Doping control analysis of GW1516 in equine plasma using liquid chromatography/electrospray ionization Q‐Exactive high‐resolution mass spectrometry

Abstract

Rationale GW1516 is a peroxisome proliferator‐activated receptor‐δ agonist in the class of hormones and metabolic modulators. The use of GW1516 is banned in both horseracing and equestrian competitions. To the best of our knowledge, this is the first metabolic study of GW1516 in horses. Methods After protein precipitation of pre‐ and post‐administration plasma GW1516 samples, the supernatants were analyzed using liquid chromatography/electrospray ionization Q‐Exactive high‐resolution mass spectrometry to detect GW1516 and its metabolites. Monoisotopic ions of GW1516 and its metabolites were monitored from the full‐scan mass spectral data of pre‐ and post‐administration samples. Quantification methods were developed and validated to establish the elimination profiles of GW1516, its sulfoxide, and its sulfone in equine plasma. Results GW1516 and its four metabolites GW1516 sulfoxide, GW1516 sulfone, 5‐(hydroxymethyl)‐4‐methyl‐2‐(4‐trifluoromethylphenyl)thiazole (HMTT), and M1 were detected in post‐administration plasma samples. GW1516 sulfoxide, GW1516 sulfone, and HMTT were identified by comparison with their respective reference standards whereas M1 was tentatively identified as 4‐methyl‐2‐[4‐(trifluoromethyl)phenyl]‐1,3‐thiazole‐5‐carboxylic acid by mass spectral interpretation. GW1516 had the longest detection time in post‐administration plasma. The elimination profiles of GW1516, its sulfoxide, and its sulfone in plasma were established. Conclusions For the purpose of doping control, GW1516 is recommended as the target analyte to be monitored in equine plasma due to its long detection time (around 1 week) and the ready availability of its reference material.

Metabolic study of GW1516 in equine urine using liquid chromatography/electrospray ionization Q‐Exactive high‐resolution mass spectrometry for doping control

“…All experimental procedures were approved by the Animal Welfare and Ethics Committee of the Equine Research Institute of the Japan Racing Association (authorization numbers 19–17). Detailed procedures were described in a previous report 12 . Urine samples were collected at 0, 1.7, 3, 6, 9, 24, 30, 48, 72, and 96 h and 1, 2, 4, 6, 8, 13, and 26 weeks following administration and all were stored at −80°C prior to analysis.…”

“…A Nexera series liquid chromatograph, consisting of binary pumps, an inline degasser, an autosampler, and a column oven (Shimadzu, Kyoto, Japan), was linked to a quadrupole Orbitrap high‐resolution mass spectrometer (Q‐Exactive HF; Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated ion spray interface and operated in both positive and negative electrospray ionization (ESI) mode. The instrumental conditions were the same as stated in our previous report 12 …”

“…The Nexera series liquid chromatograph was linked to a linear ion trap quadrupole mass spectrometer equipped with a turbo ion spray interface (QTRAP 4500; SCIEX, Framingham, MA, USA). The conditions for the quantification method were the same as those for the plasma analysis reported earlier 12 except for selected reaction monitoring (SRM) transitions of MMTC and MTTC. The SRM transitions for MMTC and MTTC in positive ion mode were m/z 302.1 → 171.9 and m/z 287.9 → 243.9, respectively; the declustering potentials were 56 V and 111 V; the collision energies (CEs) were 49 eV and 37 eV; and the collision cell exit potentials were 12 V and 10 V. Other conditions included the following: (i) analytes were separated with an ACQUITY UPLC BEH C 18 column (2.1 mm i.d.…”

“…The SRM transitions for MMTC and MTTC in positive ion mode were m/z 302.1 → 171.9 and m/z 287.9 → 243.9, respectively; the declustering potentials were 56 V and 111 V; the collision energies (CEs) were 49 eV and 37 eV; and the collision cell exit potentials were 12 V and 10 V. Other conditions included the following: (i) analytes were separated with an ACQUITY UPLC BEH C 18 column (2.1 mm i.d. × 100 mm, 1.7 μm; Waters, Milford, MA, USA) by gradient elution with mobile phases consisting of 0.1% v/v formic acid in water and acetonitrile 12 ; and (ii) 10 mmol/L glycerin in acetonitrile was added to the eluent as a post‐column modifier (PCM) to achieve better sensitivity in negative ion mode 12–14 …”

“…For equines, only detection of the intact parent drug in hair was reported by Gray et al in 2018, 11 and little was known about the metabolism of GW1516 in horses. To the best of our knowledge, we were the first group of authors to report a comprehensive analysis of GW1516 and its metabolites in equine plasma samples collected after a single nasoesophageal administration of GW1516 to a thoroughbred female horse (mare) 12 …”