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Alcohol biomarkers can monitor both recent and long-term drinking and provide information about drinking habits as a complement to self-reporting. Ethyl glucuronide (EtG) and phosphatidylethanol (PEth) are the most sensitive available biomarkers for this purpose. The present study aimed to collect data on both PEth and EtG in the same blood sample, in addition to ethanol, in order to evaluate the combined use of these biomarkers. Venous EDTA blood samples (n = 1149) sent to the laboratory as part of a clinical routine service for measuring PEth were investigated. PEth and EtG concentrations were analyzed using liquid chromatography–mass spectrometry methods and ethanol with an enzymatic method. Of the 1149 samples, 95 were positive for ethanol (range 0.11–3.12 g/L), 454 for EtG (1.0–9739 ng/mL), 635 for PEth (0.014–6.0 µmol/L), 534 for PEth ≥ 0.050 µmol/L, and 315 for PEth ≥ 0.30 µmol/L. EtG and PEth concentrations seemed largely independent as the coefficient of determination (r2) between PEth and EtG concentrations was 0.15. However, when the EtG concentrations were evaluated for different subgroups depending on ethanol or PEth concentrations a statistically significant difference between successive higher concentrations was observed. EtG and PEth are independent measures of recent alcohol drinking reflecting different time windows. Their combined measurement in the same blood sample is possible and will provide valuable information regarding recent alcohol consumption as a complement to self-reporting.

Background: Serum phosphatidylethanol (PEth) testing has emerged as a promising biomarker for assessing recent alcohol consumption, surpassing the limitations of self-reported data. Limited clinical data exists comparing PEth levels and patients' reported alcohol intake., Aims: Compare PEth testing results with self-reported alcohol intake and assesses variables associated with underreporting., Methods: Single-center retrospective cohort of patients with a diagnosis of chronic liver disease and serum PEth. A patient's first positive PEth (>/=10 ng/mL) and self-reported alcohol consumption was used. PEth results were categorized as mild (10-20), moderate (20-200), or heavy (>200). Severity measures between self-report and PEth were assessed using Bhapkar's test and Bonferroni-adjusted McNemar's tests. Demographic data was analyzed using Chi-Square tests., Results: 279 patients were included. 94 (33.7%) patients had consistency with self-report, and 185 patients had inconsistencies in their report (66.3%, p < 0.001). Of 279 patients, 161 (57.7%) underreported their alcohol consumption, and 55 (19.7%) heavy PEth patients underreported alcohol consumption as light. 58% of alcohol-related and 56.4% of non-alcohol-related cirrhotic patients underreported their alcohol use., Conclusion: In our cohort, only one third of self-reported alcohol consumption was consistent with the PEth level. Notably, 57.7% underreported alcohol intake. Our study reinforces the clinical importance of PEth testing as an objective clinical measure., Competing Interests: Conflict of interest All authors have no conflicts of interest or sources of financial support to disclose. Please reach out to the corresponding author for any questions or concerns., (Copyright © 2024 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Contingency management (CM) is an intervention where incentives are provided in exchange for biochemically confirmed alcohol abstinence. CM is effective at initiating alcohol abstinence, but it is less effective at maintaining long-term abstinence. Phosphatidylethanol (PEth), collected via a finger-stick, can detect alcohol use for 14–28 days. PEth allows for the development of a CM model that includes increasingly less frequent monitoring of abstinence to assist high risk groups, such as formerly homeless individuals, maintain long-term abstinence. Aims: Investigate whether PEth-based CM intervention targeting alcohol abstinence in formerly homeless, currently housed individuals with alcohol use disorders is: (1) acceptable and feasible for housing program tenants and personnel; and is associated with increased (2) alcohol abstinence and (3) housing tenure. Methods: Acceptability and feasibility will be assessed using a QUAL+quant mixed-methods design using qualitative interviews and quantitative measures of satisfaction and attrition. Effectiveness will be evaluated through a randomized pilot trial of 50 study participants who will receive 6 months of either treatment as usual (TAU) including incentives (e.g., gift cards) for providing blood samples (Control Condition) or TAU and incentives for negative PEth results (PEth-CM Condition). Outcomes will be assessed during the intervention and at a three-month follow-up visit. The trial will be conducted via telehealth as a result of COVID-19. Discussion: This protocol seeks to utilize a novel alcohol biomarker to evaluate the acceptability, feasibility, and initial effectiveness of a CM model that encourages long-term abstinence in a high-risk group.

