High-Throughput LC-MS/MS Method for Determination of the Alcohol Use Biomarker Phosphatidylethanol in Clinical Samples by Use of a Simple Automated Extraction Procedure—Preanalytical and Analytical Conditions

Abstract

Background: Phosphatidylethanol (PEth) is an alcohol use biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers. Since its introduction as a clinical alcohol-marker in 2006, the number of samples sent to our laboratory for the determination of PEth has shown a strong annual increase. This has prompted the need to develop a cost-effective and reliable analytical procedure with high capacity. Methods: An LC-MS/MS method for the determination of PEth 16:0/18:1 with a short turnaround time (3 min) has been evaluated with respect to accuracy, sensitivity, and precision. We compared this method with a previously used HPLC method, as well as a manual and a simplified automated method for sample workup, and investigated potential causes of analytic and preanalytic errors. Results: The method shows limits of detection and quantification of 0.0075 μmol/L (5.2 ng/mL) and <0.05 μmol/L (<35 ng/mL), respectively. During a 2.1-year period, the method has shown a total CV < 8% for control samples (n = 2808) in the range of 0.10 (70) to 3.5 μmol/L (2461 ng/mL). The simplified automated method for sample preparation works equally well as the manual one. No specific and clinically significant causes of preanalytic errors were found. Conclusions: This LC-MS/MS method with automated sample workup is well suited for a clinical laboratory with LC-MS/MS experience and has the capability, proven from several years of use, to produce reliable PEth results in a high-volume laboratory (>50 000 clinical samples/year). IMPACT STATEMENT Alcohol use disorders are a major health problem worldwide, and alcohol markers are an important tool for diagnosis, follow-up, and treatment evaluation. Phosphatidylethanol in whole blood (B-PEth) is a direct alcohol biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers. Evidence presented here showed that B-PEth can be determined by LC-MS/MS preceded by a robotized extraction procedure. The method has a high capacity and is cost-effective and clinically reliable. This information may increase interest in the marker and make it more widely used.

Inter Individual Variation and Factors Regulating the Formation of Phosphatidylethanol

“…At an EtOH concentration of 50 mmol/l, total PEth, PEth 16:0/18:1, and PEth 16:0/18:2 showed median formation rates of 0.115, 0.034, and 0.042 μ mol/l/h. Thus, PEth 16:0/18:1 and 16:0/18:2 together made up 66% of the total amount of PEth generated, which is compatible with these 2 species being the dominant forms found in blood from individuals consuming alcohol (Helander and Zheng, ; Isaksson et al., ; Nalesso et al., ). A constant EtOH concentration of 25 mmol/l for 24 hours would result in the generation of 0.480 (24 × 0.020 μ mol/l/h; Table ) and 0.576 (24 × 0.024 μ mol/l/h; Table ) μ mol/l/h of PEth 16:0/18:1 and 16:0/18:2 (median values), respectively.…”

“…This is in contrast to previously reported results where PEth formation in heparin blood with added EtOH successively decreased and essentially ceased after 2 days (Schrock et al., ). The in vitro formation of PEth in blood containing EtOH therefore seems to be self‐limiting and without clinical importance as previously shown for blood drawn in EDTA tubes (Isaksson et al., ).…”

Alcohol Consumption in Obese Patients Before and After Gastric Bypass as Assessed with the Alcohol Marker Phosphatidylethanol (PEth)

“…PEth Determination Whole blood concentration of PEth 16:0/ 18:1 was measured by liquid chromatography tandem-mass spectrometry, described in detail previously [19]. There is an agreement between Swedish laboratories on how to report and interpret a PEth value.…”

“…There is an agreement between Swedish laboratories on how to report and interpret a PEth value. Thus, no numerical results are reported < 0.05 μmol/L and such values are interpreted as low (moderate) or no consumption, whereas values > 0.30 are interpreted as corresponding to excessive consumption [19,20]. Hence, the limit of quantification was set to 0.05 μmol/ L and used as cut off in the present study.…”

Selecting and Interpreting Alcohol Biomarker Tests: Enough to Drive You to Drink

“…Only at −80°C is the in vitro formation of PEth significantly inhibited (13). However, Isaksson and colleagues (9) concluded that this degree of artefactual PEth formation was clinically insignificant in blood samples collected in EDTA, with only 1 sample showing a PEth value >35 ng/mL (moderate alcohol consumption) when exposed to 0.368 g/dL of ethanol for 3 days, which is >4 times the legal blood alcohol concentration of 0.08 g/dL for driving under the influence or driving while impaired. Nevertheless, laboratories should document the effect of transport time and temperature on this postcollection formation because samples may be received from clients worldwide with transport times of >2 days, unlike the majority of samples obtained in the article.…”