Doping control analysis of myo-inositol trispyrophosphate and 10 bisphosphonates in equine plasma by ion chromatography-mass spectrometry and its application to clodronic acid horse administration.

Bisphosphonates and myo-inositol trispyrophosphate (ITPP) are two classes of difficult-to-detect polar drugs that are prohibited under the rules of racing. ITPP is a drug capable of increasing the amount of oxygen in hypoxic tissues, and studies have shown that administration of ITPP increases the maximal exercise capacity in mice. The properties of ITPP make it an ideal candidate as a doping agent to enhance performance in racehorses. In recent years, ITPP had indeed been detected in racehorses and confiscated items. As for bisphosphonates, it is especially critical to control their use as since February 2019, the International Agreement on Breeding, Racing and Wagering (IABRW) by the International Federation of Horseracing Authorities (IFHA) had identified specific conditions on which bisphosphonates should not be administered to a racehorse. A recent review of literature shows that there is yet a simultaneous screening method for detecting ITPP and bisphosphonates in equine samples. This paper describes an efficient ion chromatography high-resolution mass spectrometry (IC-HRMS) method for the simultaneous detection of ITPP and 10 bisphosphonates at sub-parts-per-billion (ppb) to low-ppb levels in equine plasma after solid-phase extraction (SPE) and its application to an administration study of clodronic acid in horses.

Gene Doping Control Analysis of Human Erythropoietin Transgene in Equine Plasma by PCR-Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

Gene doping involves the misuse of genetic materials to alter an athlete's performance, which is banned at all times in both human and equine sports. Quantitative polymerase chain reaction (qPCR) assays have been used to control the misuse of transgenes in equine sports. Our laboratory recently developed and implemented duplex as well as multiplex qPCR assays for transgenes detection. To further advance gene doping control, we have developed for the first time a sensitive and definitive PCR-liquid chromatography high-resolution tandem mass spectrometry (PCR-LC-HRMS/MS) method for transgene detection with an estimated limit of detection of below 100 copies/mL for the human erythropoietin (hEPO) transgene in equine plasma. The method involved magnetic-glass-particle-based extraction of DNA from equine plasma prior to PCR amplification with 2'-deoxyuridine 5'-triphosphate (dUTP) followed by treatments with uracil DNA glycosylase and hot piperidine for selective cleavage to give small oligonucleotide fragments. The resulting DNA fragments were then analyzed by LC-HRMS/MS. The applicability of this method has been demonstrated by the successful detection of hEPO transgene in a blood sample collected from a gelding (castrated male horse) that had been administered the transgene. This novel approach not only serves as a complementary method for transgene detection but also paves the way for developing a generic PCR-LC-HRMS/MS method for the detection of multiple transgenes.

Targeted Metabolomics Approach To Detect the Misuse of Steroidal Aromatase Inhibitors in Equine Sports by Biomarker Profiling.

The use of anabolic androgenic steroids (AAS) is prohibited in both human and equine sports. The conventional approach in doping control testing for AAS (as well as other prohibited substances) is accomplished by the direct detection of target AAS or their characteristic metabolites in biological samples using hyphenated techniques such as gas chromatography or liquid chromatography coupled with mass spectrometry. Such an approach, however, falls short when dealing with unknown designer steroids where reference materials and their pharmacokinetics are not available. In addition, AASs with fast elimination times render the direct detection approach ineffective as the detection window is short. A targeted metabolomics approach is a plausible alternative to the conventional direct detection approach for controlling the misuse of AAS in sports. Because the administration of AAS of the same class may trigger similar physiological responses or effects in the body, it may be possible to detect such administrations by monitoring changes in the endogenous steroidal expression profile. This study attempts to evaluate the viability of using the targeted metabolomics approach to detect the administration of steroidal aromatase inhibitors, namely androst-4-ene-3,6,17-trione (6-OXO) and androsta-1,4,6-triene-3,17-dione (ATD), in horses. Total (free and conjugated) urinary concentrations of 31 endogenous steroids were determined by gas chromatography-tandem mass spectrometry for a group of 2 resting and 2 in-training thoroughbred geldings treated with either 6-OXO or ATD. Similar data were also obtained from a control (untreated) group of in-training thoroughbred geldings (n = 28). Statistical processing and chemometric procedures using principle component analysis and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) have highlighted 7 potential biomarkers that could be used to differentiate urine samples obtained from the control and the treated groups. On the basis of this targeted metabolomic approach, the administration of 6-OXO and ATD could be detected for much longer relative to that of the conventional direct detection approach.