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Alcohol consumption during pregnancy is a widespread problem and can cause severe fetal damage. As the diagnosis of fetal alcohol syndrome is difficult, the implementation of a reliable marker for alcohol consumption during pregnancy into meconium drug screening programs would be invaluable. A previously published gas chromatography mass spectrometry method for the detection of fatty acid ethyl esters (FAEEs) as alcohol markers in meconium was optimized and newly validated for a sample size of 50 mg. This method was applied to 122 cases from a drug-using population. The meconium samples were also tested for common drugs of abuse. In 73 % of the cases, one or more drugs were found. Twenty percent of the samples tested positive for FAEEs at levels indicating significant alcohol exposure. Consequently, alcohol was found to be the third most frequently abused substance within the study group. This re-validated method provides an increase in testing sensitivity, is reliable and easily applicable as part of a drug screening program. It can be used as a non-invasive tool to detect high alcohol consumption in the last trimester of pregnancy. The introduction of FAEEs testing in meconium screening was found to be of particular use in a drug-using population.
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... During the last trimester (week 28-40), its formation increases, and 75% of the meconium is produced during the last 8 weeks of pregnancy . Therefore, the results may provide an overview of the last 2-3 months before birth . Meconium expulsion is sequential and may take up to 5 days. ...
... Table 1 summarizes the confirmation method papers in meconium published from 2007 to 2014. Most of the recently published methods dealt with the determination of the alcohol biomarkers, ethyl-glucuronide (EtG), ethyl-sulfate (EtS) and/or FAEEs . ...
... Himes et al.  reported the only method able to analyze EtG, EtS and FAEEs from the same meconium aliquot (Figure 1). In the methods for alcohol biomarkers determination   6 FAEEs 50 mg 50-100 to 1000 2 ml n-heptane: acetone (5:2) Homogenization vortexing HS-SPME 65μm, PDMS/DVB GC-MS, SIM HP5-MS capillary column  3 FAEEs 500 mg 500-5000 1 ml water + 12 ml hexane Microwave assisted extraction GC-MS, FullScan/ SIM HP5-MS capillary column  4 FAEEs 500 mg 50-1000 5 ml n-heptane: ...
Consumption of drugs of abuse, tobacco and alcohol throughout pregnancy is a serious public health problem and results in an important economic cost to the health system. Drug and/or metabolites determination in biological matrices from mother and newborn is an objective measure of in utero drug exposure. We reviewed methods published for the determination of in utero drug exposure from 2007 to 2014, with special focus on meconium, placenta, umbilical cord and newborn hair. Accurate bioanalytical procedures are essential to obtain high-quality data to perform interventions and to establish correlations between analytical measures and clinical outcomes. We included a brief overview of clinical implications of in utero drug exposure to better understand the importance of this serious health issue.
... This is most likely because in meconium other species than E14:0, E16:0, E18:0 and E18:1 are included in the analysis (Table 3) and due to limited commercial available deuterated standards of those compounds. Nevertheless, in some studies, deuterated versions of the analysed FAEEs were used as internal standards, either commercially acquired or synthesized in-lab [110,119,120,. To a lesser extent, 13 C-labeled FAEEs analogues have also been reported . ...
... Additionally, almost half of 56 infants born from mothers that were prescribed methadone were exposed to alcohol during pregnancy . In 122 samples of drug-using population 20% of the samples had FAEEs levels indicating significant alcohol exposure . In another study, performed on 81 cases were the mother reported alcohol use during pregnancy, an inter-individual variability of the FAEEs levels was observed . ...
Fatty acid ethyl esters (FAEEs) are formed through the reaction of ethanol with endogenous free fatty acids, triglycerides, lipoproteins or phospholipids, catalysed by cytosolic and microsomal FAEE synthases as well as by unspecific enzymes. Since the 1960s, these compounds have been known as biotransformation products of ethanol, and in the 1990s they were proposed as promising biomarkers for monitoring ethanol exposure. Indeed, they present a linear structure which contains the intact ethyl group of ethanol, which translates into biomarkers with high specificity and sensitivity towards alcohol consumption since it excludes other reasons than alcohol consumption for their formation. The FAEEs include over 20 species, while not all the species are used as alcohol biomarkers, several species have been successfully applied as alcohol biomarkers in different biological specimens, including blood, serum, plasma, hair, meconium, and tissues such as skin surface lipids, sebum, liver and adipose tissue. The FAEEs species used as alcohol biomarkers vary in accordance with the analysed biological specimen, depending on their correlation with alcohol consumption.
In this paper, we intend to review the mechanisms of formation and structure of FAEEs to their use as alcohol biomarkers. Focusing on the developed analysis methods, fields of application and published articles, including a critical overview on the methodological challenges and considerations for their analysis; the present limitations and advantages relatively to other alcohol biomarkers; challenges and directions in the research of FAEEs as alcohol biomarkers.
Keywords: alcohol biomarkers, fatty acid ethyl esters, biological specimens, toxicological analysis
... can be predictive of adverse neurological outcome in these children (16,17). To date, most studies in term neonates have focused on correlations between FAEE and heavy alcohol consumption late in pregnancy (5,15,18). However, alcohol use is most common early in pregnancy, particularly since many pregnancies are unplanned (19). ...
... This study found that FAEEs were quantifiable via GC/MS in the meconium of VLBW newborns despite their prematurity. GC/MS has been verified as a sensitive and reproducible method to measure FAEEs in meconium (18,23). Meconium FAEEs were detectable in both Non-exposed and exposed newborns. ...
The health implications of in utero alcohol exposure have been difficult to study in very low birth weight newborns (VLBW) because of an inability to identify maternal alcohol exposure. Fatty acid ethyl esters (FAEEs) are elevated in meconium of alcohol-exposed term newborns. We hypothesized that meconium FAEEs would be similarly elevated in alcohol-exposed VLBW premature newborns.
In a retrospective cohort study of 64 VLBW neonates, newborns were classified into Non-Exposed, Any Exposure, or Weekly Exposure groups based on an in-depth structured maternal interview. Meconium FAEE concentrations were quantified via gas chromatography mass spectrometry.
