Simultaneous determination of urinary free cortisol and 6#beta#-hydroxycortisol by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry and its application for estimating hepatic CYP3A induction

摘要

A reversed-phase high-perforrnance 1iquid chromatography coupled to atmospheric pressure chemical ionization tandem lasS spectrometry,(HPLC-APCI-MS-MS) assay was developed to simultaneously determine monkey urinary free cortisol :) and 6~-hydr(}xycortisol (6~-OHC) in 8 min. Urine sample (0.5 mI) containing fludrocortisone acetate (P-C) as the Itemal standard was extracted with ethyl acetate for 5 min with an extraction efficiency of 90% and 75% for C and ~-OHC, respectively. A Perkin-Elmer Sciex API 3000 triple quadruple instrument was used for mass spectrometric etection and the column eluent was directed to a heated nebulizer probe. The assay was linear over the range 0.25-10 IJ.M )r each analyte. The intra- and inter-day relative standard deviation (RSD) over the entire concentration range for both Ilalytes was less than 10%. Accuracy determined at three concentrations (0.8, 2.0 and 8.0 IJM) ranged between 95.5 and [)8%. The method described herein is suitable for the rapid and efficient measurement of 6~-OHC/C ratio in Rhesus lonkey urine following administration of known hepatic CYP3A inducers and can be used to estimate potential CYP3A Iduction by drug candidates in the process of early drug development.