Alcohol consumption is associated with a wide risk of different diseases, injury and death, and has significant social and economic consequences worldwide. Phosphatidylethanol (PEth) is a group of promising direct alcohol biomarkers, with a significantly longer half-life in blood than ethanol, which can be measured to predict different drinking patterns, such as heavy- and social drinking. This study aimed to develop and validate an accurate and precise LC-MS/MS method for the determination of six PEth homologues in whole blood with minimal interference from unwanted phospholipids. Different organic solvent mixtures for liquid-liquid extraction were investigated to obtain satisfactory recovery of PEth homologues and removal of the lyso-phospholipids and other early eluting phospholipids. The mixture of heptane/2-propanol (80:20, v:v) gave lower phospholipid background and better signal/noise values for the PEth peaks. An LC-MS/MS TQ-S system from Waters was used for the instrumental analysis. The main part of unwanted phospholipids were separated from the PEth homologues on an Acquity BEH C 18 column (50 × 2.1 mm ID, 1.7 µm particles) using a buffer-free mobile phase of 0.025 % ammonia in Type 1 water, pH 10.7, as solvent A and methanol as solvent B. Validation and quantification of 22 authentic blood samples showed that the developed LC-MS/MS method is sensitive, precise and accurate for the determination of the six PEth homologues in whole blood. Lower limit of quantification was 10 nM for all compounds. No matrix effects were observed, possibly due to the successful strategies incorporated to avoid the influence of unwanted phospholipids., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Phosphatidylethanol (PEth) is a group of phospholipids formed exclusively in the presence of ethanol on the erythrocyte membrane, making it a direct biomarker for long-term ethanol consumption for which a clinical reference interval has been established. Here, we describe an assay for quantitation for two most abundant PEth homologues, PEth 16:0/18:1 and PEth 16:0/18:2, from human whole blood, and present challenges overcome throughout the development process. Since PEth is localized within erythrocyte membranes, a reliable sample preparation technique is an important aspect of PEth analysis. Therefore, various erythrocyte lysing agents for recovery of exogenously spiked standards and controls were evaluated to identify one that performed comparably to the recovery of endogenous analytes found in authentic samples. A supported liquid extraction (SLE) technique was employed for sample cleanup and enrichment which together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis enabled automated sample preparation, appropriate chromatographic resolution, and minimal system carryover. This resulted in a laboratory developed test with an analytical measurement range (AMR) of 10-1000 ng/mL (slope = 0.9902-1.0138, R 2  = 0.9958-0.9972), that was precise (intra-day precision: 3.4-4.1%; inter-day precision: 4.4-8.2% over the AMR), accurate when compared with an available external laboratory test (slope = 0.9943-1.0206, R 2  = 0.9635-0.9678, no lower decision point interpretation changes), with effective analyte recovery (77.2-83.5%), and established stability characteristics, while chromatographically separating the analytes to ensure no additive effects due to the isotopic distribution of the opposing analyte., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Phosphatidylethanol (PEth) is a group of phospholipids detectable in red blood cells exclusively following ethanol consumption. The primary PEth analog, PEth 16:0/18:1, has an extended half-life in red cells, providing a long window of detection and tremendous potential for the quantification of cumulative alcohol consumption. We developed and validated an LC/MS-MS method to quantify PEth 16:0/18:1 in dried blood spots (DBS) for clinical research purposes. Method development and validation followed FDA guidance but expanded on prior published methods through the evaluation of additional DBS-specific factors such as sample hematocrit, punch location, and spot volume. This method was applied to the quantification of PEth in participant samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: [Jennifer J Kiser reports financial support was provided by National Institutes of Health. Lana M Salah reports financial support was provided by National Institutes of Health.]., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Clinical and quality of life outcomes in people living with human immunodeficiency virus (PLWH) are undermined by unhealthy alcohol use (UAU), which is highly prevalent in this population and is often complicated by mental health (MH) or other substance use (SU) comorbidity. In sub-Saharan Africa, evidence-based and implementable treatment options for people with HIV and UAU are needed., Methods: We are conducting a hybrid clinical effectiveness-implementation trial at three public-sector