INTRODUCTION:

Pioglitazone is chemically designated as RS)-5-(4-[2-(5-ethylpyridin-2-yl) ethoxy ]benzyl)thiazolidine-2,4-dione is a hypoglycemic drug with a pKa value of 14.64. Its molecular formula is C₁₉H₂₀N₂O₃S and it has a molecular weight of 165.52gm/mole. It is soluble in methanol, slightly soluble in acetone and acetonitrile. It is official drug in Martindale [1], Merck index [2] and Indian Pharmacopeia 2007 and 2014 [3].

(a)

(b)

Fig 1: Chemical Structures of a) Pioglitazone and b) Glimepiride

Glimepiride is an oral hypoglycemic drug which is chemically designated as1-[[p-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) ethyl] phenyl] sulfonyl-3-(trans-4-methyl cyclohexyl) urea. The molecular formula is C₂₄H₃₄N₄O₅S and molecular weight is 490.62 gm/mol. It is a white to off-white crystalline powder. That is soluble in methanol and dimethyl formamide. It is official drug in Martindale [1], Merck index [2] and Indian Pharmacopeia 2007 and 2014[3].

Extensive literature survey proved that very few methods were reported for the determination of Pioglitazone and Glimepiride individually or in combination by RP-HPLC [4-22] but few methods had been reported for simultaneous estimation of these two drugs. So we attempted to develop an accurate, rapid, precise, stable, sensitive and economically viable liquid chromatographic method for the simultaneous determination of selected drugs in the present research.

MATERIALS AND METHODS:

Equipment used

The chromatographic separation was performed on Agilent 1120 compact liquid chromatographic system integrated with a variable wavelength programmable UV detector and a Rheodyne injector equipped with 20ml fixed loop. A reverse phase C₁₈ [Kromasil 100-5 column, 250mm x 4.6mm] was used. Lab India 3000⁺double beam UV visible spectrophotometer and Axis AGN204-PO electronic balances were used for spectrophotometric determinations and weighing purposes respectively.

Reagents and chemicals

Pharmaceutical grade pure Pioglitazone and Glimepiride gift samples were procured from Mylan Laboratories, Hyderabad. Marketed formulation Tablets with dose of 50 mg of Pioglitazone and 15 mg of Glimepiride were procured from local market (Pioglar by Ranbaxy Pharma). HPLC grade Acetonitrile and Water were procured from Merck specialties private limited, Mumbai.

Chromatographic conditions

Kromasil-C₁₈ column 5mm [250mm x 4.6mm] was used for the chromatographic separation at a detection wave length of 230 nm. Mobile phase with a composition of Phosphate buffer pH 7 and Acetonitrile in a ratio of 60:40 v/v was selected for elution and same mixture was used in the preparation of standard and sample solutions. Flow rate was adjusted to 0.8 ml/min and the injection volume was 20ml.

Preparation of Mobile phase

Phosphate buffer pH 7 was prepared by dissolve 0.504 gm of disodium hydrogen phosphate and 0.301gm of Potassium dihydrogen phosphate of HPLC grade water and adjusts the pH to 7.0 with sufficient water was added to produce100 ml filtered through 0.45m membrane filter and sonicated for 15 minutes.

Preparation of Standard solutions

25mg each of Pioglitazone and Glimepiride were accurately weighed and transferred into two 25ml volumetric flasks, dissolved using mobile phase and the volume was made up with the same solvent to obtain primary stock solutions A (Pioglitazone) and B (Glimepiride) of concentration 1000 mg/ml of each drug. From the primary stock solutions 0.8ml and 0.5 ml were pipette out from A and B respectively, transferred to a 10ml volumetric flask and the volume was made up with the mobile phase to obtain final concentrations of 80µg/ml and 50 µg/ml of Pioglitazone and Glimepiride respectively and this solution is (working stock solution A).

Preparation of Sample Solution

Twenty tablets of Pioglitazone and Glimepiride were weighed and crushed. Tablet powder equivalent to 300 mg of Pioglitazone and 40 mg of Glimepiride was weighed accurately and transferred to a 25 ml volumetric flask. The content was dissolved with 10ml of mobile phase and then sonicated for 15min. The volume was made up with the mobile phase and filtered with 0.45mmembrane filter and sonicated for 20 min. 0.8 ml of this solution was pipette out and transferred to a 10 ml volumetric flask and the volume was made up with the mobile phase to obtain a concentration of 80µg/ml of Pioglitazone and 50 µg/ml of Glimepiride (working stock solution B).

