Analytical and Bioanalytical Chemistry

, Volume 400, Issue 7, pp 1923–1931 | Cite as

Liquid chromatography–mass spectrometric determination of plasmalogens in human plasma

  • Shu-Ping Hui
  • Hitoshi ChibaEmail author
  • Takao Kurosawa
Original Paper


A new liquid chromatography–mass spectrometry (LC/MS) method has been developed for the quantitative analysis of plasmalogens in human plasma using a nonendogenous plasmalogen (1-O-1′-(Z)-tricosenyl-2-oleoyl-rac-glycero-3-phosphocholine, PLS 23:0/18:1) as an internal standard. 1-O-1′-(Z)-Tricosenyl glyceryl ether was prepared by reacting lithioalkoxyallyl with 1-iodoeicosane as the key intermediate in the formation of PLS 23:0/18:1. In LC/MS analyses, PLS 23:0/18:1 generated significant fragment ions in positive and negative modes. In positive ion mode, the [M+H]+ of PLS 23:0/18:1 yielded unique fragments with cleavages at the sn-1 and sn-2 positions of the glycerol backbone. In negative ion mode, the [M+CH3COO] of PLS 23:0/18:1 resulted in characteristic fragmentation at the sn-2 and sn-3 positions. 1-O-1′-(Z)-Hexadecenyl-2-linoleoyl-rac-glycero-3-phosphocholine (PLS 16:0/18:2) and 2-arachidonoyl-O-1′-(Z)-hexadecenyl-rac-glycero-3-phosphocholine (PLS 16:0/20:4) were chemically synthesized as PLS 23:0/18:1. The calibration curves obtained for PLS 16:0/18:2 and PLS 16:0/20:4 were linear throughout the calibration range (0.04–1.60 pmol). The LOD (S/N = 5:1) was 0.008 pmol and the LOQ (S/N = 6:1) was 0.01 pmol for both PLS 16:0/18:2 and PLS 16:0/20:4. Plasma concentrations of PLS 16:0/18:2 and PLS 16:0/20:4 were 4.0 ± 1.3 μM and 3.5 ± 1.2 μM (mean ± SD), respectively, in five healthy volunteers.


SRM chromatograms were obtained by the quantitative LC/MS system in positive mode: (A) synthetic PLS 16:0/18:2, (B) synthetic PLS 16:0/20:4, (C) IS, (A') plasma extract (precursor ion: m/z 742.6), (B') plasma extract (precursorion: m/z 766.6), (C') plasma extract after addition of the IS.


Liquid chromatography/electrospray ionization mass spectrometry LC/MS Plasmalogen Analysis Synthesis 



Electrospray ionization mass spectrometry


Internal standard


Liquid chromatography/mass spectrometry


Limit of detection


Limit of quantification

PLS 16:0/18:2


PLS 16:0/20:4


PLS 23:0/18:1



2-(Chloromethoxy) ethyltrimethylsilane


Selected reaction monitoring


Tetrabutylammonium fluoride







This study was supported by Sapporo Biocluster “Bio-S”, The Regional Innovation Cluster Program, The Ministry of Education, Culture, Sports, Science and Technology, Japan.

Supplementary material

216_2011_4921_MOESM1_ESM.pdf (57 kb)
ESM 1 (PDF 57 kb)


  1. 1.
    Khaselev N, Murphy RC (2000) Structural characterization of oxidized phospholipid products derived from arachidonate-containing plasmenyl glycerophosphocholine. J Lipid Res 41:564–572Google Scholar
  2. 2.
    Maeba R, Ueta N (2003) Ethanolamine plasmalogens prevent the oxidation of cholesterol by reducing the oxidizability of cholesterol in phospholipid bilayers. J Lipid Res 44:164–171CrossRefGoogle Scholar
  3. 3.
    Witztum JL, Steinberg D (1991) Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 88:1785–1792CrossRefGoogle Scholar
  4. 4.
    Markesbery WR (1997) Oxidative stress hypothesis in Alzheimer’s disease. Free Radic Biol Med 23:134–147CrossRefGoogle Scholar
  5. 5.
    Fonteh AN, Harrington RJ, Huhmer AF, Biringer RG, Riggins JN, Harrington MG (2006) Identification of disease markers in human cerebrospinal fluid using lipidomic and proteomic methods. Dis Markers 22:39–64Google Scholar
  6. 6.
    Han X, Holtzman DM, McKeel DW Jr (2001) Plasmalogen deficiency in early Alzheimer’s disease subjects and in animal models: molecular characterization using electrospray ionization mass spectrometry. Neurochem Res 26:771–782Google Scholar
  7. 7.
    Goodenowe DB, Cook LL, Liu J, Lu Y, Jayasinghe DA, Ahiahonu PWK, Heath D, Yamazaki Y, Flax J, Krenitsky KF, Sparks DL, Lerner A, Friedland RP, Kudo T, Kamino K, Morihara T, Takeda M, Wood PL (2007) Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer’s disease and dementia. J Lipid Res 48:2485–2498CrossRefGoogle Scholar
  8. 8.
    Schulz R, Strynadka KD, Panas DL, Olley PM, Lopaschuk GD (1993) Analysis of myocardial plasmalogen and diacyl phospholipids and their arachidonic acid content using high-performance liquid chromatography. Anal Biochem 213:140–146CrossRefGoogle Scholar
  9. 9.
    Maeba R, Ueta N (2004) Determination of choline and ethanolamine plasmalogens in human plasma by HPLC using radioactive triiodide (1−) ion (125I3-). Anal Biochem 331:169–176Google Scholar
  10. 10.
    Maeba R, Maeda T, Kinoshita M, Takao K, Takenaka H, Kusano J, Yoshimura N, Takeoka Y, Yasuda D, Okazaki T, Teramoto T (2007) Plasmalogens in human serum positively correlate with high-density lipoprotein and decrease with aging. J Atheroscler Thromb 14:12–18Google Scholar
  11. 11.
    Yang K, Zhao Z, Gross RW, Han X (2009) Systematic analysis of choline-containing phospholipids using multi-dimensional mass spectrometry-based shotgun lipidomics. J Chromatogr B 877:2924–2936CrossRefGoogle Scholar
  12. 12.
    Hui SP, Chiba H, Jin S, Nagasaka H, Kurosawa T (2010) Analyses for phosphatidylcholine hydroperoxides by LC/MS. J Chromatogr B 878:1677–1682CrossRefGoogle Scholar
  13. 13.
    Zemski Berry KA, Murphy RC (2004) Electrospray ionization tandem mass spectrometry of glycerophosphoethanolamine plasmalogen phospholipids. J Am Soc Mass Spectrom 15:1499–1508CrossRefGoogle Scholar
  14. 14.
    Hsu FF, Turk J (2007) Differentiation of 1-O-alk-1′-enyl-2-acyl and 1-O-alkyl-2-acyl glycerophospholipids by multiple-stage linear ion-trap mass spectrometry with electrospray ionization. J Am Soc Mass Spectrom 18:2065–2073Google Scholar
  15. 15.
    Nishimukai M, Yamashita M, Watanabe Y, Yamazaki Y, Nezu T, Maeba R, Hara H (2010) Lymphatic absorption of choline plasmalogen is much higher than that of ethanolamine plasmalogen in rats. Eur J Nutr. doi: 10.1007/s0039401001490 Google Scholar
  16. 16.
    Shin J, Thompson DH (2003) Direct synthesis of plasmenylcholine from allyl-substituted glycerols. J Org Chem 68:6760–6766Google Scholar
  17. 17.
    Hui SP, Murai T, Yoshimura T, Chiba H, Kurosawa T (2003) Simple chemical syntheses of TAG monohydroperoxides. Lipids 38:1287–1292CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Faculty of Health Sciences, Medical Laboratory SciencesHokkaido UniversitySapporoJapan
  2. 2.Faculty of Pharmaceutical SciencesHealth Sciences University of HokkaidoHokkaidoJapan
  3. 3.Faculty of Health SciencesHokkaido University Graduate School of Health SciencesSapporoJapan

Personalised recommendations