CHAPTER-3 A VALIDATED STABILITY-INDICATING …shodhganga.inflibnet.ac.in/bitstream/10603/4517/12/12_chapter 3.pdf · agent that acts primarily by decreasing insulin ... of the drug

103

CHAPTER-3

A VALIDATED STABILITY-INDICATING ANALYTICAL METHOD FOR THE

DETERMINATION OF IMPURITIES IN

PIOGLITAZONE HYDROCHLORIDE

104

3.1 Introduction on Pioglitazone hydrochloride and survey of

analytical methods

Pioglitazone hydrochloride is a prescription drug of the class

thiazolidinedione (TZD) with hypoglycemic (antihyperglycemic,

antidiabetic) action. Pioglitazone hydrochloride is an oral antidiabetic

agent that acts primarily by decreasing insulin resistance. It is used in

the management of type 2 diabetes mellitus (also known as non-

insulin-dependent diabetes mellitus [NIDDM] or adult-onset diabetes)

[1]. It is chemically designated as (±)-5-[4-(2-(5-Ethyl-2-

pyridinyl)ethoxy)benzyl]-2,4-thiazolidinedione hydrochloride.

Pioglitazone hydrochloride is an odorless white crystalline powder. It is

soluble in N,N-diethylformamide, slightly soluble in anhydrous

ethanol, very slightly soluble in acetone and acetonitrile, practically

insoluble in water, and insoluble in ether. The empirical formula is

C19H20N2O3S.HCl. The molecular weight of Pioglitazone hydrochloride

is 392.9.

Pioglitazone hydrochloride is marketed as trademarks ACTOS in

the USA, glustin in Europe and Zactos in Mexico by the

pharmaceutical company Takeda. Pharmacological studies indicate

that ACTOS improves sensitivity to insulin in muscle and adipose

tissue and inhibits hepatic gluconeogenesis. ACTOS improves

glycemic control while reducing circulating insulin levels.

105

Fig: 3.1 Chemical structure of Pioglitazone hydrochloride

N OS

NH H3C

O

O

. HCl

(±)-5-[4-(2-(5-Ethyl-2-pyridinyl)ethoxy)benzyl]-2,4-thiazolidinedione

hydrochloride

The empirical formula is C19H20N2O3S.HCl

The molecular weight is 392.9.

Several high performance liquid chromatographic (HPLC)

procedures for determination of Pioglitazone hydrochloride in body

fluids and pharmaceutical formulations have been reported in the

literature. HPLC with electrochemical detection has used for analysis

of the drug in human plasma [2-4], serum [5-7] and in pharmaceutical

formulations [8-10].

Organic impurities can arise during the manufacturing process

and storage of the drug substances and the criteria for their

acceptance up to certain limits are based on pharmaceutical studies

or known safety data [11]. Two unknown impurities were detected

consistently in HPLC along with four known impurities i.e. Imp-B, Imp

-C, Imp-D and Imp-E were reported in US Pharmacopeial Forum [12].

However, there was no report of these new impurities i.e., Imp-A and

Imp-F in the literature. As per regulatory guidelines, the

pharmaceutical studies using a sample of the isolated impurities can

106

be considered for safety assessment. It is, therefore, essential to

isolate and characterize unidentified impurities present in the drug

sample. Recently we have developed a process for the synthesis of

Pioglitazone hydrochloride in our laboratory. During the development

of an analytical procedure, the LC method was developed for the

determination of in-house synthesized Pioglitazone hydrochloride and

the impurities arising during its manufacturing. In the present study,

we describe a reverse phase column liquid chromatography method

for the separation and quantification of process related and

degradation impurities of Pioglitazone hydrochloride. The accuracy,

precision, limit of detection (LOD), limit of quantification (LOQ) and

robustness of the method were determined in accordance with ICH

guidelines [13]. The target is to develop a suitable stability-indicating

HPLC related substances method for Pioglitazone hydrochloride in this

chapter we describe a stability-indicating LC method for the

determination of Pioglitazone hydrochloride and its potential and

degradation impurities and also the method validation.

3.2 Development of a stability-indicating analytical method for

Pioglitazone hydrochloride

3.2.1 Materials

Reference standard of Pioglitazone hydrochloride and six

impurities namely, Imp-A, Imp-B, Imp-C, Imp-D, Imp-E and Imp-F

(Fig: 3.2) were synthesized and characterized by use of LC-MS, NMR

and IR in Aurobindo Pharma Ltd., Hyderabad, India. The commercial

107

samples of Pioglitazone hydrochloride are also manufactured by

Aurobindo Pharma Ltd. All reagents used were of analytical reagent

grade unless stated otherwise. Milli Q water, HPLC-grade acetonitrile,

HPLC-grade orthophosphoric acid (OPA) were purchased from Merck

(Darmstadt, Germany).

3.2.2 Equipment

The LC system was equipped with quaternary gradient pumps

with autosampler and auto injector (Alliance, Waters 2695, Milliford,

MA, USA) controlled with Empower software (Waters).

