Analytical Method Development and Method Validation for the Simultaneous Estimation of Metformin hydrochloride and Pioglitazone hydrochloride in Tablet Dosage Form by RP-HPLC

Jajow Swapna*, Chandaka Madhu, Mallepelli Srivani, M. Sumalatha, Y. Nehalatha, Y. Anusha

Sri Indu College of Pharmacy, Sheriguda, Hyderabad (A.P)-501 301, India.

*Corresponding Author E-mail: Srivani.pharmacy@ymail.com,

ABSTRACT:

A simple, sensitive and rapid reverse phase high performance liquid chromatographic method was developed for the estimation of Metformin HCl (MET) and Pioglitazone (PIO) in pure and in pharmaceutical dosage forms. A BDS Hypersil C18 column (250x4.6mm, 5μ) 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 1ml/min and effluents were monitored at 238nm and eluted at 2.81min (MET) and 4.57min (PIO). Calibration curve was plotted with a range from 40-240 µg / ml for MET and 12-72 µg / ml for PIO. The assay was validated for the parameters like accuracy, precision, specificity, robustness, ruggedness and system suitability parameters. The proposed method can be useful in the routine analysis for the determination on metformin and pioglitazone in pharmaceutical dosage forms.

KEYWORDS: Metformin Hydrochloride, Pioglitazone Hydrochloride, Reverse phase HPLC, Pharmaceutical dosage forms, simultaneous estimation.

INTRODUCTION:

Metformin (I, N, N-dimethyldiguanide) and Pioglitazone, (±)-5-[p-[2-(5-ethyl-2-pyridyl)-ethoxy] benzyl]-2,4-thiazolidinedione¹ are used in the treatment of type 2 diabetes. Metformin improves hepatic and peripheral tissue sensitivity to insulin without the problem of serious lactic acidosis where as Pioglitazone hydrochloride has been shown to affect abnormal glucose and lipid metabolism associated with insulin resistance by enhancing insulin action on peripheral tissues. Many patients suffering from type 2 diabetes require treatment with more than one antihyperglycemic drug to achieve optimal glycemic control. The literature reveals that there are some of the methods have been reported for metformin UV^{1, 2}, HPLC³ stability studies⁴ and potentiometry, spectrofluorimetry ⁵. For pioglitazone HPLC method in pharmaceutical dosage forms⁶ determination of its metabolites in human plasma^7,8 and simultaneous determination of metformin and pioglitazone⁹ in pharmaceutical dosage forms.

The present paper describes a simple, accurate, validated and economic method for the simultaneous determination of metformin and pioglitazone.

MATERIALS AND METHODS:

Reagents

Metformin Hydrochloride and Pioglitazone Hydrochloride were procured from CHANDRA LABS (Kukatpally, Hyderabad, A.P, India) which were claimed to contain 50mg and 3mg were used in analysis. Acetonitrile (HPLC grade, MERCK).

Other reagents were of AR grade.

Instrumentation

HPLC system (Shimadzu prominence) equipped with UV- Detector. The data acquisition was performed by Spinchrom software.

Chromatographic conditions:

Column à BDS HYPERSIL C18, 250×4.6mm, 5µ

Flow rate à 1ml/min

Wavelength à 238nm

Runtime à 10mins

Column temperature à 25⁰c

Injection Volume à 20µL

Pump mode à Isocratic

Retention time à 2.81 and 4.57 respectively

Fig. 1

Fig. 2

Preparation of standard:

Accurately weighed about 50mg and 3mg of Metformin hydrochloride and Pioglitazone hydrochloride working standards and transferred into a 25ml volumetric flask, added 15ml of diluent, and sonicated to dissolve. Cooled to room temperature and diluted to volume with diluent.

Preparation of sample:

20 tablets of Metformin hydrochloride and Pioglitazone hydrochloride were weighed and powdered in glass mortar. The powder equivalent to 114mg was transferred into a 25 ml volumetric flask, 15 ml of diluent was added to it and was shaken by mechanical stirrer and sonicated for about 30 minutes by shaking at intervals of five minutes each and was diluted up to the mark with diluent and allowed to stand until the residue settles before taking an aliquot for further dilution. 1 ml of upper clear solution was transferred to a 10 ml volumetric flask and diluted with diluent up to the mark and the solution was filtered through 0.45 m filter before injecting into the HPLC system.

