B-184 Sequential window acquisition of all theoretical spectra (SWATH) Acquisition on a linear ion trap pulsing quadrupole time-of-flight mass spectrometer improves sensitivity and selectivity in comprehensive urine drug testing

Background Liquid chromatography high-resolution mass spectrometry (LC-HRMS) on quadrupole time-of-flight (Q-TOF) systems is an established approach for general unknown drug screening and drug supply monitoring in clinical settings. This method enables identification of unknown compounds and retro-active searching for emerging illicit drugs by collecting full-scan mass spectra and tandem mass spectra (MS/MS) of high abundance compounds in patient samples. However, Q-TOF LC-HRMS systems suffer from limited intra-scan dynamic range in complex matrices like urine. In addition, information-dependent acquisition (IDA), which is the most used acquisition strategy for MS/MS spectra, is biased towards highly abundant toxicological agents or matrix constituents. Together, these factors can lead to false-negative results for lower abundance or poorly ionizing targets. To address these limitations, we investigated the value of the ZenoTOF 7600 HRMS platform (Sciex), which implements linear ion trap pulsing (i.e., Zeno trap pulsing) to improve the sensitivity of MS/MS acquisition. Further, we investigated the value of Sequential Window Acquisition of all Theoretical Spectra (SWATH) acquisition on this platform (i.e., Zeno-SWATH), which can potentially improve sensitivity by guaranteeing MS/MS acquisition for all compounds within a defined mass range. Methods To validate limits of detection (LODs), 150 toxicological compounds in three drug cocktails were spiked into drug-free urine at concentrations ranging from 1-500 ng/mL and diluted in triplicate before untargeted LC-HRMS investigation. Full-scan high-resolution mass spectra was acquired for all precursors between 125-650 m/z, and MS/MS were acquired using either Zeno-SWATH or Zeno-IDA modes (Acc. Time: 25 ms, Cycle Time: 1.25 s). Library spectra were generated by analyzing each compound in pure solvent using the MRM-HR acquisition mode. The resulting data was analyzed in SciexOS, and relevant spectra were selected for library searching based on specified retention time and extracted ion chromatogram parameters. The LOD for each compound was set as the lowest concentration at which a confident library hit was generated for all replicates. Interferences were evaluated by cross comparison with drug cocktails not containing the drug of interest, and matrix effects assessed by spiking compounds into several different blank urine matrices and at high and low concentrations, and comparing peak areas to matrix-free samples. Results Zeno-SWATH acquisition enabled the identification of more toxicological agents than Zeno-IDA at every concentration evaluated (See attached figure). In particular, Zeno-SWATH acquisition enabled the detection of 42 toxicological compounds at 1 ng/mL, while Zeno-IDA did not detect any compounds at this threshold. Furthermore, over half of the analyzed compounds exhibited LODs at least 5-fold lower when using Zeno-SWATH than when using Zeno-IDA. Interferences for both methods consisted exclusively of misidentification of closely eluting, isobaric compounds with highly similar MS/MS spectra; Zeno-IDA misidentified 8 compounds, while Zeno-SWATH acquisition misidentified 6 compounds. Matrix effects were similar between the two methods. Conclusion Linear ion trap pulsing on the Sciex 7600 ZenoTOF system, in combination with SWATH acquisition markedly improves the overall sensitivity of an LC-HRMS comprehensive urine drug test with validated 150 toxicological agents. In addition, mild selectivity improvements were documented in Zeno-SWATH mode.