51

CHAPTER 2

SIMULTANEOUS ESTIMATION OF PIOGLITAZONE,

GLIMEPIRIDE AND GLIMEPIRIDE IMPURITIES IN

COMBINATION DRUG PRODUCT BY A VALIDATED

STABILITY-INDICATING RP-HPLC METHOD

2.1 INTRODUCTION OF DOSAGE FORM AND

LITERATURE REVIEW

Type 2 diabetes is a disorder characterized by high levels of glucose in

the blood. It is the most common form of diabetes. Once affected, people have to

deal with this disorder for the rest of their lives. A combination of glimepiride,

pioglitazone hydrochloride and metformin hydrochloride extended release is used

for the management of type 2 diabetes. The primary mechanism of glimepiride in

lowering blood glucose is dependent, likely, on stimulating the release of insulin

from functioning pancreatic beta cells (Sweetman 2009). Pioglitazone

hydrochloride is a potent and highly selective agonist for peroxisome

proliferators-activated receptor gamma (Sweetman 2009). Metformin

hydrochloride helps in decreasing hepatic glucose production, decreasing

intestinal absorption of glucose and improving insulin sensitivity by increasing

peripheral glucose uptake and utilization. Thus, this combination helps in

providing better glycaemic control in the management of type 2 diabetes. It also

probably plays a role in the prevention of associated macrovascular and

microvascular complications.

Glimepiride estimation was in USP (2011). The HPLC method is

mentioned as the main method for the determination of purity of both the raw

materials and pharmaceutical formulations. The literature contains several

52

methods for the determination of glimepiride in pharmaceutical dosage forms,

including liquid chromatography (Warnjari and Gaikwad 2005) and derivative

spectroscopy (Bonfilio et al 2011). Bansal et al (2008), Kovařı́ková et al (2004)

and Khan et al (2005) have worked with glimepiride related substances and

degradation pathway methods. Similarly, Ramulu et al (2010), Shirkhedkar and

Surana (2009) and Smita Sharma et al (2010) have published the degradation

behavior of pioglitazone and stability-indicating assay methods. Jain et al (2008),

Karthik et al (2008) and Lakshmi et al (2009) have estimated the drugs in the

combination drug product.

2.1.1 Target of the Work

No stability-indicating HPLC method has been reported yet for the

simultaneous determination of pioglitazone, glimepiride and glimepiride

impurities in the combination drug product. Glimepiride and pioglitazone are

highly unstable compounds. Although several pharmaceutical companies have

marketed this combination drug product, no analytical method is available to

determine this product through routine quality control and stability sample

analysis processes. It is essential to develop a stability-indicating assay method

for the unstable molecules in glimepiride and pioglitazone. Additionally, to prove

the selectivity of the method, glimepiride major degradations of impurity B and

impurity C were injected and estimated in the combination tablets. The aim of the

present study is to develop a single HPLC method for the simultaneous estimation

of pioglitazone, glimepiride, glimepiride impurity B and impurity C from the

combination drug product.

53

2.2 EXPERIMENTAL

2.2.1 Materials and Reagents

Pharmaceutical grade standards of pioglitazone (chemically: 5-(4-[2-

(5-ethylpyridin-2-yl)ethoxy]benzyl) thiazolidine-2,4-dione) and glimepiride

(chemically: 3-ethyl-4-methyl-N-(4-[N-((1r,4r)-4-methyl cyclohexylcarbamoyl)

sulfamoyl]phenethyl)-2-oxo-2,5-dihydro-1H-pyrrole-1-carboxamide) were

supplied by M/S Pharma Lab (Baddi, India). Glimepiride impurity B (chemically:

3-Ethyl-4-methyl-2-oxo-N-[2-(4- sulphamoylphenyl) ethyl]-2,3-dihydro-1H-

pyrrole-1-carboxamide ) and impurity C (chemically: Methyl [[4-[2-[[(3-Ethyl-4-

methyl-2-oxo-2,3-dihydro-1H-pyrrol-1-yl) carbonyl]amino]ethyl]phenyl]-

sulphonyl] carbamate) were purchased from LGC Standards (Mumbai, India).

