Systematic Analysis of Diuretic Doping Agents by HPLC Screening and GC/MS Confirmation

Abstract
The simultaneous analysis of diuretic agents by reversed-phase liquid chromatography with a diode-array detector (DAD) was performed by using a gradient elution with acetonitrile and phosphate buffer on a Hypersil-ODS column. For the spiked urine the extraction recovery of solid-phase extraction (SPE) using Sep-Pak C18 cartridge was compared with that of liquid-liquid extraction (LLE) with diethyl ether at various pH. The standard calibration curves were linear from 0.20–20.0 µg/mL for all diuretic agents except amiloride, 1.0–20.0 µg/mL, and the detection limit was about 0.2 µg/mL for 3 mL of urine, except that of amiloride, which was 1.0 µg/mL. The confirmation analysis was performed by gas chromatography/mass spectrometry (GC/MS) following methylation. The characteristic mass fragment ions obtained by electron-impact (EI) ionization (70eV) provided identification of each diuretic agent.

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