Alcohol exposure during Trimester 1 (Any Exposure) occurred in ~30% of the pregnancies, while 11% of the subjects reported drinking ≥ 1 drink/week (Weekly Exposure). Meconium ethyl linolenate was higher in Any Exposure (p=0.01) and Weekly Exposure groups (p=0.005) compared to the Non-Exposed VLBW group. There was a significant positive correlation between Trimester 1 drinking amounts and the concentration of meconium ethyl linolenate (p=0.005). Adjusted ROC Curves evaluating ethyl linolenate to identify alcohol-exposed VLBW newborns generated areas under the curve of 88% with sensitivities of 86-89% and specificities of 83-88%.
Despite prematurity, meconium FAEEs hold promise to identify the alcohol-exposed VLBW newborn.Pediatric Research (2016); doi:10.1038/pr.2016.237.
... In this context, considering the responsibility of the mother, the identification of new molecular biomarkers to demonstrate prenatal exposure remains challenging for the scientific community : in different countries, there is a legal responsibility for the mother if her newborn tests positive for abused substances . The considerations about the mother's responsibility are strictly related to each country. ...
Prenatal alcohol exposure is considered one of the main causes of preventable birth disorders; however, it represents the main form of developmental delay in the world. Among the so-called secondary disabilities related to fetal alcohol spectrum disorder (FASD), there is a close connection with criminal behavior. This systematic review aims to provide up-to-date information about the relationship between FASD subjects and criminal justice analyzing different aspects. In light of the results of this review, a further goal is to provide several suggestions in order to reduce the public cost impact of FASD. A systematic review of the literature was conducted according to the PRISMA guidelines, producing 20 articles that met the inclusion criteria. Based on the results published in the selected studies, fetal alcohol syndrome (FAS) is a leading cause of preventable birth disorders and developmental disabilities in newborns. Moreover, these subjects seem to be more inclined to criminal acts compared to others. In conclusion, it should be pointed out that FASD entails high public health costs, both regarding the support measures provided to the affected individual and to their family, as well as the cost and social impact of any criminal offenses committed.
... FAEE concentrations up to 2.0 nmol/g in meconium have been internationally accepted as the threshold between low-tomoderate prenatal alcohol exposure . In addition, FAEE concentrations ten-fold above the cut-off of 2.0 nmol/g (20 nmol/g) are considered high prenatal alcohol exposure . ...
BACKGROUND | Dermatoglyphics alterations have been demonstrated to be an effective complement in the diagnosis of developmental disorders and a marker of prenatal stress. Several genetic and environmental factors can modify their morphology. Once defined, dermatoglyphics remain constant throughout life, being considered fossilized markers of the intrauterine development. Variations in bilateral morphological traits within an individual reflect developmental disturbances and can be measured by fluctuating asymmetry.
The aim of this study was to evaluate if dermatoglyphic variations can be used as a surrogate marker prenatal alcohol exposure (PAE) during foetal development. Dermatoglyphics from 58 individuals who were either exposed or non-exposed to alcohol during pregnancy (according to the levels of Fatty Acid Ethyl Ethers (FAEE) found in meconium at birth) were analyzed.
METHODS | Total a-b ridge count (TABRC) and levels of fluctuating asymmetry from the a-b ridge count (FAABRC) were obtained.
RESULTS | A significant correlation between FA and FAEE levels was found in prenatally alcohol exposed individuals (r = 0.64, p = 0.0032). Remarkably, samples with highest values of FAEEs showed greater FAABRC (6.33 ± 4.18) levels than the values of non-exposed to alcohol (2.87 ± 1.74) as well as the exposed at low concentrations (2.6 ± 1.43) (U = 61, p = 0.05 and U = 14.5, p = 0.05, respectively).
CONCLUSION | Heavy prenatal ethanol exposure (demonstrated by high levels of FAEEs) alters the neuroectoderm developmental program during pregnancy: PAE correlates with FAABRC, which behaves as a dermatoglyphic variable sensitive to FASD and deserves to be studied as a surrogate marker of neurodevelopmental damage during foetal development.
... 82,85 However, the production of meconium is nonlinear, with more than two thirds of meconium forming during the last 8 weeks of gestation, so third-trimester exposures are more readily detected. 40,59,63,85, Meconium also seems more sensitive for detection of repeated rather than sporadic drug use. 92 When considering specimens collected solely from the neonate, meconium is generally regarded as the gold standard, backed by more than 30 years of research and clinical experience. ...
Maternal substance abuse during pregnancy is a growing problem with major public health and legal concerns. In utero substance exposure may adversely affect neonatal development, pregnancy outcome and the long term behavioral, cognitive and developmental abilities of the child. Also, serious legal implications are associated with substance abuse during pregnancy, including charges of child abuse and neglect which may result in removal of the neonate from parental care and loss of custodial rights. Timely detection of in utero drug exposure is necessary for early identification and effective management of exposed newborns. Accurate identification of drugexposed newborns relies on maternal history, clinical presentation of the newborn and laboratory testing of biological maternal matrices (i.e., urine, blood, oral fluid, sweat, hair, and breast milk), neonatal matrices (i.e., urine, meconium, hair, umbilical cord blood and tissue) and/or matrices from both the mother and neonate (i.e., placenta and amniotic fluid). Evaluation of biological matrices can account for in utero exposure at various stages of gestation and approximate the time-period (recent vs chronic use) of substance exposure. Each matrix has its own unique advantages and limitation in terms of ease of collection, the window of gestational exposure and sensitivity for different parent drug analytes and metabolites, which must be carefully considered for accurate interpretation of results. Analytical approaches to sample preparation and analysis vary based on the complexity of these biological matrices. Immunoassays are routinely used for screening and chromatographic separation coupled to mass spectrometry detection methods are commonly used for definitive (confirmatory) testing. Some laboratories utilize a single technology for all testing. This review provides a discussion on approaches used to detect drug-exposed newborns, biological specimens that have been studied to identify and characterize drug exposures, example analytical methods for meconium and umbilical cord tissue and considerations surrounding the interpretation of results. A possible algorithm for testing is also proposed.
