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Clinical Chemistry 55: 1395-1405, 2009. First published May 7, 2009; 10.1373/clinchem.2008.120923
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(Clinical Chemistry. 2009;55:1395-1405.)
© 2009 American Association for Clinical Chemistry, Inc.

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Molecular Species of the Alcohol Biomarker Phosphatidylethanol in Human Blood Measured by LC-MS

Anders Helander1,a and Yufang Zheng1

1 Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden.

aAddress correspondence to this author at: Alcohol Laboratory, L7:03, Karolinska University Hospital Solna, SE-171 76 Stockholm, Sweden. Fax +46-8-51771532; e-mail anders.helander{at}ki.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The alcohol biomarker phosphatidylethanol (PEth) comprises a group of ethanol-derived phospholipids formed from phosphatidylcholine by phospholipase D. The PEth molecular species have a common phosphoethanol head group onto which 2 fatty acid moieties are attached. We developed an electrospray ionization (ESI) LC-MS method for qualitative and quantitative measurement of different PEth species in human blood.

Methods: We subjected a total lipid extract of whole blood to HPLC gradient separation on a C4 column and performed LC-ESI-MS analysis using selected ion monitoring of deprotonated molecules for the PEth species and phosphatidylpropanol (internal standard). Identification of individual PEth species was based on ESI–tandem mass spectrometry (MS/MS) analysis of product ions.

Results: The fatty acid moieties were the major product ions of PEth, based on comparison with PEth-16:0/16:0, 18:1/18:1, and 16:0/18:1 reference material. For LC-MS analysis of different PEth species in blood, we used a calibration curve covering 0.2–7.0 µmol/L PEth-16:0/18:1. The lower limit of quantitation of the method was <0.1 µmol/L, and intra- and interassay CVs were <9% and <11%. In blood samples collected from 38 alcohol patients, the total PEth concentration ranged between 0.1 and 21.7 µmol/L (mean 8.9). PEth-16:0/18:1 and 16:0/18:2 were the predominant molecular species, accounting for approximately 37% and 25%, respectively, of total PEth. PEth-16:0/20:4 and mixtures of 18:1/18:1 plus 18:0/18:2 (not separated using selected ion monitoring because of identical molecular masses) and 16:0/20:3 plus 18:1/18.2 made up approximately 13%, 12%, and 8%.

Conclusions: This LC-MS method allows simultaneous qualitative and quantitative measurement of several PEth molecular species in whole blood samples.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To aid in the early identification of people engaged in harmful alcohol consumption, and for monitoring alcohol-dependent patients during treatment, research efforts have focused on developing laboratory tests for alcohol biomarkers with a longer detection window than that offered by ethanol testing (1). The conjugated minor ethanol metabolites ethyl glucuronide (2) and ethyl sulfate(3) and fatty acid ethyl esters (4) are examples of such tests. Ethyl glucuronide and ethyl sulfate are excreted in urine for a much longer time than ethanol and hence have gained popularity as diagnostically sensitive and specific tests to spot recent alcohol consumption (5)(6)(7). For detection and follow-up of long-term risky or heavy drinking, measurement of the alcohol-related change in the serum transferrin glycoform profile known as carbohydrate-deficient transferrin (CDT)1 (8)(9) has become a standard method, often used in combination with liver function tests (e.g., {gamma}-glutamyltransferase) (1).

Phosphatidylethanol (PEth) is another indicator of high alcohol consumption identified some time ago (10)(11) that so far has received limited clinical interest. PEth is an ethanol-derived phospholipid formed from phosphatidylcholine (PC) in cell membranes by a transphosphatidylation reaction catalyzed by phospholipase D (12). Phospholipase D normally hydrolyzes PC into phosphatidic acid and choline, but because the affinity for ethanol is >1000-fold higher than for water, PEth is formed at the expense of phosphatidic acid when ethanol is present (13)(14). In clinical studies, PEth was not detected after a single high alcohol intake but after sustained drinking of more than approximately 50 g/day for 3 weeks (15). The PEth concentration in blood was reported to correlate with the amount of alcohol consumed during the previous 2 weeks (16), and PEth may be detected for up to 2–4 weeks after cessation of heavy drinking (17)(18)(19).

One reason for the limited clinical use of PEth as alcohol biomarker is probably that the analytical method employed, lipid extraction of whole blood followed by HPLC analysis using an evaporative light-scattering detector (18)(20), is not well suited to routine use in clinical laboratories. An alternative capillary electrophoresis (CE) method with UV detection was recently introduced (21), and development of an immunoassay based on a monoclonal PEth antibody has been initiated (22).

