Introduction to PEth as a direct alcohol biomarker
Professor and Dep. Director at the Institute of Forensic Medicine Bern, Switzerland
Phosphatidylethanols (PEth) are a family of direct biomarkers for alcohol consumption detectable in whole blood, since they are located in the erythrocytes. This species has been detected already approx. 40 years ago, however, modern LC-MS/MS technology was necessary to apply PEth diagnostics in many forensic and clinical fields of application, whenever time periods (middle to long-term / days to weeks) need to be monitored. Since 2009, we have used LC-MS/MS in several clinical studies for the analysis of PEth in comparison to other alcohol markers and self-reports. PEth has proven very high specificity, and due to accumulation by repeated drinking and with a half-life of up to 15 days, PEth has a high potential for the determination of alcohol consumption habits and also for abstinence monitoring. This presentation will include the following topics:
• PEth analogs detectable in human blood
• Bioformation of PEth after single drinking events – what is the minimum amount of alcohol needed for the detection of PEth
• Elimination of PEth after multiple excessive and long-term excessive alcohol consumption
• Cut-off concentrations for “abstinence” and for “excessive alcohol consumption”
• Comparison of PEth in blood and EtG in hair for abstinence monitoring
• Specificity of PEth as a direct marker of alcohol consumption
• Stability of PEth (venous blood, capillary blood, dried blood spots DBS)
• Application of PEth in different forensic and clinical fields
Increasing confidence in phosphatidylethanol measurements – The road to comparability of results over time, location and assay
Katleen van Uytfanghe
Quality manager and technical supervisor of the accredited reference laboratory (Ref4U) and routine laboratory at the Laboratory of Toxicology of the University of Ghent, Belgium
Phosphatidylethanol (PEth) measurements are used to either assess someone’s drinking behavior (by comparing a result to a decision limit) or to monitor changes in someone’s drinking behavior (by comparing new results vs previous results). As PEth measurements are more and more implemented, patient files may include results stemming from different laboratories. Next to that, there is a growing consensus to use a common decision limit to conclude on compatibility with abstinence. These types of use of laboratory results require them to be perfectly comparable over time, location and assay.
Although this prerequisite seems easy to fulfill, past experiences in the broader field of clinical chemistry have proven the opposite. Therefore, the concept of implementing “a uniform reference measurement system (RMS)” has been developed. The latter is often referred to as “standardization”. Implementing an RMS should lead to comparable laboratory results, regardless of the principle of measurement, the method, the actual measurement procedure (test kit, including the test-specific calibration), and the laboratory where the analyses are carried out. In practice, such a system comes down to (i) agreeing on a few parameters such as the definition of the analyte, and (ii) the use of a common basis for calibration.
Hence, the meaning of “standardization” in this case does not imply “agreeing on a common protocol for sample preparation and measurement”. On the contrary, in principle everybody is free to use whatever method of choice – as long as the end result can be traced back (i.e. is metrologically traceable) to the SI-units and the highest international standards available. In other words: each application laboratory can use whatever method available, provided it measures the correct analyte, and the utilized methodology is fit for purpose and well-validated.
At the onset of an increased implementation of PEth measurements, the scientific community should take its responsibility and define the anchor points of PEth standardization. This is the only way to guarantee that a result of 15 or 38 ng/mL PEth means exactly the same in each laboratory and that it is interpreted in the same way.
This presentation will explain:
the key elements of an RMS, and reflect on how to implement it for PEth measurements.
the distinction between “what is needed for standardization” and “what is expected from good laboratory practice”
Keywords: comparability of results, reference measurement system, phosphatidylethanol
#1: Formation of seven PEth-homologues after excessive use of an ethanol-based hand sanitizer
Herzog J., Skopp G., Musshoff F.
Forensic Toxicological Center (FTC) Munich, Bayerstrasse 53, 80335 Munich, Germany
Introduction and Aim(s)
Phosphatidylethanol (PEth) has become a widespread marker offering a longer retrospective window of detection than urinary ethyl glucuronide (UEtG) to detect alcohol use. For abstinence control, the main homologue 16:0/18:1 is used. In this study, six additionally homologues (16:0/18:2; 16:0/20:4; 17:0/18:1; 18:0/18:1; 18:0/18:2; 18:1/18:1) were also covered. Due to the COVID 19 pandemic, alcohol-based hand sanitizers are frequently used. It has been shown that excessive use can lead to a positive result of UEtG. The aim of this study was to prove whether the threshold of 20 ng/mL for PEth 16:0/18:1 is reached and whether other homologues are formed using an ethanol-based hand sanitizer.
