ul carryover challenge must be less the determined LOD.

Evaluating 6 negative controls spiked with a cocktail of potentially interfering substances (Table 1) assessed the specificity of each assay. The results of the 6 controls must be less than the LOD of its respective assay. Analyzing 6 LOQ controls fortified with a cocktail of potentially interfering compounds challenged the selectivity of the method. All 6 replicates must satisfy the identification criteria and the measured concentrations must be within 20% of target value.

Bias and imprecision of each assay were determined by analyzing prepared controls at three different concentrations, replicates of five over four different days. The concentrations investigated were 5, 40, and 75 ng/g for EtG and 10, 50, and 100 ng/g for POPE. The bias and imprecision challenge was considered to be successful if each intra-assay mean and the inter-assay mean were within 15% of target value and the maximum intra-and inter-assay variance must be less than 20%, respectively.

The extraction efficiency and matrix effect were determined using procedures defined by Matuszewski [50,51]. To determine the matrix effect and extraction efficiency, three sets of controls were prepared over three concentrations with five replicates each. The concentrations analyzed for each assay were 10, 100 and 200 ng/g for POPE and 5, 100, and 200 ng/g for EtG.

The first set was unextracted controls reconstituted in mobile phase A. The second set was negative UC extracts fortified with POPE or EtG after being subjected to its respective extraction procedure. The third set was negative UC controls obtained from five different sources fortified with POPE or EtG that were subjected to the extraction procedures. The extraction efficiency for each analyte was expressed as the ratio of the average peak area in set 3 to set 2. The matrix effect for each analyte was defined as the ratio of the mean peak area of set 2 to set 1. The relative matrix effect was defined as the coefficient of variation (%CV) of the standard line slopes constructed from negative UC controls obtained from five different biological sources fortified with POPE or EtG that were subjected to the extraction procedure. Five replicates of five concentrations were used that ranged from 4 to 200 ng/g. A relative matrix effect of less than 4.5% was considered acceptable [51].

Table 1. List of potentially interfering compounds used to evaluate the selectivity and specificity of PEth and EtG in UC.

The stability of prepared extracts was assessed by the re-analysis of a control set from the bias and imprecision experiment that had been stored at room temperature for five days. The stability was expressed as a ratio of the results of the incubated controls and the original measured concentrations. The stability to freeze-thaw conditions were evaluated by subjecting a control set from the precision and accuracy experiment to three daily freezethaw cycles. Freeze-thaw stability was expressed as a ratio of the observed means versus the respective target concentration.

Post-collection syntheses of EtG, POPE, and other alcohol biomarkers in specimens exposed to or contaminated with ethanol have been reported under a variety of conditions [52-55]. In the field, it is reasonable to expect that a UC specimen may be exposed to ethanol either intentionally or unintentionally. To evaluate the potential for in vitro formation of POPE and EtG in UC, we examined 2 aliquots from 3 negative UC specimens. One aliquot from each specimen was stored for 2 days at room temperature in an airtight 1000 mL beaker containing an open 5 mL vial of ethanol and the second aliquot was stored at room temperature without being exposed to ethanol vapor.

To determine if these biomarkers are present at consistent levels throughout the UC, a longitudinal study was performed on a positive and negative specimen. Six aliquots were taken at equally spaced intervals along a 6 inch section of UC. The mean, standard deviation, and %CV were calculated for each specimen.

2.12. Application of Method to Authentic Specimens

The methods were applied to 308 de-identified UC that were received by our laboratory for routine toxicological analysis.

2.13. Statistical Analysis

Statistical analysis was performed using IBM® SPSS® Statistic Version 19.0.0. Pearson correlation was used to evaluate the association between POPE and EtG observed in authentic UC. A probability of P ≤ 0.01 was considered to be significant.

