Recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production. Consequently, they are abused in human and equine sports. To deter their abuse in the horse racing industry, a sensitive and reliable method for confirmation of these agents in equine plasma has been in urgent need. Such a method by LC-MS/MS is described in this paper. The method involved analyte enrichment by immunoaffinity separation using anti-rhEPO antibody linked to magnetic beads, digestion by trypsin, and analysis by LC-MS/MS. Two specific proteotypic peptides, 46VNFYAWK52 and 144VYSNFLR150 from rhEPO and DPO were employed for confirmation of the analytes based on chromatographic retention times and major product ions. The limit of confirmation of this method was 0.2 ng/mL, and the limit of detection was 0.1 ng/mL for rhEPO and DPO in equine plasma. This method was successful in confirming the presence of rhEPO and DPO in plasma samples collected from research horses to which rhEPO or DPO was administered and from racehorses following competition and in noncompetition samples in North America. To our knowledge, this is the first LC-MS method with adequate sensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples. This method provides a powerful enforcement tool that was lacking in the fight against the abuse of rhEPO and DPO in the horse racing industry.

Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are protein-based drugs for the treatment of anemia in humans by stimulating erythrocyte production. However, these agents are abused in human and equine sports due to their potential to enhance performance. This paper describes the first method for differentiation and identification of rhEPO and DPO in equine plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method comprised analyte extraction and enrichment by immunoaffinity separation with anti-rhEPO antibodies, dual digestion by trypsin and peptide-N-glycosidase F (PNGase F), and analysis by LC-MS/MS. Two unique deglycosylated tryptic peptides, (21)EAENITTGCAEHCSLNENITVPDTK (45) (T 5) from rhEPO and (77)GQALLVNSSQVNETLQLHVDK (97) (T 9) from DPO, were employed for differentiation and identification of rhEPO and DPO via LC retention times and major product ions. The limit of identification was 0.1 ng/mL for DPO and 0.2 ng/mL for rhEPO in equine plasma, and the limit of detection was 0.05 ng/mL for DPO and 0.1 ng/mL for rhEPO. Analyte carryover problem encountered was solved by adding 20% acetonitrile to the solvent of the sample digest to increase solubility of the peptides. This method was successfully applied to identification of DPO in plasma samples collected from a research horse following DPO administration and from racehorses out of competition in North America. Thus, it provides a powerful tool in the fight against blood doping with rhEPO and DPO in the horse racing industry.

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Anabolic steroids (ABS) boldenone (BL; 1.1 mg/kg) and stanozolol (ST; 0.55 mg/kg) were administered i.m. to horses and the plasma samples collected up to 64 days. Anabolic steroids and androgenic steroids (ANS) in plasma were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The limit of detection of all analytes was 25 pg/mL. The median absorption (t1/2 partial differential) and elimination (t1/2e) half-lives for BL were 8.5 h and 123.0 h, respectively, and the area under the plasma concentration-time curve (AUCho) was 274.8 ng.h/mL. The median t1/2e for ST was 82.1 h and the was 700.1 ng.h/mL. Peak mean (X+/-SD) plasma concentrations (Cmax) for BL and ST were 1127.8 and 4118.2 pg/mL, respectively. Quantifiable concentrations of ABS and ANS were found in 61.7% of the 988 plasma samples tested from race tracks. In 17.3% of the plasma samples two or more ABS or ANS were quantifiable. Testosterone (TES) concentrations mean (X+/-SE) in racing and nonracing intact males were 241.3+/-61.3 and 490.4+/-35.1 pg/mL, respectively. TES was not quantified in nonracing geldings and female horses, but was in racing females and geldings. Plasma concentrations of endogenous 19-nortestosterone (nandrolone; NA) from racing and nonracing males were 50.2+/-5.5 and 71.8+/-4.6 pg/mL, respectively.

Anabolic steroids are structurally similar compounds, and their product-ion spectra obtained by tandem mass spectrometry under electrospray ionization conditions are quite difficult to interpret because of poly-ring structures and lack of a charge-retaining center in their chemical structures.In the present study, the fragmentation of nine anabolic steroids of interest to the racing industry was investigated by using triple quadrupole mass spectrometer, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, and a linear ion trap instrument. With the aid of an expert system software (Mass Frontier version 3.0), accurate mass measurements, and multiple stage tandem mass spectrometric (MS n ) experiments, fragmentation pathways were elucidated for boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, normethandrolone and mibolerone. Small differences in the chemical structures of the steroids, such as an additional double-bond or a methyl group, result in significantly different fragmentation pathways. The fragmentation pathways proposed in this paper allow interpretation of major product ions of other anabolic steroids reported by other researchers in a recent publication [19]. The proposed fragmentation pathways are helpful for characterization of new steroids. The approach used in this study for elucidation of the fragmentation pathways is helpful in interpretation of complicated product-ion spectra of other compounds, drugs and their metabolites. (J Am Soc Mass Spectrom 2006, 17, 477-489)