...  Because of this, meconium results may reflect just the last 2-3 months of pregnancy.  Previous studies also showed that not just the timing but also the frequency of drug exposure was critical to be able detect drugs in meconium. Frequent and chronic exposure is necessary to produce meconium positive results. ...
Drug exposure during pregnancy constitutes a major legal issue and public health concern. Drug and metabolite determination in biological matrices from mother and newborn is an objective indication of prenatal drug exposure. However, limited data are available regarding the interpretation of these analytical results in terms of window of detection and degree of exposure. We collected paired maternal hair, meconium, placenta and umbilical cord from 727 mother-newborn dyads. We analyzed these specimens by liquid chromatography tandem mass spectrometry for the determination of cocaine, opioids, methadone and amphetamines, and compared the analytical results from the four different matrices. The cases were divided in non-exposure, low and frequent exposure, based on maternal hair concentrations and segmental analysis by trimesters. For cocaine, 64 cases tested positive in hair, 9 in meconium, 6 in placenta and 7 in umbilical cord. In the case of opioids, 14 maternal hair cases were positive, 11 meconium and umbilical cord and 9 placenta samples. For methadone, 11 cases were positive in hair, 9 in meconium and 6 in placenta and umbilical cord. For amphetamines, 18 cases were positive according to maternal hair, but all meconium, placenta and umbilical cord tested negative. Maternal hair was the most sensitive specimen to detect drug exposure during pregnancy. Meconium, placenta and umbilical cord tested positive if hair concentrations showed frequent drug use during the whole pregnancy, especially during the third trimester. Meconium, placenta and umbilical cord also tested positive for morphine and metabolites, if this drug was administered during labor and delivery. This article is protected by copyright. All rights reserved.
... Most xenobiotics that the foetus is exposed to during gestation are deposited here.  Recently, Concheiro et al. compared maternal hair, meconium, umbilical cord, and placenta as alternative matrices for detecting prenatal exposure to drugs of abuse in a cohort of mother-infant dyads from Northern Spain. In agreement with what has been stated here, these authors concluded that maternal hair was the most sensitive specimen to detect drug consumption during pregnancy and meconium could detect high in utero drug exposure. ...
... ве ћи про зор де тек ци је не го дру ги ма те ри јал узет од но во ро ђен че та. Га сна хро ма то гра фи ја са ма се ном спек тро ме три јом да нас је оп ште при хва ће на хе миј скоток си ко ло шка ана ли тич ка ме то да ана ли зе ме ко ни ју ма за от кри ва ње из ло же но сти пло да опи ја ти ма и дру гим нар ко ти ци ма in ute ro . ...
Females who have developed addiction to heroin also abuse it during pregnancy. Heroin can be detected in the fetal blood-flow already an hour after maternal i.v. injection. Heroin metabolites enter the fetal blood-flow through the placental barrier by passive transport.
We present a 27-year-old female in the 5th month of pregnancy that had a miscarriage. Chemo-toxicological analysis (gas chromatography with mass spectrometry--GC/MS), showed the presence of morphine in the fetal liver (31.92 ng/g), placenta (27.94 ng/g) and meconium (136.33 ng/g). The analysis did not show the presence of 6-monoacetylmorphine.
In all cases when the autopsy of fetus or newborn is performed, with mother suspected as i.v. heroin abuser, chemo- toxicological placental analysis, placenta and meconium should be also done.
... FAEEs are formed by esterification of ethanol with endogenous free fatty acids via a non-oxidative pathway facilitated by FAEE synthase and acyl-CoA/ethanol O-acyl-transferase [4,. They can be detected in several maternal matrices: the detection of FAEEs in blood reveals only recent consumption; determination in hair can account for all the pregnancy, considering that segmental analysis in a strand of at least 9 cm corresponds to the whole gestation, but hair testing is only able to detect a daily ethanol intake of at least 30 g/day, so this biomarker is not useful for lower daily intakes [2,14,15]. ...
Ethanol consumption during pregnancy is a widespread problem which is increasing in the generation of young women. Gestational alcohol consumption causes fetal exposure to this teratogen and is associated with the onset of fetal alcohol spectrum disorders (FASD) including fetal alcohol syndrome (FAS). FASD and FAS can lead to several physical, cognitive and behavioral disabilities, whose early diagnosis is of primary importance to perform primary prevention with total abstinence from alcohol during pregnancy and secondary prevention in newborns and children for a proper follow up to reduce risk of secondary consequences. In recent years significant efforts have been made to understand the underlying mechanisms of this disease and to identify objective biological and instrumental diagnostic tools, such as exposure biomarkers in neonatal meconium and advanced magnetic resonance imaging. Nonetheless, further studies are still needed to implement our knowledge on fetal effects of ethanol, and multidisciplinary actions are necessary to raise awareness among women of childbearing age about the danger of consuming even small amounts of ethanol during pregnancy.
Fetal alcohol exposure can be assessed either retrospectively by directly asking mothers or reliable informants through structured interviews and screening questionnaires or actually by measuring biomarkers of alcohol consumption in pregnancy or in the perinatal phase. For research purposes and even in some clinical cases with a need for objective corroboration of the information given by the mother or when this information is missing, there is the possibility of laboratory assessment of alcohol biomarkers. This review will present the current state of the art of FAEE (fatty acid ethyl esters) meconium analysis. An electronic search of computerized databases was carried out on an English language database (PubMed). The searched keywords in PubMed were “fatty acid ethyl ester AND meconium”. The search was limited to the English language. The PubMed search resulted in 67 articles of which 23 were excluded by the analysis of titles and abstracts. The most frequently employed assay methods were: GC-MS (40.9 %), followed by LC-MS/MS (36.4 %), GC-FID (9.1 %), and GC-FID confirmed by GC-MS (13.6 %).
In this study, the usefulness of nail samples instead of hair for a general unknown screening (GUS) for drugs was tested. An alternative matrix for long term detection is still needed in cases where no hair is available for analysis.
Hair and nail samples from 70 postmortem cases were analyzed by liquid-chromatography quadrupole time-of-flight mass spectrometry. Hair and nail samples were ground by a ball mill and extracted twice for 18 h. Extracts were measured in Auto MS/MS mode (data dependent mode acquisition).
