Determination of diuretics in the whey protein supplements: uncovering hidden contaminants in the sports nutrition market

The widespread use of whey protein supplements raises concerns about contamination with banned diuretics, posing health risks and the potential for inadvertent doping. In this study, a liquid-liquid extraction (LLE) procedure coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous detection of 23 diuretics in whey protein supplements. The method was assessed for selectivity, linearity, matrix effect, recovery, precision, and sensitivity. Statistical evaluation using the Shapiro-Wilk and Levene's tests confirmed that most calibration residuals exhibited a normal distribution and homogeneity of variance, ensuring the reliability of the regression model. The method demonstrated excellent linearity (R² = 0.980-0.999) and low method detection limits (MDLs, 0.39-2.73 ng/g). A total of 58 commercially available whey protein supplements were analysed, and bendroflumethiazide (D4) was detected in 12 samples, with concentrations ranging from 1.4 to 40.4 ng/g. These findings highlight the applicability of the validated method for routine monitoring and underscore the urgent need for stringent quality control and regulatory oversight in the sports supplement industry to protect consumer health and maintain fair competition.