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- H ]hydroxycortisol and [1,2,4,19- C ]cortisol as internal standards for in vivo CYP3A phenotyping in humans. The lower limits of quantification were 38.513 pg/mL for 6β-OHF and 38.100 pg/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 21 days 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.
Dried blood spot (DBS) sampling is a minimally invasive method used to collect blood samples of any population for personalized medicine. We aimed to develop a sensitive and reliable analytical method for measuring 6β-hydroxycortisol (6β-OHF) and cortisol concentrations in DBS by liquid chromatography/tandem mass spectrometry so as to utilize DBS as a less invasive blood sampling method for calculating the ratio of 6β-OHF/cortisol. The lower limits of quantification obtained using four DBS were 1.08 pg/50 μl for 6β-OHF and 1.01 pg/50 μl for cortisol. The 6β-OHF and cortisol in DBS were stable for 28 days at room temperature. The intraday and interday accuracy and precision of the method was <12%. Additionally, the 6β-OHF and cortisol in DBS were measured before, during, and after 3 days of clarithromycin administration to two of the subjects. Then, their concentration was compared in the plasma and whole blood collected simultaneously. The concentrations of 6β-OHF and cortisol in four DBS ranged from 0.007 to 0.079 ng/50 μl and from 1.15 to 6.66 ng/50 μl, respectively. The 6β-OHF/cortisol ratio in DBS decreased by approximately 50% on administering clarithromycin compared with that before the administration of clarithromycin. The 6β-OHF/cortisol ratio in DBS also showed a strong correlation with that in whole blood (r = 0.9694) and plasma (r = 0.9383). This method provides high accuracy and precision for measuring 6β-OHF and cortisol in DBS. It also allows the use of DBS instead of plasma for calculating the 6β-OHF/cortisol ratio. The 6β-OHF/cortisol ratio could be an index of CYP3A activity in clinical setting.
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