ABSTRACT Meclofenoxate is a prohibited substance according to clause (S6. B: specified stimulant) by the World Anti‐Doping Agency (WADA). However, meclofenoxate is least detectable in biological matrix owing to its hydrolysis to its active metabolite 4‐chlorophenoxyacetic acid (4‐CPA) and dimethylaminoethanol (DMAE). The detection of meclofenoxate and its metabolite 4‐CPA is mandatory by the WADA Technical Letter (TL‐01). Thus, a confirmatory method for identifying meclofenoxate and 4‐CPA has been developed using a Waters Acquity BEH UHPLC C18, 1.7 µm (2.1 × 100 mm) column with a total run time of 14 min. The dilute and inject method was used as a sample preparation technique in conjunction with ultra‐high‐performance liquid chromatography–tandem mass spectrometry for analysis. The data were acquired in both positive and negative electrospray ionization. The method was validated for the detection and identification of 4‐CPA, 3‐(4‐chlorophenoxy)‐2‐hydroxypropanoic acid (4‐CPP), and chlorphenesin carbamate, with limits of identification of 50 ng/mL for 4‐CPA and 5 ng/mL for 4‐CPP and chlorphenesin carbamate. There were no interferences observed in the blank urine samples spiked with reference standards and internal standards. The matrix effect for different analytes was found to range from ‐24 to 13. The developed method was found to be precise and repeatable, as all the analytes qualified retention time (RT) and relative RT (RRT) criteria of ±0.1 min for RT & ±1% for RRT. Real urine samples of athletes were analyzed to determine the applicability of the developed method. Additionally, a study has been performed regarding the stability of meclofenoxate in spiked urine samples. The observations provided conclusive evidence that meclofenoxate is stable in acidic urine, whereas converted to 4‐CPA and DMAE in neutral or basic urine. Currently, DMAE is not included in WADA TL‐01, but can be considered altogether with 4‐CPA to conclude meclofenoxate consumption.
Sharma et al. (Sun,) studied this question.