Development and validation of analytical methods for the determination of direct ethanol metabolites in biological matrices by liquid chromatography tandem mass spectrometry. Applications in forensic toxicology

Alcohol abuse and misuse is a growing problem with relevant clinical, social, economic and administrative implications. A sensitive and specific diagnosis of the different forms of acute and chronic alcohol abuse is an essential tool to properly face this social burden. Recently, the determination of two minor non oxidative products of alcohol metabolism, ethyl glucuronide (EtG) and ethyl sulphate (EtS), in traditional (blood and derivatives, urine) as well as alternative (hair, meconium) biological matrices has been proposed as a promising approach to the efficient and effective diagnosis of alcohol abuse for forensic, clinical, and epidemiological purposes. The aim of this thesis was to (a) develop and fully validate analytical methods for the determination of these two metabolites in different biological samples (urine, blood/serum, meconium and, for EtG, hair) by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and b) to apply these methods in several diagnostic contexts and, in particular: 1) the study of the blood kinetics and blood/serum ratio of EtG and EtS in samples of heavy drinkers at the beginning of a withdrawal treatment (study carried out in collaboration with the Norwegian National Institute of Health); 2) study of the stability of EtG and EtS in post-mortem samples several years after burial; 3) evaluation of EtG in hair (HEtG) as a potential marker of chronic alcohol abuse, in comparison with other biomarkers currently used in the routine (i.e. CDT); 4) application of EtG and EtS in meconium as markers of gestational alcohol exposure. Results obtained in the different fields mentioned above showed, in some cases for the first time, or confirmed the great potential of EtG and EtS as sensitive and specific markers of alcohol misuse. Moreover they allowed to ascertain that: the elimination of EtG and EtS in heavy drinkers is similar to that occurring in social drinkers (with the important exception of subjects suffering from renal pathologies where the ethanol metabolism and consequently the metabolites elimination appear to be slowed down); EtG and EtS are to mainly distributed in serum than in blood cells, with relevant implications when the analysis is carried out on serum/plasma instead of whole blood; EtG and EtS are detectable in post-mortem biological samples even when collected several years after death; the use of an analytical, sensitive (LLOQ of 3 pg/mg), specific and fully validated method gives to HEtG a high diagnostic sensitivity and specificity; HEtG appears to qualitatively correlate with other markers ( i.e. cocaethylene in hair of cocaine users; CDT), although it exhibits better sensitivity (>90%) in ascertaining alcohol chronic abuse; EtG and EtS are detectable in meconium (even if their concentration doesn’t correlate with that of other markers of alcohol consumption such as fatty acids ethyl esters), thus offering the basis for an in-depth evaluation of the usefulness of their determination in this matrix for the diagnosis of prenatal exposure to alcohol.