PEth is not a single molecular species but a group of phospholipids with a common nonpolar phosphoethanol head group onto which 2 fatty acid moieties, typically with a chain length of 16, 18, or 20 carbons (18)(23), are attached at positions sn-1 and sn-2. Given that PEth originates from PC, the fatty acid composition of PEth is likely to mirror that of PC. The numerous combinations of chain lengths and double bonds enable formation of a large number of PC species (24), with 16:0/18:1 (nomenclature for fatty acids = [number of carbons]:[number of double bonds]) and 16:0/18:2 being the major fatty acid combinations in PC extracted from human erythrocyte membranes (25)(26)(27).

Current analytical methods for PEth based on HPLC (19)(20) and CE(21), and any immunoassay targeting the head group (22), measure the sum of all PEth species. Measurement of individual species is feasible by LC-MS (18)(28), depending on the variable length and number of unsaturated bonds of the fatty acid chains. We aimed to develop an LC-MS method for quantitative and qualitative measurement of different PEth species in human blood and determine the profile of molecular species in samples from heavy drinkers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals
We obtained PEth reference materials containing 2 palmitic acids (1,2-dipalmitoyl-sn-glycero-3- phosphoethanol; PEth-16:0/16:0) and 2 oleic acids (1,2-dioleoyl-sn-glycero-3-phosphoethanol; PEth-18:1/18:1) and phosphatidylpropanol (PProp-18:1/18:1; internal standard) from Avanti Polar Lipids; another PEth reference material containing 1 palmitic and 1 oleic acid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol; PEth-16:0/18:1) from Biomol Research Laboratories; and acetonitrile (HPLC grade), ammonium acetate, and isopropanol (analytical grade) from Merck. All other chemicals were of analytical or HPLC grade, and the water was of HPLC grade.

We prepared stock solutions of PEth reference materials (14.0 µmol/L) and PProp (1.35 µmol/L) in a 1:5 solution of 2 mmol/L ammonium acetate and acetonitrile and stored them at –20 °C until use. The stock solutions were stable for at least 3 months.

blood samples
The blood specimens used for method development were surplus volumes from the clinical samples pool sent to the Alcohol Laboratory (Karolinska University Hospital, Stockholm, Sweden) for testing of alcohol use and abuse. We obtained additional samples from healthy volunteers reporting consumption of <100 g ethanol/week. For comparison with the HPLC method for total PEth (20), we obtained blood samples from Lund University Hospital (Sweden). Blood was collected into EDTA tubes and stored at 4 °C for <3 days before analysis. PEth is stable for at least 3 weeks in refrigerated blood samples (20). The ethics committee at the Karolinska University Hospital approved the study.

sample preparation
We prepared total lipid extracts of whole blood (mainly erythrocyte membranes (19)) by stepwise addition of 100 µL blood to 600 µL isopropanol and 50 µL 1.35 µmol/L PProp internal standard under constant vortex-mixing (20)(29). Thereafter, samples were gently mixed for 10 min. We then added hexane (2x 450 µL) with mixing after each addition, and mixed again for another 10 min. The samples were finally centrifuged for 10 min at 2000g at 4 °C. The clear supernatants were transferred to new glass tubes and evaporated to dryness under a stream of nitrogen gas at 30 °C using a metal block. The final dried extract was dissolved in 50 µL hexane, followed by 50 µL acetonitrile and 75 µL isopropanol, and transferred to 0.3-mL glass autosampler vials.

lc-esi-ms quantification of peTH species
We used an Agilent 1100 series liquid chromatographic system connected to an LC/MSD SL mass spectrometric detector with the electrospray ionization (ESI) interface operated in negative ion mode together with ChemStation software. The conditions used were drying gas flow 10.0 {Psi}, nebulizer gas 20 {Psi}, drying gas temperature 350 °C, and capillary voltage 3000 V.

Chromatographic separation of the lipid extracts was achieved on a 50 by 3 mm, 5-µm HyPurity C4 column (Thermo Scientific) maintained at 25 °C. The LC system was operated in gradient mode with solvent A being 20% 2 mmol/L ammonium acetate and 80% acetonitrile and solvent B 100% isopropanol. From sample injection until 2.0 min, isocratic elution with 90% A and 10% B was used; from 2.0–3.0 min, a linear gradient to 50% B; from 3.0–6.0 min, a linear gradient to 100% B; from 6.0–7.0 min, isocratic elution with 100% B; and from 7.0–8.0 min, a linear gradient back to 90% A and 10% B. The flow rate was 200 µL/min, and the sample injection volume, 10 µL. Reconditioning the column with 90% A and 10% B for 10 min after each injection improved the reproducibility of the retention times.

