Abstract

Aims

Phosphatidylethanol (PEth) is only formed when ethanol is present in blood. This direct alcohol marker has been widely discussed, including the minimum amount of ethanol being necessary to form as much PEth as to exceed the threshold of 20 ng/mL in previously PEth negative subjects. In order to corroborate hitherto existing results, a drinking study including 18 participants after a 3-week alcohol abstinence was performed.

Methods

They consumed a pre-calculated amount of ethanol to reach a blood alcohol concentration (BAC) of at least 0.6 g/kg. Blood was drawn before and periodically seven times after alcohol administration on day 1. Blood and urine were also collected the next morning. Dried blood spots (DBS) were prepared immediately from collected venous blood. BAC was determined by head space gas chromatography and the concentrations of both PEth (16:0/18:1, 16:0/18:2 and five additional homologues) and ethyl glucuronide (EtG) were analysed using liquid chromatography–tandem mass spectrometry.

Results

Out of 18, 5 participants had concentrations of PEth 16:0/18:1 above the threshold of 20 ng/mL, and 11 out of the 18 subjects had concentrations between 10 and 20 ng/mL. In addition, four persons had PEth 16:0/18:2 concentrations above 20 ng/mL the following morning. All test subjects tested positive for EtG in DBS (≥ 3 ng/mL) and urine (≥100 ng/mL) upon 20–21 h after alcohol administration.

Conclusion

By combining both a lower cutoff of 10 ng/mL and the homologue PEth 16:0/18:2, the sensitivity to detect a single alcohol intake after a 3-week abstinence increases to 72.2%.

Introduction

According to the German Federal Office of Statistics approximately 50,000 driving licenses were suspended in 2020 because of driving under the influence (DUI) of both alcohol and drugs (Statistisches Bundesamt 2021). To retrieve a driving license, a medical-psychological assessment (MPA) has to be passed successfully. Abstinence is usually required and has to be proven by an abstinence control program involving chemical toxicological analysis. The applied regulations, including drug or biomarker thresholds to pass such programs, were set by the German Society of Traffic Medicine and the German Society of Traffic Psychology. In the fall of 2022, an updated edition was published introducing phosphatidylethanol (PEth) as a new direct alcohol parameter to assess a person’s drinking behavior. In Germany, concentrations of below 20, 20–210 and above 210 ng PEth 16:0/18:1 per mL blood are recommended to classify abstinence, social acceptable drinking behavior and riskful drinking, respectively (Helander and Hansson 2013; Brenner-Hartmann et al. 2022). An international consensus suggested 200 ng PEth 16:0/18:1 per mL blood as a threshold for harmful drinking (Luginbühl et al. 2022). In forensic cases an upper cutoff of 210 ng/mL is recommended (Musshoff et al. 2022).

PEth comprises a group of abnormal phospholipid homologues synthesised by phospholipase D (EC number 3.1.4.4) and endogenous phosphatidylcholine if alcohol is present. Even though 48 different homologues have been identified differing in saturation and length of their fatty acid chains, PEth 16:0/18:1 is the most abundant and primarily used to assess a person’s drinking behavior (Gnann et al. 2010; Kummer et al. 2016; Brenner-Hartmann et al. 2022; Luginbühl et al. 2022). Due to the absence of phosphatidylcholine-phospholipase (EC 3.1.3.75) activities in red blood cells, PEth accumulates in erythrocytes and is detectable for up to several weeks in patients undergoing alcohol withdrawal (Kobayashi and Kanfer 1987; Varga et al. 2000; Aradóttir et al. 2004; Viel et al. 2012; Kummer et al. 2016). In recently published studies, additional homologues have also been included into analysis, providing further information regarding their formation and degradation after consumption of a single dose of ethanol (Hill-Kapturczak et al. 2018; Lopez-Cruzan et al. 2018; Stöth et al. 2022).

Testing for the direct alcohol marker ethyl glucuronide (EtG) in hair (EtGH) and urine (EtGU) is well established for monitoring drinking behavior and alcohol abstinence (Andresen-Streichert et al. 2018). The detection window of this phase-II metabolite ranges from a few h in blood to 24–72 h in urine and up to several months in hair or fingernails (Hoiseth et al. 2007; Jatlow and O'Malley 2010; Albermann et al. 2012; Berger et al. 2014).

Persons undergoing alcohol abstinence control programs to positively support their MPA may choose between EtGH, EtGU, or PEth in the blood. If an EtGU or PEth control program is selected, participants are randomly asked to show up within 24 or 48 h, respectively (Brenner-Hartmann et al. 2022). These unpredictable calls were chosen to increase the chance of detecting a recent low alcohol intake. The minimum amount of a single dose of ethanol required to cause a PEth concentration above the threshold of 20 ng/mL after a previous alcohol abstinence is yet to be explored. Stöth et al. did not observe formation of PEth (cut-off: 20 ng/mL) after reaching a maximum blood alcohol concentration (BAC) of 0.63 g/kg following a previous 4-week alcohol abstinence (Luginbühl et al. 2022; Stöth et al. 2022).

One of the biggest obstacles is the post-sampling formation of PEth if alcohol is present in the sample. To minimise in vitro synthesis, whole blood samples are strongly recommended for storage at −80°C or preparation of dried blood spots (DBS; Beck et al. 2021). EtG in blood has been proven not to be influenced by a positive BAC (Schloegl et al. 2006).

The aim of this study was to investigate the formation of different PEth homologues after low to moderate alcohol consumption in volunteers who stayed alcohol abstinent for at least 3 weeks prior to the study, also including an evaluation of the threshold of 20 ng/mL for PEth 16:0/18:1 as well as of EtG in DBS and EtGU the day after the drinking experiment.

