RESEARCH PAPER A novel, simultaneous extraction of FAEE and EtG from meconium and analysis by LC-MS/MS Fabio Vaiano 1 & Donata Favretto 2 & Diego Palumbo 1 & Gail Cooper 3,4 & Helen Mactier 4,5 & Francesco P. Busardò 6 & Francesco Mari 1 & Elisabetta Bertol 1 Received: 9 December 2015 /Revised: 16 January 2016 /Accepted: 25 January 2016 /Published online: 12 February 2016 # Springer-Verlag Berlin Heidelberg 2016 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 analyt- ical 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 meco- nium. Targeted FAEEs were: ethyl-myristate (Myr), ethyl- palmitate (Pal), ethyl-oleate (Ole) and ethyl-stearate (Ste). Two hundred milligrams of meconium was sonicated with acetoni- trile, 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 spectrom- etry 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 determi- nation (R 2 ) were above 0.9961. Precision and accuracy for FAEEs and EtG were consistently ≤20 % and ±20 %, respec- tively. Ion suppression was observed for all the analytes. Matrix effect did not significantly affect the analyses. Recovery efficien- cy was 93 % for EtG and 75–85 % for FAEEs. Keywords Ethyl-glucuronide . Fatty acid ethyl esters . Meconium . LC-MS/MS . Fetal alcohol exposure Introduction Alcohol consumption during pregnancy is an important health issue since ethanol freely crosses the placenta, putting the fetus at risk of developmental problems. Fetal alcohol exposure (FAE) can result in a range of birth defects and subsequent neurodevelopmental problems, including distinctive craniofacial dysmorphology, poor growth, and cognitive and social impair- ments. The severity of effects varies widely from the full-blown clinical picture of fetal alcohol syndrome (FAS) through lesser signs and symptoms, collectively recognized as fetal alcohol spectrum disorder (FASD) [1–3]. The dose-effect correlation between alcohol intake and development of FASD varies throughout pregnancy and between individuals [4–9] but many studies have demonstrated that even low amounts of ethanol Parts of this work were presented at the 53rd TIAFT meeting 2015 – Florence, Italy. * Fabio Vaiano fabio.vaiano@unifi.it 1 Department of Health Science, Forensic Toxicology Division, University of Florence, Largo Brambilla, 50134 Florence, Italy 2 Legal Medicine and Toxicology, Department SCTV, University-Hospital of Padova, Via Falloppio 50, Padova 35121, Italy 3 Cooper Gold Forensic Consultancy Ltd, 40 Main Road, East Wemyss, Kirkcaldy, Fife KY1 4RA, Scotland, UK 4 School of Medicine, University of Glasgow, University Ave, Glasgow City G12 8QQ, Scotland, UK 5 Princess Royal Maternity Hospital, 16 Alexandra Parade, Glasgow G31 2ER, Scotland, UK 6 Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, Alfonso Borelli N 50, 00161 Rome, Italy Anal Bioanal Chem (2016) 408:2587–2594 DOI 10.1007/s00216-016-9364-8