Fig: 3.2 Chemical structures of impurities of Pioglitazone

Hydrochloride

N O

H3C NH2

O

3[(5-Ethyl-2-pyridylethoxy)phenyl]propanamide (Imp-A)

Fig: 3.2 (a)

SNH

HOO

O

5-[4-Hydroxybenzyl]-2,4-thiazolidinedione (Imp-B)

Fig: 3.2 (b)

108

SNH

OO

O

N

H3C

5-[4-[2-(5-Ethyl-2-pyridinyl) ethoxy] benzylidine]-2,4- thiazolidinedione

(Imp-C)

Fig: 3.2 (c)

SN

OO

O

N

H3C

CH2CH3

3(N)-Ethyl-5-[4-[2-(5-ethyl-2-pyridinyl)ethoxy]-benzyl]-2,4-

thiazolidinedione (Imp-D)

Fig: 3.2 (d)

SN

OO

O

N

H3C

CH3

3(N)-Methyl-5-[4-[2-(5-ethyl-2-pyridinyl)ethoxy]-benzyl]-2,4-

thiazolidinedione (Imp-E)

Fig: 3.2 (e)

C

ON

H3C H

S

COOCH3

C

ON

H3C H

COOCH3

S

2,2'-Bis[4-[2-(5-ethylpyridine-2-yl) ethoxy]-phenyl]-2,2'-dithiobisacetic acid, dimethylester (Imp-F)

Fig: 3.2 (f)

109

3.2.3 Sample Preparation

The stock solutions of Pioglitazone hydrochloride (1.0 mg/ml)

and spiked with 0.15% of Imp-A of Imp-B, Imp-D, Imp-E, Imp-F and

0.2% of Imp-C with respect to the Pioglitazone hydrochloride analyte

concentration. The stock solutions were further diluted with diluent to

obtain a standard solution of 0.0015 mg/ml (1.5 µg/ml) for related

substances determination and 0.1mg/ml (100 µg/ml) for assay

determination.

3.2.4 Generation of stress samples

One lot of Pioglitazone hydrochloride drug substance selected for

stress testing. From the ICH stability guideline: “Stress testing likely

to be carried out on a single batch of sample [14]”. Different kinds of

stress conditions (i.e., acid hydrolysis, base hydrolysis, oxidative

stress, heat, humidity and light) were employed on one lot of

Pioglitazone hydrochloride drug substance based on the guidance

available from ICH stability guideline (Q1AR2). The details of the

stress conditions are as follows:

a) Acid hydrolysis: drug in 5.0 M HCl solution was kept at 85°c for

120 mins.

b) Base hydrolysis: drug in 1 M NaOH solution was kept 85°c for

30 mins.

c) Oxidative stress: drug in 30% H2O2 solution was kept at 85°c

for 240 mins.

110

d) Thermal stress: drug was subjected to dry heat at 105°C for 120

hrs.

e) Phtolytic degradation: drug was subjected to UV at 254 nm (10

K Lux ) for 48 hrs.

3.2.5 Optimization of chromatographic conditions

The main objective of the chromatographic method was to

seperate Pioglitazone hydrochloride from Imp-A, Imp-B, Imp-C, Imp-D,

Imp-E and Imp-F impurities were coeluted using different stationary

phases such as C8, phenyl and cyano as well as different mobile

phases containing buffers like phosphate, sulfate and acetate with

different pH and using organic modifiers like acetonitrile and

methanol in the mobile phase. Apart from the co-elution of impurities,

we have also observed poor peak shapes for Pioglitazone, some

impurities and degradants. The chromatographic separation was

achieved on a Inertsil ODS-3V (150 x 4.6 mm), 5 µm particle size. The

gradient LC method employs solution A and B as mobile phase. The

solution A contains phosphate buffer pH 3.1 and acetonitrile as

solution B. The flow rate of the mobile phase was 1.5 ml/min. The

HPLC gradient program was set as: time% solution B: 0.01/25,

20/35, 30/50, 60/50, 62/25, 70/25 with a post run time of 10 min.

The column temperature was maintained at 30°C and the detection

was monitored at a wavelength of 225 nm. The injection volume was

20 µl. Standard and test solutions were prepared in mixture of 0.1%

OPA solution : acetonitrile (50:50, v/v) was used as diluent. In the

111

optimized chromatographic conditions of Pioglitazone hydrochloride,

Imp-A, Imp-B, Imp-C, Imp-D, Imp-E and Imp-F were separated with a

resolution greater than 3, typical relative retention times were

approximately 0.31, 0.58, 1.62, 1.85, 2.61, 5.21 with respect to

Pioglitazone hydrochloride eluted at 7.415 min.

No considerable degradation was observed in Pioglitazone

hydrochloride bulk samples under stress conditions such as acid

hydrolysis, photolytic, thermal conditions (Fig: 3.3, Fig: 3.6 and Fig:

3.7). The degradation of drug substance was observed during base

hydrolysis and oxidative stress condition. Pioglitazone hydrochloride

was degraded to Imp-A (1.5%) under Base conditions (1M

NaOH/85°C/90 min) and it was confirmed by co-injection with a

qualified Imp-A standard and some unknown degradation peaks

observed (12.5%). Mild degradation was observed under oxidative

environment (treated with 30% H2O2/85°C/240 min) leads to the

formation of some unknown degradation peaks (1.2%).