Material and methods

Test subjects

Eighteen subjects (18–65 years old, 5 males and 13 females) were enrolled in the study. Prerequisites were a minimum of at least two drinking events in the past 6 months and the willingness to abstain from both drinks, food and lifestyle products containing ethanol for at least 3 weeks prior the study. On day 1, the initial PEth 16:0/18:1 concentration had to be below the threshold of 20 ng/mL. Exclusion criteria were pregnancy or a nursing period, any kind of acute neurological or psychotic disease and alcohol abuse in the participants’ medical history.

All individuals had an accident insurance and provided written, informed consent. The study protocol was approved by the ethics committee of Ludwig Maximilian University Munich, Germany (project-no 21-0846). All participants were rewarded for their participation with 100 €.

Study design

To achieve a BAC of approximately 0.6–0.8 g/kg, individual volumes of alcoholic beverages (wine or vodka) for all study participants were calculated using the formula of Widmark (Widmark 1932). The resorption deficit was set to 15% for all beverages. The interval to consume the calculated amount of alcohol was set to 1.5 h. The elimination rate of ethanol was set to 0.15 g/kg per hour. A minimum BAC of 0.60 g/kg was expected 45 min after alcohol intake depending on sex, and a distribution factor (r) of either 0.7 or 0.6 was used for males and females, respectively, as all participants were of normal build (Penning 1999).

The following formula was used for calculation (Penning 1999):

$$\begin{array}{lc} &\kern-3pt\mathrm{Mass}\ \left[{\mathrm{g}}_{\mathrm{EtOH}}\right]=\left(\mathrm{BAC}+{\left(1.5+0.75\right)}\ast 0.15\right)/{\left(1-0.15\right)}\ast \\ & \left({\mathrm{weight}}\ast \mathrm{r}\right) \end{array}$$

Test subjects were asked to avoid a fat-rich breakfast and to have their last meal 2 h prior to the study.

First, they had to undergo a medical examination to prove fitness for trial, to fill in the Alcohol Use Disorders Identification Test (AUDIT) including AUDIT consumption (AUDIT-C) questionnaires (Saunders et al. 1993), and blood was drawn for pre-testing of initial PEth and EtG concentrations.

Sample collection and analysis of PEth species, BAC, EtG in blood and urine

Venous blood was collected during the pre-study medical examination. During drinking, no blood collection took place. The second venous blood was collected 15 min after the last drink. To retrace both BAC and formation of PEth and EtG over the period of up to 6 h after the end of drinking, blood was drawn hourly. Also, blood was drawn once the day after the study.

Before each blood collection (Vacutainer 10 mL, K2 EDTA, Becton Dickinson, Franklin Lakes, New Jersey, USA), an ethanol-free sanitizer was used as a disinfectant. Immediately after blood drawing, three aliquots of 20 μL of the collected blood were used to make up DBS for both PEth and EtG analysis, respectively, on Whatman 903 filter paper (Ahlstrom-Munksjö Group, Bärenstein, Germany). Urine samples were collected approximately 20–21 h after the drinking experiment. All time-points of sample collection are displayed in the flow sheet (Supplementary Fig. 1).

Urine and blood tubes were stored at 8°C before determination of EtGU and BAC, respectively. The DBS samples were dried for 3 h and stored at room temperature in the dark.

According to current German forensic guidelines, samples for determination of BAC were analysed in duplicates using two independent methods (Aderjan et al. 2011).

Detailed information about analysing PEth in DBS was published recently (Herzog et al. 2023). The spot (20 μL) was punched as a whole, extracted with methanol including all deuterated internal standards, and shaken at medium speed on a vortex shaker for 1 h. After centrifugation, 800 μL were evaporated to dryness and reconstituted in a 100 μL buffer consisting of 4 mM ammonium acetate buffer in acetonitrile/2-propanol (80/20 vol%). Analyses were performed using a high-performance liquid (HPLC) 1240 Infinity II system (Agilent, Waldbronn, Germany) coupled to a tandem mass spectrometry (MS/MS) 5500 instrument (AB Sciex, Darmstadt, Germany). In total, up to seven PEth homologues can be detected: 16:0/18:1, 16:0/18:2, 16:0/20:4, 17:0/18:1, 18:0/18:1, 18:1/18:1, 18:0/18:2. Limits of detection (LOD) and quantification (LOQ) of each homologue are listed in the supplements.

To determine the concentrations of EtG in DBS, a similar sample preparation was followed: aliquots of 240 μL of whole blank blood, respectively, were spiked with the working solution to prepare calibration standards (10–10,000 ng/mL, n = 9), and a second working solution was used to prepare quality control samples (QCs; 55, 550 and 5500 ng/mL). Aliquots of 20 μL were spotted on Whatman 903 filter paper and left to dry for at least 3 h at room temperature. Calibration, QCs and authentic samples were punched as a whole. After methanolic extraction containing EtG-d5 (5 μL, 200 ng/mL) for 1 h, 800 μL of the supernatant were evaporated under a gentle stream of nitrogen at 37°C. The residue was reconstituted in 100 μL of bi-distilled water. 10 μL were injected into the LC-MS/MS system consisting of an HPLC 1200 Infinity II system (Agilent, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer 4000 (AB Sciex, Darmstadt, Germany). Separation was achieved using a Hypercarb column (50 mm × 2.1 mm, 5 μm particle size, Thermo Fisher Scientific, Waltham, MA, USA). The flow rate was set to 750 μL/min, and the gradient elution started with an equilibration for 2 min of 98% A (0.1% formic acid (98%) in bi-distilled water), then switched to 75% B (0.1% formic acid (98%) in acetonitrile) at 1 min, 50% B from 1.5 to 1.8 min and 2% B from 2 to 3 min. Detailed information on the validation and MS parameters is provided in the supplementary material (Peters et al. 2009).