Ten volunteers (8 occasional, 2 regular drinkers) were asked to use an alcohol-based hand sanitizer 30 times over 6 hours on 5 successive days. Regular drinkers were included to study a possible increase of PEth-homologues. At the beginning and the end of the six hours’ period dried blood spots and urine samples were collected. PEth and UEtG were determined by LC/MSMS. The LOQ for PEth homologues was from 10.7 (for PEth 16:0/18:1) to 18.0 ng/mL (remaining homologues).
Results and Discussion
Before the start, all occasional drinkers were tested negative for UEtG whereas 2 of them had PEth 16:0/18:1-concentrations of 12.5 and 19.3 ng/mL. Regular drinkers showed PEth 16:0/18:1-concentrations of 242 and 354 ng/mL.
Alcohol abstaining subjects are likely of exceeding 20 ng/mL for PEth 16:0/18:1 (range: 11.6-22.1 ng/mL). Regular drinkers showed a decline of PEth-concentrations, especially of 16:0/18:1, 16:0/18:2, 16:0/20:4, 18:0/18:1 and 18:0/18:2. After six hours on every day, all volunteers showed UEtG concentrations between 152 and 1414 ng/mL.
In teetotalers, the above-mentioned threshold can be exceeded following excessive use of a hand sanitizer, whereas in cases of moderate alcohol consumption PEth homologues are not likely to be influenced.
Using an ethanol-based hand sanitizer and participating in any alcohol monitoring program respective terms and conditions of passing should carefully be considered. A previous alcohol abstinence accompanying a negative PEth value or its decline over a certain period an influence by ethanol-based hand sanitizer is not likely. However, people with a PEth concentration slightly below 20 ng/mL are endangered of exceeding this threshold using an ethanol-based hand sanitizer.
Keywords: PEth, UEtG, ethanol-based hand sanitizer
#2: “Yes, you have been drinking the past few weeks” – Increasing the confidence in a PEth 16:0/18:1 decision limit for abstinence/minor alcohol intake at 20 ng/mL
Van Uytfanghe K., Stove C. P.
Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
Introduction and Aim(s)
There is a growing international consensus to use 20 ng/mL phosphatidylethanol 16:0/18:1 (PEth) as a decision limit or cut-off value to conclude whether a person has been abstinent, or only had a minor alcohol intake in the past couple of weeks. As this conclusion can be life-changing, a high confidence in the decision limit is needed. Here we report on our efforts to seek confidence.
A large scale PEth monitoring study was set up in which over 687 participants self-reportedly refrained from alcohol consumption for one month. Participants were adults who usually drink alcohol and who were recruited via the ‘Tournée Minérale’ initiative. They took 3 finger prick samples with 2 weeks intervals via self-sampling at home using volumetric absorptive micro sampling devices (VAMS, Mitra®, Neoteryx). PEth was quantified using a validated liquid chromatography – tandem mass spectrometry method. A population-based algorithm capable of predicting abstinence with 95% probability was set up by fitting a linear mixed effect model to discern patterns in PEth elimination over time while accounting for intra- and interindividual variability in PEth scores. Based on this regression model, it can be calculated that an individual starting at 160 ng/mL PEth, will end up at 10 ng/mL PEth after 4 weeks of abstinence, with an upper limit for the prediction interval at ~19 ng/mL, here rounded to 20 ng/mL. Using a subset of the same data (477 participants), specificity and negative predictive value were calculated. If the participant’s initial PEth concentration was >160 ng/mL (n=71, PEth values ranged from 160,6 ng/mL to 1142 ng/mL), a concentration >20 ng/mL after four weeks of abstinence was considered correct (‘true positive’), while a concentration <20 ng/mL was considered wrong (‘false negative’). For those individuals with an initial PEth concentration ≤160 ng/mL (n=406, PEth values ranged from 4,14 ng/mL to 160 ng/mL), a final concentration <20 ng/mL was considered correct (‘true negative’; compatible with abstinence), whereas a concentration >20 ng/mL was considered incorrect (‘false positive’).