Peak purity test results obtained by using a PDA detector

confirmed that the Pioglitazone hydrochloride peak is homogenous

and pure in all the analyzed stress samples. The mass balance of

Pioglitazone hydrochloride in all stress samples was close to 99.5%

(%Assay + %Degradation). This clearly demonstrates that the

developed HPLC method was found to be specific for Pioglitazone

hydrochloride in presence of its impurities (Imp-A, Imp-B, Imp-C, Imp-

D, Imp-E & Imp-F) and degradation compounds.

112

Optimized liquid chromatographic conditions:

Column : Inertsil ODS-3V (150 x 4.6 mm),

5 µm particle size.

Mobile phase : The solution A contains phosphate

buffer pH 3.1 and acetonitrile as

Solution B

Pump mode : Gradient

Flow rate : 1.5 ml/min

Column oven temperature : 30°C

UV detection : 225 nm

Injection volume : 20 l

Run time : 60 min

Retention time : 7.415 min

Relative Retention Time (RRT) : Imp-A about 0.31

Imp-B about 0.58

Imp-C about 1.62

Imp-D about 1.85

Imp-E about 2.61

Imp-F about 5.21

Diluent : A mixture of 0.1% OPA solution :

acetonitrile (50:50, v/v) was used as

diluent.

113

Figures:

Fig: 3.3 to Fig 3.7 is the typical HPLC chromatograms showing

the degradation of Pioglitazone hydrochloride in various forced

degradation studies and also the corresponding peak purityplots.

Fig: 3.3 Typical HPLC chromatograms of Acid hydrolysis

Fig: 3.3 (a)

Fig: 3.3 (b)

Blank Chromatogram of Acid hydrolysis (5N HCl)

Pioglitazone HCl stressed with 5N HCl at 85°C for 240 mins

114

Fig: 3.3 (c): Peak purity plot of Acid hydrolysis

Purity Angle Purity Threshold Purity Flag Peak Purity

0.018

0.260 No Pass

Fig: 3.3 (c)

115

Fig: 3.4 Typical HPLC chromatograms of Base hydrolysis

Fig: 3.4 (a)

Fig: 3.4 (b)

Blank Chromatogram of Base hydrolysis ( 5N NaOH )

Pioglitazone HCl stressed with 5N NaOH at 85°C for 90 mins

116

Fig: 3.4 (c): Peak purity plot of Base hydrolysis

Purity Angle Purity

Threshold Purity Flag Peak Purity

0.015

0.267 No Pass

Fig: 3.4 (c)

117

Fig: 3.5 Typical HPLC chromatograms of Peroxide Degradation

Fig: 3.5 (a)

Fig: 3.5 (b)

Blank Chromatogram of Peroxide Degradation ( 30% H2O2 )

Pioglitazone HCl stressed with 30%H2O2 at 85°Cfor 240 mins

118

Fig: 3.5 (c): Peak purity plot of Peroxide Degradation

Purity Angle

Purity Threshold Purity Flag Peak Purity

0.023

0.285 No Pass

Fig: 3.5 (c)

119

Fig: 3.6 Typical HPLC chromatograms of Thermal Degradation

Fig: 3.6 (a)

Fig: 3.6 (b)

Pioglitazone HCl stressed at 105°C for 120 hours

120

Fig: 3.6 (c) Peak purity plot of Thermal Degradation

Purity Angle


0.016

0.256 No Pass

Fig: 3.6 (c)

121

Fig: 3.7 Typical HPLC chromatograms of Photolytic Degradation

Fig: 3.7 (a)

Fig: 3.7 (b)

Blank

Pioglitazone HCl stressed with 10K Lux for120 hours

122

Fig: 3.7(c) Peak purity of Photolytic Degradation

Purity Angle


0.016

0.256 No Pass

Fig: 3.7 (c)

123

3.2.6 Validation of Analytical method and its results:

The developed and optimized HPLC method was taken up to

validation. The analytical method validation was carried out is

accordance with ICH guideline [15].

3.2.6.1 System suitability : A mixture of Pioglitazone hydrochloride

reference standard, Imp-A, Imp-B, Imp-C, Imp-D, Imp-E and Imp-F

were injected into HPLC system and good resolution was obtained

between impurities and Pioglitazone hydrochloride [Fig: 3.8], and the

system suitability results are tabulated (Table: 3.1).

Fig: 3.8 Typical Blank, Pioglitazone HCl Sample and SST

Chromatograms

Fig: 3.8 (a)

Blank

124

Fig: 3.8 (b)

Fig: 3.8 (c)

Pioglitazone HCl sample spiked with impurities

Pioglitazone HCl Sample

125

Table: 3.1 System suitability results

Compound

(n=3)

No. of

theoretical plates (N)

USP Tailing factor

(T) USP Resolution(Rs)

Imp-A 12326 1.07 -

Imp-B 13668 1.11 3.02

Pioglitazone 216870 1.15 6.02

Imp-C 17630 1.39 4.36

Imp-D 26040 1.07 2.86

Imp-E 60019 1.10 4.05

Imp-F 108491 0.97 4.91

3.2.6.2 Precision:

The precision of an analytical process experiment the closeness

of agreement between a series of measurements obtained from

multiple sampling of the some homogeneous same under prescribed

conditions.

