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Clinical sensitivity and specificity of meconium fatty acid ethyl ester, ethyl glucuronide, and ethyl sulfate for detecting maternal drinking during pregnancy.

Prenatal alcohol exposure (PAE) [8] is associated with neurodevelopmental, cognitive, and behavioral disabilities in exposed infants (1). Fetal alcohol spectrum disorders (FASDs) encompass a continuum of negative consequences including growth retardation, craniofacial dysmorphology, and cognitive impairments associated with PAE, with the most extreme consequences classified as fetal alcohol syndrome (FAS). Cognitive and behavioral impairments also are prevalent in alcohol-exposed children lacking syndromal craniofacial dysmorphology (2).

Estimated drinking during pregnancy prevalence in the US is 8% (3), compared with 57% among US Northern Plains American Indians (4) and 34%-51% among mixed-ancestry women from South Africa's Western Cape (5). Alcohol consumption during pregnancy is often underreported, challenging PAE identification. Accurate PAE biomarkers available at delivery (6) would allow for early, proactive intervention for identified at-risk infants and prevent or mitigate severity of PAE-associated consequences (1, 7).

Biomarkers in meconium, the first neonatal feces, have the potential to identify in utero alcohol exposure (6, 8-18). Fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) are established in utero alcohol exposure markers (6, 8-18); although these meconium markers identify maternal alcohol consumption, data validating analyte cutoffs and interpretation criteria are limited.

FAEE are formed from endogenous free fatty acids and ethanol by specific and nonspecific enzymes in blood and several tissues (19). FAEE do not cross the placenta; therefore, meconium FAEE result exclusively from fetal synthesis (20). Some individuals recommend summing 7 meconium FAEE (ethyl linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate) and using a [greater than or equal to]2-nmol/g cutoff for heavy PAE (9, 10), whereas others sum fewer FAEE (11, 21). Summations often eliminate individual FAEE variability, although 1 report demonstrated that ethyl oleate and ethyl linoleate meconium concentrations alone distinguished women drinking [greater than or equal to]7 drinks per drinking day (DPDD) from those drinking fewer (8).

EtG is produced by UDP-glucuronosyltransferase-catalyzed conjugation of ethanol and glucuronic acid, whereas EtS results from ethanol and activated sulfate conjugation by sulfotransferases (22). Unlike FAEE, meconium EtG is primarily of maternal origin, as EtG readily crosses the placenta and fetal glucuronidation capacity is limited (23, 24). Although relatively little is known about placental EtS transfer, evidence of variable yet substantial fetal sulfotransferase activity (25) suggests that meconium EtS may be of fetal origin.

There are many published meconium FAEE reports, but far fewer for EtG and EtS. Previously proposed meconium EtG cutoffs were suggested on the basis of meconium FAEE results; therefore, meconium FAEE limitations and biases influenced these suggested cutoffs, including 111 (16), 200 (16), 274 (11), 333 (14), and 444 (12) ng/g (0.5-2 nmol/g). Meconium EtG and EtS quantification may be superior to FAEE due to improved stability in meconium (26) and insensitivity to maternal diet variation. Most FAEE in authentic meconium show degradation after 12 h at room temperature and 72 h at 4 [degrees]C, with large interindividual variability observed (26). Additionally, freeze/thaw stability experiments have shown EtG and EtS concentrations [less than or equal to]11% of initial results (13, 17, 26). These results suggest that FAEE may not be adequate long-term meconium alcohol markers due to degradation susceptibility arising from environmental conditions. Additionally, Chan et al. reported that olive oil consumption during pregnancy was associated with increased total meconium FAEE (9).

Clearly, more research is needed to validate meconium EtG and EtS cutoffs against reliable self-report measures. Our objective here was to evaluate agreement between self-reported PAE and meconium alcohol markers. With meconium results as the objective gold standard condition, we determined clinical sensitivity and specificity performance characteristics with self-reported PAE.

Materials and Methods

PARTICIPANTS

The Safe Passage Study of the Prenatal Alcohol in Sudden Infant Death Syndrome (SIDS) and Stillbirth (PASS) Network is a prospective study of 12000 mother-fetus/infant dyads, enrolled during pregnancy and followed for 1 year after birth. The primary objective is to determine the relationship between PAE, stillbirth, and SIDS (27). We recruited women at high risk for poor pregnancy outcomes and drinking during pregnancy in the US Northern Plains and Cape Town, South Africa, representing 3 diverse populations: American Indians, mixed ancestry, and white (4, 5, 27). On the basis of maternal self-report, 108 meconium samples were selected for this meconium alcohol marker investigation in the following categories: (a) no alcohol consumption at any time during pregnancy (n = 33); (b) mean third-trimester (>24 weeks) DPDD >10 (n = 14); (c) mean third-trimester DPDD 3-10 (n = 32); (d) mean third-trimester DPDD 0-<3 (n = 10); or (e) any drinking during the first or second trimesters with no drinking reported during the third trimester (n = 19). All meconium samples were collected within 48 h of birth (some at participants' homes) and refrigerated as soon as possible. Once obtained by the study sites, samples were immediately frozen ([less than or equal to] - 20[degrees]C) at study sites and remained frozen until analysis. Samples were transported on dry ice.

MATERNAL SELF-REPORT

We determined self-reported PAE by the timeline follow-back (TLFB) method (28), a structured, calendar-based interview collecting detailed ethanol consumption data for each drinking event with respect to alcohol type, container size, sharing, and duration (29). The interview was modified for PASS (27) and administered at recruitment (between 6 weeks' gestation and through but not including delivery), each prenatal visit (20-24, 28-32, and 34-38 weeks), and 1 month postdelivery. At recruitment, women were interviewed about alcohol consumption the year before pregnancy and around conception (15 days before and after last menstrual period). At other visits, the reporting period was the 30 days before the last drinking day (LDD), if the participant consumed alcohol since her last visit. We collected detailed information on alcohol brand and drink volume consumed to accurately calculate total alcohol grams consumed. We calculated the number of standard DPDD from total alcohol grams consumed each drinking day, on the basis of the US standard drink definition of 14 g ethanol (29). Local standard clinical practices determined gestational age (GA) at each site. Exposure timing was determined from GA at time of maternal report. Because meconium forms between gestational weeks 12 and 20 (30), we evaluated meconium alcohol marker detection with maternal selfreport at GA [greater than or equal to]19 weeks to account for potential GA measurement variability at the later meconium formation time (20 weeks). Additional measures were created with self-reported PAE at [greater than or equal to]12, [greater than or equal to]20, and [greater than or equal to]28 weeks' gestation (a more recent definition of the third trimester adopted by PASS after initial meconium sample selection). All measures included self-reported drinking categorization and DPDD.