Results and Discussion
Using the cut-off of 20 ng/mL, the specificity (the probability of a negative test result in an individual that should be negative, i.e. someone with a PEth starting value ≤160 ng/mL) was 99.3% and the negative predictive value (the probability of not having drunk in an individual with a negative test result) was 94.8%.
The cut-off of 20 ng/mL to score abstinence/minor alcohol intake includes all possible sources of variation, also the measurement uncertainty. The accompanying high specificity demonstrates that this cut-off can be used with high confidence to score ‘compatibility with abstinence or minor alcohol intake’.
Keywords: decision limit, cut-off value, PEth 16:0/18:1, abstinence testing
#3: Preliminary results of blood PEth16:0/18:1, PEth16:0/20:4 and ethylglucuronide simultaneous determinations in a clinical study of patients hospitalized for withdrawal
Hakim F.1-2, Wiart J. F.1, Gicquel T.3, Scliffet D.4, Cottencin O.4, Allorge D.1-2, Gaulier J. M.1-2
1CHU Lille – Laboratory of Toxicology
2University of Lille – ULR 4483 IMPECS
3CHU Rennes – Laboratory of Toxicology and Forensic
4CHU Lille – Addictology Unit
Introduction and Aim
This paper aims to report preliminary results of PETHOXICO, a monocentric non-interventional non-randomized cross-sectional clinical study (ClinicalTrials.gov NCT04557631) in a population of patients hospitalized for withdrawal.
Blood and urine were sampled on the day of admission (D0) and 7 days later (D7). Blood PEth16:0/18:1, PEth16:0/20:4 and ethylglucuronide (EtG) were analyzed simultaneously in dried blood spots using a LC-MS/MS method (LLOQ 2 µg/L). In addition, urinary EtG and blood alcohol concentration (BAC) were determined using immunoassay and GC-FID, respectively.
Results and Discussion
A total of 37 chronic and excessive ethanol consumers (ethanol per week consumptions ranging from 560 to 1750 g) were included in PETHOXICO (sex ratio 11/26 (F/M); 30 to 67 YO with a median at 46 YO).
At D0, the mean concentration of PEth16:0/18:1 was 1180 µg/L (range: 53-3889 µg/L) with 95 % of participants above the 210 µg/L threshold. PEth16:0/20:4-to-PEth16:0/18:1 mean ratio was 0.46 with PEth16:0/20:4 concentrations ranging from 9 to 2353 µg/L. The mean BAC was 1060 mg/L (range: undetected-3540 mg/L) and for 78 % of participants, urinary EtG was above the 500 µg/L threshold.
At D7, no BAC nor EtG (urine and blood) were found as expected. The mean concentration of PEth16:0/18:1 dropped to 527 µg/L (range: 16-1655 µg/L) with still 78 % of participants above the 210 µg/L threshold.
PEth16:0/20:4 concentrations dropped more significantly (range: undetected-923 µg/L) with a mean PEth16:0/20:4-to-PEth16:0/18:1 ratio of 0.26. This decreased ratio [0.46 at D0 and 0.26 at D7) is coherent with the expectation of a shortened detection window for PEth16:0/20:4. Blood elimination half-life of PEths were calculated with the assumption of a single elimination phase: mean half-life were 6.9 days (range: 2.2-18.6 days) and 4.6 days (range: 1.3-9.3 days) for PEth16:0/18:1 and PEth16:0/20:4, respectively. PEth16:0/18:1 observed half-lives are consistent with literature data (around 7 days with a significant interindividual variability). Observed PEth16:0/20:4 half-lives are higher than the very scarce data from literature (2 days).
In this study, the use of an analytical method combining 3 direct metabolites of ethanol allows, in one run, to discriminate a very recent consumption (< 48 h) from a recent consumption (< 2 weeks) and an older consumption (2-4 weeks). PEth16:0/18:1 sensitivity to detect chronic and excessive alcohol consumers was 95 % with a 210 µg/L threshold. However, these preliminary results needs yet to be confronted to detailed clinical data.