HIV clinics in Lusaka, Zambia. Adults with HIV, who report UAU, and have suboptimal HIV clinical outcomes, will be randomized to one of three arms: an alcohol-focused brief intervention (BI), the BI with additional referral to a transdiagnostic cognitive behavioral therapy (Common Elements Treatment Approach [CETA]), or standard of care. The BI and CETA will be provided by HIV peer counselors, a common cadre of lay health worker in Zambia. Clinical outcomes will include HIV viral suppression, alcohol use, assessed by audio computer-assisted self-interview (ACASI) and direct alcohol biomarkers, Phophatidylethanol and Ethyl glucuronide, and comorbid MH and other SU. A range of implementation outcomes including cost effectiveness will also be analyzed., Conclusion: Hybrid and 3-arm trial design features facilitate the integrated evaluation of both brief, highly implementable, and more intensive, less implementable, treatment options for UAU among PLWH in sub-Saharan Africa. Use of ACASI and alcohol biomarkers will strengthen understanding of treatment effects., Competing Interests: Declaration of Competing Interest All authors declare they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Purpose: To test the efficacy of two interventions to reduce alcohol use and increase viral suppression compared to a control in persons with HIV (PWH)., Methods: In a three-arm (1:1:1) randomized controlled trial (N = 269), we compared in-person counselling (45-70 minutes, two sessions over three months) with interim monthly booster phone calls (live call arm) or twice-weekly automated booster sessions (technology arm) to a brief advice control arm. We enrolled PWH self-reporting unhealthy alcohol use (Alcohol Use Disorders Identification Test - Consumption, prior three months, women ≥3, men ≥4). Primary outcomes were number of self-reported drinking days (NDD) in the prior 21 and biomarker phosphatidylethanol (PEth) at six and nine months and viral suppression (<40 copies/mL) at nine months; we adjusted for sex and baseline outcomes., Results: At baseline, mean 21-day NDDs were 9.4 (95 % CI: 9.1-9.8), mean PEth was 407.8 ng/mL (95 % CI: 340.7-474.8), and 89.2 % were virally suppressed. At follow-up, there were significant reductions in mean NDDs for the live call versus control arm (3.5, 95 % CI:2.1-4.9, p < 0.001) and for the technology versus control arm (3.6, 95 % CI: 2.2-5.1, p < 0.001). The mean PEth differences compared to the control arm were not significant, i.e. 36.4 ng/mL (95 % CI: -117.5 to 190.3, p = 0.643) for the live call and -30.9 ng/mL (95 % CI: -194.8 to 132.9, p = 0.711) for the technology arm. Nine-month viral suppression compared to the control was similar in the live call and in the technology arm., Conclusion: Intervention effects were found on self-reported NDD but not PEth or viral suppression, suggesting no treatment effect. (NCT #03928418)., Competing Interests: Conflict of interest Dr. Hahn received consulting fees from Pear Therapeutics in 2022., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

In rat skeletal muscle, calcitriol, the hormonal form of vitamin D3, rapidly stimulates the biphasic formation of diacylglycerol (DAG), the second phase being independent of phosphoinositide hydrolysis driven by phospholipase C. In this work we showed that the effect of calcitriol on the second phase of DAG formation was totally inhibited in the absence of extracellular Ca2+ and by the Ca2+-channel blockers nifedipine and verapamil, whereas the Ca2+ ionophore A23184, similar to calcitriol, increased DAG formation by 100%. GTPγS, which activates G protein-mediated signals, mimicked the effects of the hormone while GDPβS, an inhibitor of G proteins, suppressed calcitriol-induced DAG formation. To elucidate the metabolic pathway of the late phase of DAG production, we examined the contribution of phospholipase D (PLD), which acts on phosphatidylcholine (PC) generating phosphatidic acid that is converted to DAG by a phosphatidate phosphohydrolase. In [3H]arachidonate-labeled muscle, calcitriol increased [3H]phosphatidylethanol (PEt) formation in the presence of ethanol, a reaction specific for PLD. The effects of the hormone were time- and dose-dependent with maximum PEt levels achieved at 10–9 m. The phorbol ester TPA also stimulated PEt formation. The combination of calcitriol and TPA was more effective than either compound alone. In rat muscle, calcitriol increased PKC activity in a time-dependent fashion. Bisindolymaleimide, a selective inhibitor of the enzyme, completely suppressed TPA-induced PEt and attenuated the effects of the hormone. These results provide the first evidence concerning calcitriol stimulation of the hydrolysis of PC in a mammalian tissue through a phospholipase D catalyzed mechanism involving Ca2+, protein kinase C, and G proteins.—Facchinetti, M. M., R. Boland, and A. R. de Boland. Calcitriol transmembrane signalling: regulation of rat muscle phospholipase D activity. J. Lipid Res. 1998. 39: 197–204.

