Expanding the toolbox for semi-untargeted LC-MS analysis of new psychoactive substances through neutral loss and precursor ion scan

The term New Psychoactive Substances (NPS) refers to a large number of differently structured molecules of synthetic or semi-synthetic origin. These are obtained through simple structural modifications to the chemical structures of traditional drugs, allowing them to easily evade controls without losing their pharmacological effect. Since the early 2000s, 1,396 NPS have been identified globally, and they are becoming popular even among young people. The United Nations Office on Drugs and Crime (UNODC) has classified these substances based on their chemical structure, identifying 15 different families. The insight behind the present study lies in the knowledge that structurally similar substances undergo similar fragmentation, which will lead, in most cases, to charged fragments and neutral losses with the same mass. The study intends to use these fragments to conduct semi-untargeted analyses with HPLC instrumentation combined with a low-resolution triple quadrupole mass spectrometer. Currently, knowledge of the neutral losses of NPS is still limited in the literature. The few existing articles focus primarily on the class of fentanyl derivatives [1], and none utilize low-resolution mass spectrometry. The present study advances the state of the art by using low-resolution instruments for semi-untargeted analysis and focusing on four distinct classes of NPS: fentanyl derivatives, synthetic cathinones, synthetic cannabinoids, and benzodiazepines. A specific, short chromatographic gradient was optimized for each class, while maintaining the same mobile and stationary phases. For each class, a mixture of 9 to 20 substances was used, and 3 common charged fragments and 3 common neutral losses were selected. The developed methods were tested on real saliva samples. This study may be viewed as a first step toward using low-resolution instrumentations, which are generally most diffused in forensic laboratories, to conduct untargeted analyses, which are essential to keep pace with the NPS market and allow for highly reliable quantitative responses.