An integrated LC-MS/MS strategy for rapid detection of suspicious NPS in real saliva samples

New psychoactive substances (NPS) represent a large class of new drugs that are often not detected by classic screening tests. Featuring a variety of structures, they create an ever-changing landscape1. In this study, a suspicious analysis-based strategy was developed for the detection of NPS in real samples. This study aimed to develop an analytical strategy capable of detecting NPS in real samples in the absence of reference standards. The experimental design included saliva as the matrix of choice. The samples were collected in collaboration with the Public Security Forces. The samples, therefore, derive from sample checks carried out within the national territory. Saliva samples were prepared by protein precipitation, subsequently analyzed by LC-MS/MS. The mass spectra were acquired on a Sciex Qtrap 6500 mass spectrometer in IDA-EPI (Information Dependent Acquisition – Enhanced Product Ion) acquisition mode in accordance with the work carried out by Di Francesco et al. 2 ; each sample was analyzed in RP chromatography. Given the large amount of information in the matrix, an MRM programmed chromatographic method (As-MRM) was used. The mass spectrum was acquired only in positive mode. In this study, a test set comprising 86 drugs of abuse was employed to support the development of a quantitative structure-retention relationship (QSRR) model, which was subsequently used to predict the retention times (t R ) of a training set consisting of 70 NPS. The predictive model considers six molecular descriptors of known substances and their tR to predict the tR of substances for training. With this approach, it was possible to identify two NPSs of the training group within the real samples. The combination of As-MRM and the IDA-EPI acquisition mode provides mass aspects that can be directly compared to spectra in libraries. The approach based on low-resolution mass spectrometry and screening of suspects has proven effective as a preliminary tool, also providing the possibility of adding new substances to the method quickly and in parallel with alerts, even in the absence of reference standards.