MECONIUM ANALYSIS

We quantified 9 FAEE (ethyl laurate, ethyl myristate, ethyl linolenate, ethyl palmitoleate, ethyl arachidonate, ethyl linoleate, ethyl palmitate, ethyl oleate, and ethyl stearate), EtG, and EtS from a single 0.1-g meconium sample with a validated LC-MS/MS method (26). Limits of quantification (LOQs) were 25-50 ng/g for FAEE, 5 ng/g for EtG, and 2.5 ng/g for EtS. Sample preparation involved methanolic homogenization with wooden applicator sticks and solid-phase extraction (26). Meconium was thoroughly mixed before sampling to ensure a representative sample. Previously, triplicate sampling from 3 positive sources across 3 test conditions demonstrated intrasubject variability 0.7%-17.6% (26).

STATISTICAL ANALYSIS

We investigated distributions of continuous meconium biomarkers and exposure variables with Kolmogorov-Smirnov tests and visual boxplot inspection. Bivariate associations between self-reported exposure measures and meconium biomarkers were assessed with scatterplots, t tests, [chi square] tests, and Spearman correlations; among meconium biomarkers, Spearman correlations were used. Agreement between maternal self-report and meconium biomarkers was evaluated with k statistics and corresponding 95% CIs. With meconium markers as the gold-standard condition, we calculated clinical sensitivity and specificity performance characteristics and corresponding 95% CI with self-reported drinking [greater than or equal to]19 weeks' gestation as the test. Sensitivity was defined as the number of women reporting drinking [greater than or equal to]19 weeks whose infants' meconium marker concentrations were at or above the cutoff, divided by all infants with meconium marker concentrations at or above the cutoff (true positives/true positives + false negatives). Specificity was defined as the number of women who did not report drinking after 19 weeks' gestation and whose infants' meconium marker concentrations were below the cutoff, divided by all infants with meconium marker concentrations below the cutoff (true negatives/true negatives + false positives). To evaluate a dose-response effect with DPDD, 4 variables were created for reported DPDD after 19 weeks' gestation: 0 DPDD, 0 to <3 DPDD, >3-10 DPDD, and >10 DPDD. Multiple logistic regression analysis was performed associating the meconium marker with the best clinical sensitivity and specificity performance with the 4 variables representing increasing self-reported DPDD (with no DPDD as the reference group), adjusting for GA reported at LDD or nondrinking day. Model discrimination and calibration were assessed with the r-statistic and Hosmer-Lemeshow test, respectively. Analyses were performed with SAS/ STAT software, version 9.3. All analyses used a 2-sided test for statistical significance with P < 0.05, except where discussed.

Results

PARTICIPANTS

Of the 108 women selected, 107 had maternal self reported PAE information available at 19 weeks' gestation. Thirty-three women reported no alcohol consumption during pregnancy, 16 drank early in pregnancy with cessation by week 19, and 58 continued drinking beyond week 19, with 45 drinking in their third trimester (24% of women reported drinking within 1 month of delivery). Women drinking after 19 weeks reported drinking a mean (SD) of 5.6 (5.3) and 4.7 (4.7) DPDD in the second and third trimesters, respectively.

MECONIUM ALCOHOL MARKER PREVALENCE AND CONCENTRATIONS

More samples were positive for EtG (65.4%) than for EtS (21.5%), each of the 9 FAEE alone (10.3-52.3%), and the 3 FAEE summations at our LOQs (Table 1). The proposed (9, 10) meconium 7 FAEE summation (excluding ethyl laurate and myristate) cutoff [greater than or equal to] 2 nmol/g identified 25 (23.4%) infants, whereas the 4 FAEE summation (ethyl myristate, palmitate, oleate, and stearate), currently used in hair testing (31), identified 37 (34.6%).

Eighteen samples were both EtS and EtG positive; EtG concentrations (19-103716 ng/g) were 3.3-2151 times greater than EtS concentrations (2.8-408 ng/g), with a median 285.3 EtG/EtS ratio. There were 5 EtS-positive, EtG-negative samples. In 4 of the 5 cases, EtS was the only detectable marker, and concentrations ranged from 3.2 to 13 ng/g. In the fifth case, 61 ng/g EtS was detected with 13.9 nmol/g summed 7 FAEE.

Spearman correlations between individual and summed meconium alcohol marker concentrations showed that EtG was not associated with meconium EtS concentrations and only weakly or negligibly ([rho] < 0.5) (32) associated with individual and summed FAEE concentrations (Table 2). An exceptionally strong correlation ([rho] [greater than or equal to] 0.9) was seen between FAEE summations and ethyl linoleate and ethyl oleate, indicating that these analytes dominated summation calculations. Most other individual FAEE associations were strong to moderate ([rho] = 0.5-0.899) (32). Despite the many moderate/weak correlations, nearly all correlations (85.7%) were significant (n = 14; P < 0.00357), on the basis of our Bonferroni-corrected family-wise a level (Table 2).

AGREEMENT BETWEEN SELF-REPORTED EXPOSURE AND MECONIUM MARKERS

The meconium marker with the highest agreement with self-reported PAE [greater than or equal to]19 weeks was EtG [greater than or equal to]30 ng/g, with a k value of 0.57 (95% CI 0.41-0.73), signifying moderate to substantial agreement (Table 3). Individual FAEE at their LOQs had k values [less than or equal to]0.24, indicating only slight agreement. In comparison, both FAEE combinations of all 9 and 7 FAEE (excluding ethyl laurate and myristate) at [greater than or equal to]2 nmol/g demonstrated lower agreement with self-reported PAE than individual FAEE markers or the summation of 4 FAEE (ethyl myristate, palmitate, oleate, and stearate) [greater than or equal to]200 ng/g. Agreement was higher between self-reported alcohol use at [greater than or equal to]19 weeks and marker combinations that included EtG. Among the 58 women reporting drinking after 19 weeks, 48 were EtG positive with a median (range) concentration of 1101 ng/g (7.2-103716), 16 were EtS positive with a median concentration of 14 ng/g (2.8-408), and 37 were positive for 1 or more FAEE with 7 FAEE summed concentrations ranging from 0.081 to 256 nmol/g (only 13 were [greater than or equal to]2 nmol/g).

PERFORMANCE CHARACTERISTICS OF SELF-REPORTED EXPOSURE AND MECONIUM MARKERS

With EtG [greater than or equal to]30 ng/g as the gold standard condition, clinical sensitivity and specificity of self-reported drinking [greater than or equal to]19 weeks' gestation were 81.8% (95% CI 71.8-92.0) and 75.2% (95% CI 63.2-86.8), respectively. Self-reported drinking [greater than or equal to] 19 weeks' gestation yielded similar performance, with EtG and EtS at their LOQs for clinical sensitivity (68.6%, 95% CI 57.7-79.4, and 69.6%, 95% CI 50.8 -88.4, respectively), although when EtG was [greater than or equal to]5 ng/g, self-reported drinking [greater than or equal to]19 weeks had higher specificity (73.0%, 95% CI 58.7-87.3) compared with EtS [greater than or equal to]2.5 ng/g (50.0%, 95% CI 39.3-60.7). Self-reported drinking [greater than or equal to]19 weeks demonstrated 64.9% sensitivity (95% CI 49.5-80.2) and 51.4% specificity (95% CI 39.7-63.1) with the 4 FAEE summation (ethyl myristate, palmitate, oleate, and stearate) [greater than or equal to]200 ng/g; these performance measures were higher than those for the 7 and 9 FAEE summations.