The incidence and severity of exercise-induced pulmonary haemorrhage (EIPH) in the 2 most commonly raced horse breeds, Thoroughbreds (TB) and Standardbreds (STD), were studied, with particular interest in the possible influence of frusemide (F) and/or the breed (or running gait) on EIPH. The appearance of blood within the trachea was semi-quantified using a published 5-point system, with zero assigned when no blood was observed, and numbers 1-4 assigned with increasing amounts of blood. Considering each endoscopic examination as a separate event, approximately 75% of the postrace endoscopic examinations had blood-scores of 1, 2, 3, or 4, regardless of breed or F administration. For horses examined twice, the chances of finding blood-scores of 1 or greater in either of the examinations increased to approximately 95%. All horses examined 3 or more times had endoscopic blood-scores of 1 or greater following one or more races, again, irrespective of the breed or F administration. Mean +/- s.e. 'blood scores' were 1.5 +/- 0.1 and 1.8 +/- 0.2 for TB, and 1.4 +/- 0.2 and 1.2 +/- 0.1 for STD racing with and without prerace F, respectively. Therefore, there was no apparent effect of breed (or possibly racing gait) on EIPH, and no differences in the incidence or severity of EIPH were observed between horses with or without prerace frusemide administration.

effects and pharmacokinetic profile of a long-term continuous rate infusion of racemic ketamine in healthy conscious horses. J. vet. Pharmacol. Therap. 29,[477][478][479][480][481][482][483][484][485][486][487][488] Ketamine (KET) possesses analgesic and anti-inflammatory activity at subanesthetic doses, suggesting a benefit of long-term KET treatment in horses suffering from pain, inflammatory tissue injury and/or endotoxemia. However, data describing the pharmacodynamic effects and safety of constant rate infusion (CRI) of KET and its pharmacokinetic profile in nonpremedicated horses are missing. Therefore, we administered to six healthy horses a CRI of 1.5 mg/kg/h KET over 320 min following initial drug loading. Cardiopulmonary parameters, arterial blood gases, glucose, lactate, cortisol, insulin, nonesterified fatty acids, and muscle enzyme levels were measured, as were plasma concentrations of KET and its metabolites using liquid chromatographytandem mass spectrometry (LC-MS/MS). Levels of sedation and muscle tension were scored. Respiration and heart rate significantly increased during the early infusion phase. Glucose and cortisol significantly varied both during and after infusion. During CRI all horses scored 0 on sedation. All but one horse scored 0 on muscle tension, with one mare scoring 1. All other parameters remained within or close to physiological limits without significant changes from pre-CRI values. The mean plasma concentration of KET during the 1.5 mg/kg/h KET CRI was 235 ng/mL. The decline of its plasma concentration-time curve of both KET and norketamine (NKET) following the CRI was described by a twocompartmental model. The metabolic cascade of KET was NKET, hydroxynorketamine (HNK), and 5,6-dehydronorketamine (DHNK). The KET median elimination half-lives (t 1/2a and t 1/2b ) were 2.3 and 67.4 min, respectively. The area under the KET plasma concentration-time curve (AUC), elimination was 76.0 lgAEmin/mL. Volumes of C 1 and C 2 were 0.24 and 0.79 L/kg, respectively. It was concluded that a KET CRI of 1.5 mg/kg/h can safely be administered to healthy conscious horses for at least 6 h, although a slight modification of the initial infusion rate regimen may be indicated. Furthermore, in the horse KET undergoes very rapid biotransformation to NKET and HNK and DHNK were the major terminal metabolites.(Paper

A compartmental model was used to describe the pharmacokinetics of dexamethasone (DXM) and changes in the plasma concentration of endogenous cortisone (COR) and hydrocortisone (HYD) following intravenous (i.v.) administration of DXM (0.05 mg/kg) in horses. Quantification of DXM, COR and HYD in equine plasma was achieved using liquid chromatography interfaced with triple spray quadrupole quantum tandem mass spectrometry (LC/TSQ-MS/MS). The median alpha (t(1/2alpha)), beta (t(1/2beta)), and gamma (t(1/2gamma)) half-lives were 0.33, 2.2, and 10.7 h respectively. The area under the DXM plasma concentration curve (AUC) was 113.5 ng.h/mL. At 72 h post-DXM administration, the plasma concentration of DXM in all horses was below the level of quantification (100 pg/mL). The baseline plasma concentration of COR was 3.5 +/- 0.69 ng/mL and declined significantly (P < 0.02) to 2.9 +/- 0.86 ng/mL at 1 h. The nadir in COR plasma concentration was 0.65 +/- 0.12 ng/mL at 28.8 +/- 9.0 h, and the DXM plasma concentration was 0.19 +/- 0.13 ng/mL. COR concentration returned to baseline at 96 h. Baseline plasma concentration of HYD was 58.8 +/- 11.7 ng/mL and declined significantly (P < 0.001) to 41.1 +/- 14.9 ng/mL at 1 h following DXM administration but recovered to baseline at 96 h. The sensitivity of LC/TSQ-MS/MS allowed complete description of the pharmacokinetics of DXM and its effect on plasma concentrations of both COR and HYD.