It may be considered at three levels: System precision, Method

precision and Intermediate Precision. Assay method precision study

was evaluated by carrying out six independent assays of Pioglitazone

hydrochloride test sample against qualified reference standard and

RSD of six consecutive assays was 0.6% (Table: 3.2).

The results showed insignificant variation in measured

response. Which demonstrated that the assay method was repeatable

with RSD’s below 0.4%.

126

Table: 3.2 System Precision results of the Assay method

Injection ID Pioglitazone Area

1 2425249

2 2421767

3 2427876

4 2433405

5 2444339

6 2445469

Mean 2433018

SD 9967

% RSD 0.4

95% Confidence

Interval ± 10462

Table: 3.3 Method Precision results of the Assay method

Sample ID Assay (% w/w)

1 99.0

2 99.1

3 99.9

4 99.8

5 99.5

6 99.9

Mean 99.5

SD 0.4

% RSD 0.4

95% Confidence

Interval ± 0.4

127

Table: 3.4 Intermediate Precision results of the Assay method

Sample ID Assay (% w/w)

1 99.8

2 99.6

3 99.3

4 99.6

5 99.8

6 99.7

Mean 99.6

SD 0.19

% RSD 0.2

95% Confidence Interval

± 0.2

The precision of the related substance method was checked by

injecting six individual preparations of Pioglitazone hydrochloride

(1.00 mg/ml) spiked with 0.15% of Imp-A, Imp-C, Imp-D, Imp-E, Imp-

F and 0.2% of Imp-B with respect to the Pioglitazone hydrochloride

analyte concentration. The % RSD of the area percentage of each

impurity (impurities-A, -B, -C, -D, -E and -F) for six consecutive

determinations was respectively as below (Table: 3.5 to Table: 3.7).

The results showed insignificant variation in measured

response. Which demonstrated that the related substance method was

repeatable with RSD’s below 1.3%.

128

Table: 3.5 System Precision results of the Related substances

method

Injection ID Pioglitazone Area

1 44290

2 44578

3 44307

4 44430

5 44579

6 44352

Mean 44290

SD 44423

% RSD 130

95% Confidence

Interval 0.3

Table: 3.6 Method Precision results of the Related Substance

method

Preparation Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F

1 0.150 0.174 0.248 0.164 0.150 0.240

2 0.147 0.175 0.247 0.160 0.152 0.238

3 0.152 0.175 0.248 0.163 0.152 0.236

4 0.151 0.174 0.247 0.164 0.150 0.241

5 0.152 0.172 0.247 0.166 0.151 0.241

6 0.150 0.174 0.245 0.164 0.152 0.240

Mean 0.150 0.174 0.247 0.164 0.151 0.239

SD 0.0019 0.001 0.001 0.002 0.001 0.002

%RSD 1.24 0.6 0.4 1.2 0.7 0.8

95% Confidence

interval ±0.001 ±0.001 ±0.001 ±0.002 ±0.001 ±0.002

129

Table: 3.7 Intermediate Precision results of the Related substance

method

Preparation Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F

1 0.152 0.174 0.260 0.163 0.146 0.237

2 0.145 0.175 0.258 0.162 0.148 0.237

3 0.151 0.174 0.258 0.161 0.150 0.238

4 0.152 0.175 0.260 0.162 0.149 0.237

5 0.150 0.176 0.258 0.162 0.151 0.238

6 0.152 0.176 0.258 0.164 0.149 0.239

Mean 0.150 0.175 0.254 0.162 0.149 0.238

SD 0.0019 0.001 0.001 0.001 0.002 0.002

%RSD 1.24 0.6 0.4 0.6 1.3 0.4

95%

Confidence interval

±0.001 ±0.001 ±0.001 ±0.002 ±0.001 ±0.002

3.2.6.3 Limit of Detection (LOD):

The detection limit of an individual analytical procedure is the

lowest amount of analyte is a sample, which can be detected but not

necessarily quantitated as an exact value (Table: 3.8).

130

Table: 3.8 LOD values of the Pioglitazone and its impurities

Injection ID

Area

Imp-A Imp-B Pioglitazone Imp-C Imp-D Imp-E Imp-F

1 1691 1772 1281 1796 1745 1808 2182

2 1587 2127 1722 1403 1661 1393 2677

3 2013 1696 1611 1813 1656 1865 2050

4 2089 1702 1614 1794 1285 1341 2134

5 2310 1471 1619 1480 1736 1416 2185

6 1646 1638 1461 2048 1434 1646 2024

Mean 1889 1734 156 1722 1586 1578 2209

SD 290 218 10.1 239 185 227 239

% RSD 15.4 12.6 0.056 13.9 11.7 14.4 10.8

Conc. (µg/mL)

0.058 0.049 0.056 0.083 0.060 0.066 0.075

Conc. (% w/w)

0.005 0.005 0.006 0.008 0.006 0.007 0.008

131

3.2.6.4 Limit of Quantification (LOQ)

The quantitation limit (LOQ) of an analytical procedure is the

lowest amount of analyte in a sample, which can be quantitatively

determined with suitable precision and accuracy. The quantitative

limit is a parameter of quantitative assays for low levels of compounds

in sample matrices, and is used particularly for the determination of

impurities and/or degradation products (Table: 3.9).

