Development of strategies to identify new psychoactive substances and their metabolites in biological samples by LC-QTOF-MS technology

New psychoactive substances (NPS) are emerging in the illegal drug market, which has led to major challenges for analytical laboratories. Keeping screening methods up to date with all relevant drugs is hard to achieve and the risk of missing important findings in biological samples is a matter of concern. Certain groups of NPS, e.g., synthetic opioids including fentanyl analogues, are of special concern due to their high potency. This indicates the possibility of low drug concentrations in vivo and calls for sensitive analytical methods and identification of the most appropriate analytical targets. In this thesis, three studies were carried out to demonstrate the application of liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) technology in the detection of NPS and their metabolites. In Study I, a sensitive and quantitative screening method in urine with metabolites of synthetic cannabinoids that were frequently seized in Norway in the current time period (ABFUBINACA, AB-PINACA, AB-CHMINACA, AM-2201, AKB48, 5F-AKB48, BB-22, JWH-018, JWH-073, JWH-081, JWH-122, JWH-203, JWH-250, PB-22, 5F-PB-22, RCS-4, THJ-2201, and UR-144) was developed. The samples were treated with ß-glucuronidase prior to extraction and solid-phase extraction was used. Liquid handling was automated using a robot. Each sample was initially screened for identification and quantification, followed by a second injection for confirmation. The concentrations by which the compounds could be confirmed varied between 0.1 and 12 ng/ml. Overall, the validation showed that the method fulfilled the set criteria and requirements for matrix effect, extraction recovery, linearity, precision, accuracy, specificity, and stability. One thousand urine samples from subjects in drug withdrawal programs were analysed using the presented method. The metabolite ABFUBINACA M3, hydroxylated metabolite of 5F-AKB48, hydroxylated metabolite of AKB48, AKB-48 N-pentanoic acid, 5F-PB-22 3-carboxyindole, BB-22 3-carboxyindole, JWH-018 N- (5-hydroxypentyl), JWH-018 N-pentanoic acid, and JWH-073 N-butanoic acid were quantified and confirmed in 2.3% of the samples. The method was proven to be sensitive, selective, and robust for routine use for the investigated metabolites. In Study II, the in vitro metabolism of ortho-, meta-, and para-fluorofentanyl - three fluorinated derivatives of fentanyl - was investigated using human hepatocytes and compared to the results from an authentic urine sample