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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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Abstract

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.

One-point and multi-point calibration and the resulting beta-tolerance intervals from method validation

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References

  1. Birkler RID, Telving R, Ingemann-Hansen O, Charles AV, Johannsen M, Andreasen MF. Screening analysis for medicinal drugs and drugs of abuse in whole blood using ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC–TOF-MS)—toxicological findings in cases of alleged sexual assault. Forensic Sci Int. 2012;222(1):154–61. https://doi.org/10.1016/j.forsciint.2012.05.019.

    Article  CAS  Google Scholar 

  2. EMCDDA. European drug report 2016—trends and developments. Lisbon: European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) 2016.

  3. Mounteney J, Griffiths P, Sedefov R, Noor A, Vicente J, Simon R. The drug situation in Europe: an overview of data available on illicit drugs and new psychoactive substances from European monitoring in 2015. Addiction. 2016;111(1):34–48. https://doi.org/10.1111/add.13056.

    Article  Google Scholar 

  4. EMCDDA. New Psychoactive substamces in Europe. An update from the EU early warning system. Lisbon: European Monitoring Centre for Drugs and Drug Addition (EMCDDA) 2015.

  5. Maurer HH. Multi-analyte procedures for screening for and quantification of drugs in blood, plasma, or serum by liquid chromatography-single stage or tandem mass spectrometry (LC-MS or LC-MS/MS) relevant to clinical and forensic toxicology. Clin Biochem. 2005;38(4):310–8. https://doi.org/10.1016/j.clinbiochem.2005.01.014.

    Article  CAS  Google Scholar 

  6. Steuer AE, Forss AM, Dally AM, Kraemer T. Method development and validation for simultaneous quantification of 15 drugs of abuse and prescription drugs and 7 of their metabolites in whole blood relevant in the context of driving under the influence of drugs-usefulness of multi-analyte calibration. Forensic Sci Int. 2014;244:92–101. https://doi.org/10.1016/j.forsciint.2014.08.022.

    Article  CAS  Google Scholar 

  7. Johansen SS, Bhatia HM. Quantitative analysis of cocaine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;852(1–2):338–44. https://doi.org/10.1016/j.jchromb.2007.01.033.

    Article  CAS  Google Scholar 

  8. Montenarh D, Hopf M, Maurer HH, Schmidt P, Ewald AH. Development and validation of a multi-analyte LC-MS/MS approach for quantification of neuroleptics in whole blood, plasma, and serum. Drug Test Anal. 2016;8(10):1080–9. https://doi.org/10.1002/dta.1923.

    Article  CAS  Google Scholar 

  9. Steuer AE, Poetzsch M, Koenig M, Tingelhoff E, Staeheli SN, Roemmelt AT, et al. Comparison of conventional liquid chromatography-tandem mass spectrometry versus microflow liquid chromatography-tandem mass spectrometry within the framework of full method validation for simultaneous quantification of 40 antidepressants and neuroleptics in whole blood. J Chromatogr A. 2015;1381:87–100. https://doi.org/10.1016/j.chroma.2014.12.084.

    Article  CAS  Google Scholar 

  10. Musshoff F, Trafkowski J, Kuepper U, Madea B. An automated and fully validated LC-MS/MS procedure for the simultaneous determination of 11 opioids used in palliative care, with 5 of their metabolites. J Mass Spectrom. 2006;41(5):633–40. https://doi.org/10.1002/jms.1021.

    Article  CAS  Google Scholar 

  11. Fernandez Mdel M, Wille SM, Kummer N, Di Fazio V, Ruyssinckx E, Samyn N. Quantitative analysis of 26 opioids, cocaine, and their metabolites in human blood by ultra performance liquid chromatography-tandem mass spectrometry. Ther Drug Monit. 2013;35(4):510–21. https://doi.org/10.1097/FTD.0b013e31828e7e6b.

    Article  Google Scholar 

  12. Bjork MK, Nielsen MK, Markussen LO, Klinke HB, Linnet K. Determination of 19 drugs of abuse and metabolites in whole blood by high-performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem. 2010;396(7):2393–401. https://doi.org/10.1007/s00216-009-3268-9.

