In forensic toxicology, the most common techniques for screening of licit and illicit drugs are immunoassays and gas chromatography-mass spectrometry (GC-MS). These methods screen for compounds using specific target structures and chemical properties and are well established for routine drug analysis. However, NPS typically have structural modifications that can result in false positive and negative detections using these techniques. Newer techniques such as LC-QqQ-MS allow for less sample preparation, increased sensitivity, analysis of biological samples without derivatization, and separation of small, polar, and non-volatile compounds. Consequently, LC-QqQ-MS holds many advantages for screening of NPS.

This research reports the validation results for an LC-QqQ-MS dMRM method for 713 NPS and other drugs in urine using a dilute-and-shoot approach. The method was then applied for screening of several hundred authentic urine samples obtained from a drug testing laboratory. Method validation was completed using reference standards, including deuterated standards, obtained from Cayman Chemical as a neat solid or as a solution. Sixteen mixtures, each containing 25-70 analytes, were created based on the structure of the compound, retention time, drug class, and transitions that were previously collected by
LC-QqQ-MS and incorporated into an in-house database (Agilent PCDL). Mixtures were designed to minimize co-elutions and reduce interferences for reliable identification and analysis of the compounds. Validation parameters were based on Organization of Scientific Committees (OSAC) recommendations and parameters that were chosen as most applicable for screening validation included bias, carryover, limit of detection (LOD), and ion suppression/enhancement. LODs for all analytes ranged from 0.002 to 0.169 ng/mL, which are below levels typically reported for NPS in human specimens.

The validated method was used to screen for NPS in authentic urine specimens that were collected in 2013-2014 and stored at -20°C. Urines were collected from individuals in addiction treatment and pain medication monitoring programs and were supplied de-identified. Samples were processed using a dilute-and-shoot method in which 100 µL of sample was added to 500 µL of HPLC water. Urines were analyzed with and without glucuronidase treatment. Numerous positive detections of NPS, including synthetic cathinones and cannabinoids, and of other licit and illicit compounds, were determined in these specimens, indicating that the method may have value as a comprehensive NPS screen in forensic toxicological analysis.