Multiplexed analysis of steroid hormones in saliva by LC-MS/MS with 2-hydrazinopyridine derivatization

LC-MS/MS methods for multiplexed analysis of steroid hormones in saliva are useful research tools in endocrinology, but require sensitive detection.

To explore the use of hydrazide and hydrazine derivatization to improve sensitivity of detection for ketosteroids. On development and validation of a sample preparation method based on robot pipetting in the 96-well format we intended to then establish normal reference ranges for women and men, with saliva collected both in the early morning and late at night.

Four hydrazides and one hydrazine were evaluated for their effectiveness as derivatization reagents based on the comparative signal response and efficacy of LC separation of five ketosteroid hydrazones via a methanol gradient mixed with either 0.2% formic acid or 0.1% ammonium hydroxide. Processing of saliva via liquid-liquid extraction (LLE) was optimized by examining variations in both extraction solvent polarity and protein precipitation reagents intended to inhibit emulsion.

LLE using 10 % butanol in methyl tert-butyl ether (MTBE), with tannic acid as emulsion inhibitor, followed by 2-hydrazinopyridine (2-HP) derivatization enabled the multiplexed measurement of cortisol, cortisone, testosterone, dehydroepiandrosterone (DHEA), progesterone, and 17-alpha-hydroxyprogesterone (17-OHP) in the majority of saliva samples from women and men for this study.

Tannic acid is a novel and effective protein precipitation reagent in bioanalytical sample preparation. The validated method was used in the establishment of reference ranges, the success of which indicated that the method is suitable for Cushing's screening and has potential as a novel analytical research tool.