    Article  Google Scholar 

  13. Simonsen KW, Hermansson S, Steentoft A, Linnet K. A validated method for simultaneous screening and quantification of twenty-three benzodiazepines and metabolites plus zopiclone and zaleplone in whole blood by liquid-liquid extraction and ultra-performance liquid chromatography- tandem mass spectrometry. J Anal Toxicol. 2010;34(6):332–41.

    Article  CAS  Google Scholar 

  14. Dussy FE, Hamberg C, Briellmann TA. Quantification of benzodiazepines in whole blood and serum. Int J Legal Med. 2006;120(6):323–30. https://doi.org/10.1007/s00414-005-0042-1.

    Article  Google Scholar 

  15. Peters FT, Hartung M, Herbold M, Schmitt G, Daldrup 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 

  16. Tissue BM. Basics of analytical chemistry and chemical equilibria. Hoboken: Wiley; 2013.

    Book  Google Scholar 

  17. Peters FT, Maurer HH. Systematic comparison of bias and precision data obtained with multiple-point and one-point calibration in six validated multianalyte assays for quantification of drugs in human plasma. Anal Chem. 2007;79(13):4967–76. https://doi.org/10.1021/ac070054s.

    Article  CAS  Google Scholar 

  18. Roemmelt AT, Steuer AE, Kraemer T. Liquid chromatography, in combination with a quadrupole time-of-flight instrument, with sequential window acquisition of all theoretical fragment-ion spectra acquisition: validated quantification of 39 antidepressants in whole blood as part of a simultaneous screening and quantification procedure. Anal Chem. 2015;87(18):9294–301. https://doi.org/10.1021/acs.analchem.5b02031.

    Article  CAS  Google Scholar 

  19. Huang Q, Yang L, Luo J, Guo L, Wang Z, Yang X, et al. SWATH enables precise label-free quantification on proteome scale. Proteomics. 2015;15(7):1215–23. https://doi.org/10.1002/pmic.201400270.

    Article  CAS  Google Scholar 

  20. Li CX, Tan XF, Lim TK, Lin QS, Gong ZY. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human. Sci Rep-Uk. 2016;6:24329. https://doi.org/10.1038/srep24329.

    Article  CAS  Google Scholar 

  21. Loke MF, Ng CG, Vilashni Y, Lim J, Ho B. Understanding the dimorphic lifestyles of human gastric pathogen Helicobacter pylori using the SWATH-based proteomics approach. Sci Rep-Uk. 2016;6:26784. https://doi.org/10.1038/srep26784.

    Article  CAS  Google Scholar 

  22. Vowinckel J, Capuano F, Campbell K, Deery M, Lilley K, Ralser M. The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics (version 2; referees: 2 approved). F1000Res. 2014;2:272.

    Google Scholar 

  23. Hopfgartner G, Tonoli D, Varesio E. High-resolution mass spectrometry for integrated qualitative and quantitative analysis of pharmaceuticals in biological matrices. Anal Bioanal Chem. 2012;402(8):2587–96. https://doi.org/10.1007/s00216-011-5641-8.

    Article  CAS  Google Scholar 

  24. Ma Y, Tanaka N, Vaniya A, Kind T, Fiehn O. Ultrafast polyphenol metabolomics of red wines using microLC-MS/MS. J Agric Food Chem. 2016;64(2):505–12. https://doi.org/10.1021/acs.jafc.5b04890.

    Article  CAS  Google Scholar 

  25. Liu Y, Huttenhain R, Collins B, Aebersold R. Mass spectrometric protein maps for biomarker discovery and clinical research. Expert Rev Mol Diagn. 2013;13(8):811–25. https://doi.org/10.1586/14737159.2013.845089.

    Article  CAS  Google Scholar 

  26. Ortea I, Rodríguez-Ariza A, Chicano-Gálvez E, Arenas Vacas MS, Jurado GB. Discovery of potential protein biomarkers of lung adenocarcinoma in bronchoalveolar lavage fluid by SWATH MS data-independent acquisition and targeted data extraction. J Proteome. 2016;138:106–14. https://doi.org/10.1016/j.jprot.2016.02.010.

