Method Development and Validation for Quantification of Cortisol and Cortisone Using Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Cortisol and its metabolite cortisone have been used as a biological marker of stress in human psychobiological studies. Cortisol and cortisone quantitation may help to examine physiological responses towards stress. A highly sensitive and selective ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantitation of cortisol and cortisone in human serum. These two compounds were chromatographically separated using Agilent ZORBAX Eclipse Plus C 18 Rapid Resolution HD column (2.1 mm 50 mm, 1.8 m) and were eluted with (A) 0.1% formic acid in water and (B) methanol mobile phase. The sample injection volume was 2 L and the flow rate was set at 0.3 mL/min. The analytes were determined in positive ionization mode and quantitated by multiple-reaction monitoring (MRM) mode. The method was linear from 7.8 to 500 ng/mL for cortisol (r 2 = 0.986) and cortisone (r 2 = 0.990). The precision, accuracy, and recovery of the method for cortisol ranged from 1. 98-10.46%, 90.68-91.94%, 90.38-93.88%, and cortisone ranged from 1.01-11.36%, 93.12-94.79% and 90.48-95.90%, respectively. The developed method has met the quality standards of European Medicines Agency (EMA).