Procedure for assay:

20 µl of the Standard, Sample and Blank preparations in duplicate were injected separately into the HPLC system and the peak responses for Metformin hydrochloride and Pioglitazone hydrochloride were measured. The quantities from the peak area in mg of Metformin hydrochloride and Pioglitazone hydrochloride were calculated per tablet taken

RESULTS AND DISCUSSION:

A reversed-phase column procedure was proposed as a suitable method for the simultaneous determination of metformin and pioglitazone in combined dosage form. The chromatographic conditions were optimized by changing the mobile phase composition, pH, and buffers used in the mobile phase. Different ratios were experimented to optimize the mobile phase. Finally a mixture of Acetonitrile and potassium dihydrozen ortho phosphate anhydrous buffer (pH-3) in the ratio of 50:50 was used this mobile phase showed good resolution of Metformin and Pioglitazone peak. The wavelength of detection selected was 238 nm, as both the drugs showed optimum absorbance at this wavelength.

Acid Stress (0.1 M HCl)

Table 1: Specificity testing (Acid stress)

Concentration (µg/ml)		Time (hrs)	Retention time (min)
Metformin	Pioglitazone		Metformin	Pioglitazone
15	3	0	2.807	4.573
		24	2.827	4.573

Base Stress (0.1M NaOH)

Table 2: Specificity testing (Base stress)

Concentration (µg/ml)		Time (hrs)	Retention time (min)
Metformin	Pioglitazone		Metformin	Pioglitazone
15	3	0	2.807	4.573
		24	2.827	4.573

Peroxide stress (5% H2O2)

Table 3: Specificity testing (Peroxide stress)

Concentration (µg/ml)		Time (hrs)	Retention time (min)
Metformin	Pioglitazone		Metformin	Pioglitazone
15	3	0	2.807	4.573
		24	2.827	4.573

By our proposed method the retention time of metformin and Pioglitazone was about 2.803 and4.573 minute, respectively and none of the impurities were interfering in its assay (Fig. 1 and 2).

Figure 3: Linearity curve of standard Metformin HCl

Figure 4: Linearity curve of standard Pioglitazone HCl

Validation of the method:

The developed method has been validated for the assay of Metformin HCl and Pioglitazone HCl as per ICH guidelines by using following parameters.

Specificity and Selectivity:

Specificity and selectivity were studied for the examination of the presence of interfering components. It was checked by subjecting the drug solution in different stress conditions like Acid, Base, Peroxide and the degradation was noted.

Linearity:

Linearity was studied by preparing standard solutions of Metformin and Pioglitazone at different concentration levels (Fig. 3 and 4). The responses were found linear in the range of 40-240 µg / ml and 12-72 μg/ml for Metformin and Pioglitazone, respectively.

Accuracy:

Accuracy was performed in triplicate for various concentrations of Metformin and Pioglitazone equivalent to 80, 100 and 120 % of the standard amount was injected into the HPLC system per the test procedure. The average % recovery of Metformin and Pioglitazone was calculated. Table 4: Results of Analysis of Formulation and Recovery Studies.

Precision:

A) Method Repeatability

Six sample solutions of the same concentration (50%) were prepared and injected into the HPLC system as per test procedure

B) Intermediate Precision (Analyst to Analyst variability)

Two analysts as per test method conducted the study. For Analyst-1 Method Repeatability and for Analyst-2 six sample solutions of the same concentration (50%) were prepared and injected into the HPLC system as per test procedure.

Robustness and Ruggedness

Robustness was done by small deliberate changes in the chromatographic conditions and retention time of Metformin and Pioglitazone was noted. The factors selected were flow rate and variation in the mobile phase composition. The results remained unaffected by small variations in these parameters. Ruggedness of the method was checked by using different analysts and instruments. The relative standard deviation of the results obtained from different analysts and instruments was < 1.0%.

