High-resolution mass spectrometry screening in forensic toxicology

Abstract

Objective

This presentation aims to introduce high-resolution mass spectrometry (HRMS) screening techniques to new users with forensic toxicology applications.

Introduction

HRMS is becoming an increasingly used technique in forensic toxicology. This technique offers essential advantages compared to classic screening techniques in toxicology, such as immunoassays and gas chromatography-mass spectrometry. HRMS has increased specificity, sensitivity, and versatility. It can be employed in targeted screening, suspect screening, tentative identification, structure elucidation, and quantitative and confirmation methods.

Methods

This presentation will explore all the steps involved in developing, employing, and validating HRMS screening techniques, including sample preparation procedures, chromatographic separation, HRMS instrumentation and data acquisition modes, data analysis and interpretation, and validation parameters.

Results and discussion

In this screening technique, biological samples must be extracted to remove interferences and matrix components that may affect the analysis and instrument performance (downtime). The sample preparation should be unspecific to avoid losing analytes while still removing matrix components. Protein precipitation and dilute-and-shoot procedures are frequently used. Regarding chromatographic separation, reversed-phase columns such as C18 or phenylhexyl and mobile phases with formic acid, methanol, or acetonitrile are preferred. The gradient should be wide enough to allow the elution of compounds with different polarities. The current instrumentation combines a quadrupole (Q) technology with high-resolution TOF (QTOF) or orbitrap (QOT) mass filters, allowing the use of tandem mass spectrometry (MS/MS) detection modes. Data acquisition can be performed in data-dependent (DDA) or data-independent (DIA) mode, depending on the selection or not of a specific precursor before performing a product ion scan. DDA is commonly used in targeted screening and suspect screening, and DIA in non-target analysis. DDA data analysis usually is easier and faster than DIA. HRMS screening methods must be validated; however, their validation should include specific parameters of interest for this technique, such as inter-day reproducibility of the mass spectra that may affect library match and incurred sample reanalysis.

Conclusion

HRMS is becoming the state-of-the-art technique for screening analysis in clinical and forensic toxicology. This presentation will help toxicologists new to HRMS understand the technique and its advantages and address practical issues in its application.