With meconium EtG [greater than or equal to]30 ng/g as the gold standard condition, there were 45 true positives, 39 true negatives, 13 false positives, and 10 false negatives (Table 4). Among the 13 false-positive samples (EtG <30 ng/g but women reported drinking after 19 weeks), women reported LDD between 19.3 and 37.4 weeks. Three had detectable EtG (7.2-24 ng/g) below the 30 ng/g cutoff (1 had some individual FAEE detected), 2 others were EtS positive only, 2 others had some individual FAEE detected, and another had a 7 FAEE sum >2 nmol/g (10.6 nmol/g); no markers were detected in the other 5 (Table 4). Of the 10 self-report-positive, EtG-negative samples, only 5 women reported third-trimester drinking; on LDD median GA was 33.6 (30.6-37.4) weeks, and DPDD was 7.0 (0.8-13.8) standard drinks. Among the 5 women reporting second- but no third-trimester drinking, on LDD median GA was 25.9 (19.3-27.1) weeks, and DPDD was 1.6 (0.1-11.6) standard drinks. All 10 women reported 1-3 drinking days during the 31 days before LDD. In the 10 false-negative samples (EtG [greater than or equal to]30 ng/g but women reported no alcohol consumption during pregnancy or cessation before week 19), 6 samples had summed 7 FAEE meconium concentrations [greater than or equal to]2 nmol/g (Table 4).

DOSE-CONCENTRATION RELATIONSHIPS

A statistically significant dose-concentration relationship was observed between self-reported DPDD after 19 weeks' gestation and meconium EtG [greater than or equal to]30 ng/g, adjusted for GA at LDD (model P < 0.0001, all DPDD P < 0.01, GA P = 0.42); the model had good discrimination (r-statistic 0.81) and was well calibrated ([chi square] = 9.3, P = 0.23). For self-reported DPDD between >0 and [less than or equal to]3, the odds of observing meconium EtG [greater than or equal to]30 ng/g was 9.1 times higher (95% CI 1.8 -45.8) than for women reporting no drinking. Odds of meconium EtG [greater than or equal to]30 ng/g increased to 22.6 (95% CI 6.2-82.0) when considering self-reported DPDD between >3 and [less than or equal to]10 and to 29.4 (95% CI 2.9-295.6) when considering self-reported DPDD [less than or equal to]10, compared with women reporting no drinking. No significant changes were observed when the meconium exposure period was modified on the basis of maternal self-reported drinking at [greater than or equal to]12, [greater than or equal to]20, or [greater than or equal to]28 weeks.

Discussion

Less is known about EtG and EtS meconium markers in comparison to FAEE, and few studies have compared PAE detection capability among markers. Our study is the most thorough comparison to date of meconium EtG and EtS concentrations with FAEE concentrations and detailed, prospective maternal self-reported drinking during pregnancy. Five prior studies collected self-reported maternal alcohol consumption and meconium EtG, EtS, and FAEE. However, high underreporting in 4 of these studies did not permit the authors to compare self-reported PAE with meconium EtG and EtS; instead, meconium EtG and EtS cutoffs were optimized on the basis of meconium FAEE comparison (12, 14, 15, 18). In those studies, the proposed EtG meconium cutoffs (274, 333, and 444 ng/g) were derived from comparisons to summed 5-7 meconium FAEE concentrations (11, 12, 14). This approach is inadequate owing to FAEE limitations and bias, including instability and diet concerns. A recent Franconian Maternal Health Evaluation Studies report suggested that maternal self-report better agreed with meconium EtG than individual FAEE, supporting the concept that FAEE should not be used as the precedent (33). Our study is unique in its simultaneous FAEE, EtG, and EtS quantification in the same 0.1-g meconium sample, achievable owing to our combined analytical method (26), permitting accurate clinical sensitivity and specificity comparisons between self-reported exposure and meconium markers.

Our high clinical sensitivity and specificity indicate that accurate PAE classification in infants is optimal with EtG [greater than or equal to]30 ng/g. We found a significant dose-concentration relationship between maternal self-reported DPDD after 19 weeks and meconium EtG [greater than or equal to]30 ng/g, providing evidence that this marker accurately represents increasing PAE. Our results suggest that meconium EtG [greater than or equal to]30 ng/g is the most effective cutoff for PAE identification. DPDD, not drink timing after 19 weeks, was significantly associated with meconium EtG [greater than or equal to]30 ng/g.

Prior studies demonstrated that self-reported exposures could suffer from limited information regarding timing, quantity, and frequency, as well as recall bias in retrospective investigations. The TLFB method is a widely accepted tool to collect detailed and reliable self-reported exposure (29, 34). This method was tailored to our studied populations and modified to collect more precise, serial information throughout pregnancy, allowing evaluation of individual drinking patterns across time (27).

Our clinical sensitivity and specificity evaluation indicated that maternal self-reported PAE agreed best with meconium EtG [greater than or equal to]30 ng/g (81.8% sensitivity, 75.0% specificity, k = 0.569). The currently accepted 7 FAEE summation [greater than or equal to]2 nmol/g cutoff achieved only 52.0% sensitivity and 45.1% specificity. When considering marker cutoff combinations, meconium EtG alone [greater than or equal to]30 ng/g proved superior; of the 13 samples with EtG <30 ng/g from infants of women who reported any drinking beyond 19 weeks, only 1 had a 7 FAEE summed concentration [greater than or equal to]2 nmol/g. The better performance ofEtG compared with FAEE may have resulted from FAEE limitations, including postcollection meconium instability (26) and olive oil variability in maternal diet (9). Weak correlations between EtG and individual and summed FAEE concentrations (Table 2) may reflect the markers' different formation pathways and possibly different meconium incorporation mechanisms.

