INTRODUCTION:

Chemically, glimepiride is 1-(4-(2-(3-ethyl4-methyl-2-oxo-2,5-dihydro-1H-pyrrole-1-carboxamido)ethyl)phenylsulfonyl)-3-(4-methyl cyclo hexyl)urea, pioglitazone hydrochloride is (± )-5-[p- [2-(5-ethyl-2-pyridyl)-ethoxy] benzyl]-2,4-thiazolidinedione where as Metformin hydrochloride is 1,1-dimethyl biguanide hydrochloride¹ (structures shown in figure 1a, 1b and 1c). Glimepiride is a sulfonyl urea group oral anti-diabetic drug with prolonged effect and more over it maintains a more physiological regulation of insulin secretion than glibenclamide during physical exercise, suggesting that there may be less risk of hypoglycaemia with glimepiride, and act by increasing the secretion of insulin by the functioning ß-cells of the pancreas, pioglitazone has been shown to affect abnormal glucose and lipid metabolism associated with insulin resistance by enhancing insulin action on peripheral tissues where as metformin improves hepatic and peripheral tissue sensitivity to insulin without the problem of serious lactic acidosis².

This combination can be achieved by taking each of the drugs separately or alternatively fixed formulations have been developed. A combination tablet formulation is beneficial in terms of its convenience and patient compliance.

The review of literature reveals that there were analytical methods of all the three drugs individually in pharmaceutical dosage forms and even in biological samples^3-11 and a few methods reported for combination of either of the two drugs^12-18. But no stability indicating method was reported for these drugs to the date. The present paper describes stability indicating HPLC method for the determination of glimepiride (Gm), pioglitazone hydrochloride (Pg) and Metformin hydrochloride (Mt) in pharmaceutical dosage forms.

The purpose of stability testing is to provide evidence on how quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, light and enables recommendation of storage conditions, retest periods and shelf life to be established¹⁹.The two main aspects of drug products that play an important role in shelf life determinations are assay of active drug and degradants generated during the stability study. Stability-indicating methods have been reported for assays of various drugs in drug products containing only one active drug substance. Only few stability indicating methods are reported for the assay of combination drug products containing two or more active drug substances. The objective of this work was to develop an analytical LC procedure, which would serve as stability indicating assay method for combination drug products of Gm, Pg and Mt. In the present study attempts were made to develop a rapid, economical, precise and accurate method for the simultaneous determination of the Gm, Pg and Mt in the presence of their degradants.

Figure 1(A)

Figure 1(B)

Figure 1(C)

Figure 1. Structure of (A) Glimepiride (B) Pioglitazone hydrochloride and (C) Metformin hydrochloride

MATERIAL AND METHODS:

Apparatus:

A gradient high performance liquid chromatograph from younglin HPLC system, equipped with a UV detector and Autochro-3000 software was used. A reversed phase Varian C-18 (250 Χ 4.6 mm i.d, 5 μm particle size) analytical column was used for the present analysis. Shimadzu electronic balance, ultrasonic cleaner (225x125x60 mm s.s-304, 1.5 Liter capacity of tank) and pH meter LI127 (Elico Limited) were used during the study.

Reagents and materials:

Gm, Pg and Mt were obtained from Torrent Pharmaceutical Ltd (India). Market formulations Glimaday-P1 and Glimaday-P2 were obtained commercially. All solvents were of HPLC grade and all reagents were of analytical grade. Methanol, Acetonitrile, glacial acetic acid, disodium hydrogen phosphate and potassium dihydrogen phosphate were obtained from Merck (India). Sodium hydroxide, hydrochloric acid and hydrogen peroxide were obtained from S.D. Fine-Chem Ltd (Mumbai). Triple distilled water was used throughout the experiment. All solvents and solutions were filtered through a membrane filter (ultipor N66 Nylon 66, 0.2μm pore size) and degassed using ultrasonic cleaner before use.

Chromatographic conditions:

The samples were chromatographed on a reversed phase C-18 (250 Χ 4.6 mm i.d, 5 μm particle size) column with a flow rate of 1.0 ml/min. All analyses were carried out at isocratic conditions. The mobile phase consisted of a mixture of methanol : phosphate buffer(pH 4.0, adjusted with glacial acetic acid) (70 : 30, v/v). The mobile phase was filtered through a Nylon 0.2µm membrane filter and degassed before use. The volume of injection was 20µl and the detection was made at 240nm.

Preparation of solutions:

Phosphate Buffer:

Accurately weighed disodium hydrogen phosphate (5.04gm) and potassium dihydrogen phosphate (3.01gm) were dissolved in sufficient triple distilled water and then dilute upto 1000ml with the same. The pH was adjusted 4.0 using glacial acetic acid.