Table: 3.9 LOQ values of the Pioglitazone and its impurities

Injection ID Area

Imp-A Imp-B Pioglitazone Imp-C Imp-D Imp-E Imp-F

1 6956 5381 5104 5680 5268 5651 6747

2 6148 5308 4984 5594 5286 5009 6764

3 7080 5642 4993 5512 5144 5185 6665

4 7045 5423 4917 5644 5287 5052 6700

5 6630 5659 5050 5752 5475 5193 6872

6 6933 5575 5010 5509 5294 5288 6962

Mean 6799 5498 5010 5615 5292 5230 6785

SD 356 147 63 96 106 230 112

% RSD 5.2 2.7 1.3 1.7 2.0 4.4 1.7

Conc. (µg/mL)

0.175 0.150 0.170 0.251 0.181 0.200 0.226

Conc. (% w/w)

0.020 0.015 0.017 0.025 0.018 0.020 0.023

132

3.2.6.5 Linearity

Linearity of the Assay method

The linearity of an analytical procedure is its ability to obtain

test results, which are directly proportional to the concentration of

analyte in the test sample. The linearity of the assay method was

established by injecting test sample at 80%, 90%, 100%, 110% and

120% of Pioglitazone hydrochloride assay concentration (i.e.100

µg/ml). Each solution injected twice (n=2) into HPLC and the average

area at each concentration calculated (Table: 3.10).

Calibration curve drawn by plotting average area on the Y-axis and

concentration on the X-axis (Fig: 3.9).

133

Table: 3.10 Linearity results of the Assay method

%

Concentration Average area

80 1946315

90 2180238

100 2440221

110 2689004

120 2910526

Slope 24372

Intercept -3927

% Y - Intercept -0.2

Residual Sum of

Squares 11304

Correlation Coefficient 0.9996

Linearity Plot (Concentration Vs Response)

Fig: 3.9 Linearity Plot for Assay method

80.00 90.00 100.00 110.00 120.00

Aver

age

Are

a

Conc.(µg/mL)

134

Linearity of the Related Substance method

Linearity experiments were carried out by preparing the

Pioglitazone hydrochloride sample solutions containing Imp-A, Imp-B,

Imp-C, Imp-D, Imp-E, Imp-F and Imp-G from LOQ to 150% (i.e. LOQ,

10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%) with respect to

their specification limit (0.15%). Calibration curve was drawn by

plotting average area of the impurity Imp-A, Imp-B, Imp-C, Imp-D,

Imp-E, Imp-F and Imp-G) on the Y-axis and concentration on the X-

axis (Fig: 3.10 to Fig: 3.16).

135

Linearity results of the related substance method

Table: 3.11 Linearity results of the Imp-A

Imp-A

Concentration (µg/ml)

Area Statistical Analysis

0.176 5970 Slope 32494

0.249 8194 Intercept 277

0.498 16627 Residual Sum of

Squares 142

0.748 24616

0.997 32745 Correlation Coefficient

0.9999 1.246 40856

1.495 42164

1.870 53010 Response factor 1.08

2.244 63993

Linearity Plot (Concentration Vs Area)

Fig: 3.10 Linearity Plot for Imp-A

5555

17555

29555

41555

53555

65555

77555

0.151 0.501 0.851 1.201 1.551 1.901 2.251

Are

a

Con. (µg/mL)

136

Table: 3.12 Linearity results of the Imp-B

Imp-B

Concentration

(µg/ml) Area Statistical Analysis

0.151 5555 Slope 35638

0.226 8241 Intercept 321


Squares 264

0.377 14102

0.755 27359 Correlation

Coefficient 0.9999 1.132 40635

1.510 54568


2.265 80884


Fig: 3.11 Linearity Plot for Imp-B

5555

17555

29555

41555

53555

65555

77555

0.151 0.501 0.851 1.201 1.551 1.901 2.251

Are

a

Con. (µg/mL)

137

Table: 3.13 Linearity results of the Pioglitazone hydrochloride

Pioglitazone

Concentration


0.170 5010 Slope 30481

0.200 5867 Intercept -315

0.300 8951

0.400 11794

Residual Sum of

Squares 416 0.500 14542

1.000 30883

1.501 44899

Correlation

Coefficient 0.9999

2.001 60361

2.501 75688

3.001 91639


Fig: 3.12 Linearity Plot for Pioglitazone hydrochloride

5010

18510

32010

45510

59010

72510

86010

0.170 0.620 1.070 1.520 1.970 2.420 2.870

Are

a

Con. (µg/mL)

138

Table: 3.14 Linearity results of the Imp-C

Imp-C

Concentration


0.251 5615 Slope 21953

0.299 6575 Intercept -165


Squares 180

0.499 10558

0.998 21571

Correlation

Coefficient 0.9999 1.497 32609

1.996 43783


2.994 65602


Fig: 3.13 Linearity Plot for Imp-C

5615

15615

25615

35615

45615

55615

0.251 0.701 1.151 1.601 2.051 2.501 2.951

Are

a

Con. (µg/mL)

