Skip to main content

Advertisement

SpringerLink
Log in

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

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Phosphatidylethanol species (PEths) are promising biomarkers of alcohol consumption. Here, we report on the set-up, validation, and application of a novel UHPLC-ESI-MS/MS method for the quantification of PEth 16:0/18:1, PEth 18:1/18:1, and PEth 16:0/16:0 in whole blood (30 μL) and in venous (V, 30 μL) or capillary (C, 3 punches (3 mm)) dried blood spots (DBS). The methods were linear from 10 (LLOQ) to 2000 ng/mL for PEth 16:0/18:1, from 10 (LLOQ) to 1940 ng/mL for PEth 18:1/18:1, and from 19 (LLOQ) to 3872 ng/mL for PEth 16:0/16:0. Extraction efficiencies were higher than 55 % (RSD < 18 %) and matrix effects compensated for by IS were between 77 and 125 % (RSD < 10 %). Accuracy, repeatability, and intermediate precision fulfilled acceptance criteria (bias and RSD below 13 %). Validity of the procedure for determination of PEth 16:0/18:1 in blood was demonstrated by the successful participation in a proficiency test. The quantification of PEths in C-DBS was not significantly influenced by the hematocrit, punch localization, or spot volume. The stability of PEths in V-DBS stored at room temperature was demonstrated up to 6 months. The method was applied to authentic samples (whole blood, V-DBS, and C-DBS) from 50 inpatients in alcohol withdrawal and 50 control volunteers. Applying a cut-off value to detect inpatients at 221 ng/mL for PEth 16:0/18:1 provided no false positive results and a good sensitivity (86 %). Comparison of quantitative results (Bland-Altman plot, Passing-Bablok regression, and Wilcoxon signed rank test) revealed that V-DBS and C-DBS were valid alternatives to venous blood for the detection of alcohol consumption.

Passing-Bablok regression analysis of PEth 16:0/18:1 concentrations measured in blood and in capillary DBS, demonstrating equivalence of PEth concentrations in both matrices

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Joya X, Friguls B, Ortigosa S, Papaseit E, Martínez SE, Manich A, Garcia-Algar O, Pacifici R, Vall O, Pichini S (2012) Determination of maternal-fetal biomarkers of prenatal exposure to ethanol: a review. J Pharm Biomed Anal 69:209–222

    Article  CAS  Google Scholar 

  2. Cabarcos P, Álvarez I, Tabernero M, Bermejo A (2015) Determination of direct alcohol markers: a review. Anal Bioanal Chem 407(17):4907–4925

    Article  CAS  Google Scholar 

  3. Varga A, Hansson P, Johnson G, Alling C (2000) Normalization rate and cellular localization of phosphatidylethanol in whole blood from chronic alcoholics. Clin Chim Acta 299(1–2):141–150

    Article  CAS  Google Scholar 

  4. Aradóttir S, Seidl S, Wurst FM, Jönsson BAG, Alling C (2004) Phosphatidylethanol in human organs and blood: a study on autopsy material and influences by storage conditions. Alcohol Clin Exp Res 28(11):1718–1723

    Article  Google Scholar 

  5. Gnann H, Engelmann C, Skopp G, Winkler M, Auwärter V, Dresen S, Ferreirós N, Wurst F, Weinmann W (2010) Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS. Anal Bioanal Chem 396(7):2415–2423

    Article  CAS  Google Scholar 

  6. Nalesso A, Viel G, Cecchetto G, Mioni D, Pessa G, Favretto D, Ferrara SD (2011) Quantitative profiling of phosphatidylethanol molecular species in human blood by liquid chromatography high resolution mass spectrometry. J Chromatogr A 1218(46):8423–8431

    Article  CAS  Google Scholar 

  7. Helander A, Zheng Y (2009) Molecular species of the alcohol biomarker phosphatidylethanol in human blood measured by LC-MS. Clin Chem 55(7):1395–1405

    Article  CAS  Google Scholar 

  8. Zheng Y, Beck O, Helander A (2011) Method development for routine liquid chromatography-mass spectrometry measurement of the alcohol biomarker phosphatidylethanol (PEth) in blood. Clin Chim Acta 412(15–16):1428–1435

    Article  CAS  Google Scholar 

  9. Gnann H, Thierauf A, Hagenbuch F, Röhr B, Weinmann W (2014) Time dependence of elimination of different PEth homologues in alcoholics in comparison with social drinkers. Alcohol Clin Exp Res 38(2):322–326