Keywords: DBS, Phosphatidylethanol, Ethyl glucuronide, Withdrawal
#4: A New Assay for Phosphatidylethanol (PEth) in Blood via Gas Chromatography-Electron Impact-Mass Spectrometry (GC-EI-MS)
DeFrancesco J. V.1, Reichert M.1,McMillin M. T.1, Afshar M.2
1Loyola University Chicago, Forensic Science Program and Department of Chemistry and Biochemistry, 1068 W. Sheridan Rd., Chicago, IL 60660 USA
2Loyola University Chicago, Department of Medicine, Health Sciences Campus, 2160 S. First Avenue, Maywood, IL 60153 USA
Introduction and Aims
We report a new method to assay PEth in blood via derivatization and GC-EI-MS analysis. The goal was to develop a “point of care” means of detection and quantitation of PEth to identify alcohol misuse of patients in a clinical setting that was faster and less expensive than Liquid Chromatography-Tandem Mass Spectrometry (LC-MS-MS). Derivatization creates a chemical species consisting of an ethyl phosphate headgroup with two TMS moieties. Thus far, quantitation has been performed in whole and dried blood (DBS).
Samples were extracted with various combinations of methanol, isopropanol, and chloroform. DBS samples were hole punched as single units and sonicated in extracting solvent. The supernatant was filtered, evaporated to dryness, and derivatized. Derivatization of phosphatidylethanol 16:0/18:1 (PEth) and phosphatidylethanol-d5 16:0/18:1 (PEth-d5) with BSTFA or MSTFA yielded the corresponding phosphate ester-di-trimethylsilyl product. Samples were analyzed by GC-EI-MS in full scan/SIM dual mode on an Agilent 7890A/5975C GC-MSD fitted with an Rtx-5ms GC column and helium carrier gas.
Results and Discussion
Chromatographic separation of PEth-di-TMS and several other PEth phosphate ester di-TMS analogs used as internal standards was surprisingly good. Most work was focused on PEth-d5 as the internal standard. Analysis of whole and dried blood samples were linear in the range of 50-1500 ng/mL using PEth-d5 as internal standard. Method precision and accuracy have not yet been established.
This new method is effective at measuring the total amount of ethanol incorporated into the blood phospholipids. Unlike existing LC-MS-MS methods which directly measure individual PEth species, our new method captures all ethanol incorporated into the RBC phospholipids by forming a single, derivatized ethylphosphate analyte. This new approach has several advantages over prior methods: the cost of instrument purchase and operation and the flexibility to detect ethanol in whole, dried, and potentially decomposed blood (ex. postmortem).
This work is based on US patent 11,085,939 B2 titled “Quantifying Phosphatidylethanol from Blood Samples” published in 2021.
Keywords: PEth, Phosphate ester, TMS Derivatization, GC-EI-MS
#5: Ethanol dose-response of PEth levels observed following five daily drinking days in the human laboratory
Roache J. D.1, Hill-Kapturczak N.1, Lopez-Cruzan M.1, Simon T. W.2, Ginsburg B.1, and Javors M. A.1
1University of Texas Health Science Center, Department of Psychiatry & Behavioral Sciences, San Antonio, TX.
2Ted Simon, LLC, Winston, GA
Phosphatidylethanol 16:0/18:1 (PEth) is an increasingly important biomarker used to detect recent alcohol drinking. PEth is rapidly formed within hours after drinking and evidence suggests that measured PEth concentrations can be related to the amounts of alcohol consumed. Given its more gradual elimination half-life of 4-10 days, we explored the possibility that PEth levels would accumulate across days of repeated alcohol consumption.
13 female and 9 male (n=22) research volunteers who reported drinking heavily at least one day in the previous month remained abstinent (confirmed by transdermal alcohol monitoring) for one week before coming into the laboratory for five consecutive days (Mon-Fri). Participants were randomized to receive either 0.4 or 0.8 g/kg ethanol administered in the morning at 0900h over a 15 min drinking procedure. PEth levels were measured repeatedly before and 15, 30, 45, 60, 90, 120 and 360 min after drinking on Days 1 and 5 to assess initial rates of PEth formation and before and 120 min after drinking on Days 2,3,4.
Despite one week of verified abstinence, non-zero PEth values (median=52 ng/ml) were observed on Day 1. Significant (p<0.05) day effects and dose*day interactions indicated that baseline PEth levels accumulated across the five days – but more so for the 0.8 g/kg dose group. Each day, PEth levels increased from 0 to 120 min post-dose, and again the dose*day interaction was significant (p<0.03) reflecting that greater PEth increases were seen with the 0.8 g/kg group. Full pharmacokinetic time course analyses of 0-360 minute PEth levels on Days 1 vs. 5 similarly reflected PEth increases reaching a peak at about 120 min and showing day effects only within the 0.8 g/kg group. Even though there was evidence of accumulation in the baseline values within the high dose group, we unexpectedly observed that PEth levels returned towards the previous baseline value by the next day (i.e., 24 hr later) on Days 2-5.