We performed LC-ESI-MS analysis using selected ion monitoring (SIM) of the deprotonated molecules for the different PEth species and PProp (Table 1 ). We determined the PEth concentration in unknown whole blood samples from the peak area ratio between analyte and internal standard by reference to a calibration curve. The calibration curve was produced by spiking PEth-negative blood with 0.2–7.0 µmol/L PEth-16:0/18:1 or 18:1/18:1 prepared from the standard stock solutions by dilution with solvent A.


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  		Table 1. Mass spectrometric parameters for the LC-ESI-MS and LC-ESI-MS/MS methods for quantitative and qualitative measurement of different PEth molecular species in total lipid extracts of human whole blood.

lc-esi–tandem mass spectrometry identification of peTH species
The LC-ESI–tandem mass spectrometry (MS/MS) system was a Perkin-Elmer series 200 LC system connected to Sciex API 2000MS, with the ESI interface operated in negative ion mode, and Analyst 1.1 software (Applied Biosystems). The LC conditions were identical to those employed for single MS analysis.

We performed LC-ESI-MS/MS analysis using selected reaction monitoring (SRM) to detect the major deprotonated fragments from each PEth species (Table 1Up ). The conditions used were curtain gas 10 {Psi}, collision gas 3 {Psi}, ion spray voltage –3500 V, temperature 450 °C, nebulizer gas 30 {Psi}, auxiliary gas 45 {Psi}, declustering potential –61 V, focusing potential –350 V, entrance potential –10 V, and collision energy –40 V.

validation of the lc-ms method
The analytical imprecision (CV%) of the LC-ESI-MS method was determined for 4 blood samples containing 0.3–7.0 µmol/L PEth, using triplicate measurements over 5 days (Clinical and Laboratory Standards Institute EP 15-A2 guideline). We calculated linearity equations using 6 PEth-negative blood samples spiked with 0.2–7.0 µmol/L PEth-16:0/18:1 or 18:1/18:1.

To study the possible impact by matrix effects (30), PEth-16:0/16:0, 16:0/18:1, and 18:1/18:1 standards at 1.0 µmol/L were infused postcolumn at a constant rate of 10 µL/min, whereas 6 extracted PEth-negative blood samples were injected via the autosampler. Pre- and postextraction addition experiments (n = 6) compared the detector responses for standard and internal standard in blanks and blood samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
monitoring of peTH species by lc-ms and lc-ms/ms
Chemical structures for the PEth species included in this study and for PProp used as the internal standard are shown in Fig. 1 . In phospholipids isolated from human erythrocytes, saturated fatty acids (e.g., 16:0, palmitic acid) are mainly located in the sn-1 position and unsaturated fatty acids (e.g., 18:2, linoleic acid) mainly in the sn-2 position (26)(31). The corresponding deprotonated molecules used in SIM of different PEth species and PProp are listed in Table 1Up . Reference material was available for PEth-16:0/16:0, 16:0/18:1, and 18:1/18:1, whereas target masses for the other molecular species were calculated.


Figure 1
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  		Figure 1. Structures of different PEth species and phosphatidylpropanol.

Tentative structures of the molecular species of PEth and PProp [internal standard (IS)] measured by the LC-MS method. Nomenclature for fatty acids = (number of carbons):(number of double bonds). *Commercially available material.

The major product ions of the different PEth species (Table 1Up ), as identified by ESI-MS/MS, corresponded to the fatty acid chains (Fig. 2A ). The identity of the products was confirmed by analysis of PEth reference material, where PEth-16:0/16:0 and 18:1/18:1 produced 1 major product ion each (m/z 255.5 for 16:0 fatty acid and m/z 281.5 for 18:1 fatty acid) and PEth-16:0/18:1 both of them. Besides the major fatty acid product ions, 2 low-intensity ions of PEth-16:0/18:1 were detected. These corresponded to the phosphoethanol head group (m/z 125) (23) and to a loss of the 18:1 fatty acid from position sn-2 (m/z 437) (28) (Fig. 2A ).


Figure 2
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  		Figure 2. LC-MS/MS analysis of PEth-16:0/18:1.

