# Detection of Diuretics Contamination in Whey Protein-Based Dietary Supplements

## TL;DR

This study shows that many whey protein supplements contain hidden diuretics, which can lead to doping violations in athletes.

## Contribution

A new HPLC-UV method was developed to detect multiple diuretics in whey protein supplements.

## Key findings

- 42.9% of tested whey protein supplements contained at least one diuretic.
- Furosemide was the most commonly found diuretic in 28.8% of samples.
- The method was validated according to regulatory and forensic guidelines.

## Abstract

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.