We confirmed limited evidence that PEth will accumulate across days of repeated drinking, but perhaps only when higher alcohol doses such as 0.8 g/kg are consumed. These data support the value of laboratory studies of PEth formation and suggest a need to better understand the variables involved in PEth formation and incorporation into the red blood cell membrane.
#6: Assessment of Inter-Lab Reproducibility in Phosphatidylethanol Quantification using a Fully Validated Dried Blood Spot-Based Liquid Chromatography Tandem Mass Spectrometry Assay
Salah L. M.1, Bushman L. R.1, Abdelmawla F.1, Tien P. C.2, Hahn J. A.2, Kiser J. J.1
1University of Colorado Anschutz Medical Campus, Aurora, CO
2University of California San Francisco, San Francisco, CA
Introduction and Aim
There is a need for bio-analytical assays that can quantify phosphatidylethanol (PEth) in dried blood spots (DBS). This unique matrix has become prominent in research due to its ease of collection, storage, transport, and increased stability. We developed and validated a DBS-based PEth assay in our Colorado Antiviral Pharmacology (CAVP) lab per FDA guidance for bio-analytical method validation. We also systematically evaluated the effects of crucial DBS-specific parameters on PEth quantification. As part of our assay validation, we performed a cross-validation with the United States Drug Testing Laboratory (USDTL) to evaluate inter-lab reproducibility in PEth quantification.
Samples from the HCV Cure (21 samples collected in the United States from persons with Hepatitis C, 2018-2020) and ADEPT (25 samples collected in Uganda from persons with HIV, 2012-2015) studies, previously used to measure PEth in DBS by the USDTL (lower limit of quantification (LLOQ): 8 ng/mL), were quantified using the CAVP lab’s validated PEth assay (LLOQ: 5 ng/mL). PEth 16:0/18:1 was the homologue quantified in all samples. Reference materials for the USDTL and CAVP assays were obtained from Enzo Life Sciences and Cerilliant, respectively. Incurred sample reanalysis (ISR) was also conducted by the CAVP lab on 30% of the samples to assess for data reproducibility. Acceptance criteria included 67% of the retested samples being quantified within 20% of the originally quantified concentration per FDA guidance.
Results and Discussion
High correlations were observed between PEth measures at USDTL and CAVP (r2 of 0.9954 and 0.9768 for HCV Cure and ADEPT, respectively). All but one USDTL below the limit of quantification (BLQ) result were also BLQ with CAVP. The one exception was a sample quantified as 6 ng/mL by the CAVP Lab, which was below the USDTL’s LLOQ. Despite the strong correlations, CAVP PEth concentrations were consistently higher than those generated by the USDTL (median: +35.6%). ISR analysis passed with 86% (12/14) of the samples retested within the 20% acceptance criteria, which shows the CAVP PEth data were reproducible.
High correlations were observed between the CAVP lab and USDTL’s DBS-based PEth assays. However, the CAVP lab’s PEth concentrations were consistently higher than those of the USDTL. These findings are consistent with those of Fatch et al. who also identified higher PEth concentrations versus USDTL. Regioisomeric purity differences between reference materials (as previously described by Luginbühl et al.) may have contributed to the differences in concentrations between labs. This and other potential sources for the discrepant results require further study because, although similar trends in PEth measures will be observed between labs, differences in the exact PEth concentrations may result in variations in interpretations of the extent of alcohol consumption.
