Cobalt is a well-established inducer of hypoxia-like responses, which can cause gene modulation at the hypoxia inducible factor pathway to induce erythropoietin transcription. Cobalt salts are orally active, inexpensive, and easily accessible. It is an attractive blood doping agent for enhancing aerobic performance. Indeed, recent intelligence and investigations have confirmed cobalt was being abused in equine sports. In this paper, population surveys of total cobalt in raceday samples were conducted using inductively coupled plasma mass spectrometry (ICP-MS). Urinary threshold of 75 ng/mL and plasma threshold of 2 ng/mL could be proposed for the control of cobalt misuse in raceday or in-competition samples. Results from administration trials with cobalt-containing supplements showed that common supplements could elevate urinary and plasma cobalt levels above the proposed thresholds within 24 h of administration. It would therefore be necessary to ban the use of cobalt-containing supplements on raceday as well as on the day before racing in order to implement and enforce the proposed thresholds. Since the abuse with huge quantities of cobalt salts can be done during training while the use of legitimate cobalt-containing supplements are also allowed, different urinary and plasma cobalt thresholds would be required to control cobalt abuse in non-raceday or out-of-competition samples. This could be achieved by setting the thresholds above the maximum urinary and plasma cobalt concentrations observed or anticipated from the normal use of legitimate cobalt-containing supplements. Urinary threshold of 2000 ng/mL and plasma threshold of 10 ng/mL were thus proposed for the control of cobalt abuse in non-raceday or out-of-competition samples.

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The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, the performance of three sets of primers and a hydrolysis probe for hEPO were compared. One set showed adequate specificity, sensitivity, amplification efficiency, and a dynamic range of detection in the presence of horse gDNA. The assay was duplexed with the detection of horse tubulin α 4A (TUBA4A) gene as an endogenous internal control in order to prevent false-negative results due to poor recovery and storage of extracted DNA and/or qPCR experimental variation. For the extraction of hEPO-plasmid, the QIAGEN Gentra Puregene blood kit was shown to recover the majority (62%) of hEPO-plasmid from spiked horse blood cells. The specificity and limit of detection (LOD) of the duplex qPCR assay were determined in accordance with MIQE guidelines. These findings supported the application of this duplex qPCR assay to the detection of hEPO transgene in horse blood cells.

Recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO) and methoxy polyethylene glycol-epoetin beta (PEG-EPO) are synthetic analogues of the endogenous hormone erythropoietin (EPO). These erythropoiesis-stimulating agents have the ability to stimulate the production of red blood cells and are commercially available for the treatment of anaemia in humans. These drugs are understood to have performance-enhancing effects on human athletes due to their stimulation of red blood cell production, thereby improving delivery of oxygen to the muscle tissues. Although their effect on horses has not been proven, these substances were thought to be similarly performance enhancing and have indeed been applied covertly to horses. As such, these protein-based drugs are prohibited by authorities in both human and equine sports. The method officially adopted by the International Olympic Committee (IOC) and World Anti Doping Agency (WADA) for the confirmation of rhEPO and/or DPO (rhEPO/DPO) in human urine is based on electrophoresis in combination with Western blotting. A shortcoming of the WADA method is the lack of definitive mass spectral data for the confirmation of a positive finding. Recently, a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the detection and confirmation of rhEPO/DPO in equine plasma was reported. However, we have not been successful in achieving the reported sensitivity. This paper presents a method for the detection and confirmation of rhEPO/DPO, as well as the newly released PEG-EPO, in equine plasma. The procedures involve immunoaffinity extraction using anti-rhEPO antibody-coated Dynabeads followed by trypsin digestion. The injected extract was further purified and concentrated using an on-line trap column in the nano-LC system. Detection and confirmation were achieved by monitoring a unique peptide segment of rhEPO/DPO/PEG-EPO using nano-liquid chromatography-tandem mass spectrometry equipped with a nanospray ionisation source operated in the selected reaction monitoring mode. rhEPO, DPO and PEG-EPO can be confirmed at 0.1, 0.2 and 1.0 ng/mL, respectively, in equine plasma.

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