Simultaneous analysis of twenty-one glucocorticoids in equine plasma by liquid chromatography/tandem mass spectrometry.

The research is about a new method to simultaneously detect and measure the levels of 21 different glucocorticoids (a type of steroid hormone) in horse blood plasma, using a technique called liquid chromatography coupled with tandem mass spectrometry. The method was developed to test racehorse samples and has shown to be reliable, sensitive, and selective.

Methodology

• In this study, the researchers used liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) to separate, identify and quantify 21 glucocorticoids (GCC) in equine plasma.
• A sample of horse plasma was mixed with the 21 GCC and was extracted with methyl tert-butyl ether (MTBE) for analysis.
• The method used positive electrospray ionization where desoxymetasone or dichlorisone acetate were used as the internal standards. The quantification was carried out by internal standard calibration.

Results

• The research found that recovery of the 21 GCC when added into a blank plasma was between 45-200% with a coefficient of variation (CV) from 0.3-18%.
• The limits of detection (LOD) and quantification (LOQ) for most of the analytes were 50-100 pg/mL and 1 ng/mL, respectively. The limit of confirmation (LOC) was between 100-300 pg/mL, depending on the analyte.
• The method produced reasonably precise results with intra- and inter-day precisions, expressed as CV for quantification of 1 and 10 ng/mL, between 1.0-17%, and 0.51-19%, respectively.
• The accuracy of the test was between 84-110% and the linear concentration range for quantification was 0.1-100 ng/mL (r(2) > 0.997).
• The measurement uncertainty was estimated at 11-37%.

Applications

• The method was developed to allow simultaneous screening, identification, quantification and confirmation of steroids in post-race equine plasma samples.
• The technique was successfully applied to a large number of plasma samples from racehorses in competitions and in pharmacokinetic studies of dexamethasone, a type of steroid medication.

Conclusions

• The developed method of GCC detection and quantification in equine plasma using LC/TSQ-MS/MS proved to be simple, sensitive, selective and reliably reproducible.
• It could be effectively used for post-race screening, offering a comprehensive analysis of the presence and quantity of various glucocorticoids.