The project involved four main tasks. One task involved the development of a dynamic multiple reaction monitoring (dMRM) liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS) method for just over 800 NPS. The second task involved the validation of the dMRM method for screening and confirmation of the NPS, using a series of mixtures of non-coeluting standards. The third task compared and optimized NPS extraction methods for urine and whole blood. The fourth task involved screening spiked and authentic specimens to determine the real-world potential of the dMRM method. Validation was completed for the parameters of selectivity, limit of detection (LOD), limit of quantitation (LOQ), carry over, linearity, bias, precision, freeze-thaw stability, and matrix effects. A method that included 729 compounds was validated with LOD and LOQ in the pg/mL range. Through the analysis of blind spiked and authentic specimens, the applicability of the validated method as a screening and confirmatory method was demonstrated. This method will aid in the reliable identification of NPS in clinical and forensic toxicological samples. In addition, this project provided data that improves the reliability of extraction of NPS from biological matrices. 30 tables, 11 figures, and 119 references
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