Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Quantitative determination of phosphatidylethanol in dried blood spots for monitoring alcohol abstinence

Nature Protocols volume 16, pages 283–308 (2021)Cite this article

Subjects

Abstract

Phosphatidylethanol (PEth), which is formed by enzymatic reaction between ethanol and phosphatidylcholine, is a direct marker for alcohol usage. PEth has a long elimination half-life (~5–10 d) and specimens can be sampled using minimally invasive microsampling strategies. In combination with rapid analysis procedures PEth has proved to be advantageous for the detection of abstinence over other direct (e.g., ethyl glucuronide in blood, urine or hair) and indirect (e.g., carbohydrate-deficient transferrin in serum) alcohol markers. Although PEth determination is widely applied around the world, laboratory protocols are not standardized. Here we provide general guidelines for the analysis of PEth in dried blood spots (DBSs), including reference material evaluation, synthesis of a deuterated internal standard, preparation of calibration samples (reference material in teetotaller blood), and analyte separation and detection. The protocol contains information to extract the DBSs either manually or with a fully automated autosampler. Extraction of the analytes from DBS filter paper cards is performed using an organic extraction, followed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). For accurate and reliable measurement of PEth, the two most abundant analogs, PEth 16:0/18:1 and PEth 16:0/18:2, are quantified. We show data that provide guidelines on how to interpret the results for both demographic studies and forensic applications. The described protocol can be applied by experienced laboratory staff with basic LC–MS/MS knowledge and takes 2 d to perform.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: PEth threshold and decision limit, based on a normal distribution (symmetrical) of measurement results.
Fig. 2: Flowchart of the protocol.
Fig. 3: Trapping column installed at the back of the CAMAG DBS-MS 500 instrument.
Fig. 4: LC setup for the online SPE using a ten-port switching valve to switch between the trapping column (orange) and the analytical column (red): the trap is connected to the waste when the valve is set to 10_1; the trap is connected with the analytical column when the valve is set to 1_2.
Fig. 5: LC–MS/MS chromatogram for a blank blood sample spiked at 750 ng/mL recorded on a Sciex 5500 QTrap.
Fig. 6: DBS card placed with two DBS spotted inside the dashed circles.
Fig. 7: Signal intensities monitored for the PEth 16:0/18:1 MRM transition 701.3 → 255.2, recorded on a Sciex 5500 QTrap.
Fig. 8: Linear regression analysis of the calibrator’s peak area.

Similar content being viewed by others

Data availability

Previously published articles contain a summarized evaluation of the data obtained. In collaboration with the University of California, San Francisco (Robin Fatch and Judy Hahn), we recently (21 October 2020) submitted to a journal a manuscript with the title ‘Comparisons of automated determination of phosphatidylethanol (PEth) in dried blood spots (DBS) with previous manual processing and testing’. This manuscript contains further data supporting the use of the presented method.

References

  1. Varga, A., Hansson, P., Johnson, G. & Alling, C. Normalization rate and cellular localization of phosphatidylethanol in whole blood from chronic alcoholics. Clin. Chim. Acta 299, 141–150 (2000).

    Article  CAS  Google Scholar 

  2. Gnann, H. et al. Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS. Anal. Bioanal. Chem. 396, 2415–2423 (2010).

    Article  CAS  Google Scholar 

  3. Simon, T. W. Providing context for phosphatidylethanol as a biomarker of alcohol consumption with a pharmacokinetic model. Regul. Toxicol. Pharmacol. 94, 163–171 (2018).

    Article  CAS  Google Scholar 

  4. Javors, M. A., Hill-Kapturczak, N., Roache, J. D., Karns-Wright, T. E. & Dougherty, D. M. Characterization of the pharmacokinetics of phosphatidylethanol 16:0/18:1 and 16:0/18:2 in human whole blood after alcohol consumption in a clinical laboratory study. Alcohol. Clin. Exp. Res. 40, 1228–1234 (2016).

    Article  CAS  Google Scholar 

  5. Luginbühl, M., Weinmann, W., Butzke, I. & Pfeifer, P. Monitoring of direct alcohol markers in alcohol use disorder patients during withdrawal treatment and successive rehabilitation. Drug Test. Anal. 11, 859–869 (2019).

    Article  Google Scholar 

  6. Faller, A. et al. Stability of phosphatidylethanol species in spiked and authentic whole blood and matching dried blood spots. Int. J. Legal Med. 127, 603–610 (2013).

