Simultaneous quantitative analysis of seven steroid hormones in human saliva: A novel method based on O-ethylhydroxylamine hydrochloride as derivatization reagent

Rationale Saliva has been widely accepted as a more convenient alternative to serum or plasma in the field of clinical diagnosis. However, the detection of trace components in saliva has been a bottleneck problem. The aim of this work was to develop a highly sensitive and reliable method for simultaneously determining the trace steroid hormones including some with poor ionization efficiency in human saliva by liquid chromatography/tandem mass spectrometry (LC/MS). Methods Saliva was deproteinated by acetonitrile containing mixed isotope internal standards and extracted with methyl tert-butyl ether. The extraction solution was dried under a stream of nitrogen and the residue was derivatized using 50 mM O-ethylhydroxylamine hydrochloride in 80% methanol/water solution (v/v). The processed sample was determined by LC/MS in multiple reaction monitoring (MRM) mode. Results The method was successfully established for the simultaneous quantification of seven steroid hormones in human saliva and showed excellent specificity and sensitivity. The limits of quantification (LOQs) of all steroid hormones were below 5 pg/mL, in particular, the LOQ of progesterone was as low as 0.15 pg/mL. The linear correlation coefficients (r) were greater than 0.9990 in the range of 2-200 pg/mL for T, DHEA, A4, P4, P5, and 17OHP4 and in the range of 5-500 pg/mL for 17OHP5. The intra-day and inter-day variability ranged from 1.86% to 7.83% and 1.95% to 10.4%, respectively. The recovery of the method ranged from 86.9% to 111.1% for all steroid hormones using three spiked concentrations. Conclusions A novel LC/MS/MS method was developed for the simultaneous quantification of seven kinds of trace steroid hormones in human saliva. The results of the methodological study showed that the method exhibited excellent sensitivity and reliability for the evaluation of free steroid hormones in the human body. It is believed that this method could provide useful information of steroid hormone metabolism for auxiliary diagnosis of some endocrine disorders.