Only 10% of the cases showed a disagreement of results in hair and nail analysis where hair samples were tested positive and corresponding nail samples were tested negative in a general unknown screening for drugs. In most of the cases investigated the analysis of the nail clippings and whole nail samples led to results comparable to those obtained from hair analysis. The incorporation of a large number of substances into the nail matrix was proven by the detection of 89 different analytes (e.g. antidepressants, drugs of abuse or antihypertonics) in our tests.
In cases where the amount of hair available is not sufficient for a general unknown screening for drugs, nails appear to be a useful comparable matrix for the detection of long-term drug consumption due to the comparison of the qualitative GUS results from the hair and nail samples in this study.
The results of meconium specimens and fortified samples screened for drugs of abuse by both enzyme-linked immunosorbent assay (ELISA, Immunalysis) and biochip microarray (Randox) methods were compared. The ELISA method was semi-automated using a TECAN Genesis. The Randox assay used the Randox Evidence Investigator system. Previously validated gas chromatography-mass spectrometry (GC-MS), GC-GC-MS, or liquid chromatography-MS-MS methods were used for confirmation and quantitation. Results from the two techniques compared well. Agreement of the Randox assay was greater than 90% when compared to the ELISA assay for all drug classes except cannabinoids (88%). Specificity of the biochip assay was slightly better for amphetamines and cocaine.
Fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) were determined in 602 meconium samples in a maternal health evaluation study for detection of gestational alcohol consumption. A validated headspace solid phase microextraction method in combination with GC-MS was used for FAEE and the cumulative concentration of ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate with a cut-off of 500 ng/g was applied for interpretation. A new and simple method was developed and validated for quantification of EtG from 10–20 mg meconium with D
5-EtG as internal standard consisting of 30 min. extraction with methanol/water (1:1, v/v), evaporation of methanol, filtration of the aqueous solution through a cellulose filter and injection into LC-MS-MS. The limits of detection and quantification for EtG were 10 and 30 ng/g, the recovery 86.6 to 106.4% and the standard deviation of the concentrations ranged from 13% at 37 ng/g to 5% at 46,700 ng/g (N = 6). FAEE above the cut-off were found in 43 cases (7.1%) with cumulative concentrations between 507 and 22,580 ng/g and with one outlier of about 150,000 ng/g (EtG not detected). EtG was detected in 97 cases (16.3%) and concentrations between LOD and 10,200 ng/g with another outlier of 82,000 ng/g (FAEE 10,500 ng/g). Optimal agreement between the two markers was obtained with a cut-off for EtG of 274 ng/g and 547 cases with both FAEE- and EtG-negative, 33 cases with both FAEE- and EtG-positive, nine cases with FAEE-positive and EtG-negative, and seven cases with FAEE-negative and EtG-positive. Differences in physical, chemical, and biochemical properties and in the pharmacokinetic behavior are discussed as reasons for the deviating cases. In none of the 602 cases, serious alcohol consumption was reported by the mothers and no evidence for gestational ethanol exposure was observed in the medical investigation of the newborns. It is concluded that the combined use of FAEE and EtG in meconium as markers for fetal alcohol exposure essentially increases the accuracy of the interpretation and helps to avoid false positive and false-negative results.
In this study, the case of a newborn with symptoms of hyperexcitability was analyzed. After it was confirmed in the hospital
that the mother had consumed drugs during pregnancy using an enzyme multiplied immunoassay technique, samples of the newborn's
urine and meconium were sent to our laboratory to observe the evolution in the distribution of cocaine and opiates during
the days following birth. For urine analysis, screening was done with an immunoassay technique, and the confirmation was done
by gas chromatography-mass spectrometry (GC-MS) according to a published method. A GC-MS method for simultaneous analysis
of cocaine, benzoylecgonine, codeine, morphine, and 6-acetylmorphine in meconium is described. GC-MS confirmation of urine
and meconium results showed consumption of cocaine and codeine during pregnancy and also showed the levels of drugs gradually
declined, totally disappearing by the third day.
Fetal alcohol syndrome (FAS), alcohol-related birth defects (ARBDs), and alcohol-related neurodevelopment disorders (ARNDs) in neonates are often the result of maternal alcohol consumption during pregnancy. Facial characteristics are associated with FAS, but ARBDs and ARNDs are more difficult to diagnose. Fetal exposure to alcohol can cause central nervous system dysfunction, pre- and postnatal growth problems, cardiac defects in neonates, and attention deficit disorders and mental retardation in older children. To date, diagnosis of fetal alcohol effect has depended largely on maternal interview, although clinical tests are becoming more widely used. Fatty acid ethyl esters (FAEEs) are formed in the body by esterification of ethanol with free fatty acids and trans-esterification of glycerides and have been detected in the meconium of newborns. This report estimates the prevalence of fetal alcohol exposure in two populations by detecting FAEEs in meconium.
We analyzed the prevalence of FAEEs in the meconium of two separate groups of neonates by use of solid-phase extraction and analysis by gas chromatography-mass spectrometry in the chemical ionization mode. In the first study, meconium samples were taken anonymously from babies born in a large, regional perinatal center in Hawaii. In the second study, specimens were obtained from infants admitted to six different newborn intensive care units within the state of Utah.
In the first study, 73 of 436 (16.7%) meconium specimens tested were considered positive for FAEEs. When broken down into quartiles, the mean total FAEEs measured were 1,059, 3,133, 6,628, and 62,115 ng/g. In the second study, 35 of 289 (12.1%) specimens were considered positive. When broken into quartiles, the mean total FAEEs were 1,139, 3,067, 7,674, and 50,143 ng/g. The overall FAEE profiles of the two study sets were remarkably similar.
In an adequate meconium specimen, a total FAEE concentration >10,000 ng/g may indicate that the newborn has been exposed to significant amounts of alcohol during pregnancy.
