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

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

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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.

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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.

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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.

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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

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  4. Stefan Gaugler
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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.

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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

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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

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