Analytical Detection of Novel Stimulants by Immunoassay and Liquid Chromatography-High Resolution Mass Spectrometry: Case Studies on Ethylphenidate and Mephedrone.

The advent of hundreds of new compounds aimed at the substance misuse market has posed new analytical challenges. A semi-quantitative liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method has been developed to detect exposure to two novel stimulants, mephedrone and ethylphenidate, and selected metabolites. Centrifuged urine (50 µL) was diluted with LC eluent containing internal standards (mephedrone-d3, methylphenidate-d9 and ritalinic acid-d10; all 0.02 mg/L) (450 µL). Intra- and inter-assay accuracy and precision were within ±15% and <6%, respectively, for all analytes. The limit of detection was 0.01 mg/L for all analytes. Urine samples from mephedrone and ethylphenidate users were analyzed using immunoassay (amphetamine-group cloned enzyme donor immunoassay [CEDIA]) and LC-HRMS. Ethylphenidate, mephedrone and selected metabolites all had low cross-reactivity (<1%) with the immunoassay. The median (range) amphetamine-group CEDIA concentration in urine samples from mephedrone users (n = 11) was 0.30 (<0.041-3.04) mg/L, with only 1 sample giving a positive CEDIA result. The amphetamine-group CEDIA concentration in the urine sample from an ethylphenidate user was <0.041 mg/L. Improving the detection of novel compounds is of increasing importance to enable accurate diagnosis and treatment. Immunoassay methods used for drug screening may be inappropriate and lead to false-negative results. Conversely, detection of these compounds is possible through use of LC-HRMS and can provide information on the metabolites present after exposure to these drugs.