UPLCMS Method Development and Validation of Tablet Dosage form Containing Glimepiride, Metformin and Pioglitazone Using Internal Standard

Abstract

A simple, rapid, sensitive and specific Ultra performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous estimation and validation of tablet dosage form containing glimepiride (GLIM), metformin (MET) and pioglitazone (PIO) using Tolzamide (TOLAZ) as an internal standard (IS). The chromatography separation was achieved with Waters ACQUITY HSS C18, 1.8 μm, 2.1X50 mm column with mobile phase containing acetonitrile (A) & 1% ammonium acetate buffer (B) (pH 2.5 adjusted with trifluoro acetic acid) with gradient mode Gradient mode [2 min: 20 A : 80% B, 2-4 min: 70% A : 30% B, 4-5 min,80% A : 20% B, 8-10 min 90% A: 10% B]. The flow rate was 0.4 mL min -1 column maintained at 25 0 C and the injection volume was 2 μl. The selected chromatographic condition were found to effectively separate glimepiride, metformin and pioglitazone with retention time of 3.17, 0.425, 2.3 min, respectively. The proposed method was found to be rectilinear over the range of 2-12 ng mL -1 , 500-3000 ng mL -1 and 15-90 ng mL -1 for glimepiride, metformin, and pioglitazone respectively. The signal intensities obtained in both positive and negative ion mode all drugs including internal standard found to be much higher in positive ion mode (M +H)+ parent ions at m/z, 491.11, m/z 129.87, m/z 357.04 and m/z 312.04 respectively in QUATTROZQ full scan mass spectra. The present method was validated as per ICH guidelines with respect to precision, specificity, linearity, limit of detection, limit of quantification, accuracy, and robustness and it can also be used for routine quality control analysis of these drugs in biological samples either alone or in combined pharmaceutical dosage forms.