Chemical structures are shown in Figures 2.1 to 2.4. Commercially available

combination tablets containing 15 mg of pioglitazone, 2 mg of glimepiride and

500 mg of metformin hydrochloride (PRICHEK GMP®-manufactured by Indoco

Rem) were purchased. HPLC grade acetonitrile, analytical reagent grade

potassium dihydrogen phosphate and orthophosphoric acid were obtained from

Rankem (India). Millipore water manufactured by the Milli-Q plus water

purification system was used (Bedford, MA, USA).

Figure 2.1 Chemical structure of pioglitazone

(MF: C19H20N2O3S, MW: 356)

54

Figure 2.2 Chemical structure of glimepiride

(MF: C24H34N4O5S, MW: 490)

Figure 2.3 Chemical structure of glimepiride impurity B

(MF: C8H9NO2, MW: 151)

Figure 2.4 Chemical structure of glimepiride impurity C

(MF: C18H23N3O6S, MW: 409)

55

2.2.2 Instrumentation

The Waters HPLC system consisting of 2695 binary pump plus auto

sampler, a 2996 photo diode array and a 2487 UV detector (Waters Corporation,

Milford, USA) was used for the development and validation.

2.2.3 System Suitability Solution

Stock solutions of glimepiride impurity B, impurity C and glimepiride

(1000 µg/mL) were prepared by dissolving appropriate amounts in methanol.

System suitability solutions of 0.2 µg/mL of impurity B and impurity C and 0.5

µg/mL of glimepiride were prepared from the above mentioned stock solutions

with a diluent mixture of acetonitrile and water (8:2, v/v).

2.2.4 Preparation of Standard Solution

A standard solution, containing 750 µg/mL of pioglitazone and 100

µg/mL of glimepiride, was prepared by dissolving the appropriate amount of

pioglitazone and glimepiride standard in diluent.

2.2.5 Preparation of Sample Solution

Twenty tablets were weighed and powdered with the mortar and pestle

tool. Powder tablets equivalent to 10 mg of glimepiride (equivalent to 75 mg of

pioglitazone) were transferred to a 100 mL volumetric flask. About 60 mL of

diluent was added and kept on a rotatory shaker for 10 min to disperse the

material completely, sonicated for 10 min (during sonication, the bath

temperature was maintained at 25°C) and diluted to 100 mL with diluent. The

concentration of pioglitazone and glimepiride was 750 µg/mL and 100 µg/mL.

The resulting solution was centrifuged at 10000 rpm for 5 min. The supernatant

56

solution was used for the estimation of pioglitazone, glimepiride and glimepiride

impurities.

2.3 RESULTS AND DISCUSSION

2.3.1 Optimization of Chromatographic Method

The HPLC method was optimized with a view to develop a stability-

indicating method. The stability-indicating method should accurately measure the

active ingredients without any interference from degradation products and sample

matrices. As pioglitazone and glimepiride have degradation qualities, the gradient

method was preferred over the isocratic method to get a complete degradation

product as well as a good resolution between close eluting compounds. The initial

trials were taken with the pure drug forms of pioglitazone and glimepiride spiked

with glimepiride impurity B and glimepiride impurity C. Different buffer pH (2-

7) and solvent systems containing methanol and acetonitrile were tested. The

reverse phase column chemistry of C18 was applied for the preliminary trial. A

good separation was achieved in the gradient program containing solution A

(phosphate buffer at pH 3.2) and solution B (acetonitrile), with a flow rate of 0.8

mL/min. To prove the stability-indicating nature of the method, all forced

degradation samples were injected in the optimized conditions. The peak purity of

