Detection of Diuretics Contamination in Whey Protein-Based Dietary Supplements

Whey protein dietary supplements (WPDS) are widely used by athletes at all levels to improve recovery between training sessions and competitions. The increasing number of reported cases of athletes who consumed these products and subsequently tested positive in antidoping tests has raised concerns that such products may contain undeclared substances. Diuretics are among the most common adulterants found in dietary supplements, and the presence of any amount of these substances in urine or blood samples is classified as doping. This study aimed to develop a reliable method using high-performance liquid chromatography with UV detection (HPLC-UV) to simultaneously detect the presence of the diuretics chlorothiazide, hydrochlorothiazide, furosemide, amiloride, and chlorthalidone in WPDS. A simple, selective, precise, and accurate method was validated according to ANVISA, FDA, and forensic toxicology guidelines using a C8 column, a 100 mM ammonium acetate buffer, and an acetonitrile mobile phase in a gradient system, with UV detection at 276 nm. The method was applied to the analysis of 21 commercial WPDS samples, and 42.9% tested positive for at least one of the investigated diuretics. Furosemide was the most frequently detected diuretic found in 28.8% (0.20-1.82 mg/g of WPDS) of all analyzed samples, followed by hydrochlorothiazide, detected in 19.0% (0.25-1.40 mg/g of WPDS). These results highlight the importance of screening for adulterants in WPDS before consumption to ensure proper quality control, protect consumer safety, and prevent unintentional doping cases.