A Stability Study of Buthiazide, Mebutizide, and Bemethizide in Urinary and Aqueous Matrices

Rationale Diuretics are banned in sports in‐ and out‐of‐competition without applicable threshold or reporting levels, and are classified as S5 Diuretics & Masking Agents by the World Anti‐Doping Agency. In 2024, two cases reported by the Beijing Anti‐Doping Laboratory involved the simultaneous detection of buthiazide and its degradation product, 4‐amino‐6‐chloro‐1,3‐benzenedisulphonamide (ACB), in athlete urine samples. Subsequent investigations revealed that certain weight‐loss medications contained undeclared buthiazide. While instability of thiazide diuretics such as hydrochlorothiazide and chlorothiazide in urine has been previously reported, the degradation behavior of structurally related compounds, buthiazide, mebutizide, and bemethizide, remains largely unexplored. Methods This study systematically evaluated the short‐term stability of these three thiazide diuretics in human and artificial urine under various pH conditions (2.6, 5.0, 7.0, and 8.8), temperatures (−20°C, 25°C, 40°C, and 60°C), and light exposures (daylight and UV light). The degradation rate was evaluated using a single‐point quantitative method by testing samples at predetermined time points in artificial urine (pH 5.0 and 7.0) at concentrations of 200, 1000, and 2000 ng/mL, and the results were subjected to statistical analysis. Long‐term stability was evaluated after 1 week, 1 month, 3 months, and 6 months of storage at −20°C. Results Three thiazide diuretics and ACB were detected across all tested conditions, with higher degradation rates observed at higher pH values and temperatures. Single‐point quantitative analysis corroborated that degradation was most rapid at 60°C, with mebutizide and bemethizide degrading faster than buthiazide. Long‐term storage tests showed that buthiazide remained stable for 1 month at −20°C but partially degraded by 3 months, whereas mebutizide and bemethizide remained stable only for 1 week at low pH. Additionally, repeated freeze–thaw cycles promoted degradation in some samples. Conclusions These findings underscore the necessity of carefully controlled storage conditions in routine anti‐doping analyses, especially when confirmatory testing of B samples is delayed.