Detection and Identification of New Chlorphenesin Carbamate Metabolites in Human Urine and Metabolomic Characterization Using Liquid Chromatography-Q Exactive-HF-Orbitrap-Mass Spectrometry

Chlorphenesin carbamate is a skeletal muscle relaxant, and one of its metabolites is 4-chlorophenoxyacetic acid (4-CPA), which is included on the Prohibited List of the World Anti-Doping Agency (WADA). The non-prohibited substance, chlorphenesin carbamate, as a potential source of 4-CPA, needs to be identified more strongly in doping control protocols. In this paper, the metabolism of chlorphenesin carbamate in human urine was studied. Ten volunteers were recruited to take chlorphenesin carbamate tablets orally, and urine samples were collected before and after being tested. The urine samples were detected by liquid chromatography-Q Exactive HF orbitrap mass spectrometry (LC-Q Exactive HF-MS) in full MS and full MS-ddMS² scanning modes. Based on accurate molecular formulae determination and fragmentation pattern analysis by mass spectrometry, a total of 29 chlorphenesin carbamate metabolites were found, comprising 18 newly identified metabolites and 11 previously reported metabolites. There were five potential metabolic processes listed: hydroxylation, amide hydrolysis, C-oxidation, O-glucuronidation, sulfation, and their combinations. Chlorphenesin carbamate and the 29 metabolites were compared for their detection windows, and the findings indicated that the two recently reported metabolites, M9 and M10, had longer detection windows than the metabolites documented by the WADA. These metabolites are anticipated to be long-lasting biomarkers for the detection of chlorphenesin carbamate intake. By analyzing the results of metabolomic profiling, it was found that the metabolites with significant changes were mainly related to histidine metabolism and β-alanine metabolism pathways. This paper provides a comprehensive report on the metabolic profile of urinary chlorphenesin carbamate and reveals a point of view the changes in the metabolic in the human body, which is conducive to supporting the detection of chlorphenesin carbamate and meclofenoxate for doping control, as well as a better understanding of the mechanism of action of chlorphenesin carbamate as a drug.