A Fully Validated LC-QTOF-MS Screening Workflow for the Analysis of Drugs in Oral Fluid

Abstract A simple liquid-liquid extraction procedure (LLE) followed by liquid chromatography quadrupole time of flight tandem mass spectrometry (LC-QTOF-MS) analysis for drugs in oral fluid collected with the Quantisal™ device has been developed. The decision point cut-off concentrations were at or below those recommended by the National Safety Council’s Alcohol, Drugs, and Impairment Division (NSC-ADID) for toxicological investigation of driving under the influence of drugs cases (DUID). Currently ANSI/ASB standard 120 does not cover the analysis of oral fluid collected in impaired driving investigations, instead guidance from the NSC-ADID was used. The supporting mass spectral based screening library was adapted from commercially available databases and included Tier 1 and Tier 2 recommended compounds. Further, the additional inclusion of novel psychoactive substances and synthetic cannabinoids was based on the Center for Forensic Science Research and Education’s (CFSRE) quarterly publications of 2023. Metabolites from those publications were not included in this method since, with some exceptions, parent drugs are the dominant compounds in oral fluid. Briefly, Quantisal™ (1mL) was mixed with organic solvents, centrifuged, and decanted; followed by a second liquid-liquid process which extracted all the drugs in a single aliquot. A gradient liquid chromatography program using 0.1% formic acid in water and 0.1% formic acid in methanol was used and the runtime was 10 minutes. LC-QTOF-MS settings were optimized to promote greater sensitivity for a wide range of drug classes. The method was fully validated using ANSI/ASB 036 Standard Practices for Method Validation in Forensic Toxicology as guidance. Interference studies, limit of detection, precision at and around the decision points, ionization suppression/enhancement, and processed sample stability up to 96 hours were completed for each drug in the library database. While ion suppression or enhancement of the analytes varied greatly, the decision point was not significantly affected and internal standards that mimicked similar responses were chosen for each analyte. The method was applied to proficiency program samples, routine samples received into the laboratory, and blind samples screened against the search engine. The optimization of specific tune characteristics and instrument settings allowed the user to meet or exceed recommended screening limits for drugs in Quantisal™ collected oral fluid samples without the need for immunoassay testing.