Gm standard stock solution:

Accurately weighed Gl (10mg) was transferred in 100 ml volumetric flask. The drug was dissolve in methanol with sonication and final volume was adjusted with methanol upto mark to prepare a 100µg/ml stock solution.

Gm working standard solution:

From the above stock solution (100µg/ml), an accurately measured 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 ml was transfer into separate 10 ml volumetric flask and final volume was adjusted with methanol upto mark to prepare 2-12µg/ml solutions.

Pg standard stock solution:

Accurately weighed Pg (50mg) was transferred in 100 ml volumetric flask. The drug was dissolve in methanol with sonication and final volume was adjusted with methanol upto mark to prepare a 500µg/ml stock solution.

Pg working standard solution:

From the above stock solution (500µg/ml), an accurately measured 1, 2, 3, 4, 5 and 6 ml was transfer into separate 100 ml volumetric flask and final volume was adjusted with methanol upto mark to prepare 5-30µg/ml solutions.

Mt standard stock solution:

Accurately weighed Mt (50mg) was transferred in 25 ml volumetric flask. The drug was dissolve in methanol with sonication and final volume was adjusted with methanol upto mark to prepare a 2000µg/ml stock solution.

Mt working standard solution:

From the above stock solution (2000µg/ml), an accurately measured 1, 2, 3, 4, 5 and 6 ml was transfer into separate 10 ml volumetric flask and final volume was adjusted with methanol upto mark to prepare 200-1200µg/ml solutions.

Preparation of Sample solution:

Weigh and finely powder 20 tablets. Transfer powder exactly equivalent to one tablet containing 2 mg of Gm, 15mg of Pg and 500mg of Mt to a 100 ml volumetric flask. Add about 60 ml of methanol and sonicate for 15 minutes and make up volume with methanol. From this solution take 1ml and dilute upto 10ml with methanol. This solution was injected for HPLC determination.

Optimization of the solvent system:

Varying compositions of methanol : phosphate buffer(pH 4.0, adjusted with glacial acetic acid) 65 : 35, 50 : 50, 40 : 60, and 30 : 70 v/v were evaluated as mobile phase in order to achieve good peak shape and short run time. Finally, methanol : phosphate buffer (pH 4.0, adjusted with glacial acetic acid) (70 : 30, v/v) isocratic method was used.

Method Validation:

Linearity:
The linearity was evaluated by linear regression analysis. The calibration curve was obtained with concentrations of pure Gm, Pg and Mt solution ranging from 2-12µg/ml, 5-30µg/ml and 200-1200µg/ml respectively for the chromatographic method. (Figure 2)

Figure 2(A)

Figure 2(B)

Figure 2(C)

Figure 2. Calibration curve for (A) Gm (B) Pg and (C) Mt

Precision:
The precision of the procedure was determined by repeatability (intraday). Intraday precision was evaluated by assaying same concentration and during the same day. Repeatability of sample measurement was carried out in six different sample preparations from same homogenous blend of sample. Another replicate determination on three different days to estimate interday precision.

Accuracy:
Recovery studies were performed to validate the accuracy of developed method. To a preanalysed sample solution, a definite concentration of standard drug was added and recovery was studied. A 80%, 100% and 120% of pure drug solutions were added to the preanalyzed samples.

Limit of detection and limit of quantification:

For HPLC method, the limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the response and the slope by using calibration curves.

Robustness:
For the HPLC method, the robustness was determined by the analysis of the samples under a variety of conditions making small changes in the buffer pH (2.0 and 4.0), in the percentage of mobile phase compounds (methanol : phosphate buffer in the ratios 40 : 60 and 80 : 20), in the flow rate (0.9 and 1.1 ml/min), in the temperature conditions (35 and 45°C), and changing the wavelength (230 and 250 nm).

System suitability:

System suitability parameter is established to ensure that the validity of the analytical method is maintained whenever used. Typical variations are the stability of analytical solution, different equipment, and different analyzer. In case of liquid chromatography typical variations are the pH of the mobile phase, the mobile phase composition, different lots or supplier of columns, the temperature and flow rate.

Analysis of marketed formulation:

The response of sample solution was measured at 240nm using HPLC. The amount of Gm, Pg and Mt were determined by regression equation and results are shown in Table5.

Procedure for Forced Degradation Study of Drug Substances:

Forced degradation of each drug substance and the drug product was carried out under acidic, alkaline, neutral, oxidative and thermal stress conditions.

