A robust liquid chromatography–tandem mass spectrometry method for accurate quantification of omeprazole in human plasma: Integration of International Council for Harmonisation guidelines for pharmacokinetic application

Abstract

A simple, rapid, and reliable bioanalytical method based on protein precipitation coupled with LC-MS/MS was developed and validated for the quantification of omeprazole in human plasma. The method employs protein precipitation with acetonitrile for sample preparation, offering a fast and efficient extraction process. Chromatographic separation was achieved on a reverse-phase using an isocratic mobile phase composed of 2-mM ammonium formate and acetonitrile (30:70, v/v), at a flow rate of 0.2 mL min −1 , within a run time of 4 min. Mass parameters were optimized to monitor transitions at m/z [M+H] + 346.1→198 for omeprazole, and m/z [M+H] + 370→252 for lansoprazole as an internal standard (IS). The calibration curve ranged from 2 to 1,600 ng mL −1 , demonstrating excellent linearity ( R 2 = 0.998) with a lower limit of quantification of 2 ng mL −1 . The accuracy was acceptable with CV (%) < 15, matrix effect (%) < 15, and the hemolytic effect was negligible. Omeprazole was stable in human plasma for 24 h at room temperature (25 °C), for 114 days frozen at −25 °C, for 72 h in an auto-sampler at 6 °C, and for three freeze/thaw cycles. The method was further evaluated using the accuracy profile approach, a robust statistical tool that incorporates tolerance intervals to assess trueness and precision. This approach enables the construction of confidence intervals around measured concentrations, providing a quantitative measure of the method's reliability and the expected analytical performance. The validated method was applied in a pharmacokinetic study to determine the concentration of omeprazole in plasma samples. The study provides a fast and simple bioanalytical method for routine analysis and particularly useful for bioequivalence studies. The method was successfully applied to a pharmacokinetic study of fixed-dose omeprazole in ten healthy volunteers.