Simultaneous determination of hair cortisol, cortisone and DHEAS with liquid chromatography–electrospray ionization-tandem mass spectrometry in negative mode

• LC–MS/MS method with good performance was developed for analysis of 3 steroids in hair.
• Hair cortisol, cortisone and dehydroepiandrosterone sulfate were simultaneously detected.
• Matrix effect was diminished in term of reducing volume of incubation solution.
• Determination was performed in ESI negative mode for hair samples from 103 participants.

The present study aimed to develop a novel method for simultaneous assay of cortisol, cortisone and dehydroepiandrosterone sulfate (DHEAS) in human hair. The method was based on liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–MS/MS) in negative mode. Analytes were extracted from 20-mg hair incubated in 1 ml of methanol for 5 days. 100 μl non-SPE supernatant of the incubation solution was utilized in LC–MS/MS analysis. The liquid chromatography separation was performed on a reversed-phase C18 column with a mobile phase of 80:20 (v:v) methanol and deionized water containing 0.1% formic acid. The use of ESI in negative mode and the use of a small volume of the incubation solution significantly improved assay sensitivity. The linear range was 5–250 pg/mg for cortisol and cortisone, and 5–500 pg/mg for DHEAS. The limit of detection was 2 pg/mg for the three analytes in hair. The coefficients of variation for intra-day and inter-day assay were less than 10%. The method was applied to determine the three analytes mentioned above of hair samples from 103 participants. The results indicated that there was no significant effect of age and education level on the hair cortisol, cortisone and DHEAS levels. The simple treatment procedure developed in the present study may facilitate simultaneous measurement of more steroids in hair.