Quantitation of fluphenazine in equine serum following fluphenazine decanoate administration.

Fluphenazine, a potent antipsychotic used to treat schizophrenia in humans, is used in racehorses as a performance-enhancing drug, and for that reason it has been banned by the Association of Racing Commissioners International. A liquid chromatography-tandem mass spectrometry method for detecting and quantitating fluphenazine in equine serum was developed and validated. The method was then employed to quantitate fluphenazine in serum samples collected from three study horses after intramuscular injection of fluphenazine decanoate. Stability testing showed that fluphenazine is stable in unextracted and processed samples as well as samples that have been subjected to up to three freeze-thaw cycles. The limit of detection and lower limit of quantitation of fluphenazine were determined to be 0.05 and 0.1 ng/mL, respectively. Precision was evaluated based on one-way analysis of variance of replicate quality control samples and was determined to be 27.2% at the 0.2 ng/mL level and 18.1% at the 2 ng/mL level. Bias was determined to be 0.55% at the 0.2 ng/mL level and 3.66% at the 2 ng/mL level. In two of three horses, fluphenazine was detected in serum up to 14 days post-administration. The highest detected concentration of fluphenazine in serum was 1.4 ng/mL.

Pyrilamine and O-desmethylpyrilamine detection in equine serum and urine.

Pyrilamine (mepyramine) is an H1-receptor antagonist used in human and veterinary medicine. It has the potential to produce central nervous system effects in horses and therefore may have some impact on an outcome of a horse race. A single oral dose of pyrilamine (300 mg/horse) was given to three animals. Serum samples were collected before drug administration and at 0.25, 0.5, 1, 2, 4, 6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, and 13 days post-administration. Urine samples were collected at 0-1, 1-2, 2-4, 4-6, 24, 48, 72, 96, 120, and 144 h, and 7, 8, 9, 10, 11, 12, 13 days post-administration. Urine and serum samples were initially screened by the pyrilamine enzyme-linked immunosorbent assay (ELISA) kit with subsequent confirmation and quantitation utilizing a newly developed and validated gas chromatography-mass spectrometry (GC-MS) method for pyrilamine and its major metabolite O-desmethylpyrilamine with chlorpromazine as an internal standard. Prior to the basic extraction, urine specimens were hydrolyzed using beta-glucuronidase. The urine extracts as well as the serum samples were then subjected to solid-phase extraction on Bond Elut LRC-PRS columns. Pyrilamine was not found in any of the urine samples but it was present in serum in low concentrations (4-123 ng/mL) up to 6 h after drug administration. The limit of detection and limit of quantitation for the GC-MS method for pyrilamine in serum were 1.5 and 3.1 ng/mL, respectively, and for O-desmethylpyrilamine in urine were 5 and 6.2 ng/mL, respectively. Pyrilamine concentration in serum peaked at 15 min, 30 min, and 1 h in horse #1, #2, and #3, respectively. Urine specimens were screened positive for pyrilamine and its metabolites using ELISA for extended periods of time (4 days in one horse and 9 days in two other animals). Using GC-MS, O-desmethylpyrilamine was detected in urine for 11 days in horse #1, 4 days in horse #2, and 9 days in horse #3. While pyrilamine was eliminated from the bloodstream rather quickly, the metabolite level remained in the urine for days after administration. When evaluating laboratory results, regulators must take into account that a urine sample positive for O-desmethylpyrilamine does not necessarily indicate that the drug remains active in the horse's system, possibly affecting the outcome from the race.