The development and validation of a turbulent flow chromatography–tandem mass spectrometry method for the endogenous steroid profiling of equine serum

A method for the detection and quantitation of 35 endogenous steroids in equine serum was developed and validated. Androgens, estrogens, progestins and their metabolites potentially present in serum were simultaneously monitored in one method using on-line sample extraction by turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system and detected on a triple-stage quadrupole mass spectrometer by electrospray ionization. Analytes were detected and quantitated by single-reaction monitoring or selected-ion monitoring. Limits of detection (range 0.025–10 ng mL−1) and quantitation (range 0.125–25 ng mL−1) along with recovery and matrix effects were determined for each analyte. Inter- and intra-day accuracy and precision was assessed for with the majority of analytes having %CV less than 20% and accuracy within 20% of the expected concentrations. Eight of the 35 analytes were unable to meet these guidelines across all of the quality control concentrations monitored for each analyte. This method was used to determine the endogenous steroid profiles of Thoroughbred horses and has been modified for use in non-human primates and cell culture.

► Validated method for analysis of 35 endogenous steroids in equine serum.
► Androgens, estrogens, and progestins are monitored in one method.
► Turbulent flow chromatography allows for minimal sample processing.
► Allows for analysis of multiple steroids in one run from 300 μL serum.