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

A reversed-phase high-performance liquid chromatography coupled to atmosphe ric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS-MS) assay was developed to simultaneously determine monkey urinary free cortis ol (C) and 6 beta-hydroxycortisol (6 beta-OHC) in 8 min. Urine sample (0.5 mi) containing fludrocortisone acetate (F-C) as the internal standard was e xtracted with ethyl acetate for 5 min with an extraction efficiency of 90% and 75% for C and 6 beta-OHC, respectively. A Perkin-Elmer Sciex API 3000 t riple quadruple instrument was used for mass spectrometric detection and th e column eluent was directed to a heated nebulizer probe. The assay was lin ear over the range 0.25-10 mu M for each analyte. The intra- and inter-day relative standard deviation (RSD) over the entire concentration range for b oth analytes was less than 10%. Accuracy determined at three concentrations (0.8, 2.0 and 8.0 mu M) ranged between 95.5 and 108%. The method described herein is suitable for the rapid and efficient measurement of 6 beta-OHC/C ratio in Rhesus monkey urine following administration of known hepatic CYP 3A inducers and can be used to estimate potential CYP3A induction by drug c andidates in the process of early drug development. (C) 2000 Published by E lsevier Science B.V.