Detection of in utero ethanol exposure via ethyl glucuronide and ethyl sulfate analysis in umbilical cord and placenta
Alcohol exposure during pregnancy constitutes one of the leading preventable causes of birth defects, mental retardation and neurodevelopmental disorders in the exposed children. The ethanol marker ethyl glucuronide (EtG) is a specific long-term marker of ethanol in utero exposure in meconium; however, currently, there are scarce or no data about EtG and ethyl sulfate (EtS) in umbilical cord and placenta. These tissues are alternative matrices to meconium that offer critical advantages, such as always being available at birth with noninvasive and easy collection.
We developed and validated a method for the determination of EtG and EtS in umbilical cord and placenta. Tissues were homogenized in methanol, extracted using weak anion-exchange solid-phase extraction (SPE) and analyzed by liquid chromatography–tandem mass spectrometry. The umbilical cord and placenta method was applied to 59 authentic samples from newborns whose meconium samples were positive for EtG (EtG > 5 ng/g).
The method in umbilical cord and placenta was fully validated, with a limit of quantification at 5 ng/g in umbilical cord and 10 ng/g in placenta for both compounds. EtG and/or EtS were detected in 25 umbilical cord samples (4.4–529 and 4.3–39 ng/g, respectively) and in 8 placenta samples (26.5–267 and 11–24.3 ng/g, respectively). EtG and EtS showed a homogenous distribution throughout umbilical cord tissue (n = 5).
We developed and validated a sensitive and specific method for the determination of EtG and EtS in umbilical cord and placenta. To date, this is the first method to investigate both direct metabolites of ethanol in umbilical cord and placenta samples for prenatal ethanol exposure.
KeywordsEthyl glucuronide Ethyl sulfate Umbilical cord Placenta Meconium LC–MS/MS
This project was funded by the Plan Nacional Sobre Drogas, Ministerio de Sanidad, Gobierno de España, grant 2016I005. E. Lendoiro would like to thank the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, for her postdoctoral contract (ED481B2016/103-0).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
The study was approved by the Ethics Committee of the University of Santiago de Compostela (Spain). All procedures performed in this study were in accordance with the ethical standards of the Ethics Committee of the University of Santiago de Compostela (Spain) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
- 2.Centers for Disease Control and Prevention (2004) Alcohol consumption among women who are pregnant or who might become pregnant—United States, 2002. Morb Mortal Wkly Rep 53:1178–1181Google Scholar
- 6.Bailey BA, Sokol RJ (2011) Prenatal alcohol exposure and miscarriage, stillbirth, preterm delivery, and sudden infant death syndrome. Alcohol Res Health 34:86–91 (PMID: 23580045) Google Scholar
- 7.Høiseth G, Morini L, Polettini A, Christophersen A, Mørland J (2009) Ethyl glucuronide in hair compared with traditional alcohol biomarkers—a pilot study of heavy drinkers referred to an alcohol detoxification unit. Alcohol Clin Exp Res 33:812–816. https://doi.org/10.1111/j.1530-0277.2009.00900.x CrossRefGoogle Scholar
- 8.Himes SK, Dukes KA, Tripp T, Petersen JM, Raffo C, Burd L, Odendaal H, Elliott AJ, Hereld D, Signore C, Willinger M, Huestis MA (2015) Clinical sensitivity and specificity of meconium fatty acid ethyl ester, ethyl glucuronide, and ethyl sulfate for detecting maternal drinking during pregnancy. Clin Chem 61:523–532. https://doi.org/10.1373/clinchem.2014.233718 CrossRefGoogle Scholar
