Development and validation of a hydrophilic interaction liquid chromatography with tandem mass spectrometry method for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine.

A sensitive hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous detection and quantification of etilefrine and oxilofrine in equine blood plasma and urine. The method is highly sensitive and specific with good precision and accuracy. In plasma the limit of detection and limit of quantification are 0.03 and 0.1 ng/mL, respectively, for both analytes. In urine the limit of detection and limit of quantification are 0.3 and 1 ng/mL, respectively, for both analytes. The suitability of the method for doping control analysis in equine species is demonstrated by analyzing postadministration samples collected after a single intravenous administration of 50 mg etilefrine to a standardbred mare. Etilefrine was detected up to 120 h in urine and up to 48 h in plasma. Etilefrine is highly conjugated in equine urine whereas it exists in the free form in equine plasma. Therefore, enzyme hydrolysis prior to sample preparation is recommended for the detection and quantification of etilefrine and oxilofrine in equine urine.

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.

Detection of myo-inositol tris pyrophosphate (ITPP) in equine following an administration of ITPP.

Myo-Inositol tris pyrophosphate (ITPP) is a powerful allosteric modulator of haemoglobin that increases oxygen-releasing capacity of red blood cells. It is capable of crossing the red blood cell membrane unlike its open polyphosphate analog myo-inositol hexakisphosphate (IHP). Systemic administration of ITPP enhanced the exercise capacity in mice. There have been rumours of its abuse in the horse racing industry to enhance the performance of racing horses. In this paper, the detection of ITPP in equine plasma and urine after an administration of ITPP is reported. A Standardbred mare was administered 200 mg of ITPP intravenously. Urine and plasma samples were collected up to 120 h post administration and analyzed for ITPP by liquid chromatography-tandem mass spectrometry. ITPP was detected in post administration plasma samples up to 6 hours. The peak concentration was detected at 5 min post administration. In urine, ITPP was detected up to 24 h post administration. The peak concentration was detected at 1.5 h post administration.

Detection and elimination profile of cathinone in equine after norephedrine (Propalin®) administration using a validated liquid chromatography-tandem mass spectrometry method.

Cathinone is the principal psychostimulant present in the leaves of khat shrub, which are widely used in East Africa and the Arab peninsula as an amphetamine-like stimulant. Cathinone readily undergoes metabolism in vivo to form less potent cathine and norephedrine as the metabolites. However, the presence of cathine and norephedrine in biological fluids cannot be used as an indicator of cathinone administration. The metabolism of pseudoephedrine and ephedrine, commonly used in cold and allergy medications, also produces cathine and norephedrine, respectively, as the metabolites. Besides, cathine and norephedrine may also originate from the ingestion of nutritional supplemental products containing extracts of Ephedra species. In Canada, ephedrine and norephedrine are available for veterinary use, whereas cathinone is not approved for human or veterinary use. In this article, the detection of cathinone in equine after administration of norephedrine is reported. To the best of our knowledge, this is the first such report in any species where administration of norephedrine or ephedrine generates cathinone as the metabolite. This observation is quite significant, because in equine detection of cathinone in biological fluids could be due to administration of the potent stimulant cathinone or the nonpotent stimulant norephedrine. A single oral dose of 450 mg norephedrine was administered to four Standardbred mares. Plasma and urine samples were collected up to 120 h after administration. The amount of cathinone and norephedrine detected in post administration samples was quantified using a highly sensitive, specific, and validated liquid chromatography-tandem mass spectrometry method. Using these results, we constructed elimination profiles for cathinone and norephedrine in equine plasma and urine. A mechanism that generates a geminal diol as an intermediate is postulated for this in vivo conversion of norephedrine to cathinone. Cathinone was also detected in samples collected after a single intramuscular administration of 200 mg ephedrine and oral administration of 300 mg ephedrine in equine.

Phylogeography and domestication of Indian river buffalo.

BACKGROUND: The water buffalo- Bubalus bubalis holds tremendous potential in livestock sector in many Asian countries, particularly India. The origin, domestication and genetic structure of the Indian river buffalo are poorly understood. Therefore, to understand the relationship among the maternal lineages of Indian river buffalo breeds and their domestication process, we analysed mitochondrial D-loop region of 217 animals representing eight breeds from eight different locations in India along with published sequences of Mediterranean buffalo. RESULTS: The maximum parsimony tree showed one major clade with six internal branches. Reduced median network revealed expansion from more than one set of haplotypes indicating complex domestication events for this species. In addition, we found several singleton haplotypes. Using rho statistics, we obtained a time estimate of 6300 years BP for the expansion of one set of hapltoypes of the Indian domestic buffalo. A few breed specific branches in the network indicated an ancient time depth of differentiation of some of the maternal lineages of river buffalo breeds. The multidimensional display of breed pairwise FST values showed significant breed differentiation. CONCLUSION: Present day river buffalo is the result of complex domestication processes involving more than one maternal lineage and a significant maternal gene flow from the wild populations after the initial domestication events. Our data are consistent with the available archaeological information in supporting the proposition that the river buffalo was likely to be domesticated in the Western region of the Indian subcontinent, specifically the present day breeding tracts of the Mehsana, Surati and Pandharpuri breeds.