Simultaneous Determination of Mephedrone, Methylone, MDPV, and Amphetamines in Urine by LC/MS/MS Amanda Rigdon 1* , Mike Coyer 2 , Jack Cochran 1 , Ty Kahler 1 , and Paul Kennedy 3 . (1)Restek. Bellefonte, PA, USA. (2) Northern Tier Research, Mayfield, PA, USA. (3)Cayman Chemical, Ann Arbor, MI, USA. Introduction Abuse of substances marketed as ‘research chemicals’ often sold for research purposes only or added to consumer products labeled ‘not for human consumption’ has become increasingly popular. Cathinones, including mephedrone, methylone, and MDPV, are one class of compounds that have appeared on the market as part of the ‘research chemical’ movement. These compounds are commonly sold as bath salts, however drug users often snort or ingest these compounds to induce an amphetamine-like high. (Figure 1). In September 2011, mephedrone, methylone, and MDPV were placed on the DEA Schedule I list on an emergency basis. Because of this scheduling, demand for testing will increase. Figure 1: Photos of Bath Salts (left) and ‘Research Chemicals’ (right) Chromatographic Method Development and Validation: Because these compounds are very similar to amphetamines, and are used either as a substitute for or in conjunction with amphetamines [1], a chromatographic method was developed to detect both amphetamines and cathinones in a single run. In addition to the compounds mentioned in the title of this poster, several additional ‘research chemicals’ marketed to be similar to MDPV were included in the method. The final chromatographic method and results are detailed in Figure 2. Figure 2: 100 ng/mL Spiked Urine Sample Table 1: Peak List and Transitions Used Method Performance: A partial validation was performed on the three cathinones and amphetamines listed in the title of this work. Preliminary data for the remaining compounds was also collected, and is available upon request. LOD/LOQ, linearity, precision, and accuracy data were collected and are shown in Table 2. Note that at the time this work was performed, only mephedrone and its deuterated analog were available as certified reference standards. In order to evaluate the method for the remainder of the compound list, the ‘research chemicals’ shown in Figure 1 were used as mock reference standards for this work. Although a thorough characterization was not performed, all substances were analyzed for purity using LC-MS, and no additional compounds from 100 – 1000m/z were detected in the products. All samples were analyzed on a Shimadzu UFLC XR equipped with an AB SCIEX API 4000 MS/MS. Rather than being extracted, samples were prepared using a dilute-and-shoot methodology. All samples were diluted 10x in starting mobile phase containing 30 ng/mL internal standard. Table 2: Method Performance Summary Discussion: All values in the table above reflect results from the quantifier ion. MDA, MDMA, and MDEA were not evaluated using external control samples due to an error in writing the MS/MS method. These three compounds were evaluated for the remaining parameters at a later date. The calibration curve for this project was prepared from 1 ng/mL to 500 ng/mL, however, based on recent findings, urine concentrations of MDPV can range from < 10 ng/mL to > 8000 ng/mL. When samples > 1000 ng/mL were analyzed according to the method detailed above, both detector and column overloading were observed. This can be remedied by using either a higher sample dilution factor and/or a smaller injection volume, however a range such as this may exceed the linear dynamic range of the MS/MS detector. Authentic Sample Analysis: One authentic sample was obtained for this study. This sample was diluted as described above and analyzed along with a calibration curve. MDPV was found at a level of 116 ng/mL in the sample (Figure 3). Figure 3: Authentic Sample Positive for MDPV Conclusion: The method developed here is suitable for the quantitative analysis of cathinones and amphetamines in urine at low levels. Further work needs to be performed to determine performance with high-level samples. Preliminary data indicates that this method is also suitable for several of the newer designer drugs that may become used more widely in the near future due to the DEA scheduling of several cathinones. References: [1] P. Kriikku, L. Wilhelm, O. Schwarz, J. Rintatalo. Forensic Science International. 2011, 210, 1-3; 195 - 200