Development of analytical methods for the determination of amlodipine, gliclazide and perindopril in dosage forms and plasma

Abstract

Generic formulation is getting popular world-wide. Demand for reliable analytical and bio analytical technique is getting more attention from the researchers around the globe. A total analytical and bio analytical solution for generic formulations are reported in this study. Analytical and bioanalytical methods were developed and validated for the determination of antihypertensive drugs (Amlodipine (AMLO) and perindopril (PER)) and antidiabetics drug (gliclazide (GLZ)) in pharmaceutical formulations and human plasma using HPLC and LC-MSMS. Firstly, new sensitive and economic analytical method was developed and validated for the analysis of AMLO in tablet dosage form using High Performance Liquid Chromatography (HPLC) with UV detector. The degradation profile of AMLO was identified and the degradation diagram was created. In second part, an HPLC with Diode Array Detector (DAD) was developed and validated for the determination of AMLO in human plasma with cetirizine (CTZ) as internal standard. Sample preparation was optimized using DOE (Design of Experiment). Three different parameters namely solvent type, solvent volume and pH were monitored at different levels for the optimization of sample preparation that would produce best recovery of the analytes. Chromatographic separation of the analytes was obtained using Phenomenix C18 (150 × 4.5 mm, 5 µm) column. The third method was the analysis of GLZ in tablet dosage form using HPLC instrument with modern rapid resolution column. Samples were injected to C18 Waters Xselect HSS C18 (2.1 × 100 mm, 2.5 µm) column. The method showed high recovery, precision and repeatability. The degradation products were identified and some products were reported for the first time. In fourth part, we developed and validated new LC-MSMS method for the determination of GLZ in rats’ plasma using CTZ as internal standard. The method was developed using Aquity Waters H Class UPLC connected to Xevo TQD MS detector. The total run time was 5 min with a flow rate of 0.4 ml/min and a total volume of 2ml of solvent consumption making this method highly economic. The method was used to study the interaction between amlodipine and gliclazide when they are administered together. It was found that the co-administration of AMLO and GLZ led to the prolonged elimination of GLZ from rats’ plasm. This is the first report on the pharmacokinetic interaction between AMLO and GLZ. The method was extended for the simultaneous determination of AMLO and GLZ in human plasma with satisfactory results. Fifth part was to develop and validate an LC-MSMS method with Multiple Reaction Monitoring (MRM) mode for the analysis of AMLO and PER in combined tablet dosage form. Ultra-Performance Liquid Chromatography with Mass Spectrometer Detector (UPLC-MSMS) was used for the method presented in this study as the samples were injected into Waters BEH C18, 1.7 um column. The detection was performed using Xevo triple quadrupole Mass Spectrometer (TQD-MSMS) detector which was operated at MRM and electrospray positive mode. The chromatographic and detection conditions were optimized using design of experiment for the highest response. Last part was to develop and validate LC-MSMS method for the determination of AMLO and PER simultaneously in human plasma. All the methods were validated following standard guideline such as International Conference of Harmonization (ICH) for dosage forms and Centre for Drug Evaluation Research (CDER) and European Medicine Agency (EMEA) for bioanalytical method.