Highly sensitive MS/MS detection for confident identification of potent novel synthetic opioids and their metabolites

Abstract

The aim of this presentation is to demonstrate the use of high-resolution mass spectrometry as a unique tool for the untargeted detection and identification of new emerging substances, with little or no method optimization necessary. The presentation will demonstrate how accurate mass instruments are providing extra levels of confidence in analyte identification by reliably obtaining comprehensive MS/MS spectral fragment information that can be used for identification, confirmation, and/or library matching of potent drugs and metabolites present in discarded authentic case samples. NSO compounds were extracted from human whole blood using a liquid-liquid extraction (LLE) procedure. HPLC separation was performed on an ExionLC system using a Phenomenex Kinetex C18 column Mobile phases were ammonium formate and formic acid in methanol and acetonitrile. The flow rate was 0.4 mL/min with a total LC runtime of 15.5 minutes. The injection volume was 10 μL. MS and MS/MS data were collected for each sample using Zeno IDA for optimal sensitivity on the ZenoTOF 7600 system. Data acquisition consisted of a TOF MS scan to collect accurate mass precursor ions from 100 to 700 Da, followed by a TOF MS/MS full scan ranging from 25 to 700 Da to ensure all fragments were captured for identification using a maximum of 16 candidate ions. Data was acquired using SCIEX OS software 2.1. A series of 9 calibrator solutions were prepared by spiking control human whole blood samples with the 6 targeted analytes at final concentrations ranging from 10 pg/mL to 100 ng/mL. The series of calibrator solutions were injected to evaluate the quantitative performance of the system and its ability to accurately measure low level analytes with a high level of precision and accuracy in TOF MS mode. Each calibrator was injected in triplicate. LLOQs of between 10 and 50 pg/ml were observed for all compounds analysed. Accuracies at LLOQ ranged from 86–111% and imprecision was < 15% for all compounds analysed. In addition, the use of specific technologies afforded by the new SCIEX ZenoTOF 7600 system generated an average 9x sensitivity improvement on spectral data providing significantly improved library matching. A comprehensive and highly sensitive method for the screening and identification of potent NSO in human whole blood is described. The significant gains in MS/MS sensitivity on the SCIEX ZenoTOF 7600 system yielded an improvement in confident identifications of low-level analytes through spectral library matching. The observed sensitivity gains resulted in a 9x improvement, on average, in TOF MS/MS sensitivity across the drugs positively identified in the authentic case samples analyzed. This improvement enabled confident identification of key drugs and metabolites at trace levels that were not previously achievable. The MS/MS sensitivity levels afforded by ZenoTOF 7600 system provide a means to monitor low levels of ultra-potent NSO in poly-drug intake scenarios.