Quantitative Determination of Phosphatidylethanol in Dried Blood Spots for Abstinence Monitoring

The novel protocol provides general guidelines for the analysis of Phosphatidylethanol in dried blood spots including reference material evaluation, synthesis of deuterated internal standard, preparation of calibration samples, analyte separation, and detection.
Quantitative Determination of Phosphatidylethanol in Dried Blood Spots for Abstinence Monitoring

Phosphatidylethanol (PEth) is a group of abnormal phospholipids which are formed by an enzymatic reaction between ethanol and phosphatidylcholine. As PEth is only formed within the human body when ethanol is present, it serves as a direct alcohol biomarker that reflects drinking behavior. Information about a subject’s drinking habits is much needed in situations such as alcohol withdrawal treatment settings, driving aptitude assessments, issuing gun licenses, family law, and organ transplant settings. In combination with high-throughput analysis, PEth 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.


For the classification of drinking behavior, scientists agreed to apply threshold concentrations for the dominant PEth species in human blood, PEth 16:0/18:1. Generally, two threshold concentrations are applied, an upper and a lower (e.g. >200 ng/mL and <20 ng/mL), allowing the classification of analytical results into three groups: excessive consumer, moderate drinker, and teetotaler. Depending on the location of the testing laboratory, these thresholds and the classification of the groups may vary. As a general principle, we introduce the use of threshold and decision limits for PEth analysis in this manuscript.


Overall, this protocol is the latest result in our ongoing development of PEth analysis. Initially, we started measuring PEth using online SPE-LC-MS/MS analysis by manually preparing liquid blood samples and dried blood spots (DBS). Our next step was to adapt this method to perform faster PEth analysis on a more sensitive instrument. Afterward, we implemented a DBS autosampler to conduct a fully automated DBS PEth analysis. The automated DBS approach based on a CAMAG DBS-MS 500 system represents a state of the art methodology for high-throughput PEth DBS analytics and features a unique internal standard spray module. Furthermore, the protocol pays attention to the latest findings regarding the testing of the PEth reference materials’ regioisomeric purity.


Why use dried blood spots? Initially, the analysis of PEth was performed using whole blood. Nowadays, sampling blood for the analysis of PEth is shifting to the use of the DBS technique, as PEth is not stable in liquid blood unless stored at -80 ºC. The inactivation of enzymes and the removal of liquid during the DBS drying process prevents the formation and degradation of PEth, thus prolongs the stability of the sample. The use of DBS from a filter paper card is currently the cheapest and most straightforward way to assess PEth concentrations. DBS permits minimally invasive sampling procedures and excellent analyte stability. Furthermore, the remaining spots on the DBS filter paper card can be used for additional analyses such as hematocrit determination or drug of abuse screening.

Luginbühl, M., Stöth, F., Schröck, A. et al. Quantitative determination of phosphatidylethanol in dried blood spots for monitoring alcohol abstinence. Nat Protoc (2020). https://doi.org/10.1038/s41596-020-00416-x

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