Keywords: phosphatidylethanol, dried blood spots (DBS), assay, cross-validation
#7: Evaluation of changes in recent alcohol consumption using Phosphatidylethanol (PEth) 16:0/18:1 levels in dried blood samples collected virtually with a TASSO device
Javors M. A.1, Hill-Kapturczak N.1, Jett J. D.2, Sanchez J.1, Beck R. M.2, Tyutyunnyk D. O.2, Roache J. D.1, Lopez-Cruzan M.1, Ginsburg B. C.1, McDonell M.2
1Department of Psychiatry and Behavioral Sciences, UT Health Health Science Center at San Antonio, San Antonio, Texas 78229
2Department of Medical Education and Clinical Sciences, Washington State University Health Sciences, Spokane, 99202
This study assessed changes in levels of PEth in dried blood samples and urinary ethylene glucuronide (uEtG) repeatedly collected at weekly intervals for the six weeks, and thereafter monthly, to determine incentives and evaluate the success of a contingency management intervention for alcohol use disorders. A decrease in PEth from the previous visit was the primary outcome for the monetary contingency. During the period of the COVID pandemic, the TASSO device enabled supervised blood sample collection by the participants. The primary purpose of this analysis was to evaluate 3 combined biomarkers of changes in amount of recent alcohol consumption: self-reported drinking, PEth, and uEtG.
Community-dwelling participants with alcohol use disorder and baseline PEth levels ≥20 ng/mL were recruited (N=16). Participants experienced between 6 and 17 virtual visits (16 subjects=115 visits), which were accomplished with Zoom software. This approach provided observed, self-administered blood collection with TASSO-M20 devices as well as verbal self-reported drinking (daily drinks) and dipstick measurement of uEtG. PEth was quantified using HPLC/MS/MS. A specific, additional, in vitro experiment was performed using stored blood samples from a previous study to validate the analysis of PEth in TASSO plugs against liquid whole blood samples. uEtG was qualitatively tested with dip stick cards and reported as either negative (<300 ng/ml) or positive (≥300 ng/mL).
Results and Discussion
The in vitro experiment showed that PEth concentrations in 16 liquid whole blood and dried blood samples in TASSO plugs correlated with an r values of 0.994 and slope of 1.04. The contingency management study showed that the mean (SD) PEth level when uEtG was positive (N=63) was greater than when uEtG was negative (N=45) [856 (801) vs. 91.2 (186) ng/mL, respectively (p<0.0001). Increase or decrease of uEtG concentration (up or down) from one visit to the next matched the corresponding direction of PEth change 19/23 times. Strong agreement existed among changes in uEtG, blood PEth, and reported drinking.
Repeated within subject measurements at regular intervals during the contingency management study enabled objective identification of changes in drinking between virtual visits. Virtually supervised determination of reported drinking, uEtG, and PEth in blood samples using TASSO devices was essential for the continuation of this study during the COVID pandemic and proved to be very successful.
Keywords: Phosphatidylethanol, Tasso, virtual collection, contingency management intervention
#8: Phosphatidylethanol (PEth) use case: Examining PEth versus self-report in alcohol intervention studies
Hahn J. A.1, Fatch R. 1, Emenyonu E. I. 1, Camlin C. S. 1, Woolf-King S. 2, Muyindike W. R.3
1University of California, San Francisco
Introduction and Aims
Standard outcomes for randomized controlled trials (RCTs) of interventions to reduce alcohol use have traditionally relied on self-report. Some trials have used biomarkers to confirm results, but few have used PEth. Based on disparate findings between self-report and PEth, we sought to examine factors associated with under-reported alcohol use compared to PEth levels in the context of an alcohol counselling trial.
We conducted a 3-arm RCT of a 3-month counselling-based intervention among persons with HIV (PWH) in care and engaging in unhealthy alcohol use in Southwest Uganda (n=269). We collected self-reported alcohol use using the Alcohol Use Disorders Identification Test – Consumption (AUDIT-C, modified to represent the prior 3 months) and dried blood spots for PEth testing (tested at US Drug Testing Laboratories for PEth 16:0/18:1), at baseline, 6, and 9 months. AUDIT-C was categorized as low-risk: 0-2/0-3 for women/men; hazardous use: 3-7/4-7 for women/men; and high risk: ≥8. PEth was categorized as low risk: <LLQ-50 ng/mL; hazardous use 51-200 ng/mL; and high risk: >200 ng/mL. These categories are based on examinations of cutoffs for hazardous alcohol use suggesting cutoffs of PEth in the range of 35-80 ng/mL, and suggested cutoffs for excessive use of 200-210 ng/mL. We defined under-report as AUDIT-C category lower than the PEth category.