    Article  CAS  Google Scholar 

  27. Sajic T, Liu Y, Aebersold R. Using data-independent, high-resolution mass spectrometry in protein biomarker research: perspectives and clinical applications. Proteomics Clin Appl. 2015;9(3–4):307–21. https://doi.org/10.1002/prca.201400117.

    Article  CAS  Google Scholar 

  28. Roemmelt AT, Steuer AE, Poetzsch M, Kraemer T. Liquid chromatography, in combination with a quadrupole time-of-flight instrument (LC QTOF), with sequential window acquisition of all theoretical fragment-ion spectra (SWATH) acquisition: systematic studies on its use for screenings in clinical and forensic toxicology and comparison with information-dependent acquisition (IDA). Anal Chem. 2014;86(23):11742–9. https://doi.org/10.1021/ac503144p.

    Article  CAS  Google Scholar 

  29. Uges D. Hospital toxicology. In: Moffat ACOM, Widdop B, Galichet LY, editors. Clarke’s analysis of drugs and poisons in pharmaceuticals, body fluids and postmortem material. 3rd ed. London, UK: Pharmaceutical Press; 2004.

    Google Scholar 

  30. Schulz M, Iwersen-Bergmann S, Andresen H, Schmoldt A. Therapeutic and toxic blood concentrations of nearly 1,000 drugs and other xenobiotics. Crit Care. 2012;16(4):R136. https://doi.org/10.1186/cc11441.

    Article  Google Scholar 

  31. Brunton L, Chabner B, Knollman B. Goodman and Gilman’s the pharmacological basis of therapeutics. twelfth ed. New York, NY: McGraw-Hill Education; 2011.

    Google Scholar 

  32. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75(13):3019–30.

    Article  CAS  Google Scholar 

  33. Meyer GMJ, Weber AA, Maurer HH. Development and validation of a fast and simple multi-analyte procedure for quantification of 40 drugs relevant to emergency toxicology using GC-MS and one-point calibration. Drug Testing and Analysis. 2014;6(5):472–81. https://doi.org/10.1002/dta.1555.

    CAS  Google Scholar 

  34. Ma Y-C, Wang C-W, Hung S-H, Chang Y-Z, Liu C-R, Her G-R. Estimation of the measurement uncertainty in quantitative determination of ketamine and Norketamine in urine using a one-point calibration method. J Anal Toxicol. 2012;36(7):515–22. https://doi.org/10.1093/jat/bks062.

    Article  CAS  Google Scholar 

  35. Remane D, Meyer MR, Wissenbach DK, Maurer HH. Ultra high performance liquid chromatographic-tandem mass spectrometric multi-analyte procedure for target screening and quantification in human blood plasma: validation and application for 31 neuroleptics, 28 benzodiazepines, and Z-drugs. Anal Bioanal Chem. 2011;401(4):1341. https://doi.org/10.1007/s00216-011-5187-9.

    Article  CAS  Google Scholar 

  36. Di Rago M, Saar E, Rodda LN, Turfus S, Kotsos A, Gerostamoulos D, et al. Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC–MS/MS. Forensic Sci Int. 2014;243:35–43. https://doi.org/10.1016/j.forsciint.2014.03.021.

    Article  Google Scholar 

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Acknowledgments

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.

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Authors and Affiliations

  1. Department of Forensic Pharmacology and Toxicology, Institute of Forensic Medicine, University of Zurich, Winterthurerstrasse 190/52, 8057, Zurich, Switzerland

    Marco P. Elmiger, Michael Poetzsch, Andrea E. Steuer & Thomas Kraemer

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  1. Marco P. Elmiger
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  2. Michael Poetzsch
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  3. Andrea E. Steuer
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Corresponding author

Correspondence to Thomas Kraemer.

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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.

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Elmiger, M., Poetzsch, M., Steuer, A.E. et al. 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. Anal Bioanal Chem 409, 6495–6508 (2017). https://doi.org/10.1007/s00216-017-0594-1

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