Emerging new psychoactive substances (NPS) poses a great risk to public health. Analyzing these large numbers of NPS and other associated substances often relies on liquid chromatography coupled to triple quadrupole mass spectrometry (LC-QqQ-MS) with multiple-reaction monitoring (MRM) mode. However, the differentiation of critical pairs, coeluted isobaric and/or isomeric species, is one of the challenges for this analytical platform. MRM transitions with poor selectivity can jeopardize accurate quantification and lead to biased interpretation. Herein, we refined a novel workflow for developing an MRM-based method with in-house CriticalPairFinder and TransitionFinder tools for the effective identification of unique and selective MRM transitions. Transitions selected by TransitionFinder showed much better accuracies than those selected only by fragment abundance in some mixtures of critical pairs. Using the proposed analytical strategy, a method that can simultaneously determine 219 NPS and 65 other substances across a variety of NPS classes in urine samples was developed, validated and applied to analyze clinical urine samples. This automated workflow is anticipated to facilitate method development for analyzing complex analytes while considering selectivity.
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