Detection of efaproxiral (RSR13) and its metabolites in equine by liquid chromatography tandem mass spectrometry.

Efaproxiral (RSR 13) is an experimental synthetic allosteric modifier of haemoglobin (Hb) that acts by increasing the release of oxygen from Hb to the surrounding tissues. It has been shown to increase maximum oxygen uptake (VO(2max)) in a canine skeletal muscle model. The ability to increase maximal muscle oxygen uptake makes efaproxiral a potential performance-enhancing agent and is therefore prohibited by the World Anti-Doping Agency. In this study, a method for the detection and elimination of efaproxiral in equine plasma and urine after a 2.5 g intravenous administration of efaproxiral is described. Post administration plasma and urine samples were collected up to 120 h. Efaproxiral was detected up to 120 h in urine and up to 78 h in plasma. In plasma, the peak concentration was 42 µg/ml and detected at 5 min post administration. In urine, the peak concentration was 2.8 mg/ml and detected at 0-1 h post administration. A validated liquid chromatography tandem mass spectrometry method was used for the quantitation of efaproxiral in equine plasma and urine. The limit of detection of the method is 0.05 ng/ml in plasma and 0.1 ng/ml in urine. The method is highly sensitive and specific with good precision, accuracy and recovery. The manuscript also describes the systematic identification of efaproxiral metabolites detected in post administration equine urine samples. The metabolites were identified by use of enhanced mass spectra and enhanced product ion scans. Both positive and negative mode ionizations were utilized for metabolite identification and plausible fragmentation pathways were proposed for the phase 1 metabolite identified. In addition to free efaproxiral, one phase 1 metabolite and two phase 2 metabolites were identified in post administration urine.