Urine samples were analysed for creatinine by the Jaffe method and for EtG by HPLC-MS/MS as previously described (Franz et al. 2019; Herzog et al. 2023). EtG concentrations in urine are referred to 100 mg creatinine/dL.

Results

Test subjects

The AUDIT-C questionnaire did not include the three weeks of alcohol abstinence. The scores ranged from 0 to 14 points (Table 1). For participants 4, 5 and 8, scores above the threshold of 8 points were calculated, being suspicious of an alcohol-related disorder. None of the study subjects crossed the threshold of 15–20 points indicating alcoholism.

Table 1

Summary of results from 18 test subjects including sex (female (f) or male (m)), weight, Alcohol Use Disorders Identification Test including Consumption (AUDIT-C), highest BAC 45 min after drinking, concentration (c) for EtG in dried blood spots (DBS) and urine (EtGU), phosphatidylethanol (PEth) and amount (n) of detectable PEth-homologues with concentration above the limit of quantification (LOQ ≤ 20 ng/mL); subject 6: collection of venous blood only for the first and last appointment

Test subjectSexWeight (kg)AUDIT-C ScoreCalculated mass (gEtOH)BACmax (g/kg)Calculated alcohol withdrawal (g/kg/h)PEth 16:0/18:1 cinitial (ng/mL)PEth 16:0/18:1 cmax (ng/mL)PEth 16:0/18:1 clast blood collection, day 1 (ng/mL)PEth 16:0/18:1 cday 2 (ng/mL)EtG in DBS cmax (ng/mL)EtG in DBS cday 2 (ng/mL)n = PEth homologues (> LOQ)EtGU c (ng/mL), creatine100 day 2
1f63241.80.960.18< LOQ2517.214.59401623100
2f56737.10.740.15< LOQ14.614.211.282011.311740
3m85765.80.940.15< LOQ45.929.328.591079.5211,220
4m901469.60.940.15< LOQ24.922.720.4107033.4318,480
5f62941.10.720.17< LOQ14.514.2< LOQ72018.821830
6f70554.1--< LOQ40.140.140.9--220,050
7m70654.11.010.19< LOQ2822.31880011.331540
8m901269.61.030.16< LOQ50.148.646.94706.641280
9f66543.70.810.16< LOQ33.818.312114010.3488,380
10f59239.10.980.17< LOQ3021.514.286015.346870
11f67344.40.820.1816.443.424.414.696011.441550
12f74049.11.030.1712.960.641.220.9118012.1428,710
13m94572.70.870.13< LOQ33.421.312.4113017.3420,390
14f63441.80.60.19< LOQ31.28.9< LOQ5803.641980
15f59639.10.820.16< LOQ41.318.4139207043930
16f62641.10.770.1917.751.636.214.5108037.646240
17f893590.820.141137.220.513.541026.834030
18f60739.80.930.1811.132.921.611.778056.539810
Test subjectSexWeight (kg)AUDIT-C ScoreCalculated mass (gEtOH)BACmax (g/kg)Calculated alcohol withdrawal (g/kg/h)PEth 16:0/18:1 cinitial (ng/mL)PEth 16:0/18:1 cmax (ng/mL)PEth 16:0/18:1 clast blood collection, day 1 (ng/mL)PEth 16:0/18:1 cday 2 (ng/mL)EtG in DBS cmax (ng/mL)EtG in DBS cday 2 (ng/mL)n = PEth homologues (> LOQ)EtGU c (ng/mL), creatine100 day 2
1f63241.80.960.18< LOQ2517.214.59401623100
2f56737.10.740.15< LOQ14.614.211.282011.311740
3m85765.80.940.15< LOQ45.929.328.591079.5211,220
4m901469.60.940.15< LOQ24.922.720.4107033.4318,480
5f62941.10.720.17< LOQ14.514.2< LOQ72018.821830
6f70554.1--< LOQ40.140.140.9--220,050
7m70654.11.010.19< LOQ2822.31880011.331540
8m901269.61.030.16< LOQ50.148.646.94706.641280
9f66543.70.810.16< LOQ33.818.312114010.3488,380
10f59239.10.980.17< LOQ3021.514.286015.346870
11f67344.40.820.1816.443.424.414.696011.441550
12f74049.11.030.1712.960.641.220.9118012.1428,710
13m94572.70.870.13< LOQ33.421.312.4113017.3420,390
14f63441.80.60.19< LOQ31.28.9< LOQ5803.641980
15f59639.10.820.16< LOQ41.318.4139207043930
16f62641.10.770.1917.751.636.214.5108037.646240
17f893590.820.141137.220.513.541026.834030
18f60739.80.930.1811.132.921.611.778056.539810
Table 1

Summary of results from 18 test subjects including sex (female (f) or male (m)), weight, Alcohol Use Disorders Identification Test including Consumption (AUDIT-C), highest BAC 45 min after drinking, concentration (c) for EtG in dried blood spots (DBS) and urine (EtGU), phosphatidylethanol (PEth) and amount (n) of detectable PEth-homologues with concentration above the limit of quantification (LOQ ≤ 20 ng/mL); subject 6: collection of venous blood only for the first and last appointment