    Article  CAS  Google Scholar 

  10. Viel G, Boscolo-Berto R, Cecchetto G, Fais P, Nalesso A, Ferrara S (2012) Phosphatidylethanol in blood as a marker of chronic alcohol use: a systematic review and meta-analysis. Int J Mol Sci 13(11):14788–14812

    Article  CAS  Google Scholar 

  11. Aradottir S, Asanovska G, Gjerss S, Hansson P, Alling C (2006) Phosphatidylethanol (PEth) concentrations in blood are correlated to reported alcohol intake in alcohol-dependent patients. Alcohol Alcohol 41(4):431–437

    Article  CAS  Google Scholar 

  12. Gunnarsson T, Karlsson A, Hansson P, Johnson G, Alling C, Odham G (1998) Determination of phosphatidylethanol in blood from alcoholic males using high-performance liquid chromatography and evaporative light scattering or electrospray mass spectrometric detection. J Chromatogr B Biomed Sci Appl 705(2):243–249

    Article  CAS  Google Scholar 

  13. Varga A, Nilsson S (2008) Nonaqueous capillary electrophoresis for analysis of the ethanol consumption biomarker phosphatidylethanol. Electrophoresis 29(8):1667–1671

    Article  CAS  Google Scholar 

  14. Tolonen A, Lehto TM, Hannuksela ML, Savolainen MJ (2005) A method for determination of phosphatidylethanol from high density lipoproteins by reversed-phase HPLC with TOF–MS detection. Anal Biochem 341(1):83–88

    Article  CAS  Google Scholar 

  15. Faller A, Richter B, Kluge M, Koenig P, Seitz H, Thierauf A, Gnann H, Winkler M, Mattern R, Skopp G (2011) LC-MS/MS analysis of phosphatidylethanol in dried blood spots versus conventional blood specimens. Anal Bioanal Chem 401(4):1163–1166

    Article  CAS  Google Scholar 

  16. Kwak HS, Han J-Y, Ahn H-K, Kim M-H, Ryu H-M, Kim M-Y, Chung H-J, Cho D-H, Shin C-Y, Velazquez-Armenta EY, Nava-Ocampo AA (2012) Blood levels of phosphatidylethanol in pregnant women reporting positive alcohol ingestion, measured by an improved LC-MS/MS analytical method. Clin Toxicol 50(10):886–891

    Article  CAS  Google Scholar 

  17. Varga A, Hansson P, Lundqvist C, Alling C (1998) Phosphatidylethanol in blood as a marker of ethanol consumption in healthy volunteers: comparison with other markers. Alcohol Clin Exp Res 22(8):1832–1837

    CAS  Google Scholar 

  18. Jones J, Jones M, Plate C, Lewis D (2011) The detection of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol in human dried blood spots. Anal Methods 3(5):1101–1106

    Article  CAS  Google Scholar 

  19. Schröck A, Thierauf A, Wurst FM, Thon N, Weinmann W (2014) Progress in monitoring alcohol consumption and alcohol abuse by phosphatidylethanol. Bioanalysis 6(17):2285–2294

    Article  Google Scholar 

  20. Gnann H, Weinmann W, Engelmann C, Wurst FM, Skopp G, Winkler M, Thierauf A, Auwärter V, Dresen S, Bouzas NF (2009) Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC-ESI-MS/MS. J Mass Spectrom 44(9):1293–1299

    Article  CAS  Google Scholar 

  21. Bakhireva LN, Leeman L, Savich RD, Cano S, Gutierrez H, Savage DD, Rayburn WF (2014) The validity of phosphatidylethanol in dried blood spots of newborns for the identification of prenatal alcohol exposure. Alcohol Clin Exp Res 38(4):1078–1085

    Article  CAS  Google Scholar 

  22. Stove CP, Ingels AS, De Kesel PM, Lambert WE (2012) Dried blood spots in toxicology: from the cradle to the grave? Crit Rev Toxicol 42(3):230–243

    Article  CAS  Google Scholar 

  23. Bakhireva LN, Savich RD, Raisch DW, Cano S, Annett RD, Leeman L, Garg M, Goff C, Savage DD (2013) The feasibility and cost of neonatal screening for prenatal alcohol exposure by measuring phosphatidylethanol in dried blood spots. Alcohol Clin Exp Res 37(6):1008–1015

    Article  CAS  Google Scholar 

  24. Faller A, Richter B, Kluge M, Koenig P, Seitz HK, Skopp G (2012) Stability of phosphatidylethanol species in spiked and authentic whole blood and matching dried blood spots. Int J Legal Med 127(3):1–8