Procedures are given in the report for determining statistically whether the highest observation, or the lowest observation, or the highest and lowest observations, or the two highest observations, or the two lowest observations, or perhaps more of the observations in the sample may be considered to be outlying observations or discrepant values. Statistical tests of significance are useful in this connection either in the absence of assignable physical causes or to support a practical judgement that some of the experimental observations are aberrant. Both the statistical formulae and illustrative applications of the procedures to practical examples are given, thus representing a rather complete treatment of significance tests for outliers in single univariate samples.
To test the sensitivity and specificity of fatty acid ethyl esters (FAEEs) extracted from meconium to identify alcohol-using pregnant women with a sensitive and specific methodology, gas chromatography-tandem mass spectroscopy (GC/MS/MS). Study design Twenty-seven samples of meconium were obtained from infants from the mixed race community in Cape Town, South Africa, who were enrolled in a longitudinal neurobehavioral study. Maternal alcohol use was reported prospectively during pregnancy. FAEEs were isolated from meconium and quantitated by GC/MS/MS.
Ethyl oleate was the FAEE that correlated most strongly with maternal self-reported drinking, especially with the average ounces of absolute alcohol ingested per drinking day. Ethyl oleate was most strongly related to drinking in the second and third trimesters (Pearson r=.55 and.40, respectively). At a threshold of 1.5 average ounces of absolute alcohol ingested per drinking day, the area under the receiving operator characteristic curve was.92 (95% confidence interval, 0.74-0.97). Using a cut-off value of 32 ng/g, sensitivity was 84.2% and specificity was 83.3%.
Ethyl oleate concentration in meconium assayed by GC/MS/MS provides a highly sensitive and specific indicator of maternal alcohol use during pregnancy.
This study estimated in 7 Italian cities the prevalence of prenatal exposure to ethanol by determining fatty acid ethyl esters (FAEEs; palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic esters) and ethyl glucuronide (EtG) in neonatal meconium samples.
A total of 607 meconium samples were obtained from neonatal wards of 7 public hospitals: Verona and San Daniele del Friuli in the northeast of the country, Reggio Emilia in the middle east, Florence and Rome in the center, and Naples and Crotone in the southwest of the peninsula. Meconium biomarkers were assessed by a validated methodology using liquid chromatography-tandem mass spectrometry and the results categorized using the accepted cutoff of 2 nmol/g total amount of 7 FAEEs and 2 nmol/g EtG, to differentiate between heavy maternal ethanol use during pregnancy and occasional or no use at all.
On the basis of the above-reported cutoffs, the overall prevalence of newborns prenatally exposed to maternal ethanol was 7.9%: 0% in Verona, 4.0% in San Daniele del Friuli, 4.9% in Naples, 5.0% in Florence, 6.2% in Crotone, up to 10.6% in Reggio Emilia, and 29.4% in Rome. Low maternal education level and younger maternal age were associated with biomarker scores over the cutoff. There was also a significant correlation between the highest percentage of prenatal exposure in the capital and certain maternal sociodemographic characteristics.
These results indicate considerable variability in the prevalence of fetal exposure to ethanol in different Italian cities, as determined by the objective measurement of biomarkers in meconium. These data, together with previous ones obtained in Barcelona, Spain, indicate that gestational ethanol exposure is widespread, at least in parts of Europe.
Fatty acid ethyl esters (FAEEs) are nonoxidative metabolites of ethanol, and elevated levels of FAEE in meconium are a useful biomarker for heavy prenatal alcohol exposure. FAEE in meconium has been recommended as useful and cost-effective for universal screening for prenatal alcohol exposure. To support an efficient universal screening program, an analytical method to detect and quantify FAEE in meconium needs to be accurate, inexpensive, and rapid. The purpose of this study was to develop an analytical method that would satisfy these criteria and to validate this method using established laboratory guidelines. A method was developed and validated to detect and quantify four FAEEs (ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate) from 0.5 g of meconium using d(5)-ethyl esters as internal standards. The sample undergoes liquid-liquid extraction with heptane:acetone, the heptane layer is isolated and evaporated, and then, the resulting residue undergoes headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The detection limits of the four FAEEs ranged from 0.020 to 0.042 nmol/g and are 6- to 25-fold lower than the individual FAEE threshold concentrations (0.5 nmol/g). This method also has good precision with the coefficient of variation ranging from 2.6 to 19.4% for concentrations of individual FAEE between 0.5 and 2.62 nmol/g meconium (n=4). Calculated concentrations of FAEE that underwent extraction from meconium were 100-101% of the expected concentration, demonstrating the accuracy of the method. The peak shape and retention time of each FAEE were unaffected by the presence of the matrix, and there is no carryover at clinically relevant concentrations. This method was also able to produce clean chromatograms from meconium samples that could not be quantified using a previous method because of high chromatographic background. This method provides an optimal approach to detecting and quantifying FAEE in meconium that could be used in a universal screening program for prenatal alcohol exposure.
For diagnosis of chronic alcohol abuse, fatty acid ethyl esters (FAEE) were determined in hair samples from 644 individuals, mainly parents from child protection cases. The analysis for ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate was performed according to a validated procedure consisting of external degreasing by two times washing with n-heptane, extraction with a mixture of dimethylsulfoxide and n-heptane, separation and evaporation of the n-heptane layer, headspace solid phase microextraction of the residue after addition of phosphate buffer pH 7.6 and gas chromatography–mass spectrometry using deuterated internal standards. For interpretation, the sum of the concentrations of the four esters CFAEE was used with the cut-off's 0.5 ng/mg for the proximal scalp hair segment 0–3 cm or less and 1.0 ng/mg for scalp hair samples with a length between 3 and 6 cm and for body hair.
