Confirmation and quantification of clenbuterol in horse urine using liquid chromatography tandem mass spectrometry triple quadrupole.

Clenbuterol (CLE) is used in horses as a bronchodilator and for its anabolic steroid-like effects. CLE is a Class 3 drug according to current Association of Racing Commissioners International (ARCI) Uniform Classification Guidelines. The Racing Medication and Testing Consortium recommended a urine CLE threshold of 140 pg/mL after careful scientific review of the results of studies describing the disposition of CLE in the horse and this threshold was adopted by the ARCI. Enzyme-linked immunosorbent assay was previously used to screen samples for CLE in Illinois, but could not detect such low concentrations in urine. Thus, a liquid-liquid extraction of CLE from urine followed by quantification by liquid chromatography-tandem mass spectrometry was developed and validated. Method validation included testing stability, ion suppression and enhancement, precision, accuracy and uncertainty. Intra-, interday and total precision and accuracy were calculated for each control and found to be within the ±15% acceptance range. The Guide to the Expression of Uncertainty in Measurement approach was used to calculate uncertainty, which was 11% at the 95% confidence level. In the past 5 years, only 15 samples were reported as positive for CLE in Illinois. This new method was used in a pilot program to screen and confirm samples received from thoroughbred and harness horses.

Equine total carbon dioxide testing in Illinois in 2012.

During prolonged strenuous exercise, racehorses can experience acidemia. To counteract this phenomenon, trainers can administer blood alkalizing agents that raise the plasma pH and total carbon dioxide (TCO2) concentration. In Illinois, the administrative threshold for TCO2 in plasma is 37.0 mmol/L. Because accuracy in the reported measurement of TCO2 must be ensured, uncertainty measurements are often issued alongside the reported concentrations. We report a validated method for measuring TCO2 levels in equine plasma using the Beckman UniCel DxC 600. A six-point calibration curve ranging from 5 to 50 mmol/L is analyzed along with controls at four TCO2 levels with each set of samples. Using this method, we collected data from 5,199 race samples during 2012, with 134 being from thoroughbred horses and 5,065 from standardbred horses. During method validation, uncertainty was determined using the simplified Guide to the Expression of Uncertainty in Measurement approach and was found to be 3% at 99.7% confidence level with eight measurements. Additionally, to investigate other variables that could have an effect on TCO2 levels, we collected the gender, breed, Lasix(®) status, strong ion concentration, pre- or post-race collection time and track location of all horses tested during that year. The samples had an overall mean TCO2 concentration of 30.5 ± 2.0 mmol/L. The other physiological and environmental data were analyzed using analysis of covariance tables. These results indicate gender, breed, furosemide status, collection time and track location to be strongly correlated (P < 0.0001) to TCO2 levels. Thoroughbred status was found to have no effect. Finally, TCO2 concentrations were highly correlated (P < 0.0001) to sodium and chloride ion concentrations. No correlation was found between TCO2 and potassium concentrations. The results show that there are several environmental and physiological factors that can affect TCO2 concentrations. The concentration of other strong ions present in the blood may indicate doping status.

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.

Quantification of several acidic drugs in equine serum using LC-MS-MS.

The use of nonsteroidal antiinflammatory drugs in racehorses is allowed under most jurisdictions. Furosemide is administered to treat exercise-induced pulmonary hemorrhage. To help distinguish between therapeutic and illegal uses, racing regulatory bodies have set thresholds in serum for several drugs. The method for the simultaneous detection and quantification of furosemide, flunixin, ketoprofen, phenylbutazone and oxyphenbutazone using 500 µL of serum, and liquid extraction using diethyl ether : hexanes : dichloromethane followed by liquid chromatography tandem mass spectrometry quantitation, was developed and validated. Method validation included inter- and intraday precision and accuracy. Method validation also included bench-top, freeze-thaw, processed and long-term storage stability testing. For all stability testing, the compounds showed a breakdown of <15%. Inter- and intraday precision for all compounds was found to be within the acceptance interval of ±15% [±20% at the lower limit of quantitation (LLOQ)]. Accuracy data for all compounds were within the acceptance interval of ±15% (±20% at the LLOQ). Uncertainty was calculated using the simplified Guide to the Expression of Uncertainty in Measurement approach and was <30% for all drugs at 95% confidence level. The method was found to be both robust and accurate for all tested drugs.

Detection of various performance enhancing substances in specimens collected from race horses in Illinois: a five-year experience.

In order to protect the integrity of horse racing in Illinois, a complex testing of urine and blood specimens collected post-race from winning and special designation horses is continuously conducted. The initial screening by immunoassays was followed by the confirmation on presumptive positive samples. Instrumental screening was also conducted. Perimortem and postmortem specimens and special exhibits (syringes, needles, etc.) were also analyzed. The administration of alkalinizing agents was detected by measuring the total plasma carbon dioxide concentration. The laboratory analyzed specimens collected post race from winning horses and special designation horses at eight race tracks in the State of Illinois over the five-year time period (2004-2009). The total number of specimens collected was 91,808, comprising 45,210 urine specimens and 46,598 blood specimens. The total number of violations was 413 (0.45% of the total number of specimens analyzed); 207 were blood specimens (0.44% of the total blood specimens analyzed), and 206 were urine specimens (0.45% of the total urine specimens analyzed). A total of 220 violations were reported for harness horses, and 193 were reported for Thoroughbred horses. The number of reported violations of the total tested specimens in Illinois was small, but a wide variety of performance-enhancing drugs was shown.

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.

Detection and quantification of low levels of benzoylecgonine in equine urine.

Cocaine (COC) is a highly addictive plant alkaloid expressing strong psychostimulatory effect. It has no medical use in equine veterinary practice. The contamination of the environment with cocaine such as its presence on the US paper currency has been reported few times. There are anecdotal reports of low benzoylecgonine (BE) concentrations (usually much less than 100 ng/mL) being found in urine of race horses. In order to protect horsemen against harsh penalties associated with the presence of trace amounts of BE in horse urine as a result of environmental contamination, in February 2005 the Illinois Racing Board issued new medication rules that established the threshold level of 150 ng/mL for BE in equine urine. The penalties associated with this rule provide for increasing fines ($250, $500, $1000) with successive positive reports against a trainer for levels of BE below 150 ng/mL. A total of 19,315 urine samples were collected over the 2-year period of time from winning horses (both harness and thoroughbred) at race tracks in Illinois for routine drug screening (ELISA). The presence of BE was confirmed by GC/MS in 28 urine samples (0.14%). The concentration range for BE in harness horses (21 detections) was < 5-91 ng/mL, and for thoroughbred (seven detections) was 7-52 ng/mL. To date, the laboratory has not reported concentrations of BE that exceed the established threshold concentration of 150 ng/mL.