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

Tretoquinol (trimetoquinol), a ß2-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.

Plasma and urinary concentrations of trimetoquinol by LC-MS-MS following intravenous and intra-tracheal administration to horses with heaves.

Trimetoquinol (TMQ) is a very potent and fast acting bronchodilator in horses with heaves. This study assessed the plasma and urinary concentrations of TMQ in horses with heaves following administration via the intravenous (IV, 0.2 microg/kg) and intra-tracheal (IT, 2 microg/kg) routes. TMQ was administered to six horses affected with heaves (RAO - Recurrent Airway Obstruction, used interchangeably) by the above routes and plasma and urine samples collected and stored at -20 degrees C until analyzed. Solid Phase Extraction (SPE) of TMQ was followed by highly sensitive ESI(+)-LC-MS-MS (ElectroSpray Ionization, positive mode - Liquid Chromatography - Mass Spectrometry - Mass Spectrometry); with a Limit of Detection (LOD) estimated at 1 pg/mL. Following IV administration, TMQ plasma levels peaked at 1 min at 707 pg/mL, and at 9 min at 306 pg/mL following IT administration. Our results show that TMQ plasma concentrations decline rapidly following IV administration, which is consistent with the fast onset and short duration of TMQ effect that was observed in our previous studies. On the other hand, IT administration showed a very unique plasma concentration pattern. From a regulatory standpoint, the current available TMQ ELISA kit was also used in an attempt to detect TMQ from the plasma and urine samples. We report that the ELISA kit was unable to detect TMQ from any of the samples generated in these studies.