Detection of Diuretics in Horse Urine by GC/MS

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Abstract
The use of diuretics in horses subject to doping control is prohibited. Thus, a sensitive screening procedure is required to identify the chemically different diuretics. We communicate here a method to detect three commonly employed acidic diuretics: bumetanide, ethacrynic acid, and furosemide. A liquid-liquid extraction on Extrelut 3® was performed at weak acidic and basic conditions using ethyl acetate as organic solvent. For analysis by GC, the diuretics were methylated oncolumn in the presence of MSTFA/TMAH, avoiding the commonly employed highly toxic derivatizing agent methyl iodide. For identification of diuretics, we used a mass selective detector operating in the SIM (selected ion monitoring) mode. Confirmation analysis may be obtained with a full scan run. Recoveries for the individual drugs ranged from 31 to 48% at the 100-ng/mL level for 3 mL urine, using calibration curves of drug standards with linearity from 2.5 to 20 ng injected. The limit of detection amounts to 40 ng/mL for the three diuretics. The method permits rapid and sensitive detection of diuretics in horse urine and is recommended for doping control.

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