Test subjectSexWeight (kg)AUDIT-C ScoreCalculated mass (gEtOH)BACmax (g/kg)Calculated alcohol withdrawal (g/kg/h)PEth 16:0/18:1 cinitial (ng/mL)PEth 16:0/18:1 cmax (ng/mL)PEth 16:0/18:1 clast blood collection, day 1 (ng/mL)PEth 16:0/18:1 cday 2 (ng/mL)EtG in DBS cmax (ng/mL)EtG in DBS cday 2 (ng/mL)n = PEth homologues (> LOQ)EtGU c (ng/mL), creatine100 day 2
1f63241.80.960.18< LOQ2517.214.59401623100
2f56737.10.740.15< LOQ14.614.211.282011.311740
3m85765.80.940.15< LOQ45.929.328.591079.5211,220
4m901469.60.940.15< LOQ24.922.720.4107033.4318,480
5f62941.10.720.17< LOQ14.514.2< LOQ72018.821830
6f70554.1--< LOQ40.140.140.9--220,050
7m70654.11.010.19< LOQ2822.31880011.331540
8m901269.61.030.16< LOQ50.148.646.94706.641280
9f66543.70.810.16< LOQ33.818.312114010.3488,380
10f59239.10.980.17< LOQ3021.514.286015.346870
11f67344.40.820.1816.443.424.414.696011.441550
12f74049.11.030.1712.960.641.220.9118012.1428,710
13m94572.70.870.13< LOQ33.421.312.4113017.3420,390
14f63441.80.60.19< LOQ31.28.9< LOQ5803.641980
15f59639.10.820.16< LOQ41.318.4139207043930
16f62641.10.770.1917.751.636.214.5108037.646240
17f893590.820.141137.220.513.541026.834030
18f60739.80.930.1811.132.921.611.778056.539810
Test subjectSexWeight (kg)AUDIT-C ScoreCalculated mass (gEtOH)BACmax (g/kg)Calculated alcohol withdrawal (g/kg/h)PEth 16:0/18:1 cinitial (ng/mL)PEth 16:0/18:1 cmax (ng/mL)PEth 16:0/18:1 clast blood collection, day 1 (ng/mL)PEth 16:0/18:1 cday 2 (ng/mL)EtG in DBS cmax (ng/mL)EtG in DBS cday 2 (ng/mL)n = PEth homologues (> LOQ)EtGU c (ng/mL), creatine100 day 2
1f63241.80.960.18< LOQ2517.214.59401623100
2f56737.10.740.15< LOQ14.614.211.282011.311740
3m85765.80.940.15< LOQ45.929.328.591079.5211,220
4m901469.60.940.15< LOQ24.922.720.4107033.4318,480
5f62941.10.720.17< LOQ14.514.2< LOQ72018.821830
6f70554.1--< LOQ40.140.140.9--220,050
7m70654.11.010.19< LOQ2822.31880011.331540
8m901269.61.030.16< LOQ50.148.646.94706.641280
9f66543.70.810.16< LOQ33.818.312114010.3488,380
10f59239.10.980.17< LOQ3021.514.286015.346870
11f67344.40.820.1816.443.424.414.696011.441550
12f74049.11.030.1712.960.641.220.9118012.1428,710
13m94572.70.870.13< LOQ33.421.312.4113017.3420,390
14f63441.80.60.19< LOQ31.28.9< LOQ5803.641980
15f59639.10.820.16< LOQ41.318.4139207043930
16f62641.10.770.1917.751.636.214.5108037.646240
17f893590.820.141137.220.513.541026.834030
18f60739.80.930.1811.132.921.611.778056.539810

Due to poor venous access, it was not possible to draw blood from participant 6 after alcohol intake; she remained under observation, nevertheless and capillary blood was taken instead twice (Supplementary Table 1). All remaining participants (n = 17) completed the study.

In Table 1, the individual mass of ethanol estimated to achieve a BAC of 0.60 g/kg per individual is displayed. When wine was ingested, the volume was rounded to 100 mL. The volume of consumed vodka was rounded to 20 mL.

Blood alcohol concentration

The targeted BAC of at least 0.6 g/kg was reached in 17 out of 18 test subjects. The average withdrawal of ethanol was between 0.13 and 0.19 g/kg/h (mean: 0.165 g/kg/h). Figure 1 shows the mean BAC over time; BAC including standard deviation is displayed in the supplements (Supplementary Fig. 2). All participants had a negative BAC on day 2.

Mean BAC and phosphatidylethanol (PEth 16:0/18:1) concentrations of 17 participants and their decline.
Figure 1

Mean BAC and phosphatidylethanol (PEth 16:0/18:1) concentrations of 17 participants and their decline.

Synthesis of PEth homologues

All participants showed initial concentrations of PEth 16:0/18:1 below 20 ng/mL (Table 1). During the study, subjects tested positive for PEth 16:0/18:1 (> LOQ, 10.7 ng/mL) at least once. At the end of day 1, approximately 6.5 h after drinking was completed, twelve test subjects reached a PEth 16:0/18:1 concentration above 20 ng/mL (average: 29.1 ng/mL). On the next day, 20 h after alcohol administration, only five subjects including participant 6 remained above the threshold (average: 31.5 ng/mL). Five test subjects on the study day and eleven subjects on the post-drinking day reached concentrations between 10 and 20 ng/mL (mean: 16.4 and 13.6 ng/mL, respectively).

The concentration of test person 14 remained below the LOQ approximately 6.5 h after alcohol ingestion. However, the maximum BAC was 0.6 g/kg 15 min after drinking was completed. Afterwards, the BAC decreased by approximately 0.19 g/kg/h. The first negative BAC (<0.03 g/kg) occurred already approximately 4 h later. A maximum PEth concentration of 31.2 ng/mL was reached at the same time.

Only five test subjects (11, 12, 16, 17 and 18) had an initial PEth 16:0/18:1 concentration of 10–20 ng/mL at the start of the study. On the second day of the study, PEth concentrations in samples from four of these test subjects decreased to their respective initial concentration. Test subject 12 remained above 20 ng/mL on the post-drinking day.

Four test subjects with a concentration above the threshold of 20 ng/mL on the following day had an average maximum BAC of 0.98 g/kg. Test subjects with concentrations between 10 and 20 ng/mL on the following day showed an average maximum BAC of 0.86 g/kg.

The highest amount of PEth 16:0/18:1 (median: 35.2 ng/mL) was reached shortly after 4 h after the end of drinking in 16 of the 17 test subjects (subject 6 excluded). Participant 4 reached a PEth peak at 24.9 ng/mL just after 5 h.

