Validation of a novel method to identify in utero ethanol exposure: simultaneous meconium extraction of fatty acid ethyl esters, ethyl glucuronide, and ethyl sulfate followed by LC-MS/MS quantification

Abstract

Presence of fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) in meconium, the first neonatal feces, identifies maternal alcohol consumption during pregnancy. Current meconium alcohol marker assays require separate analyses for FAEE and EtG/EtS. We describe development and validation of the first quantitative liquid chromatography tandem mass spectrometry assay for 9 FAEEs, EtG, and EtS in 100 mg meconium. For the first time, these alcohol markers are analyzed in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. 100 mg meconium was homogenized in methanol and centrifuged. The supernatant was divided, and applied to two different solid phase extraction columns for optimized analyte recovery. Limits of quantification for ethyl laurate, myristate, linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate ranged from 25-50 ng/g, with calibration curves to 2,500-5,000 ng/g. EtG and EtS linear dynamic ranges were 5-1,000 and 2.5-500 ng/g, respectively. Mean bias and between-day imprecision were <15 %. Extraction efficiencies were 51.2-96.5 %. Matrix effects ranged from -84.7 to 16.0 %, but were compensated for by matched deuterated internal standards when available. All analytes were stable (within ±20 % change from baseline) in 3 authentic positive specimens, analyzed in triplicate, after 3 freeze/thaw cycles (-20 °C). Authentic EtG and EtS also were stable after 12 h at room temperature and 72 h at 4 °C; some FAEE showed instability under these conditions, although there was large inter-subject variability. This novel method accurately detects multiple alcohol meconium markers and enables comparison of markers for maternal alcohol consumption.

Fig. 1

Multiple reaction monitoring chromatograms for alcohol marker quantifier ions in a blank meconium fortified at analyte limits of quantification and in b authentic positive meconium specimens with concentrations of alcohol markers listed under the name of each marker

Fig. 2

Mean (range) percent baseline concentrations after 3 freeze/thaw cycles, 72 h at 4 °C, and 12 h at room temperature in 3 authentic positive meconium specimens (1 positive EtS source, 2 positive ethyl palmitoleate sources). Triplicate specimens were analyzed under all test conditions, including baseline; %CVs from triplicate analyses ranged from 0.7 to 17.6 %