Development and Validation of an LC-MS/MS Method for the Detection of Substituted Phenethylamines and Phenidates...

Novel psychoactive phenethylamines and phenidates are synthetically designed analogs of conventional substances such as 3,4-methylenedioxymethamphetamine (MDMA), amphetamine, and methylphenidate, developed to circumvent existing drug regulations. In Korea, the abuse of these substances has increased, particularly among habitual users and newly addicted younger individuals. To address this issue, new substances are regulated through the analog legislation and the temporary narcotic designation system. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using solid-phase extraction (SPE) was developed and validated for the simultaneous determination of 13 substituted phenethylamines and 5 substituted phenidates in human urine. The target analytes included 5-MAPDB, BDB, 4-FEA, PMEA, 3-FPM, 25iP-NBOMe, 2C-N, 2C-TFM, 30C-NBOMe, 3C-P, 4-MMA-NBOMe, DOI, and 2C-H, as well as 4-MeTMP, 4F-EPH, 4F-MPH, IPPH, and HDEP-28. No interfering peaks were observed at the retention times of analytes and internal standards. Linearity was confirmed over analyte-specific concentration within the overall range of 0.1-100 ng/mL, with coefficient of determination (r2) exceeding 0.995. The limits of detection and quantification were 0.001-0.1 ng/mL and 0.1-0.2 ng/mL, respectively. Accuracy and precision, expressed as bias and coefficient of variation (CV), were within ±10% and <10%, respectively. The CVs for matrix effects and extraction efficiencies were approximately 10%, and the extraction efficiencies exceeded 70%, confirming the reliability of the method for forensic applications. The validated method enables simultaneous determination of substituted phenethylamines and phenidates in urine and facilitates prompt positive identification in forensic casework. In addition, the structural information, MRM conditions, and analytical method validation results presented in this study can serve as a basis for expanding research on substances with similar core structures, which is expected to strengthen regulatory responses to evolving drug trends.