(A), LC-ESI-MS/MS product ion scan of PEth-16:0/18:1 reference material producing 2 major fatty acid chain fragments (m/z 255.5 for 16:0 and m/z 281.5 for 18:1) and 2 minor fragments (m/z 125 for the phosphoethanol head group [Holbrook et al. (23)] and m/z 437 for a loss of the 18:1 fatty acid chain from position sn-2 [Tolonen et al. (28)]). (B), LC-ESI-MS/MS chromatograms for the deprotonated molecules of PEth-16:0/18:1 (m/z 701.7/255.5 for 16:0; m/z 701.7/281.5 for 18:1) for a PEth-negative blood sample spiked with PEth-16:0/18:1 (top), the blood sample without addition of PEth (middle), and a pool of whole blood samples collected from heavy drinkers (total PEth concentration 7.5 µmol/L).

The identification of PEth species in blood samples was based on SRM analysis of the unique mass ratios for the fatty acid chains to the parent compound, producing similar product ion ratios as with the reference material (the m/z 281.5/255.5 ratios for PEth-16:0/18:1 were 3.0 and 3.1, respectively) (Fig. 2BUp ). Some of the examined molecular species have an identical molecular mass (e.g., m/z 727.7 for PEth-18:1/18:1 and 18:0/18:2) and also very similar retention times, and thus were not separated in the LC-MS chromatograms (Fig. 3 ). However, for mixtures of 2 or more PEth species with identical mass, SRM analysis enabled individual identification owing to the unique combinations of fatty acid chains.


Figure 3
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  		Figure 3. LC-MS measurement of different PEth molecular species in blood.

Example of LC-ESI-MS chromatograms for the deprotonated molecules of PEth molecular species and PProp (internal standard) in a whole blood sample obtained from a heavy drinker (total PEth concentration 7.5 µmol/L). IS, internal standard.

quantification of peTH species by lc-ms
The PEth species and PProp internal standard eluted over a narrow time range with retention times of around 3 min (Fig. 3Up ). In routine use (typically <40 samples/run), the intraassay imprecision for absolute and relative (PEth/PProp) retention times were typically <4% and <1%, respectively. No interfering peaks were observed on routine analysis of >100 PEth-negative clinical blood samples (data not shown).

The limit of detection (LOD) (signal-to-noise ratio ≥3) of the LC-MS method was <0.02 µmol/L for all PEth species examined, and the corresponding lower limit of quantitation (LOQ) (signal-to-noise ratio ≥10) was <0.1 µmol/L. The pre- and postextraction addition experiments showed an absolute extraction efficiency of approximately 80% for both PEth and PProp. The postcolumn infusion experiments showed no ion suppression or enhancement at the retention times of PEth (tested with 16:0/16:0, 16:0/18:1, and 18:1/18:1 reference materials) and the internal standard (data not shown). Still, in the postextraction addition experiments carried out with blood, the peak intensities for both PEth and PProp were approximately 90% compared with the blank, demonstrating approximately 10% absolute matrix effect. However, as all changes for PEth and PProp were of the same magnitude, the internal standard compensated for losses during sample extraction and analysis. Accordingly, when 5 blood samples containing 0–2.6 µmol/L total PEth were spiked with 1.0 µmol/L and 10.0 µmol/L PEth-16:0/18:1 before the extraction procedure, mean analytical recoveries were 101% and 103%, respectively.

The LC-MS method for quantification of PEth species in whole blood samples produced linear results for PEth-16:0/18:1 in the concentration range 0.2–20.0 µmol/L (see Supplemental Fig. 1, inset, in the Data Supplement that accompanies the online version of this article at www.clinchem.org/content/vol55/issue7). Calibration curves prepared by spiking PEth-negative blood samples with PEth-16:0/18:1 or 18:1/18:1 reference material showed nearly identical responses (online Supplemental Fig. 1), indicating that the MS response for different molecular species was essentially the same. Hence, for routine quantification of PEth species in whole blood, PEth-16:0/18:1, being the major species in clinical samples (see data below), was chosen for preparation of the calibration curve and 0.2–7.0 µmol/L was employed as the measuring range for each molecular species. It should be noted that this corresponded to a routine measuring range for total PEth from 0.2 µmol/L up to approximately 20 µmol/L, given that PEth-16:0/18:1 accounted for approximately 35% of total PEth in human blood.

The intraassay CV% for total PEth concentration in the measuring range was 4.5%–8.6%, and the values for interassay CV% were <11% (online Supplemental Table 1).

peTH stability and formation from ethanol on storage
In agreement with previous observations (20), all PEth species were demonstrated to be stable for at least 5 days in whole blood samples stored at 4 °C (data not shown). Furthermore, when 38 patient blood samples containing 0–20 µmol/L PEth were reanalyzed after storage for 14 months at –80 °C, there was no indication of declining total values (Passing–Bablok regression equation: y2007 = 1.0308x2008 – 0.3731) or changes in the species profile.

To study further the risk for postsampling synthesis of PEth on storage of blood samples containing ethanol (32)(33), PEth-negative samples from 4 control subjects collected in EDTA, heparin, and citrate tubes were spiked with ethanol to a final concentration of 2 g/L. Aliquots were taken at the start and after different times of storage at 20 °C, 4 °C, –20 °C, and –80 °C for analysis of PEth species by the LC-MS method. No formation of PEth was observed after storage for 4 h and 24 h, whereas concentrations ranging up to 0.75 µmol/L were found after 72 h storage at 20 °C or –20 °C (online Supplemental Fig. 2). In the blood from 1 donor, total PEth concentrations at or above 0.2 µmol/L (i.e., within the measuring range) were generated in all samples, whereas concentrations <0.1 µmol/L were found in 1 blood sample and undetectable amounts in the remaining 2.

distribution of peTH species in blood from heavy drinkers
The LC-MS method was applied for determination of the PEth species profile in blood samples collected from 38 heavy drinkers recently admitted for alcohol detoxification. When the patients tested negative for ethanol (breath test), they were transferred to an inpatient treatment ward where blood sampling took place within 2 days after admission. PEth was detected in all samples, the total concentration range being 0.1–21.7 µmol/L, with a mean of 8.9 µmol/L (median 9.6). The results demonstrated that PEth-16:0/18:1 and 16:0/18:2 were the predominant species (Fig. 4A ), accounting for on average 37% and 26%, respectively, of total PEth by this method. Owing to interindividual variations, PEth-16:0/18:2 was sometimes the major form. PEth-16:0/20:4 (m/z 723.7) and mixtures of 18:1/18:1 plus 18:0/18:2 (m/z 727.7; not separated in the SIM method because of identical mass) and 16:0/20:3 plus 18:1/18.2 (m/z 725.7) made up approximately 13%, 12%, and 8%, respectively (Fig. 4A ). When 1 outpatient was followed with serial testing over an 11-week period, including a relapse into heavy drinking, the time course for individual PEth species were similar but not identical (Fig. 4B ). In this case, PEth-16:0/18:2 became the quantitatively most important species during the relapse.


Figure 4
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  		Figure 4. Profile of PEth species in blood samples from alcohol patients.

(A), Distribution of molecular species of PEth in whole blood samples collected from 38 patients undergoing treatment for alcohol-related problems. Some species were not distinguishable in the ESI-MS method because of identical masses. Data for individual subjects are connected with lines. Inset: Mean (SD) values for relative amounts of each PEth species (% of total PEth). (B), Time course for individual PEth species in blood samples collected from 1 outpatient over 11 weeks, including a relapse into heavy drinking.

comparison of peTH results by lc-ms and hplc
In 21 blood samples where the total PEth concentration had already been determined by an HPLC method (20), LC-ESI-MS analysis was carried out for a comparison. The 2 methods produced similar total PEth values in the low concentration range (<3 µmol/L), whereas the LC-MS method generally gave higher values above this threshold (Fig. 5 ).


Figure 5
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  		Figure 5. Comparison of PEth concentrations in patient blood by LC-MS and HPLC.

Comparison of total PEth concentrations for 21 blood samples determined by the LC-ESI-MS method and an HPLC method (20) that is routinely employed for PEth testing. Inset: The same data set presented as a Bland–Altman plot.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The 9 PEth species covered in our LC-MS method with either SIM or SRM detection were selected from the composition of PC molecular species in human erythrocytes (27) and the results of previous MS studies identifying 16:0 and 18:1 as the major fatty acid chains in PEth (18)(23). In agreement with the species distribution for PC (26), the present study confirmed 16:0/18:1 and 16:0/18:2 as the predominant fatty acid combinations in PEth extracted from human whole blood samples (i.e., mainly erythrocyte membranes (19)), together making up approximately 60% of total PEth species measured by this method. However, individual variations in the molecular composition of PEth species were demonstrated both between individuals and within the same patient during a relapse. It should be pointed out that the fatty acid profile is, to some extent, also influenced by dietary factors (34).

