Integrated internal standard volumetric dried blood microsampling for the quantification of phosphatidylethanol...

Phosphatidylethanol 16:0/18:1 (PEth) is a direct biomarker of alcohol consumption, formed in red blood cell membrane in the presence of ethanol. Concentrations of 20 ng/mL and 200 ng/mL have been proposed as cut-offs to distinguish abstinence or low alcohol consumption from moderate drinking and to indicate alcohol misuse, respectively. Dried blood spots (DBS) are considered a preferred matrix for PEth determination because of its limited stability in liquid whole blood. However, conventional DBS workflows may compromise quantitative accuracy, as the internal standard does not fully compensate for extraction-related variability. In this study, we investigated the incorporation of an isotopically labelled internal standard (d5-PEth) directly into the sample disc of the Capitainer®B10 Vanadate card. A rapid UHPLC-MS/MS method based on selected reaction monitoring (SRM), with a total run time of 5 min, was developed together with an extraction protocol suitable for routine application. The method was validated in terms of calibration performance, sensitivity, accuracy, precision, matrix effect, and extraction recovery. The limit of detection was 4.2 ng/mL, moderate ion suppression was observed, and extraction recovery was 57 ± 18%. The resulting internal quantification dried blood spot (iqDBS) workflow showed satisfactory repeatability across 48 spiked samples extracted over three analytical sessions, with CV% below 15%. The stability of the device was also confirmed for up to 2.5 months after preparation under accelerated aging conditions at elevated temperature. Overall, the proposed iqDBS strategy provides a robust and promising framework for PEth quantification, improving the reliability of alcohol biomarker assessment in clinical and forensic applications.