Analytical considerations for postmortem metabolomics using GC-high-resolution MS
Metabolomics studies that aim to qualitatively and quantitatively characterize the entirety of small endogenous biomolecules in an organism are widely conducted in the clinical setting. They also become more and more popular in the field of forensics (toxicology), e.g., to assist in postmortem investigations by objective postmortem interval estimation. However, other issues in postmortem toxicology, such as the phenomenon of (time-dependent) postmortem redistribution, have not yet been tackled by metabolomics studies. Hence, the aim of the current study was to develop an (un)targeted gas chromatography-high-resolution mass spectrometry–based method for endogenous metabolites as a tool for large-scale (un)targeted human postmortem metabolomics investigations (e.g., to objectively assess PMR) with thorough analytical evaluation of this method to ensure fitness-to-purpose in terms of reliability and robustness. This was achieved by using a targeted metabolite subset (n = 56) and a targeted processing workflow. Evaluation experiments have shown that using an artificial matrix (revised simulated body fluid (rSBF) + 5% bovine serum albumin (BSA)) for calibration purposes, all parameters lay within the scope of the method (sensitivity, selectivity, calibration model, accuracy, precision, processed sample stability, and extraction efficiency). When applying this method to large-scale studies, samples should be run in randomized order if analysis time is expected to exceed 18–24 h and potential biomarkers that are found with this method should be verified by a specialized, targeted method (e.g., by using standard addition in authentic matrix for quantification purposes). Overall, the current method can be successfully used for conduction of time-dependent postmortem metabolomics investigations.
KeywordsPostmortem metabolomics GC-MS Forensic toxicology Method evaluation High resolution
The authors express their gratitude to Emma Louise Kessler, MD, for her generous legacy that she donated to the Institute of Forensic Medicine at the University of Zurich, Switzerland, for research purposes. The authors would like to thank the scientists from the Department of Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, for helpful discussions.
Compliance with ethical standards
The study was performed in full conformance with Swiss ethical laws, particularly those covering the use of human material in research. A waiver of the Cantonal Ethics Board of the Canton of Zurich was obtained (BASEC-Nr. Req-2017-00946) which states that small quantities of anonymized biological material obtained during investigations of the public prosecutor (e.g., during autopsies) can be used for research purposes without informed consent of the legally authorized representatives.
Conflict of interest
The authors declare that they have no conflict of interest.
- 4.Steuer AE, Brockbals L, Kraemer T. Metabolomic strategies in biomarker research–new approach for indirect identification of drug consumption and sample manipulation in clinical and forensic toxicology? Front Chem. 2019;7(319). https://doi.org/10.3389/fchem.2019.00319.
- 10.Kaszynski RH, Nishiumi S, Azuma T, Yoshida M, Kondo T, Takahashi M, et al. Postmortem interval estimation: a novel approach utilizing gas chromatography/mass spectrometry-based biochemical profiling. Anal Bioanal Chem. 2016;408(12):3103–12. https://doi.org/10.1007/s00216-016-9355-9.CrossRefPubMedGoogle Scholar
- 14.Brinkmann B, Madea B. Handbuch gerichtliche Medizin, vol. 2. Berlin Heidelberg NewYork: Springer-Verlag; 2004.Google Scholar
- 15.Danhof M, de Jongh J, De Lange EC, Della Pasqua O, Ploeger BA, Voskuyl RA. Mechanism-based pharmacokinetic-pharmacodynamic modeling: biophase distribution, receptor theory, and dynamical systems analysis. Annu Rev Pharmacol Toxicol. 2007;47:357–400. https://doi.org/10.1146/annurev.pharmtox.47.120505.105154.CrossRefPubMedGoogle Scholar
- 16.Peters FT, Steuer AE. Antemortem and postmortem influences on drug concentrations and metabolite patterns in postmortem species. WIREs Forensic Sci. 2019;1:e1297.Google Scholar
- 20.Staeheli SN, Poetzsch M, Kraemer T, Steuer AE. Development and validation of a dynamic range-extended LC-MS/MS multi-analyte method for 11 different postmortem matrices for redistribution studies applying solvent calibration and additional 13C isotope monitoring. Anal Bioanal Chem. 2015;407:8681–712. https://doi.org/10.1007/s00216-015-9023-5).CrossRefPubMedGoogle Scholar
- 21.Staeheli SN, Gascho D, Fornaro J, Laberke P, Ebert LC, Martinez RM, et al. Development of CT-guided biopsy sampling for time-dependent postmortem redistribution investigations in blood and alternative matrices--proof of concept and application on two cases. Anal Bioanal Chem. 2016;408(4):1249–58. https://doi.org/10.1007/s00216-015-9234-9.CrossRefPubMedGoogle Scholar
- 22.Staeheli SN, Gascho D, Ebert LC, Kraemer T, Steuer AE. Time-dependent postmortem redistribution of morphine and its metabolites in blood and alternative matrices-application of CT-guided biopsy sampling. Int J Legal Med. 2017;131(2):379–89. https://doi.org/10.1007/s00414-016-1485-2.CrossRefPubMedGoogle Scholar
- 26.Brockbals L, Habicht M, Hajdas I, Galassi FM, Rühli FJ, Kraemer T. Untargeted metabolomics-like screening approach for chemical characterization and differentiation of canopic jar and mummy samples from Ancient Egypt using GC-high resolution MS. The Analyst. 2018;143(18):4503–12. https://doi.org/10.1039/C8AN01288A.CrossRefPubMedGoogle Scholar
- 28.Peters FT, Hartung M, Herbold M, Schmitt G, Daltrup T, Musshoff F. Appendix B to the GTFCh guidelines for quality assurance in forensic-toxicological analyses. Requirements for the validation of analytical methods. Toxichem Krimtech. 2009;76:185.Google Scholar
- 29.Yang Y, Cruickshank C, Armstrong M, Mahaffey S, Reisdorph R, Reisdorph N. New sample preparation approach for mass spectrometry-based profiling of plasma results in improved coverage of metabolome. J Chromatogr A. 2013;1300:217–26. https://doi.org/10.1016/j.chroma.2013.04.030.CrossRefPubMedPubMedCentralGoogle Scholar
- 30.Bruce SJ, Tavazzi I, Parisod V, Rezzi S, Kochhar S, Guy PA. Investigation of human blood plasma sample preparation for performing metabolomics using ultrahigh performance liquid chromatography/mass spectrometry. Anal Chem. 2009;81(9):3285–96. https://doi.org/10.1021/ac8024569.CrossRefPubMedGoogle Scholar
- 34.Stuart B. Decomposition chemistry: overview, analysis, and interpretation A2 - Siegel, Jay A. In: Saukko PJ, Houck MM, editors. Encyclopedia of Forensic Sciences. Waltham: Academic Press; 2013. p. 11–5. https://doi.org/10.1016/B978-0-12-382165-2.00120-3.CrossRefGoogle Scholar
- 37.Hess C, Sydow K, Kueting T, Kraemer M, Maas A. Considerations regarding the validation of chromatographic mass spectrometric methods for the quantification of endogenous substances in forensics. Forensic Sci Int. 2018;283:150–5. https://doi.org/10.1016/j.forsciint.2017.12.019.CrossRefPubMedGoogle Scholar