Previous meconium EtG and EtS reports did not find EtS alone in meconium; when detected, EtS was always present with EtG (12, 14). However, EtS is often present without EtG in urine (35, 36). This study is the first report of EtS-positive, EtG-negative meconium samples. In 1 case, 61 ng/g EtS was detected with high FAEE (13.9 nmol/g summed 7 FAEE). In the other 4 cases, EtS was the only detectable marker. This may be explained by our low EtS LOQ compared to our other markers, although our EtS LOQwas higher than the 1 ng/g previously reported (13). EtG-negative meconium also may have resulted from reduced maternal EtG formation due to genetic UGT polymorphisms (37) or liver damage from heavy alcohol consumption. UGT polymorphism studies indicated that cannabinoids, specifically cannabidiol, negatively impacted ethanol glucuronidation activity (37). EtS-positive, EtG-negative meconium could result from increased fetal sulfotransferase activity compared to fetal glucuronidation capacity. Fetal liver sulfotransferase activity varies on the basis of isoform and gestational period (25); some isoforms demonstrate higher activity during gestation compared with infancy (25), whereas fetal UDP-glucuronosyltransferase activity is limited, with most activity beginning after birth (23). Urinary EtG/EtS studies demonstrated that bacteria-infected urine was capable of both EtG hydrolysis and formation (38); urinary bacterial meconium contamination may explain some positive self-report, EtG-negative meconium (EtG hydrolysis) and negative self-report, EtG-positive meconium (EtG formation). Urine and postmortem tissue EtS was stable (38, 39), providing a possible explanation for some EtS-only-positive urine. Most South Africa samples were collected at home within 48 h and returned to the study site; bacterial contamination during home collection could also explain these findings.

Variability in EtG and EtS formation and placental transfer of ethanol and these markers may explain the wide EtG/EtS ratio range observed (3.3-2151). The low 3.3 EtG/EtS ratio resulted from 19 ng/g EtG and 5.7 ng/g EtS from a self-reported nondrinker. The highest EtG/EtS ratio resulted from 18286 ng/g EtG and 8.5 ng/g EtS from a PAE infant exposed to 12.7 and 5.6 DPDD in the second and third trimesters, respectively.

Our study had some limitations. Our small feasibility design, as opposed to a true random selection study design, and population demographics require that the proposed meconium EtG [greater than or equal to]30 ng/g cutoff be validated in larger samples from other populations. A larger sample would also offer an opportunity to further investigate the role of EtS in identifying PAE.

Our study demonstrates the importance of identifying alcohol-exposed infants via both maternal self-report and objective biomarkers. Meconium biomarkers primarily capture drinking in the third trimester and some second-trimester exposure, whereas maternal self-report can identify earlier gestational exposure missed by meconium. In our study, most negative self-report, meconium EtG [greater than or equal to]30 ng/g cases demonstrated strong PAE evidence; EtG's validity was confirmed by the presence of additional meconium markers. These women likely underreported their drinking behavior, demonstrating limitations of self-report reliance, or results could be explained by consumption of other products containing ethanol not identified with our TLFB assessment, or bacterial contamination at collection. However, our positive self-report, meconium EtG <30 ng/g cases demonstrate that some drinking behaviors may not be accurately identified with meconium analysis. Increasing meconium formation before birth may explain these findings, as increasing amounts of negative meconium may dilute limited or previously EtG-positive meconium. Therefore, selfreport should also be included in studies using meconium biomarkers.

In conclusion, maternal self-report was correlated with meconium EtG, EtS, and FAEE concentrations in the same 0.1-g meconium sample. Optimal clinical sensitivity (81.8%) and specificity (75.0%) were observed between maternal self-reported alcohol consumption after 19 weeks' gestation and meconium EtG [greater than or equal to]30 ng/g. These data should help inform clinicians, clinical chemists, and toxicologists on meconium alcohol marker interpretation and PAE identification.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contribution to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: The PASS Research Network is supported by the National Institute on Alcohol Abuse and Alcoholism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and National Institute on Deafness and Other Communication Disorders through the Cooperative Agreements (U01 HD055154, U01 HD045935,U01 HD055155,U01 HD045991 andU01AA016501). Funding also was provided by the Intramural Research Program, National Institute on Drug Abuse, NIH. L. Burd, North Dakota Department of Health.

Expert Testimony: None declared. Patents: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: The authors gratefully acknowledge cooperation of the study participants, PASS investigators and NICHD Advisory Safety Monitoring Board members: Elizabeth Thom, PhD, (Chair); Reverend Phillip Cato, PhD; James W. Collins, Jr., MD, MPH; Terry Dwyer, MD, MPH; George Macones, MD; Philip A. May, PhD; Jeff Murray, MD; Richard M, Pauli, MD, PhD; Raymond W. Redline, MD; and Michael Varner, MD.

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(26.) Himes SK, Concheiro M, Scheidweiler KB, Huestis MA. 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 2014; 406:1945-55.

(27.) Dukes KA, Burd L, Elliott AJ, Fifer WP, Folkerth RD, Hankins GD, et al. The Safe Passage Study: design, methods, recruitment and follow-up approach. Paediatr Perinat Epidemiol 2014; 28:455-65.

(28.) Sobell LC, Sobell MB. Timeline follow-back: a technique for assessing self-reported ethanol consumption. In: Allen J, Litten RZ, eds. Measuring alcohol consumption: psychosocial and biological methods. Totowa, NJ: Humana Press; 1992:41-72.

(29.) Dawson DA. Methodological issues in measuring alcohol use. Alcohol Res Health 2003; 27:18-29.

(30.) Burd L, Hofer R. Biomarkers for detection of prenatal alcohol exposure: a critical review of fatty acid ethyl esters in meconium. Birth Defects Res A Clin Mol Teratol 2008; 82:487-93.

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Sarah K. Himes, [1] Kimberly A. Dukes, [2] Tara Tripp, [2] Julie M. Petersen, [2] Cheri Raffo, [2] Larry Burd, [3] Hein Odendaal, [4] Amy J. Elliott, [5] Dale Hereld, [6] Caroline Signore, [7] Marian Willinger, [7] and Marilyn A. Huestis, [1] * for the Prenatal Alcohol in SIDS and Stillbirth (PASS) Network

[1] Chemistry and Drug Metabolism Section, Intramural Research Program, National Institute on Drug Abuse, NIH, Baltimore, MD; [2] DM-STAT Inc., Malden, MA; [3] Department of Pediatrics, University of North Dakota School of Medicine, Grand Forks, ND; [4] Department of Obstetrics and Gynecology, Faculty of Medicine and Health Science, Stellenbosch University, Tygerberg, South Africa; [5] Center for Health Outcomes and Prevention Research, Sanford Research, Sioux Falls, SD; [6] Division of Metabolism and Health Effects, National Institute on Alcohol Abuse and Alcoholism, NIH, Rockville, MD; [7] Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD.

* Address correspondence to this author at: Chemistry and Drug Metabolism Section, IRP, NIDA, NIH, 251 Bayview Blvd, Suite 200, Rm 05A721, Baltimore, MD 21224. Fax443740-2823; e-mail mhuestis@intra.nida.nih.gov.

Received September 22, 2014; accepted December 19, 2014.

