Development and validation of a LC-MS/MS method for simultaneous determination of six glucocorticoids and its application to a pharmacokinetic study in nude mice.

ABSTRACT

In this study, a simple, rapid and sensitive LC-MS/MS analytical method for simultaneous determination of six glucocorticoids including 21-hydroxy deflazacort (21-OH DFZ), prednisolone (PNL), betamethasone (BET), beclomethasone (BEC), triamcinolone acetonide (TCA), budesonide (BUD) in nude mice plasma was developed and validated. Using testosterone as internal standard, the plasma samples were prepared by precipitation with acetonitrile and separated using an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) with a mobile phase composed of acetonitrile and 0.1 % (v/v) formic acid aqueous solution by gradient elution at a flow rate of 0.3 mL/min. Quantitation was performed on a triple quadruple tandem mass spectrometer by multiple reaction monitoring in positive electrospray ionization mode. Calibration curves were developed over the range of 1-400 ng/mL for TCA, 5-2000 ng/mL for 21-OH DFZ, BET, BEC as well as BUD, and 10-2000 ng/mL for PNL. The accuracy, precision, matrix effect, recovery and stability were validated to be within acceptable criteria. The method was successfully applied to a preclinical pharmacokinetic (PK) study of the six GCs on female nude mice after a single oral dose of 1 mg/kg. The PK profiles of all the six GCs were described by two-compartment model with first-order absorption rate.