139

Table: 3.15 Linearity results of the Imp-D

Imp-D

Concentration


0.181 5292 Slope 28436

0.224 6371 Intercept -77

0.299 8551

Residual Sum of

Squares 229 0.374 10302

0.748 20990

1.122 31987

Correlation Coefficient

0.9999 1.496 42164

1.870 53010



Fig: 3.14 Linearity Plot for Imp-D

5292

14292

23292

32292

41292

50292

59292

0.181 0.501 0.821 1.141 1.461 1.781 2.101

Are

a

Con. (µg/mL)

140

Table: 3.16 Linearity results of the Imp-E

Imp-E

Concentration


0.200 5230 Slope 26436

0.226 5941 Intercept -196


Squares 229

0.377 9764

0.754 19644

Correlation Coefficient

0.9999 1.131 29266

1.508 39470


2.262 59865


Fig: 3.15 Linearity Plot for Imp-E

5230

14230

23230

32230

41230

50230

59230

0.200 0.550 0.900 1.250 1.600 1.950

Are

a

Con. (µg/mL)

141

Table: 3.17 Linearity results of the Imp-F

Imp-F

Concentration (µg/ml)

Area Statistical Analysis

0.225 6695 Slope 30225

0.300 8917 Intercept -260

0.375 11163 Residual Sum of Squares

346 0.749 22471

1.124 33313 Correlation

Coefficient 0.9999

1.498 44439


2.248 68074


Fig: 3.16 Linearity Plot for Imp-F

6695

16695

26695

36695

46695

56695

66695

0.225 0.575 0.925 1.275 1.625 1.975

Are

a

Con. (µg/mL)

142

3.2.6.6 Accuracy/Recovery

The accuracy of an analytical procedure expresses the closeness

of agreement between the value, which is accepted either as a

conventional true value or an accepted reference value and the value

found.

Accuracy of the assay method

Accuracy of the assay method was developed by injecting three

preparations of test sample at 80%, 90%, 100% 110%, 120% and

150% of analyte concentration (i.e.100 µg/ml). Each solution was

injected twice (n=2) into HPLC and the mean peak area of Pioglitazone

hydrochloride peak was calculated. Assay (%w/w) of test solution was

determined against three injections (n=3) of qualified Pioglitazone

hydrochloride reference standard (Table: 3.18).

The method showed consistent and high absolute recoveries at

all three concentration (80%, 90%, 100% 110%, 120% and 150%)

levels with mean absolute recovery ranging from 99.3 % to 100.5%.

The obtained obsolute recoveries were normally distributed around

the mean with uniform RSD values. The method was found to be

accurate with low % bias (<1.0).

143

Table: 3.18 Acuracy results of the Assay method

S.NO % Concentration Mean recovery (%)

(n=3) %RSD

1 80 99.3 0.06

2 90 99.5 0.15

3 100 99.8 0.10

4 110 100.0 0.15

5 120 100.6 0.10

Accuracy of the related substances method established at 50% 100%

and 150% of the impurities specification limit (0.15%).

Accuracy at 50% impurity specification level:

Test solutions prepared in triplicate (n=3) with impurities (Imp-

A, B, C, D, E and F) at 0.075% (Imp-A, B, D, E, F) and 0.1% (Imp-C)

level w.r.s analyte concentration (i.e 1.00 mg/ml). Each solution was

injected thrice into HPLC (Table: 3.19).

144

Table: 3.19 Accuracy at 50% level

S.NO Impurity

name

Mean

recovery(%) SD %RSD

1 Imp-A 102.0 0.00 0.0

2 Imp-B 98.2 0.75 0.8

3 Imp-C 100.0 1.00 1.0

4 Imp-D 99.1 0.75 0.8

5 Imp-E 97.7 1.59 1.6

6 Imp-F 100.9 2.03 2.0


Test solution prepared in triplicate (n=3) with impurities (Imp-A,

B, C, D, E , F ) at 0.15% ( Imp-A, B, D, E, F) and 0.2% (Imp-C) level

w.r.s analyte concentration (i.e 1.00 mg/ml). Each solution was


145


S.NO Impurity

name

Mean

recovery(%) SD %RSD

1 Imp-A 96.6 0.65 0.7

2 Imp-B 96.6 0.65 0.7

3 Imp-C 98.8 0.29 0.3

4 Imp-D 96.1 0.65 0.7

5 Imp-E 97.6 1.40 1.4

6 Imp-F 101.7 0.75 0.7


Test solution prepared in triplicate (n=3) with impurities (Imp-A,

B, C, D, E , F and G) at 0.12% ( Imp-A, B, D, E, F) and 0.3% (Imp-C)

level w.r.s analyte concentration (i.e 1.00 mg/m l). Each solution was


146


S.NO Impurity

name

Mean

recovery(%) SD %RSD

1 Imp-A 99.7 1.40 1.4

2 Imp-B 96.6 0.67 0.7

3 Imp-C 98.9 1.33 1.3

4 Imp-D 97.8 0.45 0.5

5 Imp-E 97.3 1.17 1.2

6 Imp-F 102.1 0.23 0.2

The related substance method showed consistent and high

accurate recoveries of all six impurities at all three different

concentrations (50%, 100%, 150%) levels in drug substance.

3.2.6.7 Solution state stability

The solution state stability of Pioglitazone Hydrochloride in

diluent in the assay method was carried out by leaving both the test

solutions of sample and reference standard in tightly capped

volumetric flasks at room temperature for two days. The same sample

solutions were assayed for every one hour interval up to the study

period. The % RSD of assay of Pioglitazone during solution stability

experiments was with in 1.0%.