Previously published online at DOI: 10.1373/clinchem.2014.233718

[8] Nonstandard abbreviations: PAE, prenatal alcohol exposure; FASD, fetal alcohol spectrum disorder; FAS, fetal alcohol syndrome; FAEE, fatty acid ethyl esters; EtG, ethyl glucuronide; EtS, ethyl sulfate; DPDD, drinks per drinking day; SIDS, sudden infant death syndrome; PASS, Prenatal Alcohol in SIDS and Stillbirth Network; TLFB, timeline follow back; LDD, last drinking day; GA, gestational age; LOQ, limit of quantification.
Table 1. Meconium assay LOQ and alcohol marker concentrations in
positive samples.

                                                 Positive samples

                           Assay LOQ, ng/g               Median, ng/g
Marker                        (nmol/g)         n (%)       (nmol/g)

EtG                          5 (0.023)       70 (65.4)   208 (0.935)
EtS                        2.5 (0.020)       23 (21.5)    11 (0.087)
Ethyl laurate               50 (0.219)       11 (10.3)    81 (0.355)
Ethyl myristate             25 (0.098)       19 (17.8)    88 (0.343)
Ethyl linolenate            25 (0.049)       11 (10.3)   118 (0.385)
Ethyl palmitoleate          25 (0.053)       17 (15.9)   354 (1.250)
Ethyl arachidonate          25 (0.045)       32 (29.9)    73 (0.220)
Ethyl linoleate             25 (0.049)       42 (39.3)   156 (0.504)
Ethyl palmitate             50 (0.176)       32 (29.9)   139 (0.487)
Ethyl oleate                25 (0.048)       56 (52.3)   280 (0.902)
Ethyl stearate              50 (0.160)       30 (28.0)   165 (0.528)
Sum 9 FAEE                                   58 (54.2)   501 (1.636)
Sum 7 FAEE (excluding                        58 (54.2)   501 (1.636)
  laurate and myristate)
Sum 4 FAEE (myristate,                       58 (54.2)   375(1.219)
  palmitate, oleate, and
  stearate)

                                Positive samples

Marker                        Range, ng/g (nmol/g)

EtG                        6.2-103,716 (0.028-467)
EtS                            2.8-408 (0.022-3.23)
Ethyl laurate                   51-884 (0.223-3.87)
Ethyl myristate                25-4985 (0.098-19.4)
Ethyl linolenate                25-768 (0.082-2.51)
Ethyl palmitoleate             25-9075 (0.089-32.1)
Ethyl arachidonate             25-3511 (0.075-10.6)
Ethyl linoleate                26-9800 (0.084-31.8)
Ethyl palmitate              50-25,080 (0.176-88.2)
Ethyl oleate                 25-50,500 (0.081-163)
Ethyl stearate                 52-6688 (0.166-21.4)
Sum 9 FAEE                   25-84,064 (0.081-279)
Sum 7 FAEE (excluding        25-78,195 (0.081-256) (a)
  laurate and myristate)
Sum 4 FAEE (myristate,       25-61,194 (0.081-202) (b)
  palmitate, oleate, and
  stearate)

(a) Twenty-five cases had 7 FAEE [greater than or equal to] 2
nmol/g,thesuggested cutoff by Chan etal. (9, 10).

(b) Thirty-seven cases had 4 FAEE [greater than or equal to] 200 ng/g,
a suggested cutoffcommonly used in hair FAEE testing (31).

Table 2. Spearman correlation coefficients ([rho]) (P values in
parentheses) for meconium marker correlations and summation
calculations. (8)

                        EtG         EtS      Summed EtG     Ethyl
                                              and EtS      laurate

EtS                   0.2852
                     (0.0029)
Summed EtG EtS        0.9786#     0.3830
                     (<0.0001)   (<0.0001)
Ethyl Laurate         0.0999      0.3586       0.1468
                     (0.3061)    (0.0001)     (0.1314)
Ethyl Myristate       0.2448      0.2559       0.2728      0.6442
                     (0.0110)    (0.0078)     (0.0045)    (<0.0001)
Ethyl Linolenate      0.1634      0.3632       0.2124      0.8971#
                     (0.0927)    (0.0001)     (0.0281)    (<0.0001)
Ethyl Palmitoleate    0.2196      0.2365       0.2091      0.5033
                     (0.0230)    (0.0142)     (0.0307)    (<0.0001)
Ethyl Arachidonate    0.3259      0.3751       0.3441      0.4360
                     (0.0006)    (0.0007)     (0.0003)    (<0.0001)
Ethyl Linoleate       0.3237      0.2539       0.3400      0.5467
                     (0.0007)    (0.0083)     (0.0003)    (<0.0001)
Ethyl Pa Imitate      0.3619      0.3185       0.3760      0.4206
                     (0.0001)    (0.0008)     (0.0001)    (<0.0001)
Ethyl Oleate          0.3738      0.2665       0.3827      0.4953
                     (<0.0001)   (0.0055)    (<0.0001)    (<0.0001)
Ethyl Stearate        0.4882      0.2846       0.4783      0.3049
                     (<0.0001)   (0.0030)    (<0.0001)    (0.0014)
Summed 9 FAEE         0.4217      0.2766       0.4329      0.4908
                     (<0.0001)   (0.0039)    (<0.0001)    (<0.0001)
Summed 7 FAEE         0.4213      0.2766       0.4325      0.4888
                     (<0.0001)   (0.0039)    (<0.0001)    (<0.0001)
Summed 4 FAEE         0.4299      0.2750       0.4382      0.4733
                     (<0.0001)   (0.0041)    (<0.0001)    (<0.0001)

                       Ethyl       Ethyl         Ethyl
                     myristate   linolenate   palmitoleate

EtS

Summed EtG EtS

Ethyl Laurate

Ethyl Myristate

Ethyl Linolenate      0.7323#
                     (<0.0001)
Ethyl Palmitoleate    0.7530#      0.5080
                     (<0.0001)   (<0.0001)
Ethyl Arachidonate    0.5926       0.5201        0.5799
                     (<0.0001)   (<0.0001)     (<0.0001)
Ethyl Linoleate       0.7292#      0.5622       0.7082#
                     (<0.0001)   (<0.0001)     (<0.0001)
Ethyl Pa Imitate      0.7034#      0.4469       0.7362#
                     (<0.0001)   (<0.0001)     (<0.0001)
Ethyl Oleate          0.6722       0.5046        0.6727
                     (<0.0001)   (<0.0001)     (<0.0001)
Ethyl Stearate        0.6057       0.3881        0.6381
                     (<0.0001)   (<0.0001)     (<0.0001)
Summed 9 FAEE         0.6748       0.5024        0.6630
                     (<0.0001)   (<0.0001)     (<0.0001)
Summed 7 FAEE         0.6725       0.5004        0.6629
                     (<0.0001)   (<0.0001)     (<0.0001)
Summed 4 FAEE         0.6702       0.4871        0.6684
                     (<0.0001)   (<0.0001)     (<0.0001)

                        Ethyl         Ethyl       Ethyl       Ethyl
                     arachidonate   linoleate   palmitate    oleate

