title: Development of a new immunoassay for the detection of ethyl glucuronide (EtG) in meconium: validation with authentic specimens analyzed using LC-MS/MS. Preliminary results.
abstract: BACKGROUND
  Ethyl glucuronide (EtG) measurement in neonatal meconium has emerged as a reliable marker to objectively assess prenatal exposure to maternal ethanol complementary to fatty acid ethyl ester (FAEEs) measurement. The detection of EtG in meconium is currently a lengthy, difficult and expensive process using liquid chromatography tandem mass spectrometry (LC-MS/MS) as the analytical procedure. An enzyme-linked immunosorbent assay (ELISA) for the identification of EtG in meconium was developed, validated and applied to authentic meconium specimens from newborns collected in Europe.
  
  
  METHODS
  The ELISA procedure was calibrated using 0.45, 0.9, 1.35 and 1.8 nmol/g (100, 200 300 and 400 ng/g) standards. Meconium (0.25 g) was mixed thoroughly, with extraction buffer (pH 7.3; 0.5 mL). The tube was capped, sonicated, centrifuged and the supernatant was decanted. An aliquot of the extract (50 μL) was placed in the well of the microplate followed by enzyme conjugate (150 μL). The plate was incubated for 1 h, washed with deionized water, dried and substrate (200 μL) was added. After 30 min incubation, stop solution was added and the plate was read at 450 nm and 650 nm. Samples were also analyzed for EtG and FAEEs by validated LC-MS/MS assays.
  
  
  RESULTS
  Using an EtG cut-off of 0.9 nmol/g for both ELISA screening test and confirmatory LC-MS/MS, immunoassay sensitivity was 100%; specificity 78%; positive-predictive value (PPV) 29% and negative-predictive value (NPV) 100%.
  
  
  CONCLUSIONS
  The assay is proposed as a preliminary screening test for the meconium of newborns suspected of being born to mothers drinking alcohol during pregnancy.
authors.forename: Simona
authors.initials: S
authors.lastname: Pichini
authors.forename: Luca
authors.initials: L
authors.lastname: Morini
authors.forename: Roberta
authors.initials: R
authors.lastname: Pacifici
authors.forename: James
authors.initials: J
authors.lastname: Tuyay
authors.forename: Warren
authors.initials: W
authors.lastname: Rodrigues
authors.forename: Renata
authors.initials: R
authors.lastname: Solimini
authors.forename: Oscar
authors.initials: O
authors.lastname: Garcia-Algar
authors.forename: Juan
authors.initials: J
authors.lastname: Ramis
authors.forename: Christine
authors.initials: C
authors.lastname: Moore
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005965:Glucuronates / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D008470:Meconium / Q000737:chemistry* / Q000378:metabolism; D011247:Pregnancy; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D015415:Biomarkers; D005965:Glucuronates; C093924:ethyl glucuronide