Rapid and Trace-Level Detection of Diuretics in Equine Plasma via Non-Polar Carbon-Based Solid Phase Extraction and Ultra-High-Performance Liquid Chromatography Orbitrap Mass Spectrometry

Diuretics such as bendroflumethiazide, bumetanide, and chlorthalidone are prohibited substances in the horse racing industry due to their potential to mask the presence of banned drugs, induce weight loss, compromise fairness, and pose serious health risks to horses.This study presents the development of a method with a l screening detection limit (SDL) of 1 ng/mL and a total run time under 4.6 min for the simultaneous detection of eight diuretics in equine plasma using ultra-high-performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UHPLC-Q Exactive MS).Solid-phase extraction (SPE) using a NEXUS cartridge containing non-polar carbon resin was employed for sample preparation.Chromatographic separation was achieved on a Luna Omega HyperSil-High Strength (HSS) C18 column, with an elution gradient consisting of 5 mM ammonium formate (pH 3.0) in distilled water and 0.1% formic acid in acetonitrile at a flow rate of 0.4 mL/min.The method ensured specificity and accuracy confirmed by baseline-resolved peaks and replicate consistency (n = 10).with recoveries ranging from 9.3 2.5% (torsemide) to 62.2 8.3% (triamterene) and SDL of 0.1 ng/mL for triamterene and 1 ng/mL for the other analytes.At a concentration of 1 ng/mL, all eight test samples were detected.This method was applied to pre-race testing, highlighting its practical application as a preventive measure for detecting drug abuse and ensuring fair competition in horse racing.The developed UHPLC-Q Exactive MS method offers a valuable tool for doping control in equine sports and may be implemented in future drug testing protocols for both racing and veterinary applications.