High Throughput UPLC®-MSMS Method for the Analysis of Phosphatidylethanol (PEth) 16:0/18:1, a Specific Biomarker for Alcohol Consumption, in Whole Blood

Abstract

Phosphatidylethanol (PEth) is an alcohol biomarker formed in the presence of ethanol in the body. Both due to its specificity and because it has a detection window of up to several weeks after alcohol intake, its application potential is broader than for other ethanol biomarkers. The aim of this study was to develop and validate a robust method for PEth in whole blood with fast and efficient sample extraction and a short analytical runtime, suitable for high throughput routine purposes. A validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC®-MSMS) method for quantification of PEth 16:0/18:1 in the range 0.05-4.00 μM (R2 ≥ 0.999) is presented. PEth 16:0/18:1 and the internal standard (IS) PEth-d5 (0.55 μM), were extracted from whole blood (150 μL) by simple protein precipitation with 2-propanol (450 μL). Chromatography was achieved using a BEH-phenyl (2.1 × 30 mm, 1.7 μm) column and a gradient elution combining ammonium formate (5 mM, pH 10.1) and acetonitrile at a flow rate of 0.5 mL/min. Runtime was 2.3 min. The mass spectrometer was monitored in negative mode with multiple reaction monitoring (MRM). The m/z 701.7 > 255.2 and 701.7 > 281.3 transitions were monitored for PEth 16:0/18:1 and the m/z 706.7 > 255.3 for PEth-d5. Limit of quantification was 0.03 μM (coefficient of variation, CV = 6.7%, accuracy = 99.3%). Within-assay and between-assay imprecision were 0.4-3.3% (CV ≤ 7.1%). Recoveries were 95-102% (CV ≤ 4.9%). Matrix effects after IS correction ranged from 107% to 112%. PEth 16:0/18:1 in patient samples were stable for several days at 30°C. Repeated freezing (-80°C) and thawing did not affect the concentration. After thawing and analysis patient samples were stable at 4-8°C for at least 4 weeks. Results from a proficiency test program, showing |Z| values ≤1.2, confirm the validity of the method. Analysis of the first 3,169 samples sent to our laboratory for routine use has demonstrated its properties as a robust method suitable for high throughput purposes.

Analysis of six different homologues of phosphatidylethanol from dried blood spots using liquid chromatography–tandem mass spectrometry

“…Whereas analytical procedures including one, 3,13 two, 14,15 or three 16 homologues of PEth can be already found in literature today, parallel quantification of PEth 16:0/18:1, PEth 16:0/18:2, PEth 16:0/20:4, PEth 18:0/18:1, PEth 18:0/18:2, and PEth 18:1/18:1 has (to our knowledge) not been described before. Currently, reference materials for these six molecules are available; additional homologues could be introduced in the procedure if they become available.…”

“…Different columns for the analytical separation of different homologues have been discussed in previous methods. Andreassen et al 13 used a 30 mm long BEH‐phenyl column for high‐throughput analysis of PEth 16:0/18:1. Other authors used predominantly C4 or C8 columns, 3,15,17 which had also been considered in the present work during method development.…”

“…Whereas analytical procedures including one, 3,13 two, 14,15 The influence of Hct in DBS testing has been discussed in detail and is considered as one of the most important issues in this field. 6,18,19 In 2019 the International Association for Therapeutic Drug…”

Determination of phosphatidylethanol 16:0/18:1 in whole blood by 96‐well supported liquid extraction and UHPLC‐MS/MS

“…Protein precipitation (PPT) and solid phase extraction (SPE) have also been used for sample preparation of PEths from biological samples . Recently, Andreassen et al described a high throughput UHPLC‐MS/MS method for determination of PEth 16:0/18:1 in whole blood using 96‐well PPT with 2‐propanol as organic solvent. Supported liquid extraction (SLE) is another technique that that is fast and easy which can be used for isolation of different compounds from biological samples, and a technique that often generates cleaner extracts than what is obtained by PPT .…”

Phosphatidylethanol blood analysis

“…Le phosphatidyléthanol présente cependant deux difficultés : -du fait de sa nature chimique, il n'est pas aisé d'en réaliser l'analyse et la quantification. Cependant, les récentes avancées méthodologiques et techniques permettent aujourd'hui d'en envisager le dosage en routine [13] ; -le PEth est dit instable in vitro (dans le tube de sang) au bout de quelques jours à température ambiante [14,15]. Des travaux récents proposent une solution élégante pour stabiliser le PEth sanguin : l'utilisation de dried blood spot (DBS) [8,16].…”