Quantitative analysis of steroid hormones in human hair using a column-switching LC–APCI–MS/MS assay

•A method for the simultaneous detection of seven steroid hormones in human hair is presented.•Analyses are run on whole hair instead of pulverized hair to reduce pretreatment time.•An on-line SPE method is used to shorten sample preparation times and to increase throughput.•The method is highly sensitive, selective and fast.•All examined steroid hormones could be reliably detected a physiological concentrations. The analysis of steroid hormones in hair is increasingly used in the field of stress-related research to obtain a retrospective index of integrated long-term hormone secretion. Here, most laboratories have so far relied on immunochemical assays originally developed for salivary analyses. Although these assays are fast and easy to perform, they have a reduced reliability and specificity due to cross-reactivity with other substances and are limited to the detection of one hormone at a time. Here, we report the development of a LC–MS/MS-based method for simultaneous identification of endogenous concentrations of seven steroid hormones (cortisol, cortisone, testosterone, progesterone, corticosterone, dehydroepiandrosterone (DHEA) and androstenedione) in human hair. Hair samples were washed with isopropanol and steroid hormones were extracted from 10mg whole, nonpulverized hair by methanol incubation. A column switching strategy for on-line solid phase extraction (SPE) was applied, followed by analyte detection on an AB Sciex API 5000 QTrap mass spectrometer. Results indicated linearity of the method for all steroids over ranges of 0.09–90pg/mg (0.9–900pg/mg for DHEA) with correlation coefficients ranging between 0.9995 and 0.9999. Intra- and inter-assay coefficients of variation were between 3.7 and 9.1%. The limits of quantification (LOQ) were below (or equal to) 0.1pg/mg for all steroids, except of DHEA for which the LOQ was 0.9pg/mg. An analysis of 30 natural hair samples (15 men/15 women) using this method confirmed that all steroid hormones could be quantified at endogenous levels in each individual. In addition, the use of whole hair samples and on-line SPE resulted in a significant reduction in sample throughput times, increasing the applicability of this method for research questions where a larger number of samples needs to be processed.