A novel, simultaneous extraction of FAEE and EtG from meconium and analysis by LC-MS/MS

Abstract

Fatty acid ethyl esters (FAEEs) and ethyl-glucuronide (EtG) in meconium have been widely studied as biomarkers of maternal alcohol consumption during pregnancy. Many analytical approaches have been proposed for their analysis, mostly consisting of separated extraction procedures requiring the use of two meconium aliquots. This study aimed to validate a new analytical procedure for the simultaneous extraction of FAEEs and EtG from a meconium aliquot through a single solid-phase extraction (SPE) applied to 242 anonymized samples of meconium. Targeted FAEEs were: ethyl-myristate (Myr), ethyl-palmitate (Pal), ethyl-oleate (Ole) and ethyl-stearate (Ste). Two hundred milligrams of meconium was sonicated with acetonitrile, and a single SPE performed by means of aminopropyl columns. FAEEs were eluted with hexane, followed by EtG elution with water. Both the mixtures were dried, recovered, and analyzed by liquid chromatography-tandem mass spectrometry using C8 (FAEEs) and C18 (EtG) columns. Transitions were: m/z 257 → 57,88, Myr; m/z 262 → 57,88, Myr-d5; m/z 285 → 57, 72, Pal; m/z 290 → 57,258, Pal-d5; m/z 311 → 72,114, Ole; m/z 316 → 72,265, Ole-d5; m/z 257 → 57,72 Ste; m/z 318 → 57,286, Ste-d5; m/z 221 → 75,85, EtG; m/z 226 → 75,85, EtG-d5. Lower limit of quantification range was 10-15 ng/g for FAEEs and 10 ng/g for EtG. Linearity was evaluated for different concentration ranges; the mean coefficients of determination (R (2)) were above 0.9961. Precision and accuracy for FAEEs and EtG were consistently ≤20 % and ±20 %, respectively. Ion suppression was observed for all the analytes. Matrix effect did not significantly affect the analyses. Recovery efficiency was 93 % for EtG and 75-85 % for FAEEs.

Novel fast ultra‐performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) and extraction of ethylglucuronide in meconium samples

“…Imprecision (intra‐ and inter‐day) and accuracy values, evaluated at three different QC concentrations, were always lower than 12% (Table ). The mean analytical recovery obtained for the three different QC samples was always around 86% (Table ), in agreement with values previously reported (eg, from 71 to 104%) with an efficiency process of around 88.7%. and no significant ion suppression (range of values: 88%–107%).…”

“…EtG is a direct ethanol metabolite formed in the body by glucuronidation following exposure to ethanol. It is established as a specific biomarker in conventional and non‐conventional adult matrices (eg, blood, urine and hair) and in fetal‐neonatal matrices, such as fetal hair or meconium …”

“…Methods for the determination of EtG in meconium usually require lengthy, difficult, and expensive processes of extraction of minute amounts of the analyte from a complex biological matrix …”

“…It is established as a specific biomarker in conventional and non-conventional adult matrices 3,4 (eg, blood, urine and hair) and in fetal-neonatal matrices, such as fetal hair or meconium. [4][5][6][7][8] Since meconium is the first neonatal feces, which begins to form during weeks 12/13 of the gestational period, the eventual presence of xenobiotics in this matrix accounts for prenatal exposure during the second and third pregnancy trimester. 3 Methods for the determination of EtG in meconium usually require lengthy, difficult, and expensive processes of extraction of minute amounts of the analyte from a complex biological matrix.…”

Determining the pattern and prevalence of alcohol consumption in pregnancy by measuring biomarkers in meconium

“…A fully validated method was used for the determination of four FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) and EtG by liquid chromatography-tandem mass spectrometry 19. To summarise, 200 mg of meconium was sonicated for 15 min in the presence of 20 ng of EtG-d5 and 200 ng of each FAEE-d5.…”