DEVELOPMENT AND VALIDATION OF A LIQUID CHROMATOGRAPHIC METHOD FOR ESTIMATION OF GLIMEPIRIDE IN TABLET DOSAGE FORM

DEVELOPMENT AND VALIDATION OF A LIQUID CHROMATOGRAPHIC METHOD FOR ESTIMATION OF GLIMEPIRIDE IN TABLET DOSAGE FORM

Vaishali V. Karkhanis*¹ and Anandkumari D. Captain ²

A.R. College of Pharmacy ¹, Vallabh Vidhyanagar - 388120, Dist: Anand, Gujarat, India

G.H. Patel Institute of Pharmacy ², Vallabh Vidhyanagar - 388120, Dist: Anand, Gujarat, India

ABSTRACT: An accurate, sensitive and precise RP-HPLC method has been developed and validated for the estimation of Glimepiride (GLM) from bulk drug and Pharmaceutical Dosage form. The separation was achieved by Hypersil C18 column (250mm X 4.6mm, 5μm) in isocratic mode, with mobile phase comprises of Acetonitrile : Buffer in proportion of 60:40v/v, buffer was 0.02M Potassium Di-hydrogen Phosphate (pH 4.5 adjusted with Ortho Phosphoric Acid). The flow rate of mobile phase was 1.0ml/min and employing UV detection with 232nm wavelengths. The retention time of GLM was 5.420 min.The calibration curve was found to be linear within the concentration range of 50µg/ml to 150µg/ml. The regression data for calibration curve shows good linear relationship with r² = 0.9965. The method was validated in accordance with the requirements of ICH guidelines. Moreover, the proposed analytical method was applied to monitor the formulation commercially available.

Glimepiride (GLM), Validation, RP-HPLC, Recovery

INTRODUCTION: Glimepiride is widely used in the treatment of non-insulin dependent Type II diabetes mel-litus¹. It acts by stimulating insulin secretions from the beta cells of pancreas and is also known to increase peripheral insulin sensitivity thereby decreasing insulin resistance. It can be used in combination with metformin, thiazolidinediones, alpha-glucosidase inhibitors and in-sulin². After oral administration, it is completely absorbed from the gastrointestinal tract. Peak plasma concentration is reached 2 - 3 hrs after dosing. Its bioavailability changes a little with food and glimepiride (99.5%) are bound to proteins.

Glimepiride is completely metabolised in liver. The structure of glimepiride is shown in Figure 1.

FIGURE 1: GLIMEPIRIDE

Several different methods have been reported for qualitative and quantitative analysis of glimepiride; these include micellar electrokinetic capillary chromatography (MECC) with diode-array detection (DAD) or ultraviolet (UV) detection, high performance liquid chromatography (HPLC) with DAD and UV detection and derivative UV Spectro-photometric detection ^{3, 4}, liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) ^5-8, an HPLC method for the quantification of glimepiride in single dose formulation ^9-11, in human plasma ^12-13, the quantification of cis-isomer of glimepiride by normal phase chromatography¹⁴.A simple accurate and precise HPLC method has been developed for estimation of glimepiride in single dosageform.

MATERIALS & METHODS:

Material: The HPLC system consisted of following components: Shimadzu- Model LC20AT. Rhenodyne valve with 20μl fixed loop, isocratic system pump, Chromatographic analysis was performed on Hypersil BDS C18 column 250×4.6 mm, 5μm particle size. Analytically pure glimepiride was procured as gift samples from Hetero Labs Ltd, Andhra Pradesh, India. All other chemicals and reagents used were analytical grade and purchased from Merck Chemicals, India. Tablets were procured from the local market.