EtS

Summed EtG EtS

Ethyl Laurate

Ethyl Myristate

Ethyl Linolenate

Ethyl Palmitoleate

Ethyl Arachidonate

Ethyl Linoleate        0.7860#
                      (<0.0001)
Ethyl Pa Imitate       0.7580#       0.7979#
                      (<0.0001)     (<0.0001)
Ethyl Oleate           0.7732#       0.9279#     0.8018#
                      (<0.0001)     (<0.0001)   (<0.0001)
Ethyl Stearate         0.7093#       0.6432      0.7809#     0.6768
                      (<0.0001)     (<0.0001)   (<0.0001)   (<0.0001)
Summed 9 FAEE          0.7771#       0.9117#     0.8158#     0.9828#
                      (<0.0001)     (<0.0001)   (<0.0001)   (<0.0001)
Summed 7 FAEE          0.7790#       0.9114#     0.8169#     0.9829#
                      (<0.0001)     (<0.0001)   (<0.0001)   (<0.0001)
Summed 4 FAEE          0.7758#       0.9050#     0.8244#     0.9801#
                      (<0.0001)     (<0.0001)   (<0.0001)   (<0.0001)

                       Ethyl      9 FAEE      7 FAEE
                     stearate

EtS

Summed EtG EtS

Ethyl Laurate

Ethyl Myristate

Ethyl Linolenate

Ethyl Palmitoleate

Ethyl Arachidonate

Ethyl Linoleate

Ethyl Pa Imitate

Ethyl Oleate

Ethyl Stearate

Summed 9 FAEE         0.7390#
                     (<0.0001)
Summed 7 FAEE         0.7398#     0.9999#
                     (<0.0001)   (<0.0001)
Summed 4 FAEE         0.7542#     0.9989#     0.9990#
                     (<0.0001)   (<0.0001)   (<0.0001)

(a) For marker combinations, individual analyte concentrations
(nmol/g) were summed. Summed 7 FAEE included all except ethyl laurate
and myristate as recommended in meconium (9, 10), and summed 4 FAEE
included ethyl myristate, palmitate, oleate, and stearate, as
recommended in hair (31). Strong ([rho] = 0.7-0.899) and exceptionally
strong ([rho] = 0.9-1.0) correlations are shown in bold (32). The
Bonferroni-corrected family-wise [alpha] level for these 14
comparisons was P < 0.00357.

Note: Strong ([rho] = 0.7-0.899) and exceptionally strong ([rho] =
0.9-1.0) correlations are indicated with #.

Table 3. Clinical sensitivity and specificity of maternal self-
reported drinking during pregnancy at or beyond 19 weeks' gestation
(test condition) compared with individual and combined meconium
alcohol markers (gold standard condition).

    Meconium analytes and cutoffs       Sensitivity (95% CI)

Ethyl laurate, [greater than or equal   54.5 (25.1-84.0)
  to] 50 ng/g (LOQ)
Ethyl myristate, [greater than or       57.9 (35.7-80.1)
  equal to] 25 ng/g (LOQ)
Ethyl linolenate, [greater than or      54.5 (25.1-84.0)
  equal to] 25 ng/g (LOQ)
Ethyl palmitoleate, [greater than or    52.9 (29.2-76.7)
  equal to] 50 ng/g (LOQ)
Ethyl arachidonate, [greater than or    62.5 (45.7-79.3)
  equal to] 25 ng/g (LOQ)
Ethyl linoleate, [greater than or       61.9 (47.2-76.6)
  equal to] 25 ng/g (LOQ)
Ethyl palmitate, [greater than or       71.9 (56.3-87.5)
  equal to] 25 ng/g (LOQ)
Ethyl oleate, [greater than or equal    62.5 (49.8-75.2)
  to] 25 ng/g (LOQ)
Ethyl stearate, [greater than or        76.7 (61.5-91.8)
  equal to] 50 ng/g (LOQ)
9 FAEE, [greater than or equal to]      53.8 (34.7-73.0)
  2 nmol/g
7 FAEE, [greater than or equal to]      52.0 (32.4-71.6)
  2 nmol/g (9, 10)
4 FAEE, [greater than or equal to]      64.9 (49.5-80.2)
  200 ng/g (31)
EtS, [greater than or equal to] 2.5     69.6 (50.8-88.4)
  ng/g (LOQ)
EtG, [greater than or equal to] 5       68.6 (57.7-79.4)
  ng/g (LOQ)
EtG, [greater than or equal to] 10      72.3 (61.4-83.2)
  ng/g
EtG, [greater than or equal to] 15      73.4 (62.6-84.3)
  ng/g
EtG, [greater than or equal to]         80.4 (70.0-90.8)
  25 ng/g
EtG, [greater than or equal to]         81.8# (a) (71.6-92.0)
  30 ng/g
EtG, [greater than or equal to]         82.4 (71.9-92.8)
  50 ng/g
EtG, [greater than or equal to] 333     96.8 (90.6-100.0)
  ng/g (1.5 nmol/g) (14)
EtG, [greater than or equal to] 444     96.7 (90.2-100.0)
  ng/g (2 nmol/g) (15, 18)
EtG, [greater than or equal to] 5       66.7 (56.0-77.3)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 10      70.0 (59.3-80.7)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 15      71.0 (60.3-81.7)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 30      74.6 (63.9-85.4)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 50      74.6 (63.5-85.7)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 333     83.3 (72.1-94.6)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
9 FAEE, [greater than or equal to] 2    74.5 (62.0-86.9)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g
9 FAEE, [greater than or equal to] 2    70.4 (58.2-82.5)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g, or EtS,
  [greater than or equal to] 2.5 ng/g
7 FAEE, [greater than or equal to] 2    74.5 (62.0-86.9)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g
7 FAEE, [greater than or equal to] 2    70.4 (58.2-82.5)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g, or EtS,
  [greater than or equal to] 2.5 ng/g
4 FAEE, [greater than or equal to]      74.5 (62.5-86.5)
  200 ng/g, or EtG, [greater than or
  equal to] 1.5 nmol/g
4 FAEE, [greater than or equal to]      70.7 (59.0-82.4)
  200 ng/g, or EtG, [greater than or
  equal to] 1.5 nmol/g, or EtS,
  [greater than or equal to] 2.5 ng/g
9 FAEE, [greater than or equal to] 2    74.2 (63.3-85.1)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g
9 FAEE, [greater than or equal to] 2    70.6 (59.8-81.4)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g, or EtS,
  [greater than or equal to] 2.5 ng/g
7 FAEE, [greater than or equal to] 2    74.2 (63.3-85.1)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g
7 FAEE, [greater than or equal to] 2    70.6 (59.8-81.4)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g, or EtS, [greater
  than or equal to] 2.5 ng/g
4 FAEE, [greater than or equal to]      75.0 (64.4-85.6)
  200 ng/g, or EtG, [greater than or
  equal to] 30 ng/g
4 FAEE, [greater than or equal to]      71.4 (60.8-82.0)
  200 ng/g, or EtG, [greater than or
  equal to] 30 ng/g, or EtS, [greater
  than or equal to] 2.5 ng/g