Many ethanol dependent women also use other drugs of abuse that may affect pregnancy outcome and long-term child neurodevelopment. This study investigated the association between drugs of abuse and concurrent use of ethanol in pregnancy. A study cohort of neonates with FAEE levels above 2 nmol per gram meconium, indicative of heavy in utero ethanol exposure, was identified (n=114). Meconium and hair analyses for the presence of other drugs of abuse were obtained for some of these neonates and the rates of drug exposure were compared with the rates in a cohort of neonates who were tested negative (FAEE below 2 nmol per gram meconium) for ethanol exposure (n=622). Odds ratios (ORs) for various drugs were calculated with ethanol exposure. A 15.5% positive rate for intrauterine ethanol exposure was detected. A high rate of in utero drug exposure was detected in neonates with and without in utero ethanol exposure, 60.5% versus 62.7% respectively. Neonates with heavy in utero ethanol exposure were almost twice as likely to be exposed to narcotic opiates (OR=1.90; 95% confidence interval [CI]: 1.13-3.20) and 3.3 times as likely to be exposed to amphetamine (OR=3.30; 95% CI 1.06-10.27) when compared to neonates with no ethanol exposure. Exposure to cannabinoids predicted less likely exposure to ethanol (OR=0.61; 95% CI: 0.38-0.98) and no significant difference was noted in the exposure to cocaine (OR=1.24, 95% CI: 0.81-1.91). Neonates suspected of heavy in utero ethanol exposure should be tested for other drugs of abuse and vice versa. Early detection of drug exposures can facilitate early intervention to both the neonate and the mother, thus decreasing the risk of long-term neurodevelopmental outcomes for the child, including secondary disabilities associated with fetal alcohol spectrum disorder.
A liquid chromatography tandem mass spectrometric (LC-MS/MS) method with postcolumn addition of acetonitrile for the determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in meconium was developed and validated using pentadeuterated EtG and pentadeuterated EtS as internal standards. The analytes were extracted from the matrix by acetonitrile, concentrated by solid phase extraction, separated using a reversed-phase chromatographic column, and quantified within 9 minutes. Lower limits of quantification were 5 and 1 ng/g meconium for EtG and EtS, respectively. Calibration curves were linear from lower limits of quantifications to 500 ng/g, with a minimum r > 0.999. At 3 concentrations spanning the linear dynamic range of the assay, mean recoveries ranged between 78.7% and 96.8% for EtG and between 72.1% and 95.6% for EtS. Inaccuracy was better than 8.1%, with intra-assay and interassay imprecision better than 7.2% and 10.5%, respectively. Matrix effects (ion suppression/enhancement) were found to be negligible. The analytes of interest were stable at room temperature, at 4 degrees C, when exposed to 3 freeze-thaw cycles, and when stored at -20 degrees C for up to 6 months. This sensitive and specific method was used to assess the presence of these alcohol biomarkers in meconium samples from 2 different city cohorts.
Meconium fatty acid ethyl esters (FAEEs) are currently used as biomarkers to detect heavy prenatal alcohol exposure. We introduce a novel technique to quantify FAEEs in meconium using headspace-solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). This method improves on previous approaches by decreasing sample preparation time, eliminating the need for organic solvents, and reducing the required sample size. Using 50mg of meconium, the detection limits of FAEEs ranged from 0.05 to 0.16 nmol/g and had good reproducibility making it ideal for routine analysis of clinical samples.
Fatty acid ethyl esters (FAEEs) in meconium emerged as a reliable, direct biological marker for establishing fetal exposure to ethanol. We developed an LC-MS/MS method for ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate using ethyl heptadecanoate as the internal standard. The analytes were extracted from meconium with hexane, followed by solid-phase extraction with aminopropyl-silica columns. Chromatography was performed on a C(8) reversed-phase column using water/isopropanol/acetonitrile (20:40:40, v/v/v) as a mobile phase. A triple quadrupole mass spectrometer that monitored the transitions in multiple reaction-monitoring mode was used for the detection of the analytes. Limits of quantification (LOQs) varied between 0.12 and 0.20 nmol/g. Calibration curves were linear from LOQs to 50 nmol/g for all analytes, with a minimum r(2)>0.99. At three concentrations spanning the linear dynamic range, mean recoveries ranged between 53.6 and 86.7% for the different analytes. The validated method was applied to analysis of meconium in newborns of two European cities. The two cohorts presented with different prevalence of gestational ethanol consumption during pregnancy.
Fetal exposure to drugs has many adverse effects upon the neonate including low birthweight, small head size and an increased risk of miscarriage and death. Correct diagnosis of drug use during pregnancy is essential if the child is to receive specialized treatment and care, which will aid in learning and behavioral development. Diagnosis will also help in the prevention of subsequent drug-exposed children being born to the same mother. Meconium is the first fecal material excreted by the newborn and is an excellent depository for drugs to which the fetus has been exposed. Its analysis is widely accepted in the scientific and medical communities since it has several advantages over urinalysis, including providing a longer historical record of drug exposure and easier collection. Various drugs and their metabolites have been detected in meconium, however, the metabolic profile of drugs in meconium differs from that of neonatal and/or maternal urine. This article addresses the determination of cocaines, amphetamines, opiates, cannabinoids, phencyclidine, nicotine and methadone in meconium using several analytical procedures including immunochemical and chromatographic methods.
The analysis of meconium specimens for metabolites of substances of abuse is a relatively accurate method for the detection of fetal exposure to drugs. Most of the methods reported in the literature before the early 1990s relied on radioimmunoassays. The purpose of this study was to develop and validate methods for meconium sample preparation for the screening and gas chromatography-mass spectrometry (GC-MS) confirmation of meconium extracts for cannabinoids, cocaine, opiates, amphetamines, and phencyclidine. EMIT and TDx immunoassays were evaluated as screening methods. The sample preparation method developed for screening included extraction and purification prior to analysis. Cutoff levels were administratively set at 20 ng/g for 11-nor-delta9-THC-9-COOH (THCCOOH) and phencyclidine and at 200 ng/g for benzoylecgonine, morphine, and amphetamines, although lower levels could be detected in meconium using the EMIT-ETS system. Ninety-five meconium specimens were subjected to the screening procedure with GC-MS confirmation of presumptive positives. In addition, 30 (40 for cocaine) meconium specimens were subjected to GC-MS analysis for all analytes regardless of the screening results to determine the false-negative rate, if any, of the immunoassay. Although there were no false negatives detected, the GC-MS confirmation rate for the immunoassay-positive specimens was generally low, ranging from 0% for amphetamines to 75% for opiates. The lowest rate of confirmed positives was found with the cannabinoids, suggesting that tetrahydrocannabinol (THC) metabolites other than free 11-nor-9-carboxy-delta9-THC may be major contributors to the immunoassay response in meconium.
