Advertisement

Analytical and Bioanalytical Chemistry

, Volume 406, Issue 19, pp 4735–4744 | Cite as

Differentiation and quantification of synthetic phosphatidylethanol (PEth) homologues by 1H- and 13C-NMR in polar organic solvents

  • David Wensbo Posaric
  • Anders Andersson
  • Karl-Erik Bergquist
  • Anders Isaksson
Research Paper

Abstract

Various phosphatidylethanol (PEth) derivatives, the corresponding reversed positional isomers (RPI-PEths), lyso-PEth-16:0, and penta-deuterium-labeled PEth analogs (d5-PEths), were synthesized by enzyme-independent synthetic routes. A general solvent system consisting of a mixture of acetone-d6 and methanol-d4 (97:3; v/v) was found to provide a good solubilizing capacity and excellent hydrogen-1 NMR (1H-NMR) peak resolution of various PEth homologues. Analytical differentiation of PEth from the corresponding RPI-PEth by carbon-13 NMR (13C-NMR) was demonstrated by comparison of the 13C-NMR signals of the carbonyl groups, the allylic positions, and of the β-carbons. An exemplary stable long-term room temperature, DMSO-d6-based, and proton-sensitive quantitative nuclear magnetic resonance (1H-qNMR) independently quantified calibrator comprising PEth-16:0/18:1 for liquid chromatography (tandem) mass spectrometry (LC-MS/MS) analytical applications were prepared by employment of sodium dodecyl sulfate (SDS) as a solubilizing additive. In summary, novel hypothetically occurring PEth derivatives, e.g., RPI-PEths, have been independently synthesized with regio- and stereochemical control. Use of polar organic solvents, e.g., mixtures of acetone-d6 and methanol-d4 or DMSO-d6, improves spectral line shapes as compared to traditional hydrophobic solvents and allow for analytical differentiation between closely related PEth derivatives, as well as LC-MS/MS-independent concentration determination of dissolved single species by employment of 1H-qNMR.

Keywords

qNMR LC-MS/MS Analytical reference Phospholipids Positional isomers Lyso-PEth 

Notes

Acknowledgments

We are grateful to Innovator Skåne AB for the provision of laboratory facilities and for major sponsoring, to Can Slivo for excellent help with the resynthesis of early intermediates, to Dr. Anders Blomgren and the staff at the analytical laboratories of Skåne University Hospital for LC-MS/MS quantification of calibrators, to Dr. Johan Evenäs at Read Glead Discovery AB for the collection and processing of 1H-qNMR spectra, and to Dr. Ulf Annby for proofreading the manuscript.

Supplementary material

216_2014_7826_MOESM1_ESM.pdf (516 kb)
ESM 1 PDF 515 kb

References

  1. 1.
    Isaksson A, Walther L, Hansson T, Andersson A, Alling C (2011) Phosphatidylethanol in blood (B-Peth): a marker for alcohol use and abuse. Drug Test Anal 3:195–200CrossRefGoogle Scholar
  2. 2.
    Gnann H, Engelmann C, Skopp G, Winkler M, Auwaerter V, Dresen S, Ferreiros N, Wurst FM, Weinmann W (2010) Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS. Anal Bioanal Chem 396:2415–2423CrossRefGoogle Scholar
  3. 3.
    Nalesso A, Viel G, Cecchetto G, Mioni D, Pessa G, Favretto D, Davide Ferrara S (2011) Quantitative profiling of phosphatidylethanol molecular species in human blood by liquid chromatography high resolution mass spectrometry. J Chromatogr A 1218:8423–8431CrossRefGoogle Scholar
  4. 4.
    Zheng Y-F, 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 412:1428–1435CrossRefGoogle Scholar
  5. 5.
    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 341:83–88CrossRefGoogle Scholar
  6. 6.
    Ekroos K, Ejsing CS, Bahr U, Karas M, Simons K, Shevchenko A (2003) Charting molecular composition of phosphatidylcholins by fatty acid scanning and ion trap MS3 fragmentation. J Lipid Res 44:2181–2192CrossRefGoogle Scholar
  7. 7.
    Helander A, Zheng Y (2009) Molecular species of the alcohol biomarker phosphatidylethanol in human blood measured by LC-MS. Clin Chem 55:1395–1405CrossRefGoogle Scholar
  8. 8.
    Vernooij EAAM, Brouwers JFHM, Kettenes-Van den Bosch J, Jantina Crommelin DJA (2002) RP-HPLC/ESI MS determination of acyl chain positions in phospholipids. J Sep Sci 25:285–289CrossRefGoogle Scholar
  9. 9.
    Xia J, Hui Y-Z (1999) Synthesis of a small library of mixed-acid phospholipids from d-mannitol as a homochiral starting material. Chem Pharm Bull 47:1659–1663CrossRefGoogle Scholar
  10. 10.
    Abe M, Kitsuda S, Ohyama S, Koubori S, Murai M, Miyoshi H (2010) Concise procedure for the synthesis of cardiolipins having different fatty acid combinations. Tetrahedron Lett 51:2071–2073CrossRefGoogle Scholar
  11. 11.
    Hebert N, Beck A, Lennox RB, Just G (1992) A new reagent for the removal of the 4-methoxybenzyl ether: application to the synthesis of unusual macrocyclic and bolaform phosphatidylcholines. J Org Chem 57:1777–1783CrossRefGoogle Scholar
  12. 12.
    Sato R, Itabashi Y, Fujishima H, Okuyama H, Kuksis A (2004) Simple synthesis of diastereomerically pure phosphatidylglycerols by phospholipase d-catalyzed transphosphatidylation. Lipids 39:1025–1030CrossRefGoogle Scholar
  13. 13.
    Martin SF, Hergenrother PJ (1998) Enzymic synthesis of a modified phospholipid and its evaluation as a substrate for B. cereus phospholipase C. Bioorg Med Chem Lett 8:593–596CrossRefGoogle Scholar
  14. 14.
    Petersen G, Pedersen AH, Pickering DS, Begtrup M, Hansen HS (2009) Effect of synthetic and natural phospholipids on N-acylphosphatidylethanolamine-hydrolyzing phospholipase D activity. Chem Phys Lipids 162:53–61CrossRefGoogle Scholar
  15. 15.
    Goldring WPD, Jubeli E, Downs RA, Johnston AJS, Abdul Khalique N, Raju L, Liji W, Wafadari D, Pungente MD (2012) Novel macrocyclic and acyclic cationic lipids for gene transfer: synthesis and in vitro evaluation. Bioorg Med Chem Lett 22:4686–4692CrossRefGoogle Scholar
  16. 16.
    Alcaraz M-L, Peng L, Klotz P, Goeldner M (1996) J Org Chem 61:192–201CrossRefGoogle Scholar
  17. 17.
    Takai K, Takagi T, Baba T, Kanamori T (2008) Synthesis and monolayer properties of double-chained phosphatidylcholines containing perfluoroalkyl groups of different length. J Fluor Chem 129:686–690CrossRefGoogle Scholar
  18. 18.
    Srisiri W, Lee Y-S, O’Brien DF (1995) Chemical synthesis of a polymerizable bis-substituted phosphoethanolamine. Tetrahedron Lett 36:8945–8948CrossRefGoogle Scholar
  19. 19.
    Kihara M, Xu L, Konishi K, Kida K, Nagao Y, Kobayashi S, Shingu T (1994) Isolation and structure elucidation of a novel alkaloid, incartine, a supposed biosynthetic intermediate, from flowers of Lycoris incarnate. Chem Pharm Bull 42:289–292CrossRefGoogle Scholar
  20. 20.
    Willmann J, Thiele H, Leibfritz D (2011) Combined reversed phase HPLC, mass spectrometry, and NMR spectroscopy for a fast separation and efficient identification of phosphatidylcholines. J Biomed Biotechnol 2011:1–8CrossRefGoogle Scholar
  21. 21.
    Griffiths L, Irving AM (1998) Assay by nuclear magnetic resonance spectroscopy: quantification limits. Analyst 123:1061–1068CrossRefGoogle Scholar
  22. 22.
    Wells RJ, Cheung J, Hook JM (2004) Dimethylsulfone as a universal standard for analysis of organics by qNMR. Accred Qual Assur 9:450–456CrossRefGoogle Scholar
  23. 23.
    Lehnhardt F-G, Rohn G, Ernestus R-I, Grune M, Hoehn M (2001) 1H- and 31P-MR spectroscopy of primary and recurrent human brain tumors in vitro: malignancy-characteristic profiles of water soluble and lipophilic spectral components. NMR Biomed 14:307–317CrossRefGoogle Scholar
  24. 24.
    Vyssotski M, MacKenzie A, Scott D (2009) TLC and 31P-NMR analysis of low polarity phospholipids. Lipids 44:381–389CrossRefGoogle Scholar
  25. 25.
    McEwan AG, Hanson V, Bailey S (1998) Dimethyl sulfoxide reductase from purple phototrophic bacteria: structures and mechanism(s). Biochem Soc Trans 26:390–396Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • David Wensbo Posaric
    • 1
  • Anders Andersson
    • 2
  • Karl-Erik Bergquist
    • 3
  • Anders Isaksson
    • 2
  1. 1.Department of Clinical Sciences, Division of Biomedical EngineeringLund University, Skåne University HospitalLundSweden
  2. 2.Department of Laboratory Medicine, Division of Clinical Chemistry and PharmacologyLund University, Skåne University HospitalLundSweden
  3. 3.Centre for Analysis and Synthesis, Faculty of Science and EngineeringLund UniversityLundSweden

Personalised recommendations