Analytical and Bioanalytical Chemistry

, Volume 403, Issue 7, pp 1831–1840 | Cite as

Quantitative determination of phosphatidylcholine hydroperoxides during copper oxidation of LDL and HDL by liquid chromatography/mass spectrometry

  • Shu-Ping Hui
  • Yudai Taguchi
  • Seiji Takeda
  • Futaba Ohkawa
  • Toshihiro Sakurai
  • Shinobu Yamaki
  • Shigeki Jin
  • Hirotoshi Fuda
  • Takao Kurosawa
  • Hitoshi ChibaEmail author
Original Paper


1-Palmitoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 16:0/18:2-OOH) and 1-stearoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 18:0/18:2-OOH) were measured by liquid chromatography/mass spectrometry (LC/MS) using nonendogenous 1-palmitoyl-2-heptadecenoylphosphatidylcholine monohydroperoxide as an internal standard. The calibration curves for synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, which were obtained by direct injection of the internal standard into the LC/MS system, were linear throughout the calibration range (0.8–12.8 pmol). Within-day and between-day coefficients of variation were less than 10%, and the recoveries were between 86% and 105%. The limit of detection (LOD) and the limit of quantification (LOQ) were determined using synthetic standards. The LOD (signal-to-noise ratio 3:1) was 0.01 pmol, and the LOQ (signal-to-noise ratio 6:1) was 0.08 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. With use of this method, the concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH in the lipoprotein fractions during copper-mediated oxidation were determined. We prepared oxLDL and oxHDL by incubating native LDL and native HDL from human plasma (n = 10) with CuSO4 for up to 4 h. The time course of the PC 16:0/18:2-OOH and PC 18:0/18:2-OOH levels during oxidation consisted of three phases. For oxidized LDL, both compounds exhibited a slow lag phase and a subsequent rapidly increasing propagation phase, followed by a gradually decreasing degradation phase. In contrast, for oxidized HDL, both compounds initially exhibited a prompt propagation phase with a subsequent plateau phase, followed by a rapid degradation phase. The analytical LC/MS method for phosphatidylcholine hydroperoxides might be useful for the analysis of biological samples.

Online Abstract Figure

Quantitative determination of phosphatidylcholine hydroperoxides during copper-oxidation of LDL and HDL by liquid chromatography/mass spectrometry


Lipid hydroperoxide Oxidized low-density lipoprotein Oxidized high-density lipoprotein Liquid chromatography Liquid chromatography/mass spectrometry Mass spectrometry 



cholesterylester hydroperoxide




high-density lipoprotein


high-performance liquid chromatography


liquid chromatography


low-density lipoprotein


lipid hydroperoxide


mass spectrometry


native high-density lipoprotein


native low-density lipoprotein


phosphate-buffered saline




phosphatidylcholine hydroperoxide

PC 16:0/18:2-OOH

1-palmitoyl-2-linoleoylphosphatidylcholine monohydroperoxide

PC 16:0/17:1-OOH

1-palmitoyl-2-heptadecenoylphosphatidylcholine monohydroperoxide

PC 18:0/18:2-OOH

1-stearoyl-2-linoleoylphosphatidylcholine monohydroperoxide


oxidized high-density lipoprotein


oxidized low-density lipoprotein


reactive oxygen species


selected reaction monitoring



This study was supported in part by Sapporo Biocluster “Bio-S,” The Regional Innovation Cluster Program, The Ministry of Education, Culture, