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

• Published in: Drug testing and analysis 2021-01, Vol.13 (1), p.140-147
• Main Authors: Aboutara, Nadine, Jungen, Hilke, Szewczyk, Anne, Sterneck, Martina, Müller, Alexander, Iwersen‐Bergmann, Stefanie
• Format: Article
• Language: English
• Subjects: alcohol biomarker; Chromatography; dried blood spots; Ions; LC/MS/MS; Mass spectrometry; phosphatidylethanol; Scientific imaging
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.2910

Phosphatidylethanol (PEth) is a direct biomarker for alcohol consumption consisting of a fraction of different ethanol‐modified, homologue phospholipids. The aim of this study was to validate an ultra‐high‐performance liquid chromatography–tandem mass spectrometry method to quantitate six different homologues of PEth (16:0/18:1, 16:0/18:2, 16:0/20:4, 18:0/18:1, 18:0/18:2, and 18:1/18:1) from dried blood spots (DBSs). DBSs were prepared volumetrically (20 μL of whole blood) and extracted with 1 mL of methanol (0.02 ng/μL internal standards). PEth homologues were separated on a BEH C18 column (2.1 × 150 mm, 1.7 μm) using methanol and ammonium acetate buffer (25 mM) in a 7 min isocratic run. Multiple reaction monitoring mode was used for the detection of PEth and the internal standards. Calibrators (10–1000 ng/mL) and quality controls (40, 400, and 700 ng/mL) were prepared from spiked whole blood; external control samples were obtained from proficiency testing schemes. After a comprehensive validation of the method, quantitative patterns of the different homologues were investigated in PEth positive samples (n = 57) from patients in a transplant setting. Satisfactory chromatographic separation, sensitive detection, and reliable quantification of the PEth homologues in DBSs can be achieved using the liquid chromatography–tandem mass spectrometry (LC/MS/MS) procedure. Validation results, including accuracy, linearity, recovery, matrix effects, and in‐process stability, complied with international standards, and the analytical performance of the procedure was not affected by the hematocrit of the blood samples. Different quantitative patterns of the investigated PEth homologues were observed in authentic samples from liver transplant patients. This method will enable the study of the kinetics of six PEth homologues simultaneously and investigate the meaning of the homologues' distribution in individuals. A liquid chromatography–tandem mass spectrometry method was developed and validated to simultaneously analyze six homologues of the direct alcohol biomarker phosphatidylethanol from dried blood spots. Spots were prepared volumetrically, and the effect of hematocrit was determined. The method was used to measure samples from liver transplant patients to test for alcohol abstinence, and the occurrences of the different homologues were studied in positive samples (n = 57).