    Google Scholar 

  25. Hernandez Redondo A, Schroeck A, Kneubuehl B, Weinmann W (2013) Determination of ethyl glucuronide and ethyl sulfate from dried blood spots. Int J Legal Med 127(4):769–775

    Article  Google Scholar 

  26. Sadones N, Capiau S, De Kesel PM, Lambert WE, Stove CP (2014) Spot them in the spot: analysis of abused substances using dried blood spots. Bioanalysis 6(17):2211–2227

    Article  CAS  Google Scholar 

  27. Jager NGL, Rosing H, Schellens JHM, Beijnen JH (2014) Procedures and practices for the validation of bioanalytical methods using dried blood spots: a review. Bioanalysis 6(18):2481–2514

    Article  CAS  Google Scholar 

  28. De Kesel PM, Sadones N, Capiau S, Lambert WE, Stove CP (2013) Hemato-critical issues in quantitative analysis of dried blood spots: challenges and solutions. Bioanalysis 5(16):2023–2041

    Article  Google Scholar 

  29. Wille S, Peters F, Di Fazio V, Samyn N (2011) Practical aspects concerning validation and quality control for forensic and clinical bioanalytical quantitative methods. Accredit Qual Assur 16(6):279–292

    Article  Google Scholar 

  30. Committee for medicinal products for human use (CHMP), European medicines agency (EMEA) (2011) Guideline on validation of bioanalytical methods, London. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf. Accessed 15 June 2015

  31. Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 75(13):3019–3030

    Article  CAS  Google Scholar 

  32. Miller JN, Miller JC (2010) Statistics and Chemometrics for Analytical Chemistry 6rd edn Pearson Education Limited England

  33. Sadones N, Archer JR, Ingels AS, Dargan PI, Wood DM, Wood M, Neels H, Lambert WE, Stove CP (2015) Do capillary dried blood spot concentrations of gamma-hydroxybutyric acid mirror those in venous blood? a comparative study. Drug Test Anal 7(4):336–340

    Article  CAS  Google Scholar 

  34. Koster RA, Alffenaar JW, Botma R, Greijdanus B, Uges DR, Kosterink JG, Touw DJ (2015) The relation of the number of hydrogen-bond acceptors with recoveries of immunosuppressants in DBS analysis. Bioanalysis 7(14):1717–1722

    Article  CAS  Google Scholar 

  35. Frega NG, Pacetti D, Boselli E (2012) Characterization of phospholipid molecular species by means of HPLC-tandem mass spectrometry, tandem mass spectrometry—applications and principles, InTech. Available from: http://www.intechopen.com/books/tandem-mass-spectrometry-applications-and-principles/characterization-of-phospholipid-molecular-species-by-means-of-hplc-tandem-mass-spectrometry. Accessed 25 October 2015

  36. Denniff P, Spooner N (2010) The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs. Bioanalysis 2(8):1385–1395

    Article  CAS  Google Scholar 

  37. Stewart SH, Koch DG, Willner IR, Anton RF, Reuben A (2014) Validation of blood phosphatidylethanol as an alcohol consumption biomarker in patients with chronic liver disease. Alcohol Clin Exp Res 38(6):1706–1711

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank all the staff from the Brugmann Hospital and the Military Hospital in Brussels for their contribution and all volunteers for their collaboration.

Author information

Authors and Affiliations

  1. Federal Public Service Justice, National Institute of Criminalistics and Criminology, Chaussée de Vilvorde 100, 1120, Brussels, Belgium

    Natalie Kummer, Ann-Sofie Ingels, Sarah M. R. Wille & Nele Samyn

  2. Laboratory of Psychological Medicine and Addictology, ULB Neuroscience Institute (UNI), Université Libre de Bruxelles (ULB), Route de Lennik 800, 1070, Brussels, Belgium

    Catherine Hanak & Paul Verbanck

  3. Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000, Ghent, Belgium

    Willy E. E. Lambert & Christophe P. Stove

Authors
  1. Natalie Kummer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ann-Sofie Ingels
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sarah M. R. Wille
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Catherine Hanak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paul Verbanck
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Willy E. E. Lambert
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nele Samyn
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christophe P. Stove
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christophe P. Stove.

Ethics declarations

Financial and competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 1.01 mb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kummer, N., Ingels, AS., Wille, S.M.R. 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 408, 825–838 (2016). https://doi.org/10.1007/s00216-015-9169-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-015-9169-1

Keywords

Search

Navigation