Development and application of a supercritical fluid chromatography-tandem mass spectrometry method for the simultaneous determination of pantoprazole enantiomers in rat plasma

Abstract

The study aimed to develop a supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method for the enantioselective separation and quantification of pantoprazole in rat plasma. The method utilized protein precipitation with acetonitrile to prepare plasma samples. SFC was performed on an Acquity ultra-performance convergence chromatography (UPCC) system. An Acquity UPCC Trefoil™ CEL2 column was employed for enantioseparation, and isocratic elution was achieved using a mobile phase consisting of CO2-methanol (81:19, v/v). The detection of pantoprazole enantiomers and the internal standard phenacetin was carried out using a Xevo TQD triple quadrupole mass spectrometer in selected reaction monitoring mode with positive electrospray ionization. The developed method successfully achieved full separation of S- and R-pantoprazole enantiomers. Calibration curves were linear in the concentration range of 10–5000 ng/mL for both S- and R-pantoprazole. The method met the acceptance criteria for carry-over, accuracy, precision, extraction recovery, matrix effect, stability, and dilution integrity, demonstrating its stereoselectivity, sensitivity, accuracy, precision, and reliability. The validated method was then applied to investigate the toxicokinetics of racemic pantoprazole and evaluate the potential of chiral inversion between S- and R-pantoprazole in rats. Following oral administration of 200 mg/kg racemic pantoprazole once daily for 90 consecutive days, there was no significant difference in toxicokinetic parameters between the two enantiomers on both day 1 (single-dose experiment) and day 90 (multiple-dose experiment). This suggests that there is no enantioselectivity in the toxicokinetic behaviors of racemic pantoprazole in rat plasma. Additionally, bidirectional chiral inversion between S- and R-pantoprazole in plasma was observed after single-dose and multiple-dose oral administrations of S-pantoprazole (20, 100, and 200 mg/kg) or R-pantoprazole (200 mg/kg) to rats. Compared to single and low doses, there was no accumulation of each enantiomer in rat plasma following repeated and escalated dosing. Overall, this study provided the first report on the toxicokinetics of pantoprazole in rat plasma and presented the first SFC-MS/MS method for the simultaneous quantification of pantoprazole enantiomers in rat plasma.