Dermorphin is a unique opioid peptide that is 30-40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC-MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20-10,000 pg/mL in plasma and 50-20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.

Continuous erythropoietin receptor activator (CERA) is the third generation of recombinant human erythropoietin (rhEPO) medication that retains the effect of promoting red blood cell production but has longer duration of action in the body. CERA, rhEPO, and darbepoetin alpha (DPO) can be misused to enhance performance in both human and equine athletes. To deter such misuse, a very selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has now been developed for identification of CERA, rhEPO, and DPO in equine plasma. The method employs a new signature tryptic peptide, T8 ((54)MEVGQQAVEVWQGLALLSEAVLR(76), common to the three proteins), and improved immunoaffinity extraction. The analytes were extracted by anti-rhEPO antibodies from plasma samples that were pretreated with polyethylene glycol (PEG) 6000. The extracted analytes were digested by trypsin and analyzed by LC-MS/MS. The limit of identification was 0.5 ng/mL for CERA, 0.2 ng/mL for rhEPO, and 0.1 ng/mL for DPO in equine plasma; the limit of detection was 0.3 ng/mL for CERA, 0.1 ng/mL for rhEPO, and 0.05 ng/mL for DPO. Specificity of the method was assessed via BLAST and SEQUEST protein database searches, and the T8 is extremely specific at both peptide and product ion levels for the identification of CERA, rhEPO, and DPO. This method was successful in identifying CERA and DPO in plasma samples collected from research horses post the drug administrations. It provides a useful tool in the fight against blood doping with CERA, rhEPO, and DPO in racehorses. Additionally, the following two technical approaches adopted in this study may also be helpful in protein identifications and biomarker discoveries in a broad scope: precipitating plasma proteins with PEG 6000 to improve immunoaffinity extraction efficiency of the target proteins and making a large and more lipophilic peptide detectable at low concentrations by increasing its solubility in the sample solvent.

Summary Phenylbutazone, a non‐steroidal anti‐inflammatory drug known to produce intestinal erosions, was administered intravenously (13.46 mg/kg bodyweight) to 12 horses which were killed after 24, 48, 72 and 96 h. Eight untreated horses served as controls. Annular erosions in the duodenum and mucosal necrosis in the colon were seen after 48 h which progressed in severity. The erosions were characterised by sloughing of the surface epithelium, subepithelial cleft and bleb formation, necrosis of the lamina propria, degeneration of the walls of subsurface capillaries and microthrombosis. Large numbers of neutrophils with abundant fibrin and cellular debris were present at the erosion sites. Ultrastructurally, there was swelling of the endothelium of capillaries and small vessels, and of pericyte and smooth muscle cytoplasm in arterioles. In capillaries and post capillary venules, the endothelium ranged from swollen to lysed and necrotic. Extensive extravasation of erythrocytes and oedema were seen. These lesions were not seen in the control horses. Phenylbutazone produces a microvascular injury associated with the formation of duodenal and colonic erosions in horses. The duodenal and colonic mucosa were assayed at 48 and 96 h for prostacyclin and PGE2. There was no statistically significant difference between prostaglandin levels in the mucosa of control and treated horses. It was concluded that there was no correlation between mucosal prostaglandin levels and intestinal erosions after 48 h.

Cobalt, atomic weight 58.9, is a metallic element and environmental substance found in the animal in microgram quantities, predominantly as vitamin B12, but is also a component of at least one mammalian enzyme unassociated with B12. Cobalt is a required trace mineral and has long been administered as a dietary supplement to humans and animals. Cobalt deficiency outside of its requirement in vitamin B12 has not been reported in humans. The administration of cobalt salts was once standard treatment for anaemia in humans, owing to its ability to stimulate red blood cell synthesis. Elemental cobalt acts by stabilising hypoxia inducible factor (HIF-1α), which activates the erythropoietin gene, which in turn increases haemoglobin/red blood cell synthesis, which had led to a presumption that cobalt may be performance enhancing in athletes. Administration of cobalt in amounts sufficient to significantly increase the haematocrit are associated with risk of toxicity in humans, and the only cobalt administration study in horses showed no effect on red blood cell parameters or toxicity. Because of the perception that cobalt administration may enhance athletic performance, racing regulators have recently begun to restrict cobalt use in horseracing which has led to the introduction of cobalt thresholds in several racing jurisdictions. The International Federation of Horseracing Authorities is considering an international regulatory threshold for cobalt of 100 ng/ml in urine, based on studies performed in five different countries. In the United States, the Racing Commissioners International has recently set a primary plasma threshold of 25 ng/ml and secondary threshold of 50 ng/ml. One New York and New Jersey racetrack owner has initiated testing for cobalt and has denied his facilities to trainers whose horses tested positive for excessive quantities of cobalt. This review seeks to summarise what is known about the use of cobalt in horse racing.