Analysis of tretoquinol and its metabolites in human urine by liquid chromatography–tandem mass spectrometry

Tretoquinol (trimetoquinol), a β2‐agonist, has been explicitly listed on the World Anti‐Doping Agency Prohibited List 2019 since January 2019; however, it has been distributed as an antiasthmatic on the medical market. This study aimed to develop a liquid chromatography–tandem mass spectrometric method for the quantification of tretoquinol (free form plus glucuronide) in human urine for doping control purposes. An excretion study ( n = 6) of tretoquinol hydrochloride hydrate (6 mg) was performed, and urine samples were collected prior to oral administration and during the first 48 h, along with spot urine samples at 7 and 14 days after administration. All the urine samples were analysed using the developed method. The limit of detection for the developed method was 0.03 ng/mL. The inter‐day precision for the target analyte was excellent (2.7% to 9.2%), and the inter‐day accuracy of target analyte was −0.6% to −3.6%. In all subjects, tretoquinol (free form plus glucuronide conjugate) was identified up to 48 h after administration. The maximum concentrations were in the range of 12.4–78.8 ng/mL and the mean concentration was 55.3 ng/mL. The metabolites O‐ methylated tretoquinol, tretoquinol sulphate and O‐ methylated tretoquinol sulphate could be also identified in human urine after administration. The longest‐lasting urinary metabolite of tretoquinol currently known, O ‐methylated tretoquinol, is also likely to be a useful marker in doping controls.