The authors report testing the meconium of a newborn for the presence of FAEE. Meconium from a newborn of a woman who acknowledged drinking beer throughout pregnancy was tested. The authors also tested the meconiums of 3 newborns whose mothers did not drink at all while pregnant. The FAEE were extracted from the meconium samples using solid phase extraction (SPE), and were identified and quantitated by gas chromatography with flame ionization detection (FID). For assignment of retention times and determination of individual concentrations, authentic mixtures of FAEE were injected. The total FAEE concentration in the meconium of the alcohol-exposed infant was 13126 ng/g compared to a mean of 410 ng/g in the control meconiums. Also, in this case, palmitic, linoleic, and stearic ethyl esters were found in the alcohol-exposed infant's meconium while they were not found in the unexposed infant's meconium. In a parallel experiment, the authors spiked increasing amounts of ethyl alcohol (0-40mM) into the meconium from a newborn that was not exposed to ethanol in utero. The spiked samples were incubated for 4 hours at 37 degrees C and subsequently assayed for the presence of ethyl linoleate. In these experiments, they document for the first time that FAEE is produced in meconium. If confirmed by large studies, FAEE may become the first neonatal biologic marker for babies at risk for alcohol-related birth defects.
The detection of fatty acid ethyl esters (FAEE) in meconium may provide an objective estimate of prenatal alcohol exposure independent of maternal history. The authors report the results of the first population-based study conducted to investigate basal FAEE levels in the meconium of neonates not exposed to alcohol. Two hundred seven nondrinking women and their neonates were recruited from Toronto and Jerusalem. FAEE were extracted from meconium by solid-phase extraction and analyzed by GC/FID. Similar procedures were conducted in six neonates born to confirmed heavy drinkers. Low levels of meconium FAEE were detected from both cohorts (mean, 1.37 nmol/g vs. 2.08 nmol/g, Toronto vs. Jerusalem). Ethyl stearate, oleate, and linoleate were below the limit of detection in >80% of all samples, whereas ethyl laurate and palmitate were detected in >50% of the samples. Ethyl myristate was the FAEE most commonly detected (>80%). All six meconium samples with confirmed maternal drinking histories tested positive for FAEE at significantly higher levels (mean, 11.08 nmol/g). The use of 2 nmol total FAEE/g meconium as the positive cutoff, when lauric and myristic acid ethyl esters were excluded, yielded the greatest sensitivity (100%) and specificity (98.4%). The authors conclude that certain FAEE are present at measurable levels in the meconium of neonates not exposed to maternal drinking, and correction is needed to allow high specificity.
The detection of fatty acid ethyl esters (FAEE) in neonatal meconium has been proposed as a novel screening method for intrauterine exposure to alcohol. We investigated the potential use of meconium FAEE screening in a high-risk neonatal population in the absence of maternal drinking history. One hundred forty-two meconium samples of neonates suspected of intrauterine illicit substance exposure and referred to the Motherisk Laboratory were analyzed for the existence of drugs by enzyme-linked immunosorbent assay (ELISA) and FAEE by gas chromatography-flame ionization detection (GC-FID). A positive FAEE test was previously defined as a cumulative measurement of 7 individual FAEE > or = 2 nmol/g. Seventy-one percent of the samples tested positive for at least 1 illicit drug, with cannabis being the most prevalent (52.3%). Fourteen percent of all samples tested positive for prenatal alcohol exposure, as evidenced by cumulative meconium FAEE > or = 2 nmol/g. Ethyl oleate, linoleate, palmitate, and arachidonate were detected most often and at the highest levels. At least 3 individual FAEE were detected in 95% of all positive samples, and none could be identified by the use of 1 selected FAEE. Significantly elevated levels of FAEE above the baseline and the presence of multiple FAEE species in meconium are exclusive to neonates who have likely been exposed to excessive amounts of alcohol in utero. Babies born to mothers who are suspected to use illicit drugs in pregnancy are at elevated risk for exposure also to alcohol in utero. Meconium FAEE are emerging biologic markers that can potentially facilitate earlier diagnosis and intervention for less apparent forms of alcohol-related disabilities that cannot be confirmed in the absence of maternal drinking history.
Prenatal substance abuse is an ongoing concern with significant impact on neonatal health and development across socioeconomic lines. Meconium, passed by neonates during their first post-natal bowel movements, is a matrix unique to the developing fetus and contains a long history of prenatal metabolism. Over the last two decades, the use of meconium as a matrix for assessing prenatal exposure to drugs of abuse has yielded methods exhibiting higher sensitivity, easier collection, and a larger window of detection than traditional matrices. Recently, a method has been developed for the analysis of fatty acid ethyl esters in meconium as a biomarker of fetal alcohol exposure, potentially facilitating the future diagnosis of Fetal Alcohol Spectrum Disorder in situations where gestational alcohol consumption history is unknown. Screening for prenatal exposure to illicit and abused licit drugs in meconium is possible by use of a variety of immunoassay methods with conformational analysis usually occurring by GCMS or LCMS. In spite of increased sample preparation time relative to blood and urine, the long metabolic history, coupled with the ease and wide window of collection of meconium make it the ideal matrix for determining fetal drug exposure.
Biomarkers of fetal exposure to alcohol are important to establish so that early detection and intervention can be made on these infants to prevent undesirable outcomes. The aim of this study was to analyze long-chain fatty acid ethyl esters (FAEEs) in meconium as potential biomarkers of fetal alcohol exposure and effect.
Fatty acid ethyl esters were analyzed in the meconium of 124 singleton infants by positive chemical ionization gas chromatography/mass spectrometry (GC/MS) and correlated to maternal ethanol use.
