Development and application of a UHPLC-MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum.

The researchers have developed a simple and efficient method to identify and measure the levels of 17 steroid hormones in horse blood serum. They used a technique known as ultra-high-performance liquid chromatography-tandem mass spectrometry.

Technique Utilized

• The study was based on a technique named Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS).
• This technique is a superior method used for detecting and quantifying multiple compounds in a sample.
• The researchers used this technique to simultaneously determine the presence and the amounts of 17 different steroid hormones in the blood serum of horses.

Methodology

• The researchers began by preparing the serum samples for analysis.
• 400 µl of each sample was deproteinized with 1000 µl of acetonitrile, then evaporated and restored with a 50 µl solution of 25% methanol before undergoing the UHPLC-MS/MS procedure.

Quantitative Analysis and Recovery Percentage

• The accuracy of this method was confirmed by the high recovery percentage, which was over 85.60% in all cases.
• The coefficient of variation, another measure of accuracy, was lower than 8.37%.
• The correlation coefficients of the calibration curves for the analyzed compounds ranged between 0.9922 and 0.9986, which indicates a strong linear relationship between the measured and the actual concentrations.

Method Sensitivity

• The detection and quantification limits for the hormones were between 0.002-2 and 0.0055-5.5 ng ml, respectively.
• This makes it possible to detect even small concentrations of the hormones in the serum.

Application of the Technique

• The researchers applied this newly developed method on different horse serum samples and found it can efficiently detect and quantify the steroid hormone levels.
• The main hormones detected were corticosterone, cortisol, 17-OH-pregnenolone, dihydrotestosterone, and pregnenolone.
• The method showed higher sensitivity in testosterone detection compared to the limit admitted in geldings’ plasma.

The findings of this study could have significant implications for monitoring the health and hormonal balance of horses, as well as detecting any hormonal doping in equestrian sports.