A comprehensive analytical process, from NPS threat identification to systematic screening: Method validation and one-year prevalence study.

Several New Psychoactive Substances (NPS) enter the illicit drug market each year. This constant evolution of compounds to screen is challenging to law enforcement and drug chemists, and even more so to forensic toxicologists, who need to detect such compounds which might be at low concentrations in complex biological matrices. While some technological solutions are better suited than others to address such a challenge (e.g., high resolution mass spectrometry), laboratories with limited instrumental and financial resources are faced with a complex task: systematically screening for a rapidly evolving NPS panel using an accredited method run on standard equipment (e.g., liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). This work presents a solution to this challenge: a complete workflow from the detection of a regional NPS threat to its implementation in a method accredited under the ISO 17025:2017 norm. Initial LC-MS/MS method included 55 NPS and metabolites (31 Novel Synthetic Opioids (NSO), 22 NSO metabolites and 2 designer benzodiazepines). Following their identification as relevant territorial threats, flualprazolam, then isotonitazene, were added to the contingent. By relying on development aiming for maximal integration to the current analysis workflow, systematic NPS screening using this method was easily implemented. Between March 2019 and March 2020, the 5 079 forensic cases analyzed in the province of Québec (Canada) revealed a NPS positivity rate of 3.4%. While 94% involved designer benzodiazepines, 5% involved NSO. This process, combining high efficiency, simple detection technology, ISO accreditation and rapid response to new threats resulted in a four-fold increase in NPS detection.

A threshold LC-MS/MS method for 92 analytes in oral fluid collected with the Quantisal® device.

A study of impaired driving rates in the province of Québec is currently planned following the legalization of recreational cannabis in Canada. Oral fluid (OF) samples are to be collected with a Quantisal® device and sent to the laboratory for analysis. In order to prepare for this project, a qualitative decision point analysis method monitoring for the presence of 97 drugs and metabolites in OF was developed and validated. This high throughput method uses incubation with a precipitation solvent (acetone:acetonitrile 30:70 v:v) to boost drug recovery from the collecting device and improve stability of benzodiazepines (e.g., α-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam, nitrazepam). The Quantisal® device has polyglycol in its stabilizing buffer, but timed use of the mass spectrometer waste valve proved sufficient to avoid the glycol interferences for nearly all analytes. Interferences from OF matrices and 140 potentially interfering compounds, carryover, ion ratios, stability, recovery, reproducibility, robustness, false positive rate, false negative rate, selectivity, sensitivity and reliability rates were tested in the validation process. Five of the targeted analytes (olanzapine, oxazepam, 7-aminoclonazepam, flunitrazepam and nitrazepam) did not meet the set validation criteria but will be monitored for identification purposes (no comparison to a cut-off level). Blind internal proficiency testing was performed, where six OF samples were tested and analytes were classified as "negative", "likely positive" or "positive" with success. The final validated OF qualitative decision point method covers 92 analytes, and the presence of 5 additional analytes is screened in this high throughput analysis.

Qualitative threshold method validation and uncertainty evaluation: A theoretical framework and application to a 40 analytes liquid chromatography-tandem mass spectrometry method.

Qualitative methods hold an important place in drug testing, filling central needs in screening and analyses, among others, linked to per se legislation. Nevertheless, the bioanalytical method validation guidelines do not discuss this type of method or describe method validation procedures ill-adapted to qualitative methods. The output of qualitative methods are typically categorical, binary results, such as presence/absence or above cut-off/below cut-off. As the goal of any method validation is to demonstrate fitness for use under production conditions, qualitative validation guidelines should evaluate performance based on discrete, binary results instead of the continuous measurements obtained from the instrument (e.g. area). A tentative validation guideline for threshold qualitative methods was developed by in silico modelling of measurements and derived binary results. This preliminary guideline was applied to a liquid chromatography-tandem mass spectrometry method for 40 analytes, each with a defined threshold concentration. Validation parameters calculated from the analysis of 30 samples spiked above and below the threshold concentration (false negative rate, false positive rate, selectivity rate, sensitivity rate and reliability rate) showed a surprisingly high failure rate. Overall, 13 out of the 40 analytes were not considered validated. A subsequent examination found that this was attributable to an appreciable shift in the standard deviation of the area ratio on a day-to-day basis, a previously undescribed and unaccounted-for behaviour in the qualitative threshold method validation literature. Consequently, the developed guideline was modified and used to validate a qualitative threshold method, based on the binary results for performance evaluation and incorporating measurement uncertainty.

A Tool for Automatic Correction of Endogenous Concentrations: Application to BHB Analysis by LC-MS-MS and GC-MS.

Several substances relevant for forensic toxicology purposes have an endogenous presence in biological matrices: beta-hydroxybutyric acid (BHB), gamma-hydroxybutyric acid (GHB), steroids and human insulin, to name only a few. The presence of significant amounts of these endogenous substances in the biological matrix used to prepare calibration standards and quality control samples (QCs) can compromise validation steps and quantitative analyses. Several approaches to overcome this problem have been suggested, including using an analog matrix or analyte, relying entirely on standard addition analyses for these analytes, or simply ignoring the endogenous contribution provided that it is small enough. Although these approaches side-step the issue of endogenous analyte presence in spiked matrix-matched samples, they create serious problems with regards to the accuracy of the analyses or production capacity. We present here a solution that addresses head-on the problem of endogenous concentrations in matrices used for calibration standards and quality control purposes. The endogenous analyte concentration is estimated via a standard-addition type process. This estimated concentration, plus the spiked concentration are then used as the de facto analyte concentration present in the sample. These de facto concentrations are then used in data analysis software (MultiQuant, Mass Hunter, etc.) as the sample's concentration. This yields an accurate quantification of the analyte, free from interference of the endogenous contribution. This de facto correction has been applied in a production setting on two BHB quantification methods (GC-MS and LC-MS-MS), allowing the rectification of BHB biases of up to 30 µg/mL. The additional error introduced by this correction procedure is minimal, although the exact amount will be highly method-dependent. The endogenous concentration correction process has been automated with an R script. The final procedure is therefore highly efficient, only adding four mouse clicks to the data analysis operations.