Optimization of RP-HPLC method

The HPLC method was optimized with an aim to develop a simultaneous estimation procedure for the assay of Pioglitazone and Glimepiride. For the method optimization, different mobile phases were tried, but acceptable retention times, theoretical plates and good resolution were observed with Phosphate buffer pH 7 and Acetonitrile (60:40 v/v) using Kromasil-C₁₈ column 5mm [250mm x 4.6mm].

Validation of the RP-HPLC method

Validation of the optimized method was performed according to the ICH Q2 (B) guidelines.

System suitability

System suitability was carried out with five injections of solution of 100% concentration having 50µg/ml of Pioglitazone and 15 µg/ml of Glimepiride in to the chromatographic system. Number of theoretical plates (N) obtained and calculated tailing factors (T) were reported in table 1.

Fig 2: Optimized chromatogram of Pioglitazone and Glimepiride

Fig 3: Calibration plot of Pioglitazone

Fig 4: Calibration plot of Glimepiride

Table 1: System Suitability Parameters (n=5)

Parameters	Pioglitazone	Glimepiride
Retention time (min)	6.647	8.304
Theoretical plates (N)	7251	8652
Tailing factor (T)	1.3	1.1
Resolution (R_s)	1.657

*n= No. of determinants

Linearity

For the determination of linearity, appropriate aliquots were pipette out from working stock solution A to a series of 10ml volumetric flasks and volume was made up with the solvent to obtain concentration ranging from 10-50mg/ml of Pioglitazone and 3-15mg/ml of Glimepiride. Each solution was injected in triplicate. Calibration curves were plotted with observed peak areas against concentration followed by the determination of regression equations and calculation of the correlation coefficients. The calibration curves for Pioglitazone and Glimepiride were shown in figure 3 and figure 4 their corresponding linearity parameters were given in table 2.

Table 2: Results for Linearity (n=3)

Parameter	Pioglitazone	Glimepiride
Linearity Range (µg/ml)	10-50	3-15
Regression Equation	y = 93687x + 72226	y = 17080x + 28025
Slope (m)	93687	17080
Intercept (c)	72226	28025
Regression Coefficient (r²)	0.9999	0.9998
Limit of Detection (µg/ml)	0.658	0.544
Limit of Quantitation (µg/ml)	1.99	1.64

*n= No. of determinants

Fig.5: Assay chromatogram of Pioglitazone and Glimepiride in Tablet formulation

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The LOD and LOQ were calculated from the slope(s) of the calibration plot and the standard deviation (SD) of the peak areas using the formulae LOD = 3.3 σ/s and LOQ = 10 σ/s. The results were given in table 2.

Precision

The repeatability of the method was verified by calculating the %RSD of six replicate injections of 100% concentration (50mg/ml of Pioglitazone and 15mg/ml of Glimepiride) on the same day and for intermediate precision % RSD was calculated from repeated studies on different days. The results were given in table 3.

Table 3: Results of Precision (n=6)

Drug	Intraday Precision (%RSD)	Interday Precision (%RSD)
Pioglitazone	1.43	0.58
Glimepiride	1.64	0.7

*n= No. of determinants

Accuracy

To ensure the reliability and accuracy of the method recovery studies were carried out by standard addition method. A known quantity of pure drug was added to pre-analyzed sample and contents were reanalyzed by the proposed method and the percent recovery was reported. The results were given in table 4.

Table 5: Results for Robustness (n=3)

Parameters (n=3)	%RSD
Parameters (n=3)	Pioglitazone	Glimepiride
Detection wavelength at 228nm	0.018	0.026
Detection wavelength at 232nm	0.03	0.02
Flow rate 0.6ml/min	0.2	0.06
Flow rate 1ml/min	0.032	0.036

*n= No. of determinants

Specificity

Specificity of a method was determined by testing standard substances against potential interferences. The method was found to be specific when the test solution was injected and no interferences were found because of the presence of excipients. The optimized chromatogram of Pioglitazone and Glimepiride without any interference was shown in figure 2.

Robustness

Robustness of the method was verified by altering the chromatographic conditions like wavelength detection, flow rate, etc. and the % RSD should be reported. Small changes in the operational conditions were allowed and the extent to which the method was robust was determined. A deviation of ±2 nm in the detection wave length and ±0.2 ml/min in the flow rate, were tried individually. A solution of 100% test concentration with the specified changes in the operational conditions was injected to the instrument in triplicate. %RSD was reported in the table 5.

Ruggedness

Ruggedness of the method was verified by altering method parameters like different instruments, analysts, laboratories, reagents, days etc. The solution containing 50 μg/ml of Pioglitazone and 15 μg/ml of Glimepiride was injected into HPLC three times under different parameters like different analysts. %RSD was reported in the table 6.

