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

The researchers developed and validated a method to analyze a range of naturally occurring steroids in horse blood serum, including androgens, estrogens, and progestins, and their metabolites. This technique used turbulent flow chromatography and mass spectrometry. They assessed its accuracy and precision, and found it appropriate for use with majority of the monitored steroids.

Method Development

• The research team prepared a method to simultaneously detect and measure 35 naturally occurring steroids in horse blood serum, including androgens, estrogens, and progestins, and their metabolites.
• This procedure used a technique called turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system. TFC is a form of rapid online extraction method used to separate analytes (the components of interest in a sample) from bulk matrix.
• The separated steroids were then identified using a device called a triple-stage quadrupole mass spectrometer, by applying an electrically charged mist (or “electrospray ionization”).

Method Validation

• The researchers validated their method by determining for each steroid, the smallest amount that can be detected (limit of detection), the smallest amount that can be accurately measured (limit of quantitation), as well as the recovery and matrix-effects, which refer to the efficiency of extracting the steroids and the potential influences of other compounds in the serum, respectively.
• They also tested the technique for its consistency (precision), and the closeness of the measurements to the actual values (accuracy).
• The majority of the steroids met the predefined criteria of having a coefficient of variation (a measure of relative variability) of less than 20% and an accuracy within 20% of the expected concentrations. However, 8 out of the 35 steroids monitored could not meet these guidelines.

Applications

• This method was employed to establish the naturally occurring steroid profile of Thoroughbred horses.
• It has also been adapted for use with non-human primates and cell cultures, demonstrating its versatility in a variety of biological contexts.