Overnight, the PEth 16:0/18:1 concentration decreased by 27.7% on average (2.7–59.9%). The mean PEth 16:0/18:1 concentrations are displayed in Fig. 1; concentrations including standard deviations are displayed in the supplements (Supplementary Fig. 3).

Although it was not possible to draw venous blood from test subject 6 on the first day of the study, two capillary DBS were collected from which PEth was determined. The initial negative PEth 16:0/18:1 concentration rose to 40.1 ng/mL after 5.75 h. The day after the study, the PEth concentration was 40.9 ng/mL. Participants 6 and 8 exhibited the highest average PEth concentrations during the experiments.

PEth 16:0/18:2 was above 20 ng/mL in four of 18 test subjects the following morning. Participants 4 and 6 were positive for PEth 16:0/18:1, and subjects 13 and 16 had a PEth 16:0/18:1 concentration of 12.4 and 14.5 ng/mL, respectively.

Moreover, the concentrations of six additional PEth homologues were tested (Table 1). PEth 17:0/18:1 was not detected at all. Traces (<LOQ) of PEth 16:0/20:4 and 18:1/18:1 were occasionally observed. Test subject 2 was only positive for PEth 16:0/18:1. Nine volunteers were positive for three additional homologues (16:0/18:2, 18:0/18:1 and 18:0/18:2), four participants were positive for two additional homologues (16:0/18:2 and 18:0/18:2) and four test subjects (including subject 6) were positive for PEth 16:0/18:2 in addition to PEth 16:0/18:1. The PEth homologue concentrations, their synthesis and decline correlated (coefficient of determination, R2 > 0.7) in 14 out of 18 test subjects for PEth 16:0/18:1 and 16:0/18:2 (Fig. 2). The correlation for participants 1, 5 and 7 was below 0.7 for both homologues. PEth 16:0/18:1 and 18:0/18:2 correlated (R2 > 0.77) in 10 of 18 subjects. The correlation of PEth 16:0/18:1 and 18:0/18:1 was R2 > 0.7 in eight of the nine subjects, when PEth 18:0/18:1 was detected.

Formation of EtG in blood and urine

A second set of DBS was analysed to determine EtG. The initial EtG concentration in DBS was negative for all subjects. EtG in DBS decreased overnight in all participants but remained above the LOQ (3.0 ng/mL) the next day (Fig. 3). EtGU concentrations were also positive in all participants. No correlation was observed between BACmax and PEthmax (R2 = 0.031), PEthmax and EtGmax (R2 = 0.005), and BACmax and EtGmax (R2 = 0.006).

Correlation (coefficient of determination, R2 > 0.8) of two phosphatidylethanol homologues (PEth 16:0/18:1 and 16:0/18:2) in 14 of 18 participants.
Figure 2

Correlation (coefficient of determination, R2 > 0.8) of two phosphatidylethanol homologues (PEth 16:0/18:1 and 16:0/18:2) in 14 of 18 participants.

Mean formation and decline of EtG in DBS of 17 participants including standard deviation.
Figure 3

Mean formation and decline of EtG in DBS of 17 participants including standard deviation.

Discussion

The calculated AUDIT-C score was unexpectedly low, with one participant (subject 12) scoring 0 points. The breakdown of ethanol is in accordance with data provided in literature (Penning 1999). Participants 4 and 8 scored the highest points for the AUDIT-C questionnaire, and also had a PEth 16:0/18:1 concentration above the threshold of 20 ng/mL on the following morning.

All but one volunteer reached the expected BAC of 0.6 g/kg 45 min after drinking. As a main finding, it could be shown that after a single low to moderate dose of ethanol, it is possible to detect PEth above the threshold of 20 ng/mL on the following day. However, this outcome was limited to four test subjects, reaching BACs between 0.8 and 1.03 g/kg.

Stöth et al. previously described, that a BAC of 0.63 g/kg will not cause the threshold of 20 ng PEth 16:0/18:1/mL blood being exceeded at any point during the study (Stöth et al. 2022). In accordance with Stöth et al., none of the five test subjects with a BAC below 0.8 g/kg exceeded this threshold on the next day. However, in contrast to Stöth’s study, DBS for PEth analysis were prepared from venous blood.

A positive BAC leads to post-sampling formation in blood tubes, causing artifactual formation of PEth during storage (Aradóttir et al. 2004; Beck et al. 2021). To minimise a possible de novo synthesis, DBS were created with a very short delay in time after blood drawing. The third blood sample contained the highest mean BAC (0.82 g/kg) throughout the experiment, and also the highest mean PEth concentration (24.2 ng/mL) compared with the subsequent blood sample (20.6 ng/mL; Fig. 1). This atypical trend might be explained by post-sampling formation: the higher the BAC, the more PEth is formed post-sampling before the DBS is completely dried.

Comparing PEth concentrations formed during the latest positive BAC samples with those in the first negative BAC samples, the latter values were lower in 16 of 17 cases. However, 9 of 17 of the later incurred DBS were collected the following day.

In test subject 8, the highest average PEth concentration was found. Even though it was not possible to obtain any blood from participant 6 during the study, the collected and analysed DBS resulted in the same high amounts of over 40 ng/mL on the following day. Unfortunately, the maximum BAC could not be obtained; therefore, a comparison of alcohol consumption markers of both participants is limited. It can be seen that both participants build up PEth very quickly, and degradation is slower when compared to other participants in the study.

Two participants (12, 16) showed a decrease in maximum PEth 16:0/18:1 of ≥50% overnight (approximately 13.5 h), whereas the concentration in other participants (1, 2, 3, 4, 6, 7, 15) remained above 70%. The decline of PEth appears highly variable regarding its half-life from four to ten days in the literature (Winkler et al. 2013).