Acidic Degradation

Weigh accurately 10mg of Glimepride, transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then add 50ml of 0.1 N HCl and reflux at 80°C for 6 hours. From this solution take 0.2 ml and dilute upto 10ml with methanol (2µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Pioglitazone hydrochloride, transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then add 50ml of 5 N HCl reflux at 85°C for 24 hours. From this solution take 3 ml and dilute upto 10ml with methanol (30µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Metformin hydrochloride, transfer in 10ml volumetric flask. Dissolve in 5ml of methanol then add 5ml of 5 N HCl and reflux at 85°C for 24 hours. This solution (1000µg/ml) was injected for HPLC determination.

Alkaline Degradation:

Weigh accurately 10mg of Glimepride, transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then add 50ml of 0.1 N NaOH and reflux at 80°C for 6 hours. From this solution take 0.2ml and dilute upto 10ml with methanol (2µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Pioglitazone hydrochloride, transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then add 50ml of 0.1 N NaOH reflux at 85°C for 2 hours. From this solution take 3 ml and dilute upto 10ml with methanol (30µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Metformin hydrochloride, transfer in 10ml volumetric flask. Dissolve in 5ml of methanol then add 5ml of 5 N NaOH and reflux at 85°C for 24 hours. This solution (1000µg/ml) was injected for HPLC determination.

Oxidative Degradation:

Weigh accurately 10mg of Glimepride, transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then add 50ml of 3%H₂O₂ and kept at room temperature for 6 hours. From this solution take 0.2ml and dilute upto 10ml with methanol (2µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Pioglitazone hydrochloride, transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then add 50ml of 30% H₂O₂ and kept at room temperature for 4 hours. From this solution take 3 ml and dilute upto 10ml with methanol (30µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Metformin hydrochloride, transfer in 10ml volumetric flask. Dissolve in 5ml of methanol then add 5ml of 30% H₂O₂ and kept at room temperature for 24 hours. This solution (1000µg/ml) was injected for HPLC determination.

Dry heat Degradation:

Weigh accurately 10mg of Glimepride, kept at 105°C for 8 hours transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then dilute upto mark with methanol. From this solution take 0.2 ml and dilute upto 10ml with methanol (2µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Pioglitazone hydrochloride, kept at 105°C for 8 hours transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then dilute upto mark with methanol. From this solution take 3 ml and dilute upto 10ml with methanol (30µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Metformin hydrochloride, kept at 105°c for 8 hours transfer in 10ml volumetric flask. Dissolve in 5ml of methanol then dilute upto mark with methanol. This solution (1000µg/ml) was injected for HPLC determination.

Neutral degradation:

Weigh accurately 10mg of Glimepride, transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then add 50ml of water and reflux at 80°C for 6 hours. From this solution take 0.2 ml and dilute upto 10ml with methanol (2µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Pioglitazone hydrochloride, transfer in 100ml volumetric flask. Dissolve in 50ml of methanol then add 50ml of water and reflux at 85°C for 24 hours. From this solution take 3 ml and dilute upto 10ml with methanol (30µg/ml). This solution was injected for HPLC determination.

Weigh accurately 10mg of Metformin hydrochloride, transfer in 10ml volumetric flask. Dissolve in 5ml of methanol then add 5ml of water and reflux at 85°C for 24 hours. This solution (1000µg/ml) was injected for HPLC determination.

Procedure for Forced Degradation Study of Drug Products:

A forced degradation study of drug products in acidic, alkaline, neutral, oxidative and thermal conditions was carried out using ﬁltered solution containing 2µg/ml Gm, 30µg/ml Pg and 1000µg/ml Mt(as described in preparation of sample solution).

For thermal degradation a quantity of powder equivalent to one tablet containing 2mg of Gm, 30mg of Pg and 1000 mg of Mt was exposed. Then the solutions were prepared as described in preparation of sample.

RESULT AND DISCUSSION:

The mobile phase consisting of methanol: phosphate buffer (70:30, v/v) having pH 4.0 adjusted with glacial acetic acid, at 1ml/min flow rate which gave sharp, well-resolved peak with minimum tailing factor for Gm, Pg and Mt. The retention time for Gm, Pg and Mt were 9.95, 7.12 and 2.66 min respectively. (Figure 3)

Figure 3(A)

Figure 3(B)

Figure 3(C)

Figure 3. A typical chromatogram of (A) Gm(2µg/ml) (B) Pg(30µg/ml) and (C)Mt(1000µg/ml)

The calibration curve for Gm, Pg and Mt were found to be linear over the range of 2-12μg/ml, 5-30µg/ml and 200-1200 μg/ml respectively. The data of regression analysis of the calibration curves is shown in Table 1. The proposed method was successfully applied to the determination of Gm, Pg and Mt in market formulation.