Table 4: Results of Analysis of Formulation and Recovery Studies

Accuracy 80%

Pioglitazone					Metformin
Sl.No		Area	Amount recovered	% Amount recovered		Area	Amount recovered	% Amount recovered
1	Std	504.866			Std	1426.908
2	Accuracy 01	505.254	80.061	100.077	Accuracy 01	1425.054	79.896	99.87
3	Accuracy 02	502.922	79.691	99.615	Accuracy 02	1425.295	79.909	99.88
4	Accuracy 03	503.312	79.753	99.692	Accuracy 03	1423.756	79.823	99.78

Accuracy 100%

Pioglitazone					Metformin
Sl.No		Area	Amount recovered	% Amount recovered		Area	Amount recovered	% Amount recovered
1	Std	606.133			Std	1641.483
2	Accuracy 01	605.544	99.902	99.902	Accuracy 01	1636.265	99.682	99.68
3	Accuracy 02	600.308	99.038	99.038	Accuracy 02	1625.414	99.021	99.02
4	Accuracy 03	604.215	99.683	99.683	Accuracy 03	1632.97	99.481	99.48

Accuracy 120%

Pioglitazone					Metformin
Sl.No		Area	Amount recovered	% Amount recovered		Area	Amount recovered	% Amount recovered
1	Std	772.96			Std	1992.869
2	Accuracy 01	774.645	120.261	100.21	Accuracy 01	1991.368	119.909	99.92
3	Accuracy 02	776.519	120.525	100.46	Accuracy 02	1994.162	120.077	100.06
4	Accuracy 03	777.65	120.728	100.60	Accuracy 03	1990.781	119.874	99.89

Table 5: Results from determination of precision of analysis of Metformin and Pioglitazone

Metformin			Pioglitazone
Sl.no	Rt	Area	Sl.no	Rt	Area
1	2.82	1704.821	1	4.553	636.314
2	2.8	1677.027	2	4.52	627.06
3	2.82	1700.461	3	4.553	638.952
4	2.827	1715.753	4	4.553	640.56
5	2.82	1714.368	5	4.553	634.76
6	2.82	1714.128	6	4.553	639.042
Avg	2.817	1704.41	Avg	4.549	636.115
Std dev	0.009	14.735	Std dev	0.015	4.904
%RSD	0.325	0.864	%RSD	0.343	0.771

Table 6: Results from determination of precision of analysis of Metformin and Pioglitazone

Metformin Pioglitazone

Metformin			Pioglitazone
Sl.no	Rt	Area	Sl.no	Rt	Area
1	2.82	1675.571	1	4.56	608.141
2	2.82	1661.488	2	4.567	601.976
3	2.82	1654.72	3	4.56	604.928
4	2.82	1664.471	4	4.56	603.516
5	2.82	1672.099	5	4.56	593.603
6	2.827	1684.62	6	4.567	598.377
Avg	2.821	1668.735	Avg	4.562	601.756
Std dev	0.002	10.785	Std dev	0.003	5.135
%RSD	0.101	0.634	%RSD	0.079	0.853

Table 7: Validation parameter of HPLC method for Metformin and Pioglitazone

Validation Parameter	Metformin HCl	Pioglitazone HCl
Linearity Range (µg/ml) Regression equation Correlation Coefficient (r^2) Accuracy Precision Method Repeatability (RSD %) IntermediatePrecision (RSD %)	40-240 Y= 8.0449x+70.224 0.9999 99.02 - 100.06 0.8645 0.634	12-72 Y=10.231x+1.7976 0.9992 99.03 - 100.6 0.7709 0.853

Validation parameter

The method was validated by using the following parameters as shown in Table 7.

CONCLUSION:

The proposed method is rapid, accurate and sensitive. It makes use of fewer amounts of solvents and change of set of conditions requires a short time. Many samples can be simultaneously and suitably analysed for the routine quality control analysis of Metformin and Pioglitazone in bulk and its tablet dosage forms. It does not suffer from any interference due to common excipients present in pharmaceutical preparation and can be conveniently adopted for quality control analysis.

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Received on 25.07.2012 Accepted on 28.08.2012

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Asian J. Pharm. Ana. 2(3): July-Sept. 2012; Page 85-89