Quantitation of Diclofenac, Tolbutamide, and Warfarin as Typical CYP2C9 Substrates in Rat Plasma by UPLC-MS/MS and Its Application to Evaluate Linderane-Mediated Herb-Drug Interactions

Abstract

Linderane (LDR), the main active and distinctive component of L. aggregate, is a mechanism-based inactivator of CYP2C9 in vitro, indicating the occurrence of herb-drug interactions. However, little is known about the changes of the pharmacokinetic properties of the common clinical drugs as CYP2C9 substrates after coadministration with LDR. In this study, a selective and rapid ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of diclofenac, tolbutamide, and warfarin as CYP2C9 substrates in rat plasma has been developed. Chlorzoxazone was employed as an internal standard (IS), and protein precipitation was used for sample preparation. Chromatographic separation was achieved on a UPLC BEH-C18 (2.1 × 50 mm, 1.7 µm) with 0.1% (v:v) formic acid in water (A) and acetonitrile (B) as the mobile phase with gradient elution. The total run time was only 3.8 min. MS analysis was performed under multiple reaction monitoring (MRM) with electron spray ionization (ESI) operated in the negative mode. The bioanalytical method was validated, and the selectivity, carryover effects, linearity, precision, accuracy, matrix effect, extraction recovery, and stability were acceptable. The validated method was then successfully applied for evaluating the potential pharmacokinetic interactions when LDR was used along with diclofenac, tolbutamide, and warfarin, respectively. Results showed that the Cmax of diclofenac in the treated group was 1287.82 ± 454.16 μg/L, which was about 5-fold of that in the control group $\left(P<0.01\right)$ . The Cmax of tolbutamide in the treated group was 60.70 ± 10.70 mg/L, which was significantly decreased by about 25% when compared with the control group $\left(P<0.01\right)$ . The Vd of warfarin in the treated group was obviously increased, which was about 1.4-fold of that in the control group $\left(P<0.01\right)$ .