    Article  Google Scholar 

  7. Kummer, N. et al. Quantification of phosphatidylethanol 16:0/18:1, 18:1/18:1, and 16:0/16:0 in venous blood and venous and capillary dried blood spots from patients in alcohol withdrawal and control volunteers. Anal. Bioanal. Chem. 408, 825–38 (2016).

    Article  CAS  Google Scholar 

  8. Luginbühl, M., Gaugler, S. & Weinmann, W. Fully automated determination of phosphatidylethanol 16:0/18:1 and 16:0/18:2 in dried blood spots. J. Anal. Toxicol. 43, 489–496 (2019).

    Article  Google Scholar 

  9. Tanna, S. & Lawson, G. Self-sampling and quantitative analysis of DBS: can it shift the balance in over-burdened healthcare systems? Bioanalysis 7, 1963–1966 (2015).

    Article  CAS  Google Scholar 

  10. Luginbühl, M., et al. Variation in the relative isomer abundance of synthetic and biologically derived phosphatidylethanols and its consequences for reliable quantification. J. Anal. Toxicol. https://doi.org/10.1093/jat/bkaa034 (2020).

  11. Schröck, A., Wurst, F. M., Thon, N. & Weinmann, W. Assessing phosphatidylethanol (PEth) levels reflecting different drinking habits in comparison to the alcohol use disorders identification test–C (AUDIT-C). Drug Alcohol Depend. 178, 80–86 (2017).

    Article  Google Scholar 

  12. Schröck, A., Pfäffli, M., König, S. & Weinmann, W. Application of phosphatidylethanol (PEth) in whole blood in comparison to ethyl glucuronide in hair (hEtG) in driving aptitude assessment (DAA). Int. J. Legal Med. 130, 1527–1533 (2016).

    Article  Google Scholar 

  13. Schröck, A., Thierauf-Emberger, A., Schürch, S. & Weinmann, W. Phosphatidylethanol (PEth) detected in blood for 3 to 12 days after single consumption of alcohol—a drinking study with 16 volunteers. Int. J. Legal Med. 131, 153–160 (2017).

    Article  Google Scholar 

  14. Schröck, A., Thierauf, A., Wurst, F. M., Thon, N. & Weinmann, W. Progress in monitoring alcohol consumption and alcohol abuse by phosphatidylethanol. Bioanalysis 6, 2285–2294 (2014).

    Article  Google Scholar 

  15. Luginbühl, M., Willem, S., Schürch, S. & Weinmann, W. Formation of phosphatidylethanol from endogenous phosphatidylcholines in animal tissues from pig, calf, and goat. Forensic Sci. Int. 283, 211–218 (2018).

    Article  Google Scholar 

  16. Luginbuhl, M. & Gaugler, S. The application of fully automated dried blood spot analysis for liquid chromatography-tandem mass spectrometry using the CAMAG DBS-MS 500 autosampler. Clin. Biochem. 82, 33–39 (2020).

    Article  CAS  Google Scholar 

  17. Andresen-Streichert, H. et al. Improved detection of alcohol consumption using the novel marker phosphatidylethanol in the transplant setting: results of a prospective study. Transpl. Int. 30, 611–620 (2017).

    Article  CAS  Google Scholar 

  18. Fleming, M. F. et al. Phosphatidylethanol detects moderate-to-heavy alcohol use in liver transplant recipients. Alcohol. Clin. Exp. Res. 41, 857–862 (2017).

    Article  CAS  Google Scholar 

  19. Schröck, A., Hernández Redondo, A., Martin Fabritius, M., König, S. & Weinmann, W. Phosphatidylethanol (PEth) in blood samples from “driving under the influence” cases as indicator for prolonged excessive alcohol consumption. Int. J. Legal Med. 130, 393–400 (2016).

    Article  Google Scholar 

  20. Ulwelling, W. & Smith, K. The PEth blood test in the security environment: what it is; why it is important; and interpretative guidelines. J. Forensic Sci. 63, 1634–1640 (2018).

    Article  Google Scholar 

  21. Helander, A. & Hansson, T. National harmonization of the alcohol biomarker PEth. Lakartidningen 110, 1747–1748 (2013).