Table 1. Data of regression analysis and calibration curve

Drug	Linearity range	Y=mx+c		r²
Drug	Linearity range	Slope	Intercept	r²
Gm	2-12µg/ml	6812.9	243.33	0.9994
Pg	5-30µg/ml	15519	426.8	0.9990
Mt	200-1200 µg/ml	4122.5	34944	0.9996

n=6

The LOD for Gm, Pg and Mt were found to be 0.36 μg/ml, 1.70 μg/ml and 1.944 μg/ml respectively, while LOQ were 1.11 μg/ml, 5.17 μg/ml and 5.891 μg/ml respectively. The results for system suitability test parameters and recovery study are summarized in Table 2and3. The summary of validation parameters for analysis of Gm, Pg and Mt were shown in Table 4.

Table 2. Result of system suitability parameters

Parameter	RT	AUC	No. of theoretical plates	Tailing factor
Gm	9.90	26518.20	4904.4	0.97
Pg	5.29	241205.2	3637.4	1.08
Mt	2.69	2202496	1363.12	0.96

n=6

Table 3. Results of recovery studies of Gm, Pg and Mt

Drug	Conc. Of Form. (µg/ml)	Conc. Of Std. added (µg/ml)	Conc. Recovery (µg/ml)	% recovery
Gm	1	0.8	1.79	99.44%
	1	1	1.97	97.50%
	1	1.2	2.19	99.54%
Gm	2	1.6	3.59	99.72%
	2	2	3.96	99.00%
	2	2.4	4.39	99.54%
Pg	15	12	26.89	99.59%
	15	15	29.91	99.70%
	15	18	32.87	99.48%
Mt	500	400	901.05	100.1167
	500	500	1000.09	100.009
	500	600	1100.03	100.0027

n=6

Table 4. Summary of Validation Parameters

Parameter	Gm	Pg	Mt
Wavelength	240 nm	240 nm	240 nm
Range	2 -12 µg/ml	5 -30 µg/ml	200-1200 µg/ml
Linearity	0.9994	0.9990	0.9996
Intercept	6812.9	15519	34944
Slope	243.33	426.8	4122.5
Intra day precision	%RSD < 2	%RSD < 2	%RSD < 2
Inter day precision	%RSD < 2	%RSD < 2	%RSD < 2
LOD	0.367µg/ml	1.706µg/ml	1.944µg/ml
LOQ	1.113µg/ml	5.172µg/ml	5.891µg/ml

Figure 4(A)

Figure 4(B)

Figure 4(C)

Figure 4(D)

Figure 4(E)

Figure 4. Forced degradation of drug product under (A) acidic (B) alkaline (C) oxidative (D) dry heat (E) neutral conditions

Table 5. Result of Market formulation of Gm, Pg and Mt

Brand Name	Drug	Amount taken (µg/ml)	Amount found (µg/ml)	%Amount found
Glimaday-P1	Gm	1	0.987	99.35±0.22
	Pg	15	14.89	98.93±0.28
	Mt	500	499.89	99.92±0.11
Glimaday-P2	Gm	2	1.987	99.35±0.22
	Pg	15	14.89	99.26±0.43
	Mt	500	499.89	99.92±0.11

n=6

Table 6. Result of Forced degradation study of drug product

Degradation condition	Drug	Conc. Of drug(µg/ml)	RT of observed peak	AUC	%of drug	%of degrdation
acidic	Gm	2	9.95	13148.3	97.45%	0.00%
			9.12(I)	214.89	-	1.21%
			8.1(II)	132.28	-	0.98%
	Pg	30	5.31	464789	99.98%	-
	Mt	500	2.66	2002012	99.65%	-
alkaline	Gm	2	10.01	12954.2	96.24%	-
			9.12(I)	214.89	-	1.21%
			8.1(II)	132.28	-	0.98%
			8.54(III)	199.76	-	1.04%
	Pg	30	7.12	468469	98.21%	-
	Pg	30	6.87	8468	-	1.40%
	Mt	500	2.68	1997854	99.57%	-
oxidative	Gm	2	10.01	12976.4	96.04%	-
			9.12(I)	188.12	-	1.54%
			8.1(II)	132.28	-	0.98%
			8.54(III)	199.76	-	1.04%
	Pg	30	5.31	464789	99.98%	-
	MET	500	2.66	2013267	99.84%	-
dry heat	Gm	2	9.96	13468	99.90%	-
	Pg	30	5.31	463264	99.88%	-
	MET	500	2.66	2013267	99.84%	-
neutral	Gm	2	9.95	13223.84	97.94%	-
			8.1(II)	132.28	-	0.98%
			8.54(III)	128.54	-	0.96%
	Pg	30	5.31	463264	99.88%	-
	Mt	500	2.66	2013267	99.84%	-

n=6