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.
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.
A simple, rapid, sensitive and reproducible method for enantiomer analysis of methamphetamine, amphetamine, cathinone and methcathinone was developed and validated. The compounds were extracted from equine plasma and urine using a fast liquid-liquid extraction procedure. Only one milliliter plasma and one hundred microliter urine sample is needed for analysis. The extraction procedure had good recovery (>70%) and the matrix effect was negligible. Enantiomer differentiation and confirmation were achieved using liquid chromatography with chiral stationary phase and mass spectrometry detection. The method demonstrated excellent reproducibility with intraday and inter-day precision of lower than 5%. The lower limits of detection for all of the compounds studied here were at low pg/mL level for both plasma and urine. This is the first report of the analysis of four chiral compounds in equine plasma and urine. Routine application was demonstrated for (S)-and (R)-enantiomer differentiation.
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