 0.41 (mean SD) L/kg of body weight
[124]. Pioglitazone is extensively protein bound (>99%) in human serum,
principally to serum albumin. Pioglitazone also binds to other serum pro-
teins, but with lower affinity. Metabolites M-III and M-IV also are exten-
sively bound (>98%) to serum albumin [124].
7.6 Metabolism
Pioglitazone is extensively metabolized in the liver, with the majority
excreted as inactive metabolites in the feces. Pioglitazone undergoes signif-
icant hepatic metabolism by hydroxylation of aliphatic methylene groups to
form three metabolites M-I, M-II, and M-IV (hydroxy derivatives of
pioglitazone), by the oxidation of the methyl group to form an additional
metabolite (M-V), and by oxidation of metabolite M-IV to metabolite
M-VI [124]. Three of the metabolites, M-III, M-IV, and to a lesser extent
M-II (keto derivative of pioglitazone), were shown to have pharmacological
activity in diabetic animal models. In rats, the relative hypoglycaemic
potency (ED50) of these metabolites was 40–60% of that of pioglitazone.
The potency of the triglyceride-lowering effect of M-II is nearly twice that
of the parent compound, while the potency of metabolites M-III and M-IV
is slightly less than that of pioglitazone [124]. The major active metabolites
M-III and M-IV have considerably longer terminal half-lives than the parent
compound (approximately 26–28 h) [124]. Multiple CYP isoforms were
Pioglitazone 431
reported to contribute to pioglitazone metabolism with CYP2C8 and

CYP3A4 as the major pioglitazone metabolizing enzymes [124].
7.7 Excretion
Following oral administration, approximately 15–30% of the pioglitazone
dose is recovered in the urine. Renal elimination of pioglitazone is negligi-
ble, and the drug is excreted primarily as metabolites and their conjugates. It
is presumed that most of the oral dose is excreted into the bile either
unchanged or as metabolites and eliminated in the feces [124].
7.8 Elimination Half-Life

The mean serum half-life (t1/2) of pioglitazone and its metabolites (M-III
and M-IV) range from 3 to 7 h and 16 to 24 h, respectively. Pioglitazone
apparent clearance, CL/F, has been calculated to be 5–7 L/h [124,126].
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CHAPTER FIVE

7.4 Absorption 430

1.1.2 Nonpropriety Name

Pioglitazone C19H20N2O3S 356.44 g/ [111025-46-8] [2]

1.2.2 Structural Formula

1.3 Elemental Analysis

2. USES AND APPLICATIONS

Scheme 1 Fischer and Les method for synthesis of pioglitazone 1.

condensation of 6 with thiazolidine-2,4-dione in basic medium afforded 5-[-

Scheme 2 Synthesis of pioglitazone 1 from (intermediate methyl 2-bromo-3-(4-(2-(5-

Scheme 3 Synthesis of pioglitazone 1 from the Knoevenagel condensation of

Huber [13] reported synthesis of pioglitazone 1 that involves a reduction

Scheme 4 Synthesis of pioglitazone from (Z)-5-(4-(2-(5-propylpyridin-2-yl)ethoxy)

Scheme 5 Synthesis of pioglitazone from (Z)-5-(4-(2-(5-propylpyridin-2-yl)ethoxy)

Scheme 6 Synthesis of pioglitazone 1 from intermediate halohydrin compounds.

dimethylglyoxime to furnish compound 8. Chlorination of 8 with PCl5,

Scheme 7 Synthesis of pioglitazone 1 from intermediate (Z)-4-((2,4-dioxothiazolidin-5-

Scheme 8 Synthesis of pioglitazone 1 from intermediate (Z)-4-((2,4-dioxothiazolidin-5-

followed by deprotection of trityl group in the presence of hydrochloric acid

4.2 Melting Point

4.3 Dissociation Constant

4.4 Octanol/Water Partition Coefficient

4.6 Vapor Pressure

4.7 X-Ray Powder Diffraction Pattern

4.8 Thermal Analysis

4.8.2 Differential Scanning Calorimetry

4.8.3 Thermogravimetric Analysis

4.9.2 Infrared Spectroscopy

Figure 2 Infrared absorption spectrum of pure pioglitazone HCl.

4.10 Mass Spectroscopy

4.11 NMR Spectrometry

×102 +Scan(0.297 min) pg00002.d

δ-values (ppm) relative to TMS as an internal standard. Coupling constants

4.11.2 13C NMR Spectrometry

Table 1 1H NMR of Pioglitazone (DMSO-d6)

Signal Location (δ) Shape Integration Correspondences

Figure 4 (A) 1H NMR spectrum of pioglitazone. (B) 1H NMR spectrum of pioglitazone

Figure 4—Cont'd (C) 1H NMR spectrum of pioglitazone (aromatic region).

• System suitability stock solution: 0.5 mg/mL of USP Pioglitazone

Signal Location (δ) Correspondences

• Samples: System suitability solution and standard solution

• Resolution: NLT 15 between pioglitazone and benzophenone,

rU ¼ peak response from the Sample solution

CU ¼ Concentration of Pioglitazone Hydrochloride in the Sam-

rU ¼ peak response of each individual impurity from the

Adhikari et al. [26] used a spectroscopic method to quantify three anti-

pioglitazone HCl, metformin HCl, and glibenclamide were found to be

Mohd et al. [34] developed another UV spectroscopic method for the

The proposed method was applied to the determination of pioglitazone in

Abdelmonem et al. [43] represented simple atomic absorption spectro-

acidic condition by using 3 N HCl and product degradation was found to

5.2.1.2 Atomic Absorption Spectroscopic

and bismuth (III) tetraiodide in acidic medium to form orange-red ion-pair

5.2.1.3 Spectrofluorimetric Method

5.3 Electrochemical Method

5.3.2 Potentiometric Titration

Faridbod et al. [54] selected pioglitazone–tetraphenyl borate as a suitable

5.3.3 Voltammetric Method

Wang et al. [59] developed a method that uses flow-through

5.4.2 Capillary Electrophoresis

5.4.3 HPLC Methods

a bioequivalence study after administration of pioglitazone as 30 mg tablets

Swapna et al. [70] developed an RP-HPLC method for the estimation of

with glimepiride. This method uses a Eurosphere-100 C18 (250 Ã—