Identification criteria, in-house compound libraries and SWATH: A benchmark implementation of quadrupole time-of-flight mass spectrometry in routine toxicological casework

Abstract

To establish the identification criteria sufficient for the daily and routine reporting of compounds using Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) in forensic casework. Liquid chromatography coupled with High Resolution Mass Spectrometry (LC-HRMS) provides sensitive full-spectrum MS and MS/MS data for the identification of known and previously unknown compounds. However, the daily and routine implementation of methods that use LC-HRMS techniques in routine analysis can be challenging due to the establishment of identification criteria and confidence in reporting suspected positive detections. With the large amount of data that is collected during SWATH, it is important to have a well-developed and validated identification criterion, that is also high throughput. To identify previously published identification criterion, literature searches were conducted for the keywords Quadrupole Time of Flight, Toxicology, QTOF, Forensic Toxicology, Sequential Window Acquisition, SWATH and Mass Spectrometry. A total of 11 articles were evaluated regarding the use of SWATH in clinical or forensic toxicology. Based off these findings, we performed internal studies to validate identification criteria. Curation of an internal library were conducted using available reference standards. For compound identification and confirmation, criterion acceptability of exact mass ±5 ppm, retention time (RT) ±0.5 min, and library match for MS and MSMS including comparison of isotopic pattern and product ion pattern to the library. A combined weighted score (CWS) for each candidate species was calculated based on these criteria with a reporting score of >75% set. In reviewing the literature of SWATH methods that have already been developed, it has been found that the most common target drug classes are stimulants, benzodiazepines, opiates, NPS’, cannabinoids and anti-depressants. In the acquisition process, the number of SWATH windows ranges from 12-30. Mass error, isotopic ratio difference, retention time error and library scores were used as the identification criteria for all studies. HRMS acquisition is data-rich, voluminous and complicated. We have adopted categories to delineate the identification and reportion of compounds through the QTOF Method: Target Qualitative, Target Quantitative, Target Monitored, Suspect Screening and Unknown Screening. Each category must pass a pre-set identification criterion that resulted from identification criteria studies. The described method uses five levels of identification and confirmation, and three types of mass spectral libraries consisting of a main of an in-house curated library, updated quarterly. New targets are added through a vigorous process including interference studies, as many NPS show similar fragmentation and identical molecular weights. Interference studies may result in the grouping of compounds that have the same exact mass, identical fragmentation, and/or small retention time differences. For HRMS analysis, robust assessment of accurate mass information and reference to in-house libraries are essential in the accurate identification and reporting of drugs in forensic toxicological casework.