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Optimization of chromatography to overcome matrix effect for reliable estimation of four small molecular drugs from biological fluids using LC-MS/MS.

The article describes a systematic study to overcome matrix effect during chromatographic analysis of gemfibrozil, rivastigmine, telmisartan and tacrolimus from biological fluids using LC-ESI-MS/MS. All four methods were thoroughly developed by appropriate choice of analytical column, elution mode and pH of mobile phase for improved chromatography and overall method performance. Matrix effect was assessed by post column analyte infusion, slope of calibration line approach and post extraction spiking. The best chromatographic conditions established were, Acquity BEH C18 (50 × 2.1 mm, 1.7 μm) column with 5.0 mm ammonium acetate, pH 6.0: methanol as the mobile phase under gradient program for gemfibrozil; Luna CN (50 × 2.0 mm, 3 μm) column with a mobile phase consisting of acetonitrile-10 mM ammonium acetate, pH 7.0 (90:10, v/v) for rivastigmine; Inertsustain C18 (100 × 2.0 mm, 5 μm) column using methanol-2.0 mM ammonium formate, pH 5.5 (80: 20, v/v) as the mobile phase for isocratic elution of telmisartan; and Acquity BEH C18 (50 × 2.1 mm, 1.7 μm) with methanol-10 mM ammonium acetate, pH 6.0 (95:5, v/v) as mobile phase for tacrolimus. The methods were thoroughly validated as per EMA and USFDA guidance and were successfully applied for pharmacokinetic studies in healthy subjects.