Development and validation of a method for the simultaneous analysis of fatty acid ethyl esters, ethyl sulfate and ethyl glucuronide in neonatal meconium: application in two cases of alcohol consumption during pregnancy

Alcohol consumption during pregnancy constitutes one of the leading preventable causes of birth defects and neurodevelopmental disorders in the exposed children. Fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG) and ethyl sulfate (EtS) have been studied as potential biomarkers of alcohol consumption. However, most analytical approaches proposed for their analysis in meconium samples consist of separated extraction procedures requiring the use of two meconium aliquots, which is costly in terms of both time and materials. Therefore, the aim of this study was to develop and validate a method for the simultaneous extraction of 9 FAEEs, EtG and EtS from one meconium aliquot. The sample was homogenized using methanol, and then FAEEs were extracted with hexane while EtG and EtS were isolated using acetonitrile. Then, extracts were applied to solid-phase extraction columns and analysed by gas chromatography mass spectrometry (FAEEs) and liquid chromatography tandem mass spectrometry (EtG and EtS). Calibration curves were linear with r values greater than 0.99. The LODs ranged from 0.8 to 7.5 ng/g for FAEEs and were 0.2 ng/g and 0.8 ng/g for EtS and EtG, respectively. LOQs ranged from 5 to 25 ng/g for FAEEs and were 1 ng/g and 2.5 ng/g for EtS and EtG, respectively. Accuracies and precisions were between 93.8 and 107% and between 3.5 and 9.7%, respectively. The recovery values ranged from 89.1 to 109%. The method proved to be sensitive, specific, simple and fast and allowed for the reduction of the amount of organic solvent used for extraction compared to other published data while higher recoveries were obtained. The method was used for analysis of meconium samples in two cases of mothers who were consuming alcohol during pregnancy.