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The combination of sulfadoxine (SDO) with trimethoprim (TMP) is widely used in veterinarian medicine. The aim of the present study was to compare excretion profiles and detection time windows of SDO and TMP in plasma and urine by means of a validated quantitative method. Eight horses received a single intravenous (i.v.) dose of 2.7 mg TMP and 13.4 mg SDO per kg bodyweight. Plasma and urine samples were collected up to 15 and 70 days post-administration, respectively. While urine samples underwent an enzymatic hydrolysis, plasma samples were proteolysed before further analysis. After solid-phase extraction, samples were analysed by liquid chromatography/electrospray ionisation tandem mass spectrometry in positive ionisation mode. The applied multiple reaction monitoring (MRM) method allowed the detection of SDO and TMP with a lower limit of detection of 0.03 ng/mL in plasma and 0.2 (SDO) and 0.4 ng/mL (TMP) in urine, respectively. In the present study, detection times for SDO were 15 days in plasma and 49 days in urine, respectively. TMP was detected for up to 7 days in plasma and up to 50 days in urine, respectively. The detection via the TMP metabolite 3-desmethyl-trimethoprim was possible for 70 days in urine. Detection times of the other confirmed metabolites N 4 -acetylated sulfadoxine, hydroxytrimethoprim, trimethoprim-1-oxide and trimethoprim-3-oxide were significantly lower. In order to postulate reasonable screening limits (SLs) to control specific withdrawal times, a Monte Carlo simulation was performed for SDO. The proposed SL of 10 ng/mL SDO in blood and 300 ng/mL urine corresponds to a detection time of 4 days., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Methylxanthines positives in competition samples have challenged doping control laboratories and racing jurisdictions since methylxanthines are naturally occurring prohibited substances and often constituents of feed. For theobromine, an international threshold (renamed in International Residue Limit, IRL) of 2 µg/mL in urine has been established. On the basis of the data presented herein, a threshold or rather an IRL for theobromine in plasma of 0.3 µg/mL was proposed and was thereupon approved by the International Federation of Horseracing Authorities (IFHA). Official recommendations for reporting caffeine and theophylline are still lacking. The aim of the study was to investigate IRLs for theobromine in blood and for caffeine and theophylline in blood and urine. Therefore, a set of six administrations were carried out including both single i.v. and single oral administrations of caffeine, theobromine and theophylline. Plasma and urine concentrations were determined using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). Applying the Toutain model approach an effective plasma concentration (EPC) of caffeine was estimated at 3.05 µg/mL, irrelevant concentrations in blood (IPC) and urine (IUC) approached 6 and 12 ng/mL, respectively. EPC of theobromine was calculated with 3.80 µg/mL, and irrelevant concentrations of theobromine were determined at 8 ng/mL in plasma and at 142 ng/mL in urine. Toutain modelling of the theophylline data produced an EPC, IPC, and IUC of 3.20 µg/mL, 6 ng/mL, and 75 ng/mL, respectively. The obtained irrelevant concentrations were used to postulate IRLs for theobromine in plasma and for caffeine and theophylline in plasma and urine. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)