The solution state stability of Pioglitazone hydrochloride related

substance method was carried out by leaving sample solution in

tightly capped volumetric flask at room temperature for two days.

Content of Imp A, B, C, D, E, and F were checked for every six hours

internal up to the study period. No significant change was observed in

147

the content of all six impurities drug solution stability experiments up

to the study period. Hence Poiglitazone hydrochloride sample

solutions are stable for atleast 48 hours in the developed method.

In assay method the standard and sample solution injected at

each 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12hr (Table:

3.22).

Table: 3.22 Solution stability results of the assay method

S.No Time in Hours Assay (% w/w)

1 initial 99.8

2 1 99.7

3 2 99.2

4 3 99.4

5 4 99.5

6 5 99.6

7 6 99.7

8 7 99.4

9 8 99.3

10 9 99.1

11 10 99.4

12 11 99.6

13 12 99.5

% RSD 0.21

In related substances method the stability of Pioglitazone

hydrochloride in diluent was established for 15 hr by injecting test

solution for every one hour interval up to the study period. The

impurity profiles obtained at different interval were very consistent

and matched with initial value.

148

3.2.6.8 Robustness

The robustness of an analytical procedure is a measure of its

capacity to remain unaffected by small, but deliberate variations in

method parameters and provides an indication of its reliability during

normal usage. To determine the robustness of the developed method

experimental conditions were purposely altered and the resolution

between Imp-C and Imp-D was evaluated. In each of the deliberately

altered chromatographic condition (flow rate 1.3 ml/min and

1.7ml/min, acetonitrile 23% and 27% in the mobile phase, column

temperature 25C and 35C) the resolution between Imp-B, Imp-C and

Imp-D, Imp-E and Imp-F was greater than 2.0, illustrating the

robustness of the method.

3.2.6.9 Mass balance of the analytical method

The mass balance is a process of adding together the assay

value and the levels of degradation products to see how closely these

add up to 100% of the initial value, with due consideration of the

margin of analytical error. Its establishment hence is a regulatory

requirement. The mass balance is very closely linked to the

development of stability-indicating assay method as it acts as an

approach to establish its validity. The stressed samples of Pioglitazone

hydrochloride bulk drug were assayed against the qualified reference

standard and the results of mass balance obtained were very close to

99.8%. The results of mass balance obtained in each condition is

presented below [Table 3.23).

149

Table: 3.23 Mass balance of the Assay method

Degradation

Mechanism

Degradation

Condition

% Assay of

active substance

Mass balance

(% Assay+ %

impurities+ %

degradants)

Remarks

Acid 5M HCl / 85°C

/ 240 min 99.5 99.8

No

degradation

observed

Base 1M NaOH /

85°C /90min 85.9 99.9

Degraded to Imp-A and

some

unknown degradants

observed

Peroxide

30% H2O2 /

85°C / 240

min

98.6 99.8

some

unknown degradants

observed

Thermal 105°C / 120

Hours 99.5 99.7

No

degradation observed

Photolytic 10K Lux / 120

Hours 99.2 99.5

No

degradation

observed

3.3 Analysis of Pioglitazone hydrochloride drug substance

stability samples

One manufacturing lot of Pioglitazone hydrochloride drug

substance was placed on stability study in chambers maintained at

ICH set conditions. The analysis of stability samples were carried up

to 24 months period using the above optimized method. The stability

data results obtained are presented in Table: 3.24 & Table: 3.25. The

developed HPLC method performed satisfactorily for the quantitative

evaluation of stability samples.

150

Table: 3.24 Accelerated stability data ( Storage conditions:

40°C/75%RH)

Batch No: MHK(684)90 Packing & storage conditions: Each sample packed in a polyethylene bag in a triple

laminated bag and kept in a HDPE drum

Stability study duration: 6 months Temperature

%Relative humidity 40°C/75%RH

Tests Description Loss on drying

(%w/w)

Identification

Assay

(By HPLC,

%w/w, on dried basis,)

Specifications

A white to almost

white

powder

NMT 1.0

IR spectrum

should

concordant with that of

standard

NLT 98.5 and

NMT 101.5

Initial A white

powder 0.13 Complies 99.8

1M A white powder

0.12 Complies 100.4

2M A white


3M A white


6M A white powder

0.19 Complies 100.1

Related substances details on next page.

151

Related

Substances

LOQ

(%w/w)

LOD

(%w/w)

Related Substances (By HPLC, %w/w)

Initial 1M 2M 3M 6M

Imp-A 0.015 0.005 Below

LOQ ND ND ND ND

Imp-B 0.014 0.005 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-C 0.025 0.008 0.06 0.04 0.04 0.04 0.04

Imp-D 0.018 0.006 Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Imp-E 0.020 0.007 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-F 0.023 0.008 0.03 0.03 0.03 0.02 0.02

Highest

unknown - - ND ND ND ND ND

Total unknown

- - NA NA NA NA NA

Total RS - - 0.09 0.07 0.07 0.06 0.06

ND: Not detected NA: Not applicable

152

Table: 3.25 Long term stability data ( Storage conditions

25°C/60%RH)

Batch No: MHK(684)90 Packing & storage conditions: Each sample packed in a polyethylene bag in a

triple laminated bag and kept in a HDPE drum

Stability study duration: 24 months Temperature %Relative humidity

25°C/60%RH

Tests Description

Loss on

drying (%w/w)

Identification

Assay (By HPLC,

%w/w, on

dried basis,)

Specifications

A white to

almost white

powder

NMT 1.0

IR spectrum should

concordant

with that of standard

NLT 8.5 and NMT 101.5

Initial A white


3M A white powder

0.12 Complies 100.0

6M A white


9M

A white


12M

A white powder

0.21 Complies 99.8

24M A white


Related substances details on next page.

