Assessment of simpler calibration models in the development and validation of a fast postmortem multi-analyte LC-QTOF quantitation method in whole blood with simultaneous screening capabilities using SWATH acquisition
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In postmortem toxicology, fast methods can provide a triage to avoid unnecessary autopsies. Usually, this requires multiple qualitative and quantitative analytical methods. The aim of the present study was to develop a postmortem LC-QTOF method for simultaneous screening and quantitation using easy sample preparation and reduced alternative calibration models. Hence, a method for 24 highly relevant substances in forensic toxicology was fully validated using the following calibration models: one-point external, one-point internal via corresponding deuterated standards, multi-point external daily calibration, and multi-point external weekly calibration. Two hundred microliters of postmortem blood were spiked with internal deuterated standard mixture and extracted by acetonitrile protein precipitation. Analysis was performed on a Sciex 6600 QTOF instrument with ESI+ mode using data-independent acquisition (DIA) namely sequential window acquisition of all theoretical mass spectra (SWATH). Validation of the different calibration models included selectivity, autosampler stability, recovery, matrix effects, accuracy, and precision for 24 substances. In addition, corresponding deuterated analogs of 52 substances were included to the internal standard mix for semi-quantitative concentration assessment. The simple protein precipitation provided recoveries higher than 55 and 75% for all analytes at low and high concentrations, respectively. Accuracy and precision criteria (bias and imprecision ± 15 and ± 20% near the limit of quantitation) were fulfilled by the different calibration models for most analytes. The validated method was successfully applied to more than 100 authentic postmortem samples and 3 proficiency tests. Furthermore, the one-point internal calibration via corresponding deuterated standard proved to be a considerably time saving technique for 76 analytes.
KeywordsLC-ESI-QTOF SWATH One-point internal calibration Postmortem toxicology Calibration models
The authors express their gratitude to Emma Louise Kessler, MD for her generous legacy she donated to the Institute of Forensic Medicine at the University of Zurich, Switzerland for research purposes.
Compliance with ethical standards
Authentic human blood samples were collected and analyzed anonymously from healthy volunteers who provided written informed consent. Postmortem blood samples were used after anonymization. According to Swissethics (Human Research Act, HRA), no further ethical approval from the cantonal ethic commission is necessary if the research is not aiming to investigate diseases or functions of the human body as has been the case in the current study.
Conflict of interest
The authors declare that they have no potential conflict of interest.
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