    PubMed  Google Scholar 

  22. World Anti-Doping Agency. WADA Technical Document TD2019DL v. 2.0. https://www.wada-ama.org/sites/default/files/resources/files/td2019dl_v2_finalb.pdf (2019).

  23. Cowan, J. M., Weathermon, A., McCutcheon, J. R. & Oliver, R. D. Determination of volume of distribution for ethanol in male and female subjects. Anal. Toxicol. 20, 287–290 (1996).

    Article  CAS  Google Scholar 

  24. Wurst, F. M. et al. Ethanol metabolites: their role in the assessment of alcohol intake. Alcohol. Clin. Exp. Res. 39, 2060–2072 (2015).

    Article  CAS  Google Scholar 

  25. Hartmann, S. et al. Phosphatidylethanol as a sensitive and specific biomarker—comparison with gamma-glutamyl transpeptidase, mean corpuscular volume and carbohydrate-deficient transferrin. Addict. Biol. 12, 81–84 (2007).

    Article  CAS  Google Scholar 

  26. Stewart, S. H., Koch, D. G., Willner, I. R., Anton, R. F. & Reuben, A. Validation of blood phosphatidylethanol as an alcohol consumption biomarker in patients with chronic liver disease. Alcohol. Clin. Exp. Res. 38, 1706–1711 (2014).

    Article  CAS  Google Scholar 

  27. Afshar, M. et al. Cut-point levels of phosphatidylethanol to identify alcohol misuse in a mixed cohort including critically ill patients. Alcohol. Clin. Exp. Res. 41, 1745–1753 (2017).

    Article  CAS  Google Scholar 

  28. Aradottir, S., Asanovka, G., Gjerss, S., Hansson, P. & Alling, C. Phosphatidylethanol (PEth) concentrations in blood are correlated to reported alcohol intake in alcohol-dependent patients. Alcohol Alcohol. 41, 431–437 (2006).

    Article  CAS  Google Scholar 

  29. Luginbühl, M., Bekaert, B., Suesse, S. & Weinmann, W. Detox shampoos for EtG and FAEE in hair—results from in vitro experiments. Drug Test. Anal. 11, 870–877 (2019).

    Article  Google Scholar 

  30. Luginbühl, M., Nussbaumer, S. & Weinmann, W. Decrease of ethyl glucuronide concentrations in hair after exposure to chlorinated swimming pool water. Drug Test. Anal. 10, 689–693 (2018).

    Article  Google Scholar 

  31. Reisfield, G. M. et al. The roles of phosphatidylethanol (PEth), ethyl glucuronide (EtG), and ethyl sulfate (EtS) in identifying alcohol consumption among participants in professionals’ health programs. Drug Test. Anal. 12, 1102–1108 (2020).

    Article  CAS  Google Scholar 

  32. Jones, J., Jones, M., Plate, C. & Lewis, D. The detection of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol in human dried blood spots. Anal. Methods 3, 1101–1106 (2011).

    Article  CAS  Google Scholar 

  33. Wang, E. J., et al. HemaApp: noninvasive blood screening of hemoglobin using smartphone cameras. in UbiComp 2016—Proceedings of the 2016 ACM International Joint Conference on Pervasive and Ubiquitous Computing 593–604 (2016).

  34. Capiau, S., Wilk, L. S., De Kesel, P. M. M., Aalders, M. C. G. & Stove, C. P. Correction for the hematocrit bias in dried blood spot analysis using a nondestructive, single-wavelength reflectance-based hematocrit prediction method. Anal. Chem. 90, 1795–1804 (2018).

    Article  CAS  Google Scholar 

  35. Oostendorp, M., El Amrani, M., Diemel, E. C., Hekman, D. & Van Maarseveen, E. M. Measurement of hematocrit in dried blood spots using near-infrared spectroscopy: robust, fast, and nondestructive. Clin. Chem. 62, 1534–1536 (2016).

    Article  Google Scholar 

  36. Velghe, S., Delahaye, L. & Stove, C. P. Is the hematocrit still an issue in quantitative dried blood spot analysis? J. Pharm. Biomed. Anal. 163, 188–196 (2019).

    Article  CAS  Google Scholar 

  37. Abu-Rabie, P., Denniff, P., Spooner, N., Chowdhry, B. Z. & Pullen, F. S. Investigation of different approaches to incorporating internal standard in DBS quantitative bioanalytical workflows and their effect on nullifying hematocrit-based assay bias. Anal. Chem. 87, 4996–5003 (2015).