There were no differences related to the formation and breakdown of either PEth or EtG due to sex, which is also in accordance with the literature (Wurst et al. 2010).

Sixteen out of 18 participants tested positive for recent alcohol intake if a lower threshold of 10 ng/mL was applied, as suggested by Aboutara et al. (Aboutara et al. 2022).

In a recent study of Lopez-Cruzan et al., PEth 16:0/20:4 was described as a homologue to detect very recent alcohol consumption. Therefore, its concentration was expected to exceed the LOQ of 16.0 ng/mL; however, this homologue could only be detected in traces above the LOD of 2.1 ng/mL (Lopez-Cruzan et al. 2018). In the present study, all participants were asked to abstain from alcohol for at least 3 weeks, and their initial PEth concentration was below 20 ng/mL, whereas Lopez-Cruzan’s volunteers refrained from alcohol for seven days only and had an initial PEth 16:0/18:1 concentration of over 50 ng/mL (Lopez-Cruzan et al. 2018). To detect PEth 16:0/20:4 above the LOQ, a higher BAC or a repeated alcohol intake over several days is probably a prerequisite.

Both PEth homologues 18:0/18:1 and 18:0/18:2 were detected in this study, with the latter being the third most common one. This suggests that 18:0/18:2 may be another PEth homologue suitable for plausibility evaluation and higher sensitivity for assessing recent alcohol intake.

On the following morning, participants 13 and 16 showed PEth 16:0/18:2 concentrations above 20 ng/mL, whereas PEth 16:0/18:1 declined below the threshold. Including additional homologues will support detection of recent alcohol consumption, even if PEth 16:0/18:1 is below the threshold. However, only two standards (PEth 16:0/18:1 and 16:0/18:2) are commercially available, for which a regioisomeric purity of 16:0 located at the sn1 position is guaranteed (Luginbühl et al. 2021). If 18:0/18:2 will be used to increase the sensitivity toward uncovering a recent moderate alcohol ingestion, the regioisomeric purity of this homologue is considered a prerequisite.

As expected, EtGU exceeded the threshold of 100 ng/mL for all participants. EtG in DBS was positive (> LOQ) for all participants the following day. The half-life of EtG in blood is about 2.5 h and breaks down according to zero-order kinetics (Høiseth et al. 2007; Neumann et al. 2020). Before the last urine and blood collection as well as DBS preparation, the participants were unsupervised for approximately 13.5 h, and were not allowed to consume any ethanol. During this time frame, the EtG half-time passed about five times. Only test subject 3 showed a higher final concentration (79.5 ng/mL, expected ~27 ng/mL, Table 1), including a PEth 16:0/18:1 concentration above 20 ng/mL. Upon request with the participant to confirm alcohol abstinence overnight, the high concentration of EtG can be explained by a higher half-life of EtG and the PEth concentration above the threshold can unambiguously be traced back to the BAC during the first study day.

To increase the sensitivity for detecting recent alcohol consumption, the analysis of EtG might be helpful as well. In this study, the additional detection of EtG in DBS revealed a sensitivity of 100% for detecting recent alcohol consumption, independent of the consumed amount.

Conclusion

After drinking a moderate amount of alcohol following a previous 3-week abstinence and reaching a minimum BAC of 0.93 g/kg (n = 8), the threshold of 20 ng/mL for PEth 16:0/18:1 was exceeded by four individuals (5, if participants 6 is included) on the following morning. Eleven test subjects reached a PEth 16:0/18:1 concentration between 10 and 20 ng/mL on the following morning, thus increasing the sensitivity for a single alcohol intake from 27.7 to 61.1% after 3 weeks of alcohol abstinence. By considering PEth 16:0/18:2 at a threshold of 20 ng/mL, the sensitivity rises to even 72.2%. The concentrations, including the formation and breakdown of different PEth homologues correlated with R2 < 0.8 in 14 out of 18 participants. Including the detection of EtG in blood, the sensitivity to prove recent single alcohol intake increased to 100%. EtGU was positive (≥ 100 ng/mL) in all samples the following day. To further investigate if a lower cutoff of 10 ng PEth 16:0/18:1 per mL blood may preferably be applied, the abstinence period should be prolonged or participants with initial PEth concentration of 10 ng/mL or above be excluded. Moreover, the time of blood collection should be extended as well.

Author contributions

Josefine Herzog (Conceptualization-Equal, Data curation-Lead, Formal analysis-Lead, Investigation-Equal, Methodology-Equal, Project administration-Equal, Validation-Lead, Visualisation-Lead, Writing—original draft-Lead), Gisela Skopp (Conceptualization-Equal, Methodology-Equal, Project administration-Equal, Writing—review and editing-Equal), Frank Musshoff (Conceptualization-Equal, Funding acquisition-Equal, Methodology-Equal, Project administration-Equal, Resources-Equal, Writing—review and editing-Equal), Benno Hartung (Conceptualization-Equal, Funding acquisition-Equal, Investigation-Equal, Methodology-Equal, Project administration-Equal, Resources-Equal, Supervision-Lead, Writing—review and editing-Equal).

Conflict of Interest statement: None of the authors have conflict of interests regarding this article.

Funding

This study was funded by the Bund gegen Alkohol und Drogen im Straßenverkehr e.V. (BADS), Hamburg (4,000 €, grant number 73102).

Data Availability

Data required for this article is available in the article and its supplementary material.

References

Aboutara
 
N
,
Jungen
 
H
,
Szewczyk
 
A
 et al.   
PEth 16:0/18:1 and 16:0/18:2 after consumption of low doses of alcohol- a contribution to cut-off discussion
.
Drug Test Anal
 
Epub ahead of print 30 September 2022
 
2022
;
15
:
104
14
. https://doi-org.libproxy.ucl.ac.uk/10.1002/dta.3376.

