Detection of 4‐CPA and Its Related Metabolites Based on LC‐QE‐HF‐MS Technology: A Study on the Impact of Doping Test on the Intake Pathways of Chlorphenesin Carbamate and Chlorphenesin

This study focused on meclofenoxate and its metabolite 4-CPA in the field of doping control and examined the urinary metabolism of chlorphenesin carbamate and chlorphenesin, two substances that may produce 4-CPA. A liquid chromatography-Q Exactive-HF-Orbitrap-mass spectrometry (LC-QE-HF-MS)-based assay was developed and validated for the accurate detection of the metabolites of 4-CPA, chlorphenesin carbamate, and chlorphenesin and verified by human subject investigations. Human subjects were studied for three different modes of ingestion (chlorphenesin carbamate administration, sunscreen application containing chlorphenesin, and sunscreen spray application), and urine samples were collected before and after the administration to study the excretion profile of metabolites such as 4-CPA. The developed assay meets the routine testing requirements of the World Anti-Doping Agency. Following oral administration of chlorphenesin carbamate, 4-CPA levels in the urine ranged from 390 to 6929 ng·mL-1, reaching their maximum concentrations in 12-24 h, with two volunteers having values over the reporting limit. The highest excretion happened 12-24 h after the treatment and continued until 168-264 h later. The Cmax of 4-CPA was 1354-2063 and 340 ng·mL-1 after application of sunscreen and spray sunscreen spray, respectively. Maximum excretion of other relevant metabolites was also concentrated at 12-24 h post-dose. The results suggest that when measuring 4-CPA, the target analyte of meclofenoxate, in sports drug testing, special consideration needs to be given to whether volunteers have ingested normal therapeutic drugs, such as chlorphenesin carbamate, to avoid misreporting of meclofenoxate results. This finding offers crucial decision support for doping control.