Copyright © 2006 John Wiley & Sons, Ltd. Biomed. Chromatogr. 20: 1043–1048 (2006) Simultaneous estimation of six anti-diabetic drugs 1043 ORIGINAL RESEARCH ORIGINAL RESEARCH Copyright © 2006 John Wiley & Sons, Ltd. BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr. 20: 1043–1048 (2006) Published online 28 February 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/bmc.635 Simultaneous estimation of six anti-diabetic drugs— glibenclamide, gliclazide, glipizide, pioglitazone, repaglinide and rosiglitazone: development of a novel HPLC method for use in the analysis of pharmaceutical formulations and its application to human plasma assay † P. Venkatesh, 1 T. Harisudhan, 1 Hira Choudhury, 1 Ramesh Mullangi 1 * and Nuggehally R. Srinivas 2 1 Department of Drug Metabolism and Pharmacokinetics, Discovery Research, Dr Reddy’s Laboratories Ltd, Miyapur, Hyderabad-500 049, India 2 Department of Drug Development, Discovery Research, Dr Reddy’s Laboratories Ltd, Miyapur, Hyderabad-500 049, India Received 21 November 2005; accepted 16 December 2005 ABSTRACT: This paper describes a convenient method for the separation and simultaneous determination of six anti-diabetic drugs viz., glibenclamide (GLB), gliclazide (GLC), glipizide (GLZ), pioglitazone (PGL), repaglinide (RPG) and rosiglitazone (RGL) in pharmaceutical formulations. Also, the assay has been shown applied to support quantification of the six anti-diabetic drugs in human plasma. The analytes were either injected directly onto the column after suitable dilution (pharmaceutical formu- lation analysis) or a simple extraction procedure, using acetonitrile, from human plasma spiked with anti-diabetic drugs and internal standard (IS). Ternary gradient elution at a flow rate of 1 mL/min was employed on an Intertisl ODS 3V column (4.6 × 250 mm, 5 μm) at ambient temperature. The mobile phase consisted of 0.01 m formic acid (pH 3.0), acetonitrile, Milli Q water and methanol. Celecoxib was used as an IS. The six anti-diabetic drugs were monitored at a wavelength of 260 nm. The nominal reten- tion times of RGL, PGL, GLZ, GLC, GLB, IS and RGL were 11.4, 13.3, 14.8, 17.6, 20.78, 22.1 and 25.4 min, respectively. The assay developed for formulation analysis was found to be accurate and precise. The calibration curves ranged from 0.1 to 100 μg/mL for all analytes with the exception of GLB, where the range was 0.3–100 μg/mL. The plasma assay was validated for parameters such as specificity, accuracy and extraction recovery. The proposed method is simple, selective and can be extended for routine analysis of anti-diabetics in pharmaceutical preparations and in biological matrices. Copyright © 2006 John Wiley & Sons, Ltd. KEYWORDS: glibenclamide; gliclazide; glipizide; pioglitazone; repaglinide; rosiglitazone; pharmaceutical formulations; human plasma INTRODUCTION Drugs belonging to class such as biguanides (e.g. metformin), sulfonyl ureas (e.g. glipizide, glibenclamide, gliclazide) and thiazolidinedione (TZD) derivatives (pioglitazone, rosiglitazone) are commonly prescribed hypoglycemic drugs for the treatment of non-insulin- dependent type II diabetes mellitus. The use of com- bination of sulfonyl ureas and TZDs is commonly observed in clinical practice. The six anti-diabetic drugs chosen for this study were glibenclamide (GLB), gliclazide (GLC), glipizide (GLZ), pioglitazone (PGL), repaglinide (RPG) and rosiglitazone (RGL). GLB, GLC and GLZ are sulfonylurea drugs, which act by increasing the secretion of insulin by the functioning β-cells of the pancreas. RPG, which belongs to the meglitinide class, also acts by stimulating insulin secre- tion of β-cells, but it binds to sites distinct from the sulfonylurea binding sites (Fuhlendroff et al., 1998). Both PGL and RGL are members of the thiazoli- dinedione class, which exert their glucose-lowering effect by binding to peroxisome proliferator-activated receptors gamma (PPARγ ), thus increasing the receptor sensitivity to insulin (Lehman et al., 1995; Wilson et al., 1996; Young et al., 1998). Ho et al. (2004) developed an LC-MS/MS method for simultaneous de- tection of 10 anti-diabetic drugs comprising mainly sulfonylureas and thiazolidinediones in equine plasma and urine, Aburuz et al. (2005) developed an HPLC method in human plasma for the determination of metformin with three sulfonylurea, namely gliben- clamide, glipizide and gliclazide. Other literature infor- mation is confined to estimation of single components and are not suitable for determination of two or more anti-diabetic drugs (Courtois et al., 1999; Noguchi et al., 1992; Radhakrishna et al., 2002). Except these *Correspondence to: R. Mullangi, Department of Drug Metabolism and Pharmacokinetics, Discovery Research, Dr Reddy’s Laboratories Ltd, Miyapur, Hyderabad-500 049, India. E-mail: mullangiramesh@drreddys.com † DRL publication no. 532. Abbreviations used: GLB, glibenclamide; GLC, gliclazide; GLZ, glipizide; PGL, pioglitazone; RPG, repaglinide; RGL, rosiglitazone; TZD, thiazolidinedione.