The identity of the PEth species was partly based on comparison with reference material that was available for 3 molecular species, whereas theoretical target masses for the other species were calculated. The chromatographic peaks were finally assigned to distinct PEth species based on MS/MS monitoring of the corresponding fatty acid fragment ions, albeit not identifying the exact positioning of double bonds. In contrast to the stable polar head group of PC that is usually used as the target product in SRM analysis, producing a characteristic fragment of the phosphocholine moiety at m/z 184 in positive ion mode, the nonpolar phosphoethanol head group of PEth (m/z 125 in negative ion mode) (23) is unstable. Accordingly, the main fragment ions of PEth were the fatty acid chains that were readily detected in negative ion mode. For chromatographic peaks that could represent mixtures of 2 or more PEth species with identical mass, SRM analysis enabled individual quantification due to the unique combinations of fatty acid chains. For routine measurement of the 9 PEth species covered in this study, LC-MS analysis in SIM mode was used. It should be noted that monitoring at least 4 PEth species in SIM mode, or 1 precursor ion and 2 product ions in SRM mode, could both meet proposed requirements for confirmatory LC-MS analysis (i.e., a minimum of 4 identification points) (35) and thereby produce legally defendable results.

The LC-MS method allowed simultaneous measurement of the major and some minor PEth species in human whole blood samples, as well as determination of the total concentration (i.e., sum of all species covered) in a clinically relevant concentration range. This represents a methodological development compared with HPLC and CE methods that can only determine a total amount (20)(21). To reduce the analysis time of the LC-MS method in routine use, a 2-column switching setup can be applied. For clinical application of PEth as alcohol biomarker, previous studies have used cutoffs for total PEth in the range of approximately 0.2–0.7 µmol/L, depending on the LOQ of the HPLC method at the time. In Sweden, 0.7 µmol/L is currently used as the routine clinical threshold. Based on previous studies, the lower limit of the measuring range for the LC-MS method was set to 0.2 µmol/L for each PEth species. However, the method can detect at least 20 times lower concentrations, thereby possibly also allowing for detection of lower drinking levels. This hypothesis is supported by observations that the apparent alcohol consumption cutoff (based on self-report) detectable by PEth was approximately 50 g/day at an LOQ of 0.7 µmol/L total PEth by HPLC (15), whereas amounts below 40 g/day were detectable when the LOQ was reduced to approximately 0.2 µmol/L (16).

For use as an alcohol biomarker, an advantage of PEth over some conventional analytes (e.g., liver function tests) is the theoretical high specificity for alcohol, being a direct ethanol metabolite. Still, observations of individual PEth formation rates (32) indicated that it might not be possible to link the PEth concentration in blood to a precise drinking level. A main drawback with PEth is the risk for postsampling production on storage of ethanol-containing samples, also when frozen at –20 °C as demonstrated in this and previous publications (32)(33), which could generate false-positive results. This risk is especially serious in postmortem examinations, because even if the deceased subject had not ingested alcohol before death, artifactual ethanol formation between time of death and autopsy due to microbial action is a common problem (36)(37). For that reason, special precaution related to handling and storage of samples before analysis is required if PEth results are to be used for medicolegal matters.

In conclusion, the LC-MS method allowed for simultaneous qualitative and quantitative analysis of several PEth species in human whole blood. The fatty acid composition observed for the different molecular species agreed with that reported for PC, indicating that PEth formation from PC by action of phospholipase D is a general process and not limited to certain molecular species. To simplify future analysis of PEth as alcohol biomarker and allow for standardization of measurement, it should be advantageous to focus on distinct molecular species, which is possible by LC-MS, instead of the total concentration. Based on the patient data, the 2 predominant PEth species to monitor clinically would be 16:0/18:1 and 16:0/18:2, as these together accounted for approximately 60% of the total amount in blood from heavy drinkers.


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

Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: A. Helander, financial support through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institute.

Expert Testimony: None declared.

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

Acknowledgments: The authors thank Dr Therese Hansson for providing blood samples used for comparison with the routine HPLC method, and Professor Olof Beck for valuable comments on the manuscript.


   Footnotes
 
1 Nonstandard abbreviations: CDT, carbohydrate-deficient transferrin; PEth, phosphatidylethanol; PC, phosphatidylcholine; CE, capillary electrophoresis; PProp, phosphatidylpropanol; ESI, electrospray ionization; SIM, selected ion monitoring; MS/MS, tandem mass spectrometry; SRM, selected reaction monitoring; LOD, limit of detection; LOQ, lower limit of quantitation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Helander A. Biological markers in alcoholism. J Neural Transm Suppl 2003;(66):15-32.
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