Aderjan
 
R
,
Daldrup
 
T
,
Käferstein
 
H
 et al.   
Richtlinien zur Bestimmung der Blutalkoholkonzentration (BAK) für forensische Zwecke: – BAK-Richtlinien
.
Blutalkohol
 
2011
;
48
:
137
43
.

Albermann
 
ME
,
Musshoff
 
F
,
Doberentz
 
E
 et al.   
Preliminary investigations on ethyl glucuronide and ethyl sulfate cutoffs for detecting alcohol consumption on the basis of an ingestion experiment and on data from withdrawal treatment
.
Int J Leg Med
 
2012
;
126
:
757
64
. https://doi-org.libproxy.ucl.ac.uk/10.1007/s00414-012-0725-3.

Andresen-Streichert
 
H
,
Müller
 
A
,
Glahn
 
A
 et al.   
Alcohol biomarkers in clinical and forensic contexts
.
Dtsch Ärztebl Int
 
2018
;
115
:
309
15
. https://doi-org.libproxy.ucl.ac.uk/10.3238/arztebl.2018.0309.

Aradóttir
 
S
,
Moller
 
K
,
Alling
 
C
.
Phosphatidylethanol formation and degradation in human and rat blood
.
Alcohol Alcohol
 
2004
;
39
:
8
13
. https://doi-org.libproxy.ucl.ac.uk/10.1093/alcalc/agh003.

Beck
 
O
,
Mellring
 
M
,
Löwbeer
 
C
 et al.   
Measurement of the alcohol biomarker phosphatidylethanol (PEth) in dried blood spots and venous blood-importance of inhibition of post-sampling formation from ethanol
.
Anal Bioanal Chem
 
2021
;
413
:
5601
6
. https://doi-org.libproxy.ucl.ac.uk/10.1007/s00216-021-03211-z.

Berger
 
L
,
Fendrich
 
M
,
Jones
 
J
 et al.   
Ethyl glucuronide in hair and fingernails as a long-term alcohol biomarker
.
Addiction
 
2014
;
109
:
425
31
. https://doi-org.libproxy.ucl.ac.uk/10.1111/add.12402.

Brenner-Hartmann
 
J
,
Fastenmeier
 
W
,
Graw
 
M
.
Urteilsbildung in der Fahreignungsbegutachtung: Beurteilungskriterien
.
Bonn
:
Kirschbaum Verlag
,
2022
.

Statistisches Bundesamt
(
2021
)
Unfälle unter dem Einfluss von Alkohol oder anderen berauschenden Mitteln im Straßenverkehr 2020
.
Available at:
 https://www.destatis.de/DE/Themen/Gesellschaft-Umwelt/Verkehrsunfaelle/Publikationen/Downloads-Verkehrsunfaelle/unfaelle-alkohol-5462404207004.pdf?__blob=publicationFile  
(14 November 2022, date last accessed)
.

Franz
 
S
,
Skopp
 
G
,
Boettcher
 
M
 et al.   
Creatinine excretion in consecutive urine samples after controlled ingestion of water
.
Drug Test Anal
 
2019
;
11
:
435
40
. https://doi-org.libproxy.ucl.ac.uk/10.1002/dta.2514.

Gnann
 
H
,
Engelmann
 
C
,
Skopp
 
G
 et al.   
Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS
.
Anal Bioanal Chem
 
2010
;
396
:
2415
23
. https://doi-org.libproxy.ucl.ac.uk/10.1007/s00216-010-3458-5.

Helander
 
A
,
Hansson
 
T
.
Nationell harmonisering av alkoholmarkören PEth
.
Lakartidningen
 
2013
;
110
:
1747
8
.

Herzog
 
J
,
Skopp
 
G
,
Musshoff
 
F
.
Development and validation of seven phosphatidylethanol-homologues in dried blood spots including preliminary results after excessive use of an ethanol-based hand sanitizer
.
J Anal Toxicol
 
2023
;
47
:
245
52
. https://doi-org.libproxy.ucl.ac.uk/10.1093/jat/bkac086.

Hill-Kapturczak
 
N
,
Dougherty
 
DM
,
Roache
 
JD
 et al.   
Differences in the synthesis and elimination of phosphatidylethanol 16:0/18:1 and 16:0/18:2 after acute doses of alcohol
.
Alcohol Clin Exp Res
 
2018
;
42
:
851
60
. https://doi-org.libproxy.ucl.ac.uk/10.1111/acer.13620.

Hoiseth
 
G
,
Karinen
 
R
,
Christophersen
 
AS
 et al.   
A study of ethyl glucuronide in post-mortem blood as a marker of ante-mortem ingestion of alcohol
.
Forensic Sci Int
 
2007
;
165
:
41
5
. https://doi-org.libproxy.ucl.ac.uk/10.1016/j.forsciint.2006.02.045.

Høiseth
 
G
,
Bernard
 
JP
,
Karinen
 
R
 et al.   
A pharmacokinetic study of ethyl glucuronide in blood and urine: applications to forensic toxicology
.
Forensic Sci Int
 
2007
;
172
:
119
24
. https://doi-org.libproxy.ucl.ac.uk/10.1016/j.forsciint.2007.01.005.

Jatlow
 
P
,
O'Malley
 
SS
.
Clinical (nonforensic) application of ethyl glucuronide measurement: are we ready?
 
Alcohol Clin Exp Res
 
2010
;
34
:
968
75
. https://doi-org.libproxy.ucl.ac.uk/10.1111/j.1530-0277.2010.01171.x.

Kobayashi
 
M
,
Kanfer
 
JN
.
Phosphatidylethanol formation via transphosphatidylation by rat brain synaptosomal phospholipase D
.
J Neurochem
 
1987
;
48
:
1597
603
. https://doi-org.libproxy.ucl.ac.uk/10.1111/j.1471-4159.1987.tb05707.x.