3. Results

3.1. Validation Results

The parameters and transitions for the mass spectrometry were consistent with previous reports [43,56,57]. The precursor ions for the PEth analytes were the deprotonated molecular weight ions m/z 702.0 and m/z 733.0 for POPE and POPE-d31, respectively [37,56,57]. The product ion(s) for each POPE corresponded to its fatty acid moiety(s), m/z 255 of the palmitic acid, m/z 281 of the oleic acid, and m/z 281 of the per-deuterated palmitic acid (Figure 1) [37,56,57]. The precursor ions for the EtG and EtG-d5 were the deprotonated molecular weight ions m/z 221.0 and m/z 226.1 for EtG and EtG-d5, respectively (Figure 2). The product ion used for quantitation (m/z 75) has been proposed to be the 2-hydroxyethanoate ion or the 2-hydroxy-1-propoxide ion and the qualifying ion for EtG (m/z 85) has been proposed to be the 2-hydroxy-3-buten-1-oxide anion [58]. Both fragments are remnants of a complex ring opening and multiple step fragmentation [58]. The number of identification points for both compounds was 4, satisfying the com-

Figure 1. Structure and fragmentation pattern of POPE.

Figure 2. Structure of EtG.

monly accepted recommendation of at least 3 identification points [49]. Extracted ion chromatograms of a LOQ control for POPE and EtG are presented in Figures 3 and 4.

The determined LOD and LOQ for POPE were 2.0 and 4.0 ng/mL, respectively. The determined LOD and LOQ for EtG were 1.0 and 3.0 ng/mL, respectively. The specificity of each assay was considered acceptable because POPE and EtG were not detected (

The relative matrix effects were 3.4% and 4.3% for POPE and EtG, respectively. The accuracy, precision and linearity calculations are presented in Table 2. The absolute matrix effect and extraction efficiency results are posted in Table 3. The stability data are listed in Table 4. All bias determinations of the validation were within 14.7% of target concentration. All imprecision calculations were less than 7.6%.

The 3 ethanol-vapor-exposed aliquots formed POPE and EtG in vitro after standing at room temperature for 2 days. POPE and EtG were not detected in the 3 non-exposed segments. The exposed aliquots produced between 208 and 1029 ng/g of POPE and between 11 and 201 ng/g of EtG. The negative specimens were negative

Figure 3. Selected ion chromatogram of POPE and POPEd31 LOQ control (4 ng/g).

Figure 4. Selected ion chromatogram of EtG and EtG-d5 LOQ control.

Table 2. Precision, accuracy, and linearity of methods for the detection of POPE and EtG in UC.

Table 3. Matrix effect and extraction efficiency data for the detection of POPE and EtG in UC.

Table 4. Stability data for POPE and EtG in UC.

throughout the entire length of specimen tested. The longitudinal study for the positive specimen demonstrated that POPE (719 ± 273 ng/g; %CV = 38%) and EtG (2742 ± 85.9 ng/g; %CV = 3%) were consistently found along the length of UC.

3.2. Application of Method

A group of 308 UC specimens that had been received by our laboratory were tested using the newly validated methods. Two hundred and ninety five (295) specimens did not contain detectable amounts of POPE or EtG. Four specimens contained both POPE and EtG. Eight specimens contained EtG and no detectable POPE. One specimen that contained POPE did not contain a detectable amount of EtG.

Five (1.6%) specimens contained a detectable amount of POPE. The POPE mean concentration was 11.4 ng/g ± 9.4 ng/g and the median was 11.0 ng/g. Twelve (3.9%) specimens contained a detectable amount of EtG. The EtG mean concentration was 127.2 ng/g ± 227.7 ng/g and the median was 21.0 ng/g. The Pearson product-moment correlation coefficient (r) was 0.576 (P = 0.07, n = 13). Results of the 13 specimens that contained either POPE or EtG are charted in Figure 5.

4. Discussion

We have presented fully validated assays for the detection of two direct alcohol biomarkers, POPE and EtG, in human UC. In addition, we have analyzed 308 authentic UC that had been received into our laboratory for routine analysis for the presence of POPE and EtG. The positiveity rates for POPE and EtG in the authentic specimen survey were 1.6% and 3.9%, respectively. When detected, the mean concentration of POPE was 11.4 ng/g ± 9.4 ng/g and the mean concentration of EtG was 127.2 ± 227.7

Figure 5. Comparison of results for authentic UC specimens with detectable levels of POPE or EtG.

ng/g. The measured concentrations of POPE and EtG were positively associated (r = 0.5174) but the association was insignificant (P = 0.07).