    Article  CAS  Google Scholar 

  38. Schröck, A., Henzi, A., Bütikofer, P., König, S. & Weinmann, W. Determination of the formation rate of phosphatidylethanol by phospholipase D (PLD) in blood and test of two selective PLD inhibitors. Alcohol 73, 1–7 (2018).

    Article  Google Scholar 

  39. Thompson, P. M. et al. Phosphatidylethanol in postmortem brain and serum ethanol at time of death. Alcohol. Clin. Exp. Res. 40, 2557–2562 (2016).

    Article  CAS  Google Scholar 

  40. Weinmann, W., Schröck, A. & Wurst, F. M. Commentary on the paper of Thompson P. et al.: Phosphatidylethanol in postmortem brain and serum ethanol at time of death. Alcohol. Clin. Exp. Res. 41, 501–503 (2017).

    Article  CAS  Google Scholar 

  41. Gustavsson, L. & Alling, C. Formation of phosphatidylethanol in rat brain by phospholipase D. Biochem. Biophys. Res. Commun. 142, 958–963 (1987).

    Article  CAS  Google Scholar 

  42. Gomez-Cambronero, J. and Henkels, K. M. in Encyclopedia of Signaling Molecules (ed. Choi, S.) 3999–4010 (Springer International Publishing, 2018).

  43. Gu, H., Liu, G., Wang, J., Aubry, A.-F. & Arnold, M. E. Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance. Anal. Chem. 86, 8959–8966 (2014).

    Article  CAS  Google Scholar 

  44. US Food and Drug Administration. Bioanalytical Method Validation: Guidance for Industry. https://www.fda.gov/media/70858/download (2018).

Download references

Author information

Authors and Affiliations

  1. CAMAG DBS Laboratory, CAMAG, Muttenz, Switzerland

    Marc Luginbühl & Stefan Gaugler

  2. Institute of Forensic Medicine Bern, University of Bern, Bern, Switzerland

    Frederike Stöth & Wolfgang Weinmann

  3. Institute of Forensic Medicine Aargau, Kantonsspital Aarau, Aarau, Switzerland

    Alexandra Schröck

Authors
  1. Marc Luginbühl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frederike Stöth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexandra Schröck
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stefan Gaugler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wolfgang Weinmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.L. initiated the project. M.L., S.G. and W.W. planned and developed the project. A.S. initiated PEth measurements at the Institute of Forensic Medicine in Bern during her PhD thesis and wrote the first standard operating procedure for PEth analysis. M.L., S.G. and W.W. developed the fully automated method for PEth determination. F.S. has been responsible for the long-term application of the fully automated method for scientific research and analysing routine samples during the last 1.5 years of her PhD thesis. M.L., F.S., A.S., S.G. and W.W. wrote the manuscript; all authors contributed to the writing. M.L. and W.W. supervised the project.

Corresponding authors

Correspondence to Marc Luginbühl or Wolfgang Weinmann.

Ethics declarations

Competing interests

M.L. and S.G. are employees of CAMAG, which focuses on providing fully automated DBS analysis solutions.

Additional information

Peer review information Nature Protocols thanks Gisela Skopp, Christophe Stove and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Luginbühl, M. et al. J. Anal. Toxicol. 43, 489–496 (2019): https://academic.oup.com/jat/article-abstract/43/6/489/5486352

Luginbühl, M. et al. J. Anal. Toxicol. bkaa034 (2020): https://academic.oup.com/jat/advance-article-abstract/doi/10.1093/jat/bkaa034/5815966

Fatch, R. et al. Comparisons of automated determination of phosphatidylethanol (PEth) in dried blood spots (DBS) with previous manual processing and testing. Submitted.

Luginbühl, M. et al. Drug Test Anal. 11, 859–869 (2019): https://onlinelibrary.wiley.com/doi/abs/10.1002/dta.2567

Key data used in this protocol

Luginbühl, M. et al. J. Anal. Toxicol. 43, 489–496 (2019): https://academic.oup.com/jat/article-abstract/43/6/489/5486352

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Luginbühl, M., Stöth, F., Schröck, A. et al. Quantitative determination of phosphatidylethanol in dried blood spots for monitoring alcohol abstinence. Nat Protoc 16, 283–308 (2021). https://doi.org/10.1038/s41596-020-00416-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-00416-x

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing