Sensitive and simultaneous quantitation of 6β-hydroxycortisol and cortisol in human plasma by LC-MS/MS coupled with stable isotope dilution method.

Abstract

CYP3A phenotyping provides a means for personalized drug therapy. We focused our attention on the plasma 6β-hydroxycortisol (6β-OHF) to cortisol ratio as an index for CYP3A phenotyping. In the present study, we developed a sensitive and reliable method for the simultaneous determination of 6β-OHF and cortisol in human plasma using high-performance liquid chromatography/tandem mass spectrometry together with picolinylester derivatization or nonderivatization methods and 6β-[9,11,12,12-2 H4 ]hydroxycortisol and [1,2,4,19-13 C4 ]cortisol as internal standards for in vivo CYP3A phenotyping in humans. The lower limits of quantification were 38.513pg/mL for 6β-OHF and 38.100pg/mL for cortisol. The relative error and relative standard deviation of the lower limits of quantification were<5% for both methods. The intra-day and inter-day assay reproducibilities of the determined 6β-OHF and cortisol concentrations were consistent with the actual amounts added as relative errors and relative standard deviations for both methods, which were <5.4% and <3.9%, respectively. Both methods were applied for the quantification of plasma 6β-OHF and cortisol concentrations in healthy subjects taking oral contraceptives. The absolute concentrations and time course of 6β-OHF and cortisol were found to be consistent when measured using the 2 methods. The ratio as an index for in vivo CYP3A activity decreased after 21days of taking oral contraceptives for both methods. To the best of our knowledge, this is the first study reporting the detailed investigation of accuracy and precision in the simultaneous measurement of 6β-OHF and cortisol in human plasma using liquid chromatography/tandem mass spectrometry coupled with stable isotope dilution method, which can be applied to CYP3A phenotyping.