Validation of a rapid semi-automated method for monitoring alcohol abuse by phosphatidylethanol (16:0/18:1) LC-MS/MS determination in dried blood spots.

Phosphatidylethanol (PEth) is currently considered the most specific direct biomarker of alcohol consumption in blood, with a detection window of up to four weeks. Its clinical and occupational applications have expanded considerably, leading to increasing demand for reliable PEth determination. Although Dried Blood Spot (DBS) sampling provides clear logistical and pre-analytical advantages, sample preparation remains labor-intensive and prone to variability. This study describes the development and validation of a semi-automated LC-MS/MS method for PEth quantification using DBS, designed for high-throughput routine use. The analytical workflow combines automated DBS identification and punching in 96-well extraction plate and LC-MS/MS analysis with automatic and secured data transfer to the Laboratory Information Management (LIM) system. Method validation was conducted in accordance with EMA and ISO 15189 requirements and then applied to authentic venous patient DBS. The calibration curve covered a concentration range from 10 to 1000 µg/L with accuracy and precision of + /-15% for all levels. The low limit of quantification (LLOQ) was 10 µg/L and the upper limit of quantification linearity (ULOQ) was 1000 µg/L while the quantification range was extended up to 2500 µg/L, maintaining acceptable accuracy and precision. Analytical run time was 9 min, with no detectable interferences, carry-over or matrix effect, demonstrating high selectivity. Among 346 patient samples analyzed, 67 exceeded the 200 µg/L threshold defined by the Basel consensus to be classified as chronic alcohol consumers. This validated semi-automated DBS LC-MS/MS method provides a robust, sensitive, and high-throughput approach for rapid routine PEth determination, enhancing analytical standardization, reliability, and traceability in clinical and forensic laboratories.