glimepiride and pioglitazone was not successful because of the interference of

degradation compounds. To rectify this problem, a little adjustment in gradient,

column temperature and flow rate was made, but these trials were not coming up

with the desired results. So, different column chemistry was tried. Initially, the C8

column was selected, and a known compound was merged. While using the

phenyl column, one degradation peak came out from the glimepiride peak. The

glimepiride peak purity was satisfactory, but the pioglitazone peak purity

remained the same. Finally, the cyano column was used for development. The

main base degradation peak came with more than 2.0 resolutions from the

57

pioglitazone peak. To our knowledge, this is the first method, where in spite of lot

of degradation peaks being reported, the known compound got a very good

resolution. Pioglitazone, glimepiride, glimepiride impurity B and impurity C were

found with adequate response at 230 nm. In the case of a stressed sample,

chromatogram was extracted with the entire range of 200-400 nm to check a new

impurity at different wavelengths, but no extra peak was found except at 230 nm

wavelength observed peaks. The required LOQ value of glimepiride impurity B

and impurity C was found by using 100 µg/mL of glimepiride sample preparation

with 25 µL injection volume. During the development, it was observed that the

impurity B gets formed very fast, and to get a consistent result, a fresh sample

preparation was prepared and used. Sonicator bath temperatures were maintained

at less than 25°C while preparing the sample solution. The critical close eluting

impurity of glimepiride impurity B and impurity C was found at a better

resolution compared to the current USP monograph glimepiride tablet method.

The optimized chromatographic method is shown in Table 2.1.

58

Table 2.1 Optimized chromatographic method

Mobile phase-A 20 m mol/L potassium dihydrogen phosphate, pH adjusted to 3.2 using dilute ortho phosphoric acid

Mobile phase-B Acetonitrile

Diluent Mixture of acetonitrile and water (8:2, v/v)

Column Zorbax cyano, 250 mm x 4.6 mm, 5 micron Column oven temperature

25°C

Detection wavelength

230 nm

Injection volume 25 µL

Flow rate 0.8 mL/min

Gradient programme

Time (min)

Mobile phase-A (%)

Mobile phase-B (%)

0.01 80 20

13 80 20

50 50 50

55 20 80

60 20 80

63 80 20

70 80 20

2.3.2 Method Validation

The developed chromatographic method was validated for system

suitability, selectivity, specificity, linearity, precision, accuracy, LOD, LOQ and

robustness as per ICH and FDA guidelines.

2.3.2.1 System Suitability

The observed analyte retention time (RT) and relative retention time

(RRT) are presented in Table 2.2. The resolution between the close eluting pair of

glimepiride impurity B and glimepiride impurity C was set as the system

59

suitability parameter (> 6.0). Also, the % RSD of the peak area of pioglitazone

and glimepiride was calculated. The system suitability chromatogram is shown in

Figure 2.5.

Figure 2.5 System suitability chromatogram (Containing glimepiride,

glimepiride impurity B and glimepiride impurity C)

Table 2.2 System suitability results

Parameter Pioglitazone Glimepiride Impurity B Impurity C

% RSD

Retention time

Relative retention

time

USP resolution

USP tailing factor

USP theoretical

Plates

1.1

31.93

- -

1.01

15011

1.3

38.73

1.00 -

0.99

18123

4.1

21.99

0.57

6.50

1.22

8012

3.2

19.82

0.51 -

1.13

7532

60

2.3.2.2 Specificity and Selectivity

The specificity of the developed method was assessed by performing

forced degradation studies. The specificity of the developed HPLC method was

determined in the presence of its degradation products and other sample matrices.