    Meconium analytes and cutoffs       Specificity (95% CI)

Ethyl laurate, [greater than or equal   45.8 (35.9-55.8)
  to] 50 ng/g (LOQ)
Ethyl myristate, [greater than or       46.6 (36.2-57.0)
  equal to] 25 ng/g (LOQ)
Ethyl linolenate, [greater than or      45.8 (35.9-55.8)
  equal to] 25 ng/g (LOQ)
Ethyl palmitoleate, [greater than or    45.6 (35.3-55.8)
  equal to] 50 ng/g (LOQ)
Ethyl arachidonate, [greater than or    49.3 (38.0-60.6)
  equal to] 25 ng/g (LOQ)
Ethyl linoleate, [greater than or       50.8 (38.6-62.9)
  equal to] 25 ng/g (LOQ)
Ethyl palmitate, [greater than or       53.3 (42.0-64.6)
  equal to] 25 ng/g (LOQ)
Ethyl oleate, [greater than or equal    54.9 (41.2-68.6)
  to] 25 ng/g (LOQ)
Ethyl stearate, [greater than or        54.5 (43.4-65.7)
  equal to] 50 ng/g (LOQ)
9 FAEE, [greater than or equal to]      45.7 (34.8-56.5)
  2 nmol/g
7 FAEE, [greater than or equal to]      45.1 (34.4-55.9)
  2 nmol/g (9, 10)
4 FAEE, [greater than or equal to]      51.4 (39.7-63.1)
  200 ng/g (31)
EtS, [greater than or equal to] 2.5     50.0 (39.3-60.7)
  ng/g (LOQ)
EtG, [greater than or equal to] 5       73.0 (58.7-87.3)
  ng/g (LOQ)
EtG, [greater than or equal to] 10      73.8 (60.5-87.1)
  ng/g
EtG, [greater than or equal to] 15      74.4 (61.4-87.5)
  ng/g
EtG, [greater than or equal to]         74.5 (62.5-86.5)
  25 ng/g
EtG, [greater than or equal to]         75.0# (a) (63.2-86.8)
  30 ng/g
EtG, [greater than or equal to]         71.4 (59.6-83.3)
  50 ng/g
EtG, [greater than or equal to] 333     63.2 (52.3-74.0)
  ng/g (1.5 nmol/g) (14)
EtG, [greater than or equal to] 444     62.3 (51.5-73.2)
  ng/g (2 nmol/g) (15, 18)
EtG, [greater than or equal to] 5       75.0 (60.0-90.0)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 10      75.7 (61.9-89.5)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 15      76.3 (62.8-89.8)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 30      75.0 (62.2-87.8)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 50      70.8 (58.0-83.7)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 333     64.6 (53.0-76.2)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
9 FAEE, [greater than or equal to] 2    61.7 (49.4-74.0)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g
9 FAEE, [greater than or equal to] 2    62.3 (49.2-75.3)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g, or EtS,
  [greater than or equal to] 2.5 ng/g
7 FAEE, [greater than or equal to] 2    61.7 (49.4-74.0)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g
7 FAEE, [greater than or equal to] 2    62.3 (49.2-75.3)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g, or EtS,
  [greater than or equal to] 2.5 ng/g
4 FAEE, [greater than or equal to]      64.3 (51.7-76.8)
  200 ng/g, or EtG, [greater than or
  equal to] 1.5 nmol/g
4 FAEE, [greater than or equal to]      65.3 (52.0-78.6)
  200 ng/g, or EtG, [greater than or
  equal to] 1.5 nmol/g, or EtS,
  [greater than or equal to] 2.5 ng/g
9 FAEE, [greater than or equal to] 2    73.3 (60.4-86.3)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g
9 FAEE, [greater than or equal to] 2    74.4 (60.7-88.1)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g, or EtS,
  [greater than or equal to] 2.5 ng/g
7 FAEE, [greater than or equal to] 2    73.3 (60.4-86.3)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g
7 FAEE, [greater than or equal to] 2    74.4 (60.7-88.1)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g, or EtS, [greater
  than or equal to] 2.5 ng/g
4 FAEE, [greater than or equal to]      76.7 (64.1-89.4)
  200 ng/g, or EtG, [greater than or
  equal to] 30 ng/g
4 FAEE, [greater than or equal to]      78.4 (65.1-91.6)
  200 ng/g, or EtG, [greater than or
  equal to] 30 ng/g, or EtS, [greater
  than or equal to] 2.5 ng/g

    Meconium analytes and cutoffs              [kappa] (95% CI)