Table 6: Results for Ruggedness (n=6)

Drug	%RSD
Drug	Analyst 1	Analyst 2
Pioglitazone	0.05	0.02
Glimepiride	0.61	0.28

Assay of Marketed Formulations

20 ml of sample solution of concentration 50 mg/ml of Pioglitazone and 15mg/ml of Glimepiride was injected into chromatographic system and the peak responses were measured. The solution was injected three times in to the column. The amount of drug present and percentage purity was calculated by comparing the peak areas of the standards with that of test samples. A typical chromatogram for assay of marketed formulation was shown in figure 5 and the obtained values were reported in the table 7.

Table 7: Results for Assay (n=3) of Marketed formulation

Drug	Label claim (mg/tab)	Amount recovered	% Amount found in drug
Pioglitazone	50	48.2	96.4%
Glimepiride	15	14.8	98%

*n= No. of determinants

RESULTS AND DISCUSSION:

After a number of trials with mobile phases of different composition, Phosphate buffer pH 7 and Acetonitrile in the ratio 60:40v/v was selected as mobile phase because of better resolution and symmetric peaks. Pioglitazone and Glimepiride were found to show appreciable absorbance at 230nm when determined spectrophotometrically and hence it was selected as the detection wavelength. An optimized chromatogram showing the separation of Pioglitazone and Glimepiride at different Rts was shown in figure 2.

System suitability was carried out by injecting 5 replicate injections of 100% test concentration, number of theoretical plates, HETP and resolution were satisfactory. The chromatograms confirm the presence of Pioglitazone and Glimepiride at 6.7min and 8.38min respectively without any interference. The parameters were given in table 1.

Concentration range of 10-50 µg/ml for Pioglitazone and 3-15 µg/ml of Glimepiride were found to be linear with correlation coefficients 0.999 and 0.999 for Pioglitazone and Glimepiride respectively. The results were given in table 2.

The limits of detection for Pioglitazone and Glimepiride were found to be 0.658 µg/ml and 0.54 µg/ml respectively and the limit of Quantitation were 1.99 µg/ml and 1.64 µg/ml respectively. Values were represented in table 2.

The proposed method was found to be precise and reproducible with %RSD of 0.58 and 0.77 for Pioglitazone and Glimepiride respectively. %RSD was reported in table 3.

Accuracy of the method was verified by performing recovery studies by standard addition method. The percent recovery of the standard added to the pre-analysed sample was calculated and it was found to be 96% to 97% and 93 to 96% for Pioglitazone and Glimepiride respectively. This indicates that the method was accurate. Values obtained were given in table 4. The method was found to be robust after changing the conditions like detection wavelength (± 1nm) and flow rate (± 0.2 ml). %RSD was calculated for each variation and reported. Values obtained were given in table 5.

The method was found to be specific for the combination of interest after verifying the chromatograms showing no interference of the excipients present. Hence, the method was well suitable for the estimation of the commercial formulations of the selected combination with a percentage purity of 96% for Pioglitazone and 98% for Glimepiride. The typical chromatogram for assay of marketed formulations was shown in figure.5 and Values obtained were given in table 6.

CONCLUSION:

The RP-HPLC method developed and validated allows a simple and fast quantitative determination of Pioglitazone and Glimepiride from their formulations. All the validation parameters were found to be within the limits according to ICH guidelines. The proposed method was found to be specific for the drugs of interest irrespective of the excipients present and the method was found to be simple, accurate, precise, rugged and robust. So the established method can be employed in the routine analysis of the marketed formulations.

ACKNOWLEDGMENTS:

The authors are thankful to the Mylan Pharmaceuticals ltd. for providing the gift samples of drugs and also to the management of Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Chowdavaram, Guntur for providing facilities and great support to carry out the research work.

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Received on 18.03.2017 Accepted on 28.05.2017

Asian J. Pharm. Ana. 2017; 7(3): 145-150.

DOI: 10.5958/2231-5675.2017.00023.0

Recovery Level	*Pioglitazone*				*Glimepiride*
	Amount Added (µg/ml)		Amount Found (mg)	%Recovery	Amount Added (µg/ml)		Amount Found (mg)	% Recovery (w/w)
	Std.	Test	Amount Found (mg)	%Recovery	Std	Test	Amount Found (mg)	% Recovery (w/w)
80%	10	30	38.5	96.2	2	10	11.2	93
100%	20	30	48.6	97.2	5	10	14.3	94
120%	30	30	58.6	97.6	8	10	17.3	96