Opiorphin analysis in equine plasma and urine using hydrophilic interaction LC-MS.

BACKGROUND: Due to opiorphin's analgesic and antidepressant functions, its illicit use is rumored in some racing jurisdictions. Opiorphin is very difficult to detect due to its hydrophilic nature and rapid degradation in plasma and urine samples. METHODOLOGY & RESULTS: We have developed a sensitive, reliable method for opiorphin detection and confirmation in equine samples, using EDTA to inhibit analyte degradation between the time of collection and analysis. Opiorphin was extracted by weak cation exchange followed by analysis using HILIC-MS/MS. The method was validated and the LOD was determined to be 50 pg/ml in equine plasma and urine. CONCLUSION: The method has good selectivity and precision and is the first reported method for the detection of opiorphin in equine plasma and urine.

Fast and sensitive analysis of dermorphin and HYP6-dermorphin in equine plasma using liquid chromatography tandem mass spectrometry.

Dermorphin and HYP(6) -dermorphin are hepta-peptides and natural opioids originally isolated from the skin of South American frogs. They are more potent than morphine but less likely to produce drug tolerance and addiction. These properties make them ideal candidates for the doping of racehorses to enhance performance during competition. Dermorphin was recently classified as a Class I drug by Racing Commissioners International (RCI), indicating that it is a banned substance in equine athletes. To enforce this ban, a fast and sensitive method was developed for dermorphin and HYP(6)-dermorphin analysis in equine plasma. Equine plasma (2 ml) was extracted on a mixed mode cation exchange solid-phase column. After extraction, dermorphin and HYP(6)-dermorphin were separated and detected using a liquid chromatography (LC) triple quadrupole linear ion trap mass spectrometry in positive multiple-reaction-monitoring (MRM) mode. Each analysis was 3.5 min. Four MRM transitions were used for identification of each compound. The extraction procedure was efficient and the limits of detection (LOD) were 2 pg/ml and 10 pg/ml for dermorphin and HYP(6)-dermorphin, respectively. The method has good selectivity and precision. Results of stability studies showed that both analytes were stable at low temperature. This is the first report of dermorphin and HYP(6)-dermorphin analysis in equine plasma, which could be adopted as a regular screening or confirmation method for controlling the abuse of these compounds in equine sports.

Rapid and sensitive analysis of 3,4-methylenedioxypyrovalerone in equine plasma using liquid chromatography-tandem mass spectrometry.

3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive drug with potent stimulant properties and potential for abuse and drug dependency. MDPV was recently classified as a Class I drug by Racing Commissioners International, indicating that it is a banned substance in equine athletes because it lacks therapeutic value in horses. To enforce this ban, a sensitive and fast liquid chromatography-tandem mass spectrometry method was needed. It is for this reason that this method was developed for quantification and confirmation of MDPV in equine plasma. Sample preparation involved liquid-liquid extraction. The analyte was analyzed by a triple-quadrupole linear ion trap mass spectrometer in positive multiple-reaction-monitoring and enhanced product ion scan modes. The method was validated for precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), linearity, stability, extraction recovery, matrix effect, dilution accuracy and selectivity. The extraction recovery was >90%. The linearity range was from 5 to 15,000 pg/mL. LOD and LOQ were 2 and 5 pg/mL, respectively. Intra-day and inter-day accuracies were nearly 100%. The method is suitable for screening, quantification and confirmation of MDPV in equine plasma and has been successfully used to detect and confirm the presence of MDPV in equine plasma obtained post-competition.