Methods:

1. Preparation of standard stock solution and solutions for calibration curve: Stock solutions of Glimepiride were prepared by dissolving 100 mg of Glimepiride in 100 ml of volumetric flask with diluent. Aliquot of 5.0ml of the standard stock solution of glimepiride was transferred into 50 ml volumetric flask and from that appropriate aliquots were taken to give concentration range of 50-150µg/ml for calibration curve.
2. Chromatographic conditions: Chromato-graphic estimation was performed using a Hypersil BDS C18 column (250mm×4.6mm i.d.), mobile phase consisting of Acetonitrile: Buffer in proportion of 60:40v/v, buffer was 0.02M Potassium Di-hydrogen phosphate (pH 4.5 adjusted with Ortho Phosphoric Acid 5%). Detection was done at wavelength of 232nm. The sample was injected using a 20μl fixed loop, flow rate 1ml/min and the total run time was 8 minutes.
3. Validation: The method was validated as per the ICH guideline.

i.            Regression analysis: Regression of analytical method is expressed in terms of correlation co-efficient of the regression analysis. Accuracy- For determination of Accuracy, recovery study was carried out. That was performed by standard addition method at three different levels (80%, 100%, and 120%), to the pre-analyzed samples and the subsequent solutions were re-analyzed. At each level, three determinations were performed.

ii.            Precision: The precision of analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogeneous samples. Intraday precision- Intraday variance for the Glimepiride was done at the interval of 3 hrs. Interday precision- Interday variance for the Glimepiride was done at the interval of one day.

iii.            Limit of Detection (LOD): LOD was found out based on the standard deviation of the response and the slope method. Limit Of Quantification (LOQ) - LOQ was foundout based on the standard deviation of the response and the slope method. Specificity- Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.

1. Determination of Glimepiride in Tablet Dosage Form: Twenty tablets were weighed, finely powdered, and an accurately weighed sample of powdered tablets equivalent to 10mg of Glimepiride was treated with mobile phase in a 100mL volumetric flask using ultra sonicator. This solution was filtered through 0.45 μm filter paper. Suitable aliquot of the filtered solution was added to a volumetric flask and make up to volume with mobile phase to get appropriate concentration in range.

RESULTS AND DISCUSSION: Several mobile phase compositions were tried to resolve the peak of GLM. The mobile phase containing Acetonitrile: Buffer in proportion of 60:40v/v, buffer was 0.02M Potassium Di-hydrogen (pH 4.5 adjusted with Ortho Phosphoric Acid) was found ideal to resolve the peak of GLM. Retention time of GLM was 5.420 min (Figure 2).

FIGURE 2: CHROMATOGRAM OF GLM STANDARD

Linear regression data showed a good liner relationship over a concentration range of 50-150μg/ml for GLM. The correlation coefficient (r²) was 0.9965 (Figure 3).

FIGURE 3: CALIBRATION CURVE OF GLM

The system suitability parameters are shown in Table 1.

TABLE 1: SYSTEM SUITABILITY PARAMETERS

The accuracy of the method was evaluated by carrying out recovery studies, were performed by standard addition method at three different levels I, II and III (80%, 100%, and 120%), to the pre-analyzed samples and the subsequent solutions were re-analyzed. At each level, three determinations were performed (Table 2).

TABLE 2: RECOVERY STUDY DATA FOR GLM

The limit of detection and limit of quantification were found to be 1.82ug/ml and 5.52ug/ml respectively. The intra-day and inter day precision was determined by analyzing standard solution of 50, 100 and 150 µg/mL and the results are reported in terms of relative standard deviation (Table 3).

TABLE 3: PRECISION DATA

The assay result was repeated for three times which was found to be 99.76-98.11 % of labelled claim (Table 4).

TABLE 4: ASSAY RESULT

CONCLUSIONS: A method of quantitative determination of Glimepiride using HPLC has been developed. The validation results have demonstrated that this method is accurate, precise and linear. The method can also be applied for drug content in pharmaceutical preparations.

ACKNOWLEDGEMENTS: The authors are thankful to Hetero Labs, Andhra Pradesh, India, for providing gift sample of drugs. We are also thankful to the Principal, A.R. College of Pharmacy and G.H. Patel Institute of Pharmacy, who had helped us throughout the entire work.

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How to cite this article:

Karkhanis VV and Captain AD: Development and validation of a Liquid chromatographic method for estimation of Glimepiride in Tablet Dosage form. Int J Pharm Sci Res 2013: 4(7); 2742-2745. doi: 10.13040/IJPSR. 0975-8232.4(7).2742-45

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