Reaching new sensitivity levels for the detection of fentanyl analogs and highly potent novel synthetic opioids in blood

Abstract

The aim of this presentation is to demonstrate a sensitive quantitative workflow for the analysis of 32 of potent substances in poly-drug, discarded postmortem case samples at low (pg/mL) levels. The presentation will demonstrate that the unparalleled quantification performance of the method enabled accurate detection of potent substances in poly-drug, discarded postmortem case samples at trace levels that were not previously achievable, providing toxicologists a valuable insight into the causation of accidental overdoses. A total of 32 NSO including 17 fentanyl analogs and 15 newly emerging non-fentanyl opioids were selected for this panel. NSO were extracted from the 200 μL of spiked whole blood mixtures using a liquid-liquid extraction (LLE) procedure. Analytes were chromatographically separated using a Phenomenex Kinetex C18 column (50 × 3.0 mm, 2.6 μm, 00B-4462-Y0). Mobile phase A (MPA) and mobile phase B (MPB) were ammonium formate with formic acid and formic acid in methanol and acetonitrile, respectively. The injection volume was 10 μL and the total LC runtime was 15.5 minutes. A single acquisition method consisting of 68 MRM transitions (64 for the NSO and 4 for the internal standards) was created using the Scheduled MRM Algorithm. Control human whole blood samples spiked with the 32 target analytes were prepared at concentrations ranging from 1 pg/mL to 100 ng/mL. Excellent linearity was observed across the concentration ranges analyzed with R2 values greater than 0.99 for all of the NPS in the panel. Reported LLOQ values ranged between 10 to 50 pg/mL for the 32 analytes in the panel. The accuracy and precision of measurements ranged from 80.50–116.64% and from 0.42–17.80%, respectively. The excellent accuracy and precision were observed over the entire concentration range, including at the LLOQ. Overall, the developed method showed excellent reproducibility and linearity, proving the robustness of the developed method and quantitative performance of the SCIEX 7500 System even at low concentration levels for each of the targeted NSO in this study. The applicability of the method to analyze discarded postmortem case samples from subjects suspected of NPS ingestion resulting in accidental overdoses was investigated. For example, the results from the analysis of one of these case samples showed the successful detection of one NSO and its metabolite: isotonitazene and 5-aminoisotonitazene, as well as fentanyl and four of its analogs/metabolites: beta-hydroxy-fentanyl, norfentanyl, 4-ANPP and acetyl fentanyl at concentrations of 1434.33, 7.93, 599.10, 9756.67, 147.69 and 1465.00 pg/mL, respectively. The results demonstrate that the developed method enabled accurate detection of potent substances in poly-drug, authentic case samples at trace levels that were not previously achievable. A robust and sensitive drug screening workflow for the analysis of 32 potent NSO was successfully developed using the SCIEX 7500 System. Overall, the remarkable quantification performance of the SCIEX 7500 System enabled accurate detection of potent NSO at concentrations that were not previously achievable, providing a means to monitor ultra-potent NSO in overdose scenarios. The sensitivity levels afforded by the SCIEX 7500 system provided the ability to detect low levels of NSO in postmortem case samples that would normally go undetected, providing a clearer picture for help in determining the cause of death.