UC Davis

Synthetic thyroid hormones, particularly levothyroxine, are a concern in horse racing due to their performance-enhancing effects. Regulating these substances is essential to prevent malpractice and protect horse safety. However, current analytical methodologies struggle to differentiate between natural and synthetic thyroid hormones. This study developed a sensitive method to simultaneously detect thyroid hormones, their acid metabolites, precursors, and a derivative in equine athlete serum using solid phase extraction using a mixed-mode anion exchange sorbent and liquid chromatography mass spectrometry using parallel ion reaction monitoring based detection and positive and negative mode electrospray ionization. Limits of detection and limits of quantitation, measured in pg/mL, were determined for 3,5,3’,5’-tetraiodothyronine (100, 1000), 3,3’,5- triiodothyronine (10, 100), 3,3’,5’-triiodothyronine (10, 100), 3,5-diiodothyronine (2.5, 5), 3,3’-diiodothyronine (1.25, 2.5), 3-iodothyroacetic acid (7.5, 5), 3,3’,5-triiodothyroacetic acid (2.5, 5), 3,3’,5,5’-tetraiodothyroacetic acid (5, 50), monoiodotyrosine (2.5, 5), diiodotyrosine (1, 5), and 3-iodothyronamine (1.25, 5). Accuracy, precision, recovery, stability, and matrix effects were determined as validation parameters and were all acceptable. Using this method, reference ranges were derived for serum from a population of pre-race geldings, mares, and stallions. These reference ranges, measured in pg/mL, for geldings, mares, and stallions were 3,5,3’,5’-tetraiodothyronine (18100 ± 14200, 18200±15600, 14000±12500), 3,3’,5- triiodothyronine (1340 ± 1500, 1180 ± 1230, 1030 ± 1200), 3,3’,5’-triiodothyronine (665 ± 521, 722 ± 653, 631 ± 581), 3,5-diiodothyronine (5.19 ± 8.41, 6.40 ± 7.04, 5.82 ± 6.58), 3,3’-diiodothyronine (66.8 ± 52.6, 66.9 ± 62.1, 62.6 ± 57.8), monoiodotyrosine (41.2 ± 96.5, 50.6 ± 125, 42.4 ± 121), diiodotyrosine (70.4 ± 162, 54.3 ± 127, 56.2 ± 157). 3-iodothyroacetic acid, 3,3’,5-triiodothyroacetic acid, and 3,3’,5,5’-tetraiodothyroacetic acid were below the quantitation range for all sexes and 3-iodothyronamine was not detected.