Kummer
 
N
,
Ingels
 
A-S
,
Wille
 
SMR
 et al.   
Quantification of phosphatidylethanol 16:0/18:1, 18:1/18:1, and 16:0/16:0 in venous blood and venous and capillary dried blood spots from patients in alcohol withdrawal and control volunteers
.
Anal Bioanal Chem
 
2016
;
408
:
825
38
. https://doi-org.libproxy.ucl.ac.uk/10.1007/s00216-015-9169-1.

Lopez-Cruzan
 
M
,
Roache
 
JD
,
Hill-Kapturczak
 
N
 et al.   
Pharmacokinetics of phosphatidylethanol 16:0/20:4 in human blood after alcohol intake
.
Alcohol Clin Exp Res
 
2018
;
42
:
2094
9
. https://doi-org.libproxy.ucl.ac.uk/10.1111/acer.13865.

Luginbühl
 
M
,
Young
 
RSE
,
Stoeth
 
F
 et al.   
Variation in the relative isomer abundance of synthetic and biologically derived phosphatidylethanols and its consequences for reliable quantification
.
J Anal Toxicol
 
2021
;
45
:
76
83
. https://doi-org.libproxy.ucl.ac.uk/10.1093/jat/bkaa034.

Luginbühl
 
M
,
Wurst
 
FM
,
Stöth
 
F
 et al.   
Consensus for the use of the alcohol biomarker phosphatidylethanol (PEth) for the assessment of abstinence and alcohol consumption in clinical and forensic practice (2022 consensus of Basel)
.
Drug Test Anal
 
Epub ahead of print 18 July 2022
 
2022
;
14
:
1800
2
. https://doi-org.libproxy.ucl.ac.uk/10.1002/dta.3340.

Musshoff
 
F
,
Böttcher
 
M
,
Graw
 
M
 et al.   
Comment on the upper cutoff level for the alcohol biomarker phosphatidylethanol (PEth) for the assessment of alcohol consumption in forensic practice
.
Drug Test Anal
 
Epub ahead of print 30 December 2022
 
2022
. https://doi-org.libproxy.ucl.ac.uk/10.1002/dta.3432.

Neumann
 
J
,
Beck
 
O
,
Helander
 
A
 et al.   
Sensitive determination of ethyl glucuronide in serum and whole blood: detection time after alcohol exposure compared with urine
.
J Lab Med
 
2020
;
44
:
211
9
. https://doi-org.libproxy.ucl.ac.uk/10.1515/labmed-2019-0203.

Penning
 
R
.
Rechtsmedizin Systematisch
.
Bremen, Lorch/Württemberg
:
UNI-MED-Verl
,
1999
.

Peters
 
F
,
Hartung
 
M
,
Herbold
 
M
 et al.   
Appendix B to the guidelines for quality assurance in forensic-toxicological analyses: requirements for the validation of analytical methods
.
Toxichem Krimtech
 
2009
;
76
:
185
208
.

Saunders
 
JB
,
Aasland
 
OG
,
Babor
 
TF
 et al.   
Development of the Alcohol Use Disorders Identification Test (AUDIT): WHO Collaborative Project on Early Detection of Persons with Harmful Alcohol Consumption-II
.
Addiction
 
1993
;
88
:
791
804
. https://doi-org.libproxy.ucl.ac.uk/10.1111/j.1360-0443.1993.tb02093.x.

Schloegl
 
H
,
Dresen
 
S
,
Spaczynski
 
K
 et al.   
Stability of ethyl glucuronide in urine, post-mortem tissue and blood samples
.
Int J Leg Med
 
2006
;
120
:
83
8
. https://doi-org.libproxy.ucl.ac.uk/10.1007/s00414-005-0012-7.

Stöth
 
F
,
Kotzerke
 
E
,
Thierauf-Emberger
 
A
 et al.   
Can PEth be detected with a cutoff of 20 ng/mL after single alcohol consumption?
 
J Anal Toxicol
 
2022
;
46
:
e232
8
. https://doi-org.libproxy.ucl.ac.uk/10.1093/jat/bkac069.

Varga
 
A
,
Hansson
 
P
,
Johnson
 
G
 et al.   
Normalization rate and cellular localization of phosphatidylethanol in whole blood from chronic alcoholics
.
Clin Chim Acta
 
2000
;
299
:
141
50
. https://doi-org.libproxy.ucl.ac.uk/10.1016/S0009-8981(00)00291-6.

Viel
 
G
,
Boscolo-Berto
 
R
,
Cecchetto
 
G
 et al.   
Phosphatidylethanol in blood as a marker of chronic alcohol use: a systematic review and meta-analysis
.
Int J Mol Sci
 
2012
;
13
:
14788
812
. https://doi-org.libproxy.ucl.ac.uk/10.3390/ijms131114788.

Widmark
 
E
.
The Theoretical Basis and Practical Application of Medical-Legal Determination of Alcohol
.
Berlin
:
Urban und Schwarzenberg
,
1932
.

Winkler
 
M
,
Skopp
 
G
,
Alt
 
A
 et al.   
Comparison of direct and indirect alcohol markers with PEth in blood and urine in alcohol dependent inpatients during detoxication
.
Int J Leg Med
 
2013
;
127
:
761
8
. https://doi-org.libproxy.ucl.ac.uk/10.1007/s00414-012-0812-5.

Wurst
 
FM
,
Thon
 
N
,
Aradottir
 
S
 et al.   
Phosphatidylethanol: normalization during detoxification, gender aspects and correlation with other biomarkers and self-reports
.
Addict Biol
 
2010
;
15
:
88
95
. https://doi-org.libproxy.ucl.ac.uk/10.1111/j.1369-1600.2009.00185.x.

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Supplementary data