Forced degradation studies were performed on the tablet sample to indicate the

proposed method’s stability-indicating property and specificity. The sample

solutions were subjected to acid and base hydrolysis (using 0.1 N HCl and 0.1 N

NaOH respectively for 2 hours), oxidation (using 3 % H2O2 for 2 hours) and UV

radiation (254 nm for 48 hours). When the drug was exposed to acid and a

peroxide condition, minor degradation was observed, but when exposed to base

condition, major degradation was observed. Impurity B was increased in all acid,

base and peroxide stressed samples, but impurity C was found only in the

peroxide condition. The drugs were not affected by photolysis, and no

degradation was observed. In all the stressed samples, peak purity was found

within the acceptable limits (the purity angle is less than the purity threshold)

indicating the specificity of the method. Results are shown in Table 2.3.

Table 2.3 Forced degradation results

Condition Time % Assay of

Glimepiride

% Assay of

Pioglitazone

Unstressed sample

Acid hydrolysis (0.1 N HCl)

Base hydrolysis (0.1 N NaOH)

Oxidation (3 % H2O2)

Light (254 nm)

-

2 hours

2 hours

2 hours

48 hours

99.2

96.0

91.9

95.3

100.2

99.0

97.2

82.3

96.3

99.2

61

To prove the selectivity of the method, all individual compounds, i.e.,

pioglitazone, glimepiride, metformin, glimepiride impurity B and glimepiride

impurity C were injected in the optimized method. Blank interference was

checked by injecting the sample diluents. No interference was found with the

discussed compounds. Specificity chromatograms are shown in Figures 2.6 to

2.15.

Figure 2.6 Blank chromatogram

Figure 2.7 Chromatogram of glimepiride impurity B injection

62

Figure 2.8 Chromatogram of glimepiride impurity C injection

Figure 2.9 Chromatogram of metformin injection

Figure 2.10 Chromatogram of glimepiride injection

63

Figure 2.11 Chromatogram of pioglitazone injection

Figure 2.12 Chromatogram of unstressed sample injection

64

Figure 2.13 Chromatogram of acid degradation sample injection

Figure 2.14 Chromatogram of peroxide degradation sample injection

65

Figure 2.15 Chromatogram of base degradation sample injection

2.3.2.3 Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD and LOQ were determined by measuring the magnitude of

analytical background. To estimate the LOD and LOQ, serial dilutions of

glimepiride impurity B and impurity C solutions were used. The signal-to-noise

ratio was then determined. Signal-to-noise ratios of 3 and 10 were considered as

LOD and LOQ respectively. By injecting six preparations of the LOD and LOQ

solutions of glimepiride impurity B and glimepiride impurity C, 0.005 % (i.e.,

0.005 µg/mL) and 0.02 % (i.e., 0.02 µg/mL) for 25 µL injection volume were

achieved. The precision at the LOQ concentration (six individual preparations)

for glimepiride impurity B and glimepiride impurity C was less than 5.0 %. The

results are shown in Table 2.4.

66

Table 2.4 LOQ level precision for impurities

Peak Area

Injection Impurity B Impurity C

1

2

3

4

5

6

Mean

SD

% RSD

16950

16985

17001

17500

16680

17100

17036

267.34

1.57

15591

15659

16000

15350

15455

15377

15572

241.71

1.55

2.3.2.4 Linearity

The linearity of the assay method was evaluated by determining five

concentration levels at three preparations from 50 % to 150 % of analyte

concentration i.e., 750 µg/mL for pioglitazone and 100 µg/mL for glimepiride.

Correlation obtained was found to be more than 0.9999 for both the compounds.

For impurity B and impurity C, six concentration levels from LOQ to

200 % (LOQ, 25 %, 50 %, 100 %, 150 % and 200 %) were prepared by diluting

the impurity stock solution to the required concentrations. The correlation

coefficient obtained was greater than 0.9999. The results are shown in Table 2.5.

The linearity plots are shown in Figures 2.16 to 2.19.

