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

• Published in: Journal of chromatographic science 2023-02, Vol.61 (2), p.130-139
• Main Authors: Sun, Lu-Ning, Shen, Ye, Yang, Yu-Qing, Chen, Xiang-Long, Huang, Feng-Ru, Wang, Dun-Jian, Zhang, Ye, Wang, Da-Wei, Wang, Yong-Qing
• Format: Article
• Language: English
• Subjects: Chromatography, High Pressure Liquid - methods; Chromatography, Liquid - methods; Cytochrome P-450 CYP3A; Humans; Hydrocortisone; Tandem Mass Spectrometry - methods
• ISSN: 0021-9665; 1945-239X
• DOI: 10.1093/chromsci/bmac038

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