Qualitative Method Validation and Extraction of Synthetic Cannabimimetics from Equine Urine

The synthetic cannabimimetics bind to CB1 and CB2 receptors. In horse racing, the potential to abuse synthetic cannabimimetics lies in its potential to mediate pain. This research was conducted to determine if synthetic cannabimimetics could be extracted from equine urine and detected and differentiated by the Liquid Chromatography Tandem Mass Spectrometry Triple Quadrupole. A qualitative method validation was performed for the Animal Forensic Toxicology Laboratory in Chicago, IL. The compounds used to validate the method include AM2201, AM2201 4-hydroxypentyl metabolite, JWH-018, JWH-018 5-pentazoic acid, JWH-210 5-hydroxypentyl metabolite, RCS-4 and UR-144. The results of the method validation indicate that the LOD for AM2201 and AM2201 4-hydroxypentyl metabolite is 1 ng/mL. The LOD for JWH-018, JWH-018 5-pentazoic acid, JWH-210 5-hydroxypentyl metabolite, RCS-4 and UR-144 was found to be 2 ng/mL. The matrix effects were found to be -94.5% to -99.4%. The extraction recovery ranged from 62.3% to 543.4%. The samples that were placed in the oven, caused the extraction efficiency results to be skewed. Due to a large amount of the drug being suppressed by the urine, the process efficiency ranged from 0.55% to 9.43%. The stability results indicate that the synthetic cannabimimetics present in the urine are unstable and begin to degrade in the bench top samples and the freeze thaw samples. However, the synthetic cannabimimetics present in the long term and processed urine samples were stable. The specificity results indicate that no compounds that naturally occur in urine will register as a false positive for the synthetic cannabimimetics. After the method was validated, ten additional compounds were analyzed. The compounds included JWH-073, JWH-073 N-(3-hydroxybutyl) metabolite, JWH-250, JWH-250 5-hydroxypentyl metabolite, JWH-122, JWH-122 5-methylnaphthyl isomer, JWH-122 7-methylnaphthyl isomer, JWH-398, JWH-398 N-(5-hydroxypentyl) metabolite and JWH-210. All of the compound could successfully be extracted from the equine urine and detected by the LC-MS/MS. Due to the fact that JWH-122 and the JWH-122 isomers had the same molecular weight and product ions, these three compounds could not be differentiated from one another on the LC-MS/MS.