POPE was previously reported in 10 types of postmortem tissue (kidney, lung, spleen, liver, heart, skeletal muscle, small intestine, fat, cerebellum, and brain cortex) from known alcoholics [55]. The measured concentrations of POPE in these tissues were several orders of magnitude higher (9.8 g/g to 937 g/g) than those found in our UC (3 ng/g to 27 ng/g). The elevated levels of the autopsy tissues were proposed to be due in part to in vitro synthesis from the significant blood alcohol content of the decedents at the time of death (121 mg/dL to 364 mg/dL) and subsequent freezing of the harvested tissue which further concentrates the ethanol in the tissues [55]. In our study, UC presented a unique opportunity to evaluate the presence of POPE in human tissue other than post-mortem analysis.

The detection of EtG in post-mortem tissues has been suggested as a useful tool to gain insight into a decedent’s alcohol history [59]. Once again, EtG levels much higher than our observations were reported presumably influenced by elevated blood alcohol concentrations (106 mg/dL to 183 mg/dL) at the time of death or formed from ethanol due to putrefaction. Morini et al. [43] reported the presence of EtG in placenta and the fetal remains of pregnancies that had been voluntarily terminated in the 12th week. The fetal study reported EtG concentrations between 78 ng/g and 1299 ng/g, which were consistent with the levels we observed in our UC survey (4 ng/g to 666 ng/g).

The measured concentrations of EtG in UC in our study (4 ng/g to 666 ng/g; mean 127 ng/g) were found to be similar to concentrations found in recent meconium studies. Bakdash et al. [15] reported concentrations between 10 ng/g and 10,230 ng/g (mean 601 ng/g) in a study originating from Erlangen, Germany. Morini el al [41,42] and Pichini et al. [44] reported concentrations between 6.9 ng/g and 1443 ng/g from five cities in Italy and Barcelona, Spain.

The BRFSS reported that 7.4% of women self-reported the use of alcohol during their pregnancy [5]. A very similar prevalence of self-reported drinking specifically during the 3rd trimester (6.5%) was reported by the NBDPS [4]. The BRFSS and NBDPS further reported that 1.4% self-reported binge drinking at some point during the pregnancy and 0.5% self-reported binge drinking during the 3rd trimester. We understand that these findings are underestimated due to obvious limitations of using self-report but provide context for evaluating the prevalence of direct alcohol biomarkers in newborn tissue. The prevalence of POPE and EtG in UC at 1.6% and 3.9%, respectively, may be more consistent with those reporting binge drinking and therefore a more risky behavior.

Our study found that detectable levels of POPE and EtG may be formed in vitro by exposure to ethanol vapor. Historically, caution has been advised when interpreting FAEE, urine ETG, and whole blood POPE for medicolegal issues due to post-collection synthesis. That caution applies to these analyses as well. Therefore, it is very important for the collection staff to be aware of this observation and ensure that ethanol containing products are not used on or near the specimen during the collection process.

A limitation of this study was the absence of experimentally determined pharmacokinetics of POPE and EtG in UC, due to obvious ethical concerns. A second limitation of this study was the lack of accurate detailed selfreport data concerning maternal consumption of ethanol. Accurate pharmacokinetic and self-report data would have provided insight to the correlation, clinical sensitiveity and clinical specificity of these two assays in relation to risky alcohol behavior. Another limitation of this study was that due to productivity concerns in an operational reference laboratory, the two assays were developed at different times on different analytical platforms and the target concentrations chosen for each validation study were not identical.

5. Conclusion

This study provides evidence that umbilical cord tissue is a suitable specimen type to identify in utero exposure to ethanol. Umbilical cord tissue is an ideal specimen type for newborn screening programs and large scale epidemiological studies because, when compared to other newborn toxicology specimen types, it is truly a universal specimen and very simple to collect. Recently, detection of the direct alcohol biomarkers, POPE and EtG, have been gaining popularity to monitor risky alcohol drinking behavior in areas such as professional health programs, substance abuse treatment evaluation, and chronic disease management. These assays provide another tool for the neonatal health professional to identify candidates in need of further evaluation.

6. Acknowledgements

Portions of this study were sponsored by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) grant R 43 AA016702. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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NOTES

*Declaration of interest: The authors are employed by United States Drug Testing Laboratories, Inc (USDTL). USDTL is a commercial reference laboratory that analyzes biological specimens for the presence of alcohol and drug biomarkers. This does not alter our adherence to all the journal’s policies on sharing data and materials.

#Corresponding author.

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