67

Table 2.5 Linearity data for drug substances and impurities

Compound Range (µg/mL)

Regression Parameters (n = 3)

Equation of

regression line R

2 value

Pioglitazone 375-1125 Y = 332706x - 479991 0.9999

Glimepiride 50-150 Y = 123014x + 160007 0.9999

Impurity B 0.02-0.4 Y = 867436x - 115.18 0.9999

Impurity C 0.02-0.04 Y = 780417x - 524.47 0.9999

Glimepiride Linearity Graph

y = 123014x + 160007

R2 = 0.9999

0

5000000

10000000

15000000

20000000

0 20 40 60 80 100 120 140 160

Concentration (ppm)

Peak

Are

a

Figure 2.16 Linearity graph for glimepiride

Pioglitazone Linearity Graph

y = 332706x - 479991

R2 = 0.9999

0

10000000

20000000

30000000

40000000

50000000

60000000

0 20 40 60 80 100 120 140 160

Concentration (ppm)

Peak

Are

a

Figure 2.17 Linearity graph for pioglitazone

68

Glimepiride Impurity B Linearity Graph

y = 332706x - 479991

R2 = 0.9999

0

10000000

20000000

30000000

40000000

50000000

60000000

0 20 40 60 80 100 120 140 160

Concentration (ppm)

Peak

Are

a

Figure 2.18 Linearity graph for glimepiride impurity B

Glimepiride Impurity C Linearity Graph

y = 780417x - 524.47

R2 = 0.9999

0

50000100000

150000200000

250000300000

350000

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

Concentration (ppm)

Peak

Are

a

Figure 2.19 Linearity graph for glimepiride impurity C

2.3.2.5 Precision

The % RSD of six sample preparations assay value was 1.1 for

pioglitazone and 0.9 for glimepiride. The average assay was found to be 98.2 %

for pioglitazone and 100.2 % for glimepiride. The intermediate precision of the

assay method was evaluated by different columns, systems, and analysts. On

different days, % RSDs were within 2.0 for both pioglitazone and glimepiride.

69

Assay value was found between 98 % and 102 %, confirming the ruggedness of

the method.

The precision of impurity B and impurity C was verified by injecting

six individual preparations (in single injection) of 100 µg/mL glimepiride, spiked

with 0.2 % of the above mentioned impurities. The % RSDs of impurity B and

impurity C were 3.2 and 2.9 respectively. In the intermediate precision, the %

RSDs for impurities were well within the limit of (<5.0). The results are shown in

Tables 2.6 and 2.7.

Table 2.6 Summary of method precision

Injection Pioglitazone

(%)

Glimepiride

(%)

Impurity B

(%)

Impurity C

(%)

1

2

3

4

5

6

Mean

SD

% RSD

98.5

97.5

97.3

98.0

97.5

100.5

98.22

1.20

1.22

99.0

99.2

100.1

101.0

101.1

100.5

100.15

0.89

0.89

0.26

0.27

0.27

0.27

0.25

0.27

0.27

0.01

3.16

0.21

0.22

0.21

0.21

0.22

0.22

0.21

0.01

2.87

70

Table 2.7 Summary of intermediate precision

Injection Pioglitazone

(%)

Glimepiride

(%)

Impurity B

(%)

Impurity C

(%)

1

2

3

4

5

6

Mean

SD

% RSD

98.9

99.3

99.1

98.4

98.0

100.1

98.97

0.73

0.74

97.1

98.3

99.2

99.5

99.8

99.1

98.83

0.99

1.00

0.27

0.26

0.26

0.26

0.27

0.26

0.26

0.01

1.96

0.20

0.21

0.20

0.19

0.20

0.20

0.20

0.01

3.16

2.3.2.6 Accuracy

The recovery of three sample preparations at five concentration levels,

i.e., 50 %, 75 %, 100 %, 125 %, and 150 % of working concentration levels for

pioglitazone, glimepiride and glimepiride impurities were determined. The

recovery of pioglitazone and glimepiride was obtained within the acceptable

range, i.e., from 98 % to 102 %. The recovery of impurity B and impurity C

ranged from 96.1 % to 101.3 % and 98.1 % to 102.1 % respectively. The recovery

results are shown in Table 2.8.

