An effective DBS-LC-MS/MS strategy for the monitoring of alcohol biomarkers

Workplace monitoring of alcohol consumption habits is highly advisable especially in the healthcare framework, due to the serious consequences alcohol use and abuse could bring. Within this project, two highly selective alcohol consumption biomarkers were selected, namely ethyl glucuronide (EtG) and ethyl sulfate (EtS), to perform a reliable quali-quantitative analysis as a tool to investigate alcohol consumption behaviours. The two analytes, as medium-term biomarkers, are suitable for workplace monitoring. To this aim, a minimally invasive biosampling strategy has been proposed and developed within this research, also allowing simplified sample storage and shipping of numerous samples: a dried blood spots (DBS) microsampling approach. It is based on haematic collection through a finger prick, avoiding an invasive procedure (i.e. phlebotomy). An accurate blood volume (10 µL) was collected from a fingertip and spotted on special cellulose DBS cards; after spot drying, the cards were stored at room temperature without the need for cryopreservation. The use of a miniaturised, dried biological matrix also ensures enhanced EtG and EtS stability in blood, when compared to the same analytes in classic fluid blood samples. On the other hand, the reduced volumes and the low expected analyte levels required the development and validation of a highly sensitive and selective instrumental analytical method: the microsampling approach was coupled to an original LC-MS/MS method. In the first phase of the project, an in-depth optimisation of mass spectrometry parameters was carried out and led to the definition of electrospray ionization (ESI) polarity, acquisition mode, fragmentation parameters, selection of mass ion transitions leading to the highest selectivity and sensitivity. Chromatographic conditions were also optimised for the simultaneous determination of both analytes in a short time (3.5 minutes) and validated according to the main International Guidelines. In the second phase of the research, the developed method was applied to evaluate the analyte levels in DBS from volunteers, after alcohol controlled dosing and aiming at discriminating between abstinence, accidental alcohol intake, acute and chronic consumption. At the same time, an attempt was made to define EtG and EtS baseline levels, as well as effective cut-off reference values. The original high-throughput analytical methodology proposed herein combines the advantages of microsampling for sample collection, transportation and storage with the high sensitivity and selectivity of LC-MS/MS. It has granted the successful analysis of samples from healthcare professionals, recruited in the last phase of the study, for the monitoring of workplace alcohol consumption. This study was funded by the Italian Ministry of Health within the Finalized Research Project 2011-2012 (RF-2011-02352096) and performed at the Pharmaco-Toxicological Analysis Laboratory (PTA Lab) of the Department of Pharmacy and Biotechnology (FaBiT), Alma Mater Studiorum - University of Bologna.