Simultaneous Determination of Cortisol and 6β-Hydroxycortisol in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

The feasibility of taking the ratio of 6β-hydroxycortisol (6β-OHCOR) to cortisol (COR) in plasma as a biomarker to reflect CYP3A4 activity needs to be verified, but the low concentration of 6β-OHCOR which is an endogenous substance in plasma presents a challenge for determination. In this study, a Liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was established to simultaneously quantify the COR and 6β-OHCOR in plasma with COR-d4 and 6β-OHCOR-d4 as internal standards (ISs). Plasma samples were treated by protein precipitation using acetonitrile. Separation with a gradient elution within 5 min was achieved on C18+ column utilizing 5 mM ammonium formate and methanol. An API 4,000 MS in multiple reaction monitoring mode with transitions of 407.1 → 361.1 and 423.1 → 347.1 was utilized. Albumin solution was used as a surrogate matrix, with good linearities over the concentration of 1.20-300 ng/mL for COR and 0.0400-10.0 ng/mL for 6β-OHCOR. The precisions for intrarun and interrun were < 6.8%, and the accuracy was fell in the interval of -5.2 to 3.5%. Matrix effect was not found. Recovery was close to 100.0%. Stability was confirmed under the storage and processing conditions. The validated method was applied to evaluate the inhibitory effect of voriconazole to CYP3A by the ratio of 6β-OHCOR to COR.