Liquid chromatography–tandem mass spectrometry for fatty acid ethyl esters in meconium: Assessment of prenatal exposure to alcohol in two European cohorts

Abstract

Fatty acid ethyl esters (FAEEs) in meconium emerged as a reliable, direct biological marker for establishing fetal exposure to ethanol. We developed an LC–MS/MS method for ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate using ethyl heptadecanoate as the internal standard. The analytes were extracted from meconium with hexane, followed by solid-phase extraction with aminopropyl-silica columns. Chromatography was performed on a C₈ reversed-phase column using water/isopropanol/acetonitrile (20:40:40, v/v/v) as a mobile phase. A triple quadrupole mass spectrometer that monitored the transitions in multiple reaction-monitoring mode was used for the detection of the analytes. Limits of quantification (LOQs) varied between 0.12 and 0.20 nmol/g. Calibration curves were linear from LOQs to 50 nmol/g for all analytes, with a minimum r² > 0.99. At three concentrations spanning the linear dynamic range, mean recoveries ranged between 53.6 and 86.7% for the different analytes. The validated method was applied to analysis of meconium in newborns of two European cities. The two cohorts presented with different prevalence of gestational ethanol consumption during pregnancy.

Introduction

Consumption of ethanol during pregnancy is the leading preventable cause of neurodevelopmental delay in North America. Maternal alcohol use during pregnancy can cause Fetal Alcohol Spectrum Disorder (FASD), which is estimated to affect 1% of all North American live births [1]. FASD is an umbrella term used to describe a wide range of potentially lifelong effects that include physical, mental, behavior, and learning disabilities [2]. In order to diagnose the disorder and institute early intervention, preferably before the development of secondary disabilities, early detection of in utero exposure is of utmost importance [3].

In recent years, fatty acid ethyl esters (FAEEs) found in neonatal matrices such as meconium and neonatal hair emerged as a reliable, direct biological markers for the assessment of gestational alcohol exposure [4], [5], [6], [7], [8].

Although ethanol is the most widely consumed teratogen, most of the research on the prevalence of Fetal Alcohol Spectrum Disorder has come from North America.

In Europe, despite abundant alcohol consumption, the issue of maternal drinking during pregnancy is largely ignored.

For the first time in Europe, the “Meconium Project” was initiated in order to estimate the prevalence of drug use by pregnant women and the effects of chronic illicit drug exposure on the fetus and infant. This was achieved by meconium analysis, maternal structured interview and clinical observations. The first mother–infant dyads cohort was recruited in Barcelona, Spain, and a high prevalence of opiates (8.7%), cocaine (4.4%) and cannabis (5.3%) in meconium specimens was reported [9], [10]. The assessment of fetal exposure to alcohol was the next step of the project, together with the addition of a second cohort of mother–infant dyads from Reggio Emilia, Italy.

Published methods for the analysis of FAEEs in meconium are mainly based on gas chromatographic separation of the compounds, coupled either with flame ionization detection (FID) or mass spectrometry [8], [4], [11], [13]. In this latter case, chemical ionization has been applied for improved detection and because electron impact ionization of these compounds yielded identical fragments for the various FAEEs [8], [13].

New ionization techniques have made liquid chromatography–mass spectrometry (LC–MS) and LC–tandem MS (LC–MS/MS) extremely effective for the specific determination of different analytes in complex biological matrices. Within the framework of the “Meconium Project” LC–MS methodologies have been developed and validated for the detection of several drugs of abuse and metabolites in meconium [14], [15], [16], [17].

We describe here an LC–MS/MS assay for the determination of the fatty acid ethyl esters ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate and ethyl arachidonate (Fig. 1) in meconium and its application to assess fetal exposure to alcohol in the above-reported cohorts.