Results and Discussion
The study sample was 65% male with median age 39.5 (interquartile range [IQR]: 33-46). Prior to receipt of any intervention, baseline median AUDIT-C was 7 (IQR: 4-9) and median PEth was 259 ng/mL (IQR: 73-522). AUDIT-C declined over the study period to a median of 3 (IQR: 1-6) at 9 months, while PEth stayed relatively stable (median 217 ng/mL, IQR: 68-490)). Under-report increased from 27% at baseline to 54% at 9 months. In multivariable logistic regression analyses accounting for repeated measures, variables associated with under-reporting included follow up visits compared to baseline visits (adjusted odds ratio [aOR]: 9.46; 95% CI: 4.89- 18.29), being in either intervention arm (versus control) (aOR: 3.79; 95% CI: 1.77-8.11), having higher baseline PEth (aOR per log10 PEth: 6.75; 95% CI: 3.58-12.75), being older (aOR per 10 years: 1.90; 95% CI: 1.27-2.85), and having lower body mass index (aOR per BMI point 0.93; 95% CI: 0.86-0.99). We also adjusted for hemoglobin, sex, and social desirability score, but these were not associated with under-report. These finding suggest substantial levels of under-report that increase after receipt of behavioral counselling, especially for those receiving interventions, although BMI has an impact on PEth/self-report discrepancies as well.
Counselling-based alcohol intervention trials may have substantial levels of under-reporting, likely due to social desirability bias previously observed in this population. BMI may impact PEth sensitivity due to increased body fat, as previously observed. PEth results should be included as primary outcome; adjustment for BMI as a covariate in statistical models comparing PEth levels between intervention groups may also be needed.
Keywords: Phosphatidylethanol, intervention trial, under-report
#9: Microplate liquid-liquid extraction: a rapid technique for routine analysis of phosphatidylethanol in whole blood
Dumitrascu C.1,* , Gys C.1 , D’Hondt D.2 , Jacobs W.2 , Neels H.1 , Covaci A.1 , van Nuijs A. L. N.1
1Toxicological Centre, University of Antwerp, Antwerp, Belgium
2Department of Forensic Medicine and Pathology, University Hospital Antwerp, Antwerp, Belgium
Phosphatiadylethanol (PEth) in whole blood is a direct alcohol biomarker that can provide evidence of alcohol consumption over the last two to four weeks. Therefore, clinical/forensic laboratories started to include PEth routine analysis. Often, these laboratories are challenged by laborious sample preparation, and the use of relatively high solvent and sample volumes. This study proposes liquid-liquid microextraction as alternative sample preparation for PEth analysis.
A microplate liquid-liquid extraction was optimised based on previous experience with classical liquid-liquid extraction. Briefly, to each microplate well, 70 µL whole blood was added to 420 µL of isopropanol (IPA), and subsequently spiked with 35 µL PEth-D5 (200 ng/mL). After vortexing, 630 µL n-hexane was added. The 96-Well Plate was placed on a shaker for 30 min at 950 rpm. The supernatant was evaporated to dryness with nitrogen (40 °C). The dried extract was reconstituted in 140 µL of MeOH:IPA (1:1) and transferred and filtered in a 96 Filtration Plate (0.2 µm). Five µL of the extract was injected in an Agilent 1290 Infinity LC coupled to an Agilent 6495C triple quadrupole MS. Validation was done according to the European Medicines Agency (EMA) guidelines for bioanalytical method validation. Precision, accuracy, calibration range, carry over, matrix effect and recovery, as well as lower limit of quantification (LLOQ) were investigated during the validation process.
Results and Discussion
The linearity of the calibration curve was established for a range of 5 to 2000 ng/mL. Within- and between-run accuracy and precision of the method was determined by analysing four quality control levels [LLOQ (5 ng/mL), QC low (15 ng/mL), medium (150 ng/mL) and high (1500 ng/mL)] over different days. The carry-over was addressed by injecting a blank sample after the highest calibration point. Matrix effects and recovery efficiency were assessed in whole blood at QC low and high concentrations. The validated method was applied on 30 authentic samples collected in a medico-legal context and compared with the traditional LLE method. Validation results were in accordance with the EMA guidelines.
The analysis of PEth in whole blood is an asset for clinical/forensic laboratories. Therefore, there is a need of a rapid technique for the routine analysis of PEth. The presented micro-extraction uses 30% less solvent and sample volume, and sample preparation time is up to 50% less. This study shows that micro-extraction can be a green, time- and cost-efficient alternative to classical LLE, to facilitate the analysis of PEth.
Key words: PEth, whole blood, 96 well plates, rapid technique