Ethyl laurate, [greater than or equal    0.001 (-0.106 to 0.109)
  to] 50 ng/g (LOQ)
Ethyl myristate, [greater than or        0.025 (-0.111 to 0.161)
  equal to] 25 ng/g (LOQ)
Ethyl linolenate, [greater than or       0.001 (-0.106 to 0.109)
  equal to] 25 ng/g (LOQ)
Ethyl palmitoleate, [greater than or    -0.008 (-0.138 to 0.123)
  equal to] 50 ng/g (LOQ)
Ethyl arachidonate, [greater than or     0.096 (-0.069 to 0.261)
  equal to] 25 ng/g (LOQ)
Ethyl linoleate, [greater than or        0.119 (-0.060 to 0.298)
  equal to] 25 ng/g (LOQ)
Ethyl palmitate, [greater than or        0.204 (0.043-0.366)
  equal to] 25 ng/g (LOQ)
Ethyl oleate, [greater than or equal     0.174 (-0.012 to 0.361)
  to] 25 ng/g (LOQ)
Ethyl stearate, [greater than or         0.243 (0.087-0.399)
  equal to] 50 ng/g (LOQ)
9 FAEE, [greater than or equal to]      -0.003 (-0.159 to 0.152)
  2 nmol/g
7 FAEE, [greater than or equal to]      -0.02 (-0.173 to 0.134)
  2 nmol/g (9, 10)
4 FAEE, [greater than or equal to]       0.144 (-0.028 to 0.316)
  200 ng/g (31)
EtS, [greater than or equal to] 2.5      0.126 (-0.018 to 0.271)
  ng/g (LOQ)
EtG, [greater than or equal to] 5        0.386 (0.214-0.558)
  ng/g (LOQ)
EtG, [greater than or equal to] 10       0.448 (0.279-0.617)
  ng/g
EtG, [greater than or equal to] 15       0.468 (0.300-0.636)
  ng/g
EtG, [greater than or equal to]          0.550 (0.391-0.708)
  25 ng/g
EtG, [greater than or equal to]          0.569# (a) (0.413-0.725)
  30 ng/g
EtG, [greater than or equal to]          0.535 (0.376-0.693)
  50 ng/g
EtG, [greater than or equal to] 333      0.476 (0.334-0.619)
  ng/g (1.5 nmol/g) (14)
EtG, [greater than or equal to] 444      0.459 (0.317-0.601)
  ng/g (2 nmol/g) (15, 18)
EtG, [greater than or equal to] 5        0.362 (0.192-0.531)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 10       0.424 (0.255-0.593)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 15       0.444 (0.276-0.612)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 30       0.488 (0.322-0.654)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 50       0.453 (0.284-0.623)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
EtG, [greater than or equal to] 333      0.449 (0.289-0.610)
  ng/g, or EtS, [greater than or
  equal to] 2.5 ng/g
9 FAEE, [greater than or equal to] 2     0.352 (0.180-0.525)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g
9 FAEE, [greater than or equal to] 2     0.327 (0.148-0.505)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g, or EtS,
  [greater than or equal to] 2.5 ng/g
7 FAEE, [greater than or equal to] 2     0.352 (0.180-0.525)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g
7 FAEE, [greater than or equal to] 2     0.327 (0.148-0.505)
  nmol/g, or EtG, [greater than or
  equal to] 1.5 nmol/g, or EtS,
  [greater than or equal to] 2.5 ng/g
4 FAEE, [greater than or equal to]       0.386 (0.213-0.559)
  200 ng/g, or EtG, [greater than or
  equal to] 1.5 nmol/g
4 FAEE, [greater than or equal to]       0.36 (0.183-0.537)
  200 ng/g, or EtG, [greater than or
  equal to] 1.5 nmol/g, or EtS,
  [greater than or equal to] 2.5 ng/g
9 FAEE, [greater than or equal to] 2     0.47 (0.302-0.637)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g
9 FAEE, [greater than or equal to] 2     0.426 (0.256-0.596)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g, or EtS,
  [greater than or equal to] 2.5 ng/g
7 FAEE, [greater than or equal to] 2     0.47 (0.302-0.637)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g
7 FAEE, [greater than or equal to] 2     0.426 (0.256-0.596)
  nmol/g, or EtG, [greater than or
  equal to] 30 ng/g, or EtS, [greater
  than or equal to] 2.5 ng/g
4 FAEE, [greater than or equal to]       0.506 (0.342-0.670)
  200 ng/g, or EtG, [greater than or
  equal to] 30 ng/g
4 FAEE, [greater than or equal to]       0.463 (0.297-0.628)
  200 ng/g, or EtG, [greater than or
  equal to] 30 ng/g, or EtS, [greater
  than or equal to] 2.5 ng/g

(a) Bold text indicatesoptimal clinical sensitivity and specificity
parameter agreement.

Note: Bold text indicatesoptimal clinical sensitivity and specificity
parameter agreement indicated with #.

Table 4. Meconium alcohol marker results and maternal self-reported
alcohol consumption for true and false positives and negatives
considering meconium EtG [greater than or equal to] 30 ng-g as the
gold standard condition and maternal self-reported alcohol consumption
beyond 19 weeks as the test condition. (a)

                   Meconium EtG       Self-reported      n
                  (gold standard      alcohol intake
                    condition)        after week 19
                                     (test condition)

True positive    [greater than or          Yes          45
                 equal to] 30 ng/g

True negative        <30 ng/g               No          39

False positive       <30 ng/g              Yes          13

False negative   [greater than or           No          10
                 equal to] 30 ng/g

                   Meconium EtG      Additional meconium marker results
                      (ng/g)

True positive    1215 (30-103,716)   14 (31.1%) also EtS-positive; 12
                                       (26.7%) also FAEE-positive with
                                       sum 7 >2 nmol/g; 8 (17.8%)
                                       positive for all 3 markers

True negative         0(0-26)        9 EtG only; 2 EtS only; 4 FAEE
                                       only; 1 EtS and FAEE; 2 EtG and
                                       EtS; 1 EtG, EtS, and FAEE

False positive        0(0-24)        3 of 13 had detectable EtG below
                                       cutoff (7.2, 21, 24 ng/g); (e) 2
                                       others were EtS-positive only
                                       (3.2,13 ng/g); 1 other FAEE-
                                       positive only, with sum 7 FAEE >
                                       2 nmol/g; given positive
                                       maternal self-report, these
                                       cases should be classified as
                                       exposed

False negative    79.5 (51-6230)     5 also with 7 FAEE sum [greater
                                       than or equal to] 2 nmol/g and 1
                                       EtS- and FAEE-positive (6230 ng/g
                                       EtG, 13 ng/g EtS, 4.44 sum 7 FAEE
                                       nmol/g); (f) strong intrauterine
                                       alcohol exposure evidence in 6
                                       of 10 cases as multiple markers
                                       were in agreement; women likely
                                       underreported drinking behavior

                   GA at last reported      Standard drinks at
                       drinking day            last reported
                       (weeks) (a)             drinking day

True positive    34.1 (20.1-40.9)           3.7 (0.06-23.3) (c)

True negative    1.1 (-23.7 to 10.3) (d)    5.6 (1.6-15.1) (d)

False positive   33.4(19.3-37.4)            2.3(0.14-13.8)

False negative   5.3 (-7.9 to 16.9)s        5.0 (0.5-12.0) (g)

                  Number of drinking
                 days in past 31 days
                  from last reported
                     drinking day

True positive    2 (1-31)

True negative    2 (1-28) (d)

False positive   1 (1-9)

False negative   2 (1-31) (g)

(a) Data are median (range) unless noted otherwise.

(b) GA at birth based on fetal ultrasound (n = 73,68.2%) or last
reported menstrual cycle (n = 34,31.8%) at prenatal enrollment visit;
GA at last reported drinking calculated from subtracting last reported
drinking day from GA at birth. Negative numbers indicate last drinking
day was preconception.

(c) Data available for 44 (97.8%).

(d) Data available for 17 (43.6%). The 22 remaining women reported no
drinking in any trimester.

(e) The 3 EtG-positive samples were from women who reportedly all
stopped drinking during week 34. Women whose infants had the 3.2 and
13 ng/g EtS-positive samples stopped drinking during week 20 and 37,
respectively. The woman whose infant had meconium 7 FAEEsum >2 nmol/g
stopped drinking at27.1 weeks.

(f) The false negative with 6230 ng/g EtG, 13 ng/g EtS, and high FAEE
reportedly stopped drinking at 5 weeks' gestation. During the 31 days
before stopping, she drank every day with an average of 7 drinks/day
reported.

(g) Data from 7 women (70%) were included here. The remaining 3 women
reported no drinking in any trimester.
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Title Annotation:Drug Monitoring and Toxicology
Author:Himes, Sarah K.; Dukes, Kimberly A.; Tripp, Tara; Petersen, Julie M.; Raffo, Cheri; Burd, Larry; Ode
Publication:Clinical Chemistry
Article Type:Report
Date:Mar 1, 2015
Words:10053
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