153

Related Substances

LOQ (%w/w)

LOD

(%w/w)

Related Substances (By HPLC, %w/w)

Initial 3M 6M 9M 12M 24M

Imp-A 0.015 0.005 Below LOQ

ND ND ND ND ND

Imp-B 0.014 0.005 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-C 0.025 0.008 0.06 0.04 0.04 0.05 0.05 0.05

Imp-D 0.018 0.006 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-E 0.020 0.007 Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Imp-F 0.023 0.008 0.03 0.03 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Highest

unknown - - ND ND ND ND ND ND

Total unknown

- - NA NA NA NA NA NA

Total RS - - 0.09 0.07 0.06 0.05 0.05 0.05

ND: Not detected NA: Not applicable

154

3.4 Summary and conclusions

Validated stability-indicating HPLC method was developed for

Pioglitazone hydrochloride after subjecting the samples to stress

testing under ICH recommendes conditions. The RPLC method

developed for quantitative and related substance determination of

Pioglitazone hydrochloride is rapid precise, accurate linear and

selective. The method was completely validated showing satisfactory

data for all the method validation parameters tested. The developed

method was found ‘specific’ to the drug, as the peaks of the

degradation products did not interfere with the degradation peak.

Thus the proposed method can be employed for assessing the stability

of Pioglitazone hydrochloride bulk drug samples.

155

Table: 3.26 Summary of Analytical method validation data

Test

Parameter

Related Substances method Assay

method

Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F

Precision

(RSD) 0.6 0.4 1.2 0.7 0.8 0.6 0.4

LOD(µg/ml) 0.049 0.033 0.034 0.033 0.033 0.034 N/A

LOQ(µg/ml) 0.150 0.101 0.103 0.100 0.100 0.102

N/A

Linearity

(corre

coefficient

0.9999 0.9999 0.9999 0.9999 0.9999 0.9999

0.9999

Accuracy (%) 96.6-98.2 96.6-98.2 98.8-100.0 96.1-99.1 97.3-97.7 100.9-102.1 99.3-100.5

Robustness Resolution

b/w Imp-A&

Imp-B>2

Resolution b/w Imp-A&

Imp-B>2


Imp-B>2


Imp-B>2


Imp-B>2


Imp-B>2


Imp-B>2

Solution

stability

Stable up to

15hr

Stable up to

15hr

Stable up to

15hr

Stable up to

15hr

Stable up to

15hr

Stable up to

15hr

Stable up to

15hr

Mobile phase stability

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

156

3.5 References:

1. Sean. C. S.; Martindale-The complete drug reference,

35th edition, 2007, Vol.2, p.414.

2. Lin, Z. J.; Ji, W.; Desai-Karieger, D.; Shum, L.; J. Pharm.

Biomed. Anal., 2003, 33, 101.

3. Souri, E.; Jalalizadeh, H.; Saremi, S.; J. Chromatogr. Sci. 2008,

46, 80.

4. Sripalakit, P.; Neamhom, P.; Saraphanchotiwittaya, A.;

J.Chromatogr. B, 2006, 843, 164.

5. Zhong, W. Z.; Williams, M. G.; J. Pharm. Biomed. Anal., 1996,

14, 465.

6. Xue, Y. J.; Turner, C. K.; Meeker, B. J.; Pursley, J.; Arnold, M.;

Unger, S.; J. Chromatogr.B, 2003, 795, 215.

7. Yamashita, K.; Murakani, H.; Okuda, T.; Motohashi, M.;

J. Chromatogr B, 1996, 677, 141.

8. Mehta, R. S.; Patel, D. M.; Bhatt, K. K.; Shankar, M. B.;

Ind. J. Pharm Sci., 2005, 67, 487.

9. Jedlicka, A.; Kimes, J.; Grafnetterova, T.; Pharmazie. 2004, 59,

178.

10. Radhakrishna, T.; Sreenivas Rao, D.; Om Reddy, G.; J. Pharm.

Biomed. Anal., 2002, 29, 593.

11. International Conferences on Hormonization Q3A (R2): Draft

Revised Guidance on impurities in New Drug Substances,

2006.

12. US Pharmacopeial Forum, 2010, 36, 126.

157

13. ICH, Validation of analytical Procedures: Text and methodology

Q2 (R1), International Conferences on Hormonization, IFPMA,

Geneva, 2005.

14. Steven, W. Baertschi Pharmaceutical Stress Testing Predicting

Drug Degradation.

15. Validation of Analytical Procedures: Methodology Q2B – ICH

Guidelines.

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