Phosphatidylethanols (PEths) are specific, direct alcohol biomarkers with a substantially longer half-life than ethanol, and can be used to distinguish between heavy- and social drinking. More than forty PEth homologues have been detected in blood from heavy drinkers, and PEth 16:0/18:1 is the predominant one. Since PEths are phospholipids it can be difficult to isolate them from unwanted phospholipids during sample preparation. To minimize possible matrix effects it is therefore important to separate PEths from other phospholipids during LC-MS/MS analysis. In this study, we have investigated how the retention and chromatographic separation of eight PEth homologues and the phospholipid background are influenced by changes in mobile phase composition using two different LC columns, the Acquity BEH C 18 column (50 × 2.1 mm ID, 1.7 µm particles) and the Kinetex biphenyl column (100 × 2.1 mm ID, 1.7 µm particles). Our findings show that the buffer concentration of the aqueous part of the mobile phase had a huge effect on the retention of PEth homologues and separation of PEths from unwanted phospholipids. By using a buffer-free mobile phase consisting of 0.025% ammonia in Type 1 water, pH 10.7, as solvent A and methanol as solvent B, all eight PEth homologues were separated from both the early eluting lyso-phospholipids and the later eluting phospholipids with two fatty chains using the BEH C 18 column. The knowledge obtained in this study can be of great importance for those seeking to develop reliable and robust bioanalytical LC-MS/MS methods for determination of PEth homologues., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background and Aims: Phosphatidylethanol (PEth) is a direct alcohol biomarker. Aim of the study was to evaluate the performance of six homologues of PEth in comparison to other alcohol markers in patients with liver diseases., Methods: The study included 234 patients with liver disease, who gave statements about alcohol consumption during the three months prior to the doctor's appointment. Ethylglucuronide in urine (uEtG) and in hair (hEtG) and carbohydrate-deficient transferrin (CDT) were analyzed in addition to PEth., Results: Of all patients 47% stated to have drunk alcohol during the past three months. UEtG, hEtG and CDT showed a sensitivity of 29% and a specificity of 92% together for ingestion of at least two standard drinks (24 g) per week. With PEth 16:0/18:1 in addition, sensitivity increased to 59%. For consumption in the last week uEtG's sensitivity and specificity was 28% and 100%, respectively. PEth's was 75% and 93%. When looking at patients who consumed at least two standard drinks per week during the past three months and of which a hair sample could be obtained, hEtG's sensitivity was 37% and specificity 90%. PEth had a sensitivity of 53% and specificity of 100%. Quotients of PEth 16:0/18:1 with 16:0/18:2, 16:0/20:4 and 18:0/18:2 were smaller when alcohol had been consumed more recently., Conclusion: Despite the rather poor overall sensitivity of alcohol biomarkers in this study, PEth showed best sensitivity for all time periods of alcohol consumption., (Copyright © 2021. Published by Elsevier B.V.)

Background: Stimulated by the increased recognition of phosphatidylethanol (PEth) as sensitive direct marker of alcohol intake, the Ghent University's Laboratory of Toxicology and the National Institute of Criminalistics and Criminology combined their efforts to develop a quantitative method. To facilitate implementation the focus was on the use of a sampling technique which allows quick and easy blood collection, without the need of dedicated personnel at any place/any time. In the meantime the cooperation of the two labs should also allow to initiate a Belgian network of laboratories capable of quantifying PEth., Methods: Dried blood microsamples were collected via volumetric absorptive microsampling (VAMS). PEth 16:0/18:1 was quantified after liquid-liquid extraction using two independent isotope dilution - liquid chromatography - tandem mass spectrometry methods. A systematic review of the entire process at both sites was performed before the final method comparison using samples from 59 routine toxicology cases collected within a one-year time interval., Results: Initial differences between both laboratories were solved by focusing on important methodological aspects: (i) trueness verification of the calibration protocol focusing on the primary material, preparation of the stock solutions and adequate equilibration of calibrators and QCs, and (ii) verification of comparability of results obtained with different m/z transitions. Several of these aspects could only be verified by critically assessing spiked and native samples. After a final validation good average comparability of the two methods was observed. The average bias was -0.4%, with 85% of the differences within 20%. Moreover, the methods proved to be reproducible and robust within a one-year time interval., Conclusion: This study is the first to develop a quantitative volumetric absorptive microsampling based method for PEth measurements, in addition it is the first to perform a systematic comparison of PEth measurements between two laboratories. From the discussion on the encountered pitfalls it is clear that also on a global scale, more efforts are needed to improve interlaboratory agreement., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Most bioanalytical LC-MS/MS methods are developed for determination of single drugs or classes of drugs, but a multi-compound LC-MS/MS method that can replace several methods could reduce both analysis time and costs. The aim of this study was to develop a high-throughput LC-MS/MS method for determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 (PEth 16:0/18:1) and 33 other compounds from eight different drug classes in whole blood., Methods: Whole-blood samples were prepared by 96-well supported liquid extraction (SLE). Chromatographic separations were performed on a biphenyl core shell column with a mobile phase consisting of 10 mM ammonium formate, pH 3.1 and methanol. Each extract was analyzed twice by LC-MS/MS, injecting 0.4 μL and 2 μL, in order to obtain narrow and symmetrical peaks and good sensitivity for all compounds. Stable isotope-labeled internal standards were used for 31 of the 34 compounds., Results: A 96-well SLE reversed phase LC-MS/MS method for determination of PEth 16:0/18:1 and 33 other compounds from eight different drug classes was developed and validated. By using an organic solvent mixture of isopropanol/ methyl tert-butyl ether (1:5, v:v), all compounds, including the polar and ampholytic compounds pregabalin, gabapentin and benzoylecgonine, was extracted by 96-well SLE., Discussion/conclusion: For the first time an LC-MS/MS method for the determination of alcohol biomarker PEth 16:0/18:1 and drugs and metabolites from several different drug classes was developed and validated. The developed LC-MS/MS method can be used for high-throughput analyses and sensitive determinations of the 34 compounds in whole blood., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this article, we discuss a recent publication by Casati et al. (2018) focusing on the sample preparation that is described for the analysis of phosphatidylethanols and whether or not their proposed method of 'automated filtration' and the advantages it brings is really just a standard solid phase extraction protocol., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Phosphatidylethanols (PEths) are currently under investigation as highly sensitive and specific direct biomarkers of long-term alcohol abuse. PEths belong to a group of aberrant phospholipids formed in erythrocyte membranes in presence of ethanol by the catalytic action of the enzyme phospholipase D on phosphatidylcholine. Compared to other alcohol biomarkers, a higher sensitivity (94.5-100%) and specificity (100%) characterizes PEth species., Method: Prior to detection, an important practical aspect in the work-flow of PEths analysis is the sample preparation step. To date, traditional techniques such as liquid-liquid extraction (LLE) and solid phase extraction (SPE) require multiple steps to remove blood