- 10.Bakdash A, Burger P, Goecke TW, Fasching PA, Reulbach U, Bleich S, Hastedt M, Rothe M, Beckmann MW, Pragst F, Kornhuber J (2010) Quantification of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) in meconium from newborns for detection of alcohol abuse in a maternal health evaluation study. Anal Bioanal Chem 396:2469–2477. https://doi.org/10.1007/s00216-010-3474-5 CrossRefGoogle Scholar
- 11.Goecke TW, Burger P, Fasching PA, Bakdash A, Engel A, Häberle L, Voigt F, Faschingbauer F, Raabe E, Maass N, Rothe M, Beckmann MW, Pragst F, Kornhuber J (2014) Meconium indicators of maternal alcohol abuse during pregnancy and association with patient characteristics. Biomed Res Int 2014:702848. https://doi.org/10.1155/2014/702848 Google Scholar
- 12.Morini L, Groppi A, Marchei E, Vagnarelli F, Algar OG, Zuccaro P, Pichini S (2010) Population baseline of meconium ethyl glucuronide and ethyl sulfate concentrations in newborns of nondrinking women in 2 Mediterranean cohorts. Ther Drug Monit 32:359–363. https://doi.org/10.1097/FTD.0b013e3181d5f14a CrossRefGoogle Scholar
- 13.Pichini S, Marchei E, Vagnarelli F, Tarani L, Raimondi F, Maffucci R, Sacher B, Bisceglia M, Rapisardi G, Elicio MR, Biban P, Zuccaro P, Pacifici R, Pierantozzi A, Morini L (2012) Assessment of prenatal exposure to ethanol by meconium analysis: results of an Italian multicenter study. Alcohol Clin Exp Res 36:417–424. https://doi.org/10.1111/j.1530-0277.2011.01647.x CrossRefGoogle Scholar
- 14.Morini L, Marchei E, Vagnarelli F, Garcia Algar O, Groppi A, Mastrobattista L, Pichini S (2010) Ethyl glucuronide and ethyl sulfate in meconium and hair-potential biomarkers of intrauterine exposure to ethanol. Forensic Sci Int 196:74–77. https://doi.org/10.1016/j.forsciint.2009.12.035 CrossRefGoogle Scholar
- 19.Morini L, Falcón M, Pichini S, Garcia-Algar O, Danesino P, Groppi A, Luna A (2011) Ethyl-glucuronide and ethyl-sulfate in placental and fetal tissues by liquid chromatography coupled with tandem mass spectrometry. Anal Biochem 418:30–36. https://doi.org/10.1016/j.ab.2011.06.038 CrossRefGoogle Scholar
- 20.Swortwood MJ, Bartock SH, Scheidweiler KB, Shaw S, Filis P, Douglas A, O’Shaughnessy PJ, Soffientini U, Lucendo-Villarin B, Iredale JP, Hay DC, Fowler PA, Huestis MA (2018) Quantification of ethyl glucuronide, ethyl sulfate, nicotine, and its metabolites in human fetal liver and placenta. Forensic Toxicol 36:102–112. https://doi.org/10.1007/s11419-017-0389-2 CrossRefGoogle Scholar
- 21.Concheiro M, Lendoiro E, de Castro A, Gónzalez-Colmenero E, Concheiro-Guisan A, Peñas-Silva P, Macias-Cortiña M, Cruz-Landeira A, López-Rivadulla M (2017) Bioanalysis for cocaine, opiates, methadone, and amphetamines exposure detection during pregnancy. Drug Test Anal 9:898–904. https://doi.org/10.1002/dta.2087 CrossRefGoogle Scholar
- 22.Himes SK, Concheiro M, Scheidweiler KB, Huestis MA (2014) 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. Anal Bioanal Chem 406:1945–1955. https://doi.org/10.1007/s00216-013-7600-z CrossRefGoogle Scholar
- 32.Concheiro M, Gonzalez-Colmenero E, Lendoiro-Belio E, Concheiro-Guisan A, de Castro A, Cruz-Landeira A, López- Rivadulla M (2013) Alternative matrices for cocaine, opioids and methadone in utero drug-exposure detection. Ther Drug Monit 35:502–509. https://doi.org/10.1097/FTD.0b013e31828a6148 CrossRefGoogle Scholar
- 33.de Castro A, Jones HE, Johnson RE, Gray TR, Shakleya DM, Huestis MA (2011) Methadone, cocaine, opiates, and metabolite disposition in umbilical cord and correlations to maternal methadone dose and neonatal outcomes. Ther Drug Monit 33:443–452. https://doi.org/10.1097/FTD.0b013e31822724f0 CrossRefGoogle Scholar