71

Table 2.8 Accuracy results for developed HPLC method

Compound Level

(%)

Amount added

(µg/mL)

Recovery

(%)

% RSD

(n = 3)

Pioglitazone

Glimepride

Impurity B

Impurity C

50

75

100

125

150

50

75

100

125

150

50

75

100

125

150

50

75

100

125

150

375

563

750

938

1125

50

75

100

125

150

0.10

0.15

0.20

0.25

0.30

0.10

0.15

0.20

0.25

0.30

98.3

98.5

100.1

100.3

99.2

98.1

99.3

99.1

98.7

100.2

100.3

101.2

96.1

100.1

101.3

99.1

98.1

98.7

102.1

101.5

1.1

1.3

0.9

1.2

0.8

0.9

1.1

1.2

0.8

0.5

1.1

1.4

3.1

0.8

0.9

1.3

1.2

2.0

1.9

0.9

72

2.3.2.7 Robustness

Chromatographic parameters of the method were intensely altered to

measure the robustness of the method. The system suitability parameters as well

as the recovery for the main ingredients in the sample solution were examined.

The parameters altered were the flow rate (± 0.1 mL/min), the buffer’s pH (± 0.2)

and the organic composition (± 5 %) in the mobile phase. The results obtained

from the deliberate changes were well within the limits. The adequate resolution

obtained between impurity B and impurity C in all the changes was greater than

5.0. The assay value of pioglitatone and glimepiride was obtained between 98 %

and 102 %, confirming the robustness of the method. The robustness results are

shown in Tables 2.9 to 2.11.

Table 2.9 Robustness result for flow rate variation

Compound

0.7

mL

/min

0.8

mL

/min

0.9

mL

/min

Resolution between impurity B and impurity C

Pioglitazone (%)

Gimepiride (%)

Impurity B (%)

6.8

98.3

99.1

0.06

6.6

99.1

99.5

0.07

6.5

98.9

98.6

0.07

73

Table 2.10 Robustness result for buffer pH variation

Compound

pH

3.0

pH

3.2

pH

3.4

Resolution between impurity B and impurity C

Pioglitazone (%)

Gimepiride (%)

Impurity B (%)

6.6

98.8

99.5

0.07

6.6

99.1

99.5

0.07

6.7

99.2

98.2

0.07

Table 2.11 Robustness result for organic concentration variation

Compound

Ace

ton

itri

le

(95 %

)

Ace

ton

itri

le

(100

%)

Ace

ton

itri

le

(105

%)

Resolution between impurity B and impurity C

Pioglitazone (%)

Gimepiride (%)

Impurity B (%)

7.1

98.2

99.1

0.06

6.6

99.1

99.5

0.07

6.1

99.7

98.4

0.07

74

2.3.2.8 Application of the Developed Method to Commercial Tablets

To evaluate the application of the developed method, commercial

preparations (PRICHEK GMP®-manufactured by Indoco Rem-Tablets

containing 15 mg of pioglitazone, 2 mg of glimepiride and 500 mg of metformin

hydrochloride) were analysed. The commercial samples were prepared six times,

and the contents of pioglitazone, glimepiride, glimepiride impurity B and

glimepiride impurity C were calculated. The average assay values of pioglitazone,

glimepiride and glimepiride impurity B were 98.2 %, 100.1 % and 0.07 %

respectively. Glimepiride impurity C was not detected in the analysed

commercial sample.

2.3.2.9 Conclusion

The single reversed phase stability-indicating RP-HPLC method has

been established for the simultaneous estimation of pioglitazone, glimepiride,

glimepiride impurity B and impurity C from the combination drug product. The

method was fully validated and the data found to be satisfactory for all the

method validation parameters tested. The developed method can be conveniently

used by both quality control departments for routine analysis to determine the

compound and commercial sample purity checks.