A total of 124 mother/infant dyads were enrolled in the study: 31 were in the control group and 93 were in the alcohol-exposed group. The incidence (28% vs 9.7%, p = 0.037) of ethyl linoleate detected in meconium was significantly higher in the alcohol-exposed groups than the control groups. Similarly, when the concentrations of ethyl linoleate in meconium were grouped (trichotomized), there was a significant linear by linear association between alcohol exposure and group concentrations of ethyl linoleate (p = 0.013). Furthermore, only alcohol-exposed infants were found in the group with the highest ethyl linoleate concentration. The sensitivity of ethyl linoleate in detecting prenatal alcohol exposure was only 26.9%, and its specificity and positive predictive value were 96.8 and 96.2%, respectively. There was no significant correlation between the concentration of ethyl linoleate in meconium and absolute alcohol consumed (oz) per drinking day across pregnancy, although a trend toward a positive correlation is seen at lower amounts of alcohol consumed. Among the polyunsaturated, long-chain FAEEs, there was weak evidence that the incidence (21.5% vs 6.5%, p = 0.057) and concentration (p = 0.064) of ethyl arachidonate (AA) were significantly higher in the alcohol-exposed groups than the control groups. Ethyl linolenate and ethyl docosahexanoate (DHA) in meconium were found only in the alcohol group, although not at statistically significant levels. Highly significant correlations were found among the concentrations of ethyl linoleate, ethyl linolenate, ethyl AA, and ethyl DHA in meconium (correlations ranged between rs = 0.203, p = 0.024; and rs = 0.594, p < 0.001).
We conclude that FAEEs in meconium, particularly ethyl linoleate and ethyl AA, are biomarkers of high specificity for prenatal exposure to alcohol in newborn infants. We also propose that ethyl AA and DHA could be potential biomarkers of fetal alcohol effects on the developing fetal brain and should be investigated further.
In utero exposure to alcohol can have numerous adverse effects on a developing fetus. These effects represent a spectrum of structural anomalies and neurocognitive and behavioral disabilities that have recently been termed fetal alcohol spectrum disorders (FASD). Children at the most severe end of this spectrum and displaying the complete phenotype of characteristic facial anomalies, growth retardation and developmental abnormalities of the central nervous system are defined as having fetal alcohol syndrome (FAS). While FAS is the most readily clinically recognized form of FASD, other categories within the continuum of adverse effects due to prenatal alcohol exposure are becoming better defined. These include partial fetal alcohol syndrome (PFAS), alcohol-related birth defects (ARBD) and alcohol-related neurodevelopmental disorder (ARND). As more is learned regarding the exact manifestations of alcohol on brain development, these classifications may be expanded and/or refined. Because FASD represents a major public health concern, early recognition of at-risk children is important for initiating interventional strategies. Thus, the purpose of this report is to educate practicing physicians about the recognizable phenotypes of FASD in order to accurately identify these children and implement the most appropriate management plans.
Meconium has become the specimen of choice for determining fetal exposure to drugs of abuse, but its physical complexity can cause interferences from matrix effects. A new method to determine 9-carboxy-11-nor-Delta(9)-THC (9-THCA) and 11-hydroxy-Delta(9)-THC (11-OH-THC) using two-dimensional (2D) GC-MS was developed to reduce interferences and carryover. The method was validated using 70 spiked samples prepared in drug-free meconium and 46 residual patient specimens that were confirmed to contain cannabinoids. Ten patient specimens that failed to confirm due to interferences using the previous GC-MS method were analyzed using the new 2D method and 9-THCA was quantitated in all ten samples. The 2D GC-MS method improved chromatography which significantly reduced interferences and carryover when compared to the previous GC-MS method.
Challenges in identifying children exposed prenatally to ethanol necessitate the development of a biomarker for neonates at risk for fetal alcohol spectrum disorder. Meconium fatty acid ethyl esters (FAEE), products of nonoxidative ethanol metabolism, have been established as a novel biomarker of fetal ethanol exposure. We present the first application of this biomarker to a population-based sample in Canada. Six-hundred eighty-two meconium specimens were anonymously collected in the region of Grey Bruce, Ontario, Canada. Meconium FAEE were extracted by liquid-liquid and solid-phase extraction and analyzed by gas chromatography with flame-ionization detection confirmed by gas chromatography with mass spectrometry. We measured ethyl palmitate (E16:0), ethyl palmitoleate (E16:1), ethyl stearate (E18:0), ethyl oleate (E18:1), ethyl linoleate (E18:2), ethyl linolenate (E18:3), and ethyl arachidonate (E20:4). Seventeen of 682 meconium samples tested positive for significant prenatal ethanol exposure (>2.0 nmol/g). FAEE analysis detected fivefold more ethanol-exposed pregnancies than standard postpartum questionnaires in this population (2.5% versus 0.5%) (P < 0.001). The prevalence of ethanol-exposed pregnancies was consistent with Centers for Disease Control and Prevention estimates of "frequent" prenatal drinking and previously published estimates of fetal alcohol spectrum disorder disease prevalence in the general North American population. The FAEE concentrations of negative (95% confidence interval, 0.38-0.49 nmol/g) versus positive (95% confidence interval, 7.74-151.28 nmol/g) samples were distinct, further demonstrating the specificity of this biomarker in determining significant prenatal ethanol exposure. Meconium FAEE analysis demonstrates a fivefold increase in sensitivity over currently used methods of self-report-based screening in Ontario for the detection of ethanol-exposed pregnancies in a clinical setting.
The objective of this study was a review of published studies utilizing measurement of fatty acid ethyl esters (FAEE) in meconium as biomarkers for prenatal alcohol exposure.
We completed a literature search of PubMed using the terms meconium, fatty acid ethyl esters, biomarkers, and prenatal alcohol exposure. We included only peer reviewed studies utilizing analysis of meconium for the presence of FAEE in humans through the year 2007.
We found 10 articles reporting on original research examining the relationship of FAEE from meconium and prenatal alcohol exposure (PAE). The 10 articles used six different PAE assessment strategies and four different analytical techniques for determining FAEE endpoints. The articles included 2,221 subjects (range 4 to 725) with 455 (20.5%) subjects identified as exposed using the methods stated in the articles. FAEE levels above the studies' respective cutoffs were reported for 502 (22.6%) subjects.
The accurate identification of alcohol-exposed pregnancies represents a significant challenge in the development of FAEE detection cutoffs to maximize the sensitivity and specificity of the test. We present several options for the improvement of exposure assessment in future studies of FAEE as biomarkers for PAE.
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