interferences. Due to the simplicity of use and the possibility of automation, sample filtration is also a widespread technique in biomedical laboratories. In this work, a reliable sample preparation method based on an automated filtration with Phree™ Phospholipid Removal Plates (Phenomenex, California, USA) was developed to extract PEths from human whole blood. Surface characteristics of Phospholipids Removal material allow phospholipids retention on the filter and a suitable PEths recovery after elution. The blood samples were added with internal standard (IS) and purified in acetonitrile (1 mL). After centrifugation, supernatants were applied to the Phospholipids Removal Plates in an automated workstation. After washing, the phospholipids retained on the filter were eluted with 1-mL 2-propanol 1% ammonia. PEth 16:0/18:1, PEth 16:0/16:0 and PEth 18:1/18:1 were extracted using the proposed method and detected by LC-MS/MS operated in electron spray ionization (ESI). The detection of all compounds was based on multiple reaction monitoring (MRM) transitions. This method was validated for the quantitative profiling of PEth molecular species in human blood collected from heavy and social drinkers., Results: The method was validated according to Food and Drug Administration (FDA) guidelines. Linearity was observed in the 25-1250 (PEth 16:0/18:1) and 5-250 (PEth 16:0/16:0 and PEth 18:1/18:1) ng/mL range with a correlation coefficient (r²) between 0.997 and 0.999 for all three compounds. Moreover, the nominal concentrations of non-zero calibrators were ±15%. Variation coefficient (%CV) was < 10% for all the analytes, while lowest limit of quantitation (LLOQ) was found to be 1.25 ng/mL for PEth 16:0/18:1, 0.50 ng/mL for PEth 16:0/16:0 and 0.50 ng/mL for PEth 18:1/18:1. Intra- and inter-day precision and accuracy were always lower than 14% and 11%, respectively. Analytical recovery was higher than 68.8% for all analytes. Sample stability at 4 °C and -20 °C showed a concentration drop lower than 20% up to 4 weeks. Extracts were stable for 7 days in the autosampler and 30 days at -20 °C and 4 °C in a closed vial. The procedure was successfully applied to blood samples collected from heavy drinkers (n = 8), social drinkers (n = 5), and teetotalers (n = 7)., Conclusions: Due to the simplicity of application and the possibility of automation, sample filtration is well suited for a clinical and forensic laboratory. To monitor alcohol consumption, an analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) with novel and automated sample preparation was developed and validated for the simultaneous quantification of PEth 16:0/18:1, PEth 16:0/16:0 and PEth 18:1/18:1 in whole blood samples, characterized by a fast sample preparation and lower pre-analysis costs than other extraction procedures., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Phosphatidylethanol (PEth) is increasingly used as a biomarker of heavy drinking. Many different forms of PEth can form in red blood cell membranes from the action of the enzyme phospholipase D. PEth has a very long duration in blood because, in contrast to other tissues, RBCs lack the enzymes that degrade PEth. Because this biomarker is relatively new, interpretations of the analytical measurements of PEth may be misinterpreted and the resulting predictions of actual alcohol consumption inaccurate. Hence, a simple pharmacokinetic model of PEth was developed to provide a means of contextualizing these analytical results. A number of alcohol consumption scenarios and current clinical screening levels were examined with the model., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phosphatidylethanol (PEth) is a group of phospholipids formed in cell membranes following alcohol consumption. PEth measurement in whole blood samples is established as a specific alcohol biomarker with clinical and medico-legal applications. This study further evaluated the usefulness of dried blood spot (DBS) samples collected on filter paper for PEth measurement. Specimens used were surplus volumes of venous whole blood sent for routine LC-MS/MS quantification of PEth 16:0/18:1, the major PEth homolog. DBS samples were prepared by pipetting blood on Whatman 903 Protein Saver Cards and onto a volumetric DBS device (Capitainer). The imprecision (CV) of the DBS sample amount based on area and weight measurements of spot punches were 23-28%. Investigation of the relationship between blood hematocrit and PEth concentration yielded a linear, positive correlation, and at around 1.0-1.5μmol/L PEth 16:0/18:1, the PEth concentration increased by ~0.1μmol/L for every 5% increase in hematocrit. There was a close agreement between the PEth concentrations obtained with whole blood samples and the corresponding results using Whatman 903 (PEth DBS =1.026 PEth WB +0.013) and volumetric device (PEth DBS =1.045 PEth WB +0.016) DBS samples. The CV of PEth quantification in DBS samples at concentrations≥0.05μmol/L were ≤15%. The present results further confirmed the usefulness of DBS samples for PEth measurement., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Phosphatidylethanol (PEth) is an interesting biomarker finding increased use for detecting long term alcohol abuse with high specificity and sensitivity. Prior to detection, sample preparation is an unavoidable step in the work-flow of PEth analysis and new protocols may facilitate it. Solid-phase extraction (SPE) is a versatile sample preparation method widely spread in biomedical laboratories due to its simplicity of use and the possibility of automation. In this work, SPE was used for the first time to directly extract PEth from spiked human plasma and spiked human blood. A library of polymeric SPE materials with different surface functionalities was screened for PEth extraction in order to identify the surface characteristics that control PEth retention and recovery. The plasma samples were diluted 1:10 (v/v) in water and spiked at different concentrations ranging from 0.3 to 5μM. The library of SPE materials was then evaluated using the proposed SPE method and detection was done by LC-MS/MS. One SPE material efficiently retained and recovered PEth from spiked human plasma. With this insight, four new SPE materials were formulated and synthesized based on the surface characteristics of the best SPE material found in the first screening. These new materials were tested with spiked human blood, to better mimic a real clinical sample. All the newly synthetized materials outperformed the pre-existing commercially available materials. Recovery values for the new SPE materials were found between 29.5% and 48.6% for the extraction of PEth in spiked blood. A material based on quaternized 1-vinylimidazole with a poly(trimethylolpropane trimethacrylate) backbone was found suitable for PEth extraction in spiked blood showing the highest analyte recovery in this experiment, 48.6%±6.4%., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Phosphatidylethanol (PEth) is a special phospholipid that is only formed in the presence of ethanol, and therefore, serves as a promising biomarker for alcohol intake. In this study, a simple, rapid and precise method based on LC-MS/MS combined with ultrasound-assisted dispersive liquid-liquid microextraction was developed and validated for the measurements of PEth (16:0/18:1, 16:0/18:2, 16:0/16:0, and 18:1/18:1) in human blood. The influences of several variables for sample extraction and MS detection were carefully investigated. The extraction efficiencies for all the four PEth species were markedly increased compared with the traditional extractions. A limit of detection below 0.56ngmL -1 was obtained. This high sensitivity makes it possible to monitor various alcohol consumption levels in light to heavy drinkers. Good linearity was obtained for all the analytes without interference from the sample matrix. The imprecisions of the intra-run and total assays were lower than 3.1% and 6.5%, respectively, with an average recovery of 99.87%. In addition, the utility of the method was evaluated in an alcohol intake status study. The results indicate that the developed protocol is simple, precise, and sensitive, and can be easily adapted for objective and reliable assessments of alcohol intake in clinical research., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: In Cambodia, most of the female sex workers (FSW) work in venues where unhealthy alcohol use is ubiquitous and potentially contributing to the HIV epidemic. However, no accurate data exists. We compare self-reported unhealthy alcohol consumption to a biomarker of alcohol intake in Cambodian FSW and male clients, and determine factors associated with unhealthy alcohol use., Methods: A cross-sectional study was conducted among FSW (n=100) and male clients (n=100) in entertainment and sex work venues in Cambodia. Self-reported unhealthy alcohol use (AUDIT-C) was compared to phosphatidylethanol (PEth) positive (≥50ng/ml), a biomarker of alcohol intake. Sociodemographics data was collected. Correlates of self-reported unhealthy alcohol use and PEth positive were determined., Results: The prevalence of PEth positive in FSW was 60.0%. Self-reported unhealthy alcohol consumption was reported by 85.0% of the women. Almost all women (95.0%) testing PEth positive also reported unhealthy alcohol use. Prevalence of unhealthy alcohol consumption (self-report and PEth positive) was higher in FSW working in entertainment establishments compared to other sex work venues (p<0.01). Among male clients, 47.0% reported unhealthy alcohol consumption and 42.0% had a PEth positive. However, only 57.1% of male clients with PEth positive reported unhealthy alcohol use., Conclusions: Unhealthy alcohol consumption is prevalent in Cambodian sex work settings. Self-reported unhealthy alcohol use is well reported by FSW, but less by male clients. These findings highlight the urgency of using accurate measures of unhealthy alcohol consumption and integrating this health issue into HIV prevention interventions., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)