Determination of Cortisol in Human Serum Using Ultrasonic Extraction Coupled with Liquid Chromatography-Isotope Dilution Mass Spectrometry

Abstract

A liquid chromatography-tandem mass spectrometric(LC-MS/MS) method was developed for the determination of cortisol in human serum.Serum was spiked with corisol-d4 as internal standard and then equilibrated for 30 min.Acetonitrile was employed for deproteinization and the mixture was extracted by ultrasonication for 10 min followed by centrifugation at 3000 r/min for 15 min.The supernatant fluids were evaporated to dryness with nitrogen flow and redissolved in water and subsequently extracted with ethyl acetate for twice.The extracts were evaporated with nitrogen flow and redissolved in 200 μL mobile phase.Chromatographic separation was performed on Zorbax Eclipse Plus C18 column with the mobile phase consisted of methanol and water containing 0.1% formic acid(43 ∶ 57,V/V).Good precision was achieved with inter-day relative standard deviations(RSDs) of 0.3%-0.7% and intra-day CVs of 0.3%-0.6%.The recoveries of spiked samples ranged from 99.6% to 101.0%,limit of quantitation was 1.68 μg/kg.This method was applied to the determination of three kinds of human serum cortisol and the stability of cortisol in serum was also studied.