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

Author:

Sun Lu-Ning1234,Shen Ye12,Yang Yu-Qing12,Chen Xiang-Long12,Huang Feng-Ru12,Wang Dun-Jian12,Zhang Ye5,Wang Da-Wei67,Wang Yong-Qing1234ORCID

Affiliation:

1. Research Division of Clinical Pharmacology , , Nanjing 210029 , China

2. First Affiliated Hospital of Nanjing Medical University , , Nanjing 210029 , China

3. School of Pharmacy , , Nanjing 210029 , China

4. Nanjing Medical University , , Nanjing 210029 , China

5. Department of Pharmacy, Nanjing Jinling Hospital , Nanjing 211166 , China

6. Affiliated Hospital of Integrated Traditional Chinese and Western Medicine , , Nanjing 210028 , China

7. Nanjing University of Chinese Medicine , , Nanjing 210028 , China

Abstract

Abstract 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.

Funder

First Affiliated Hospital

Nanjing Medical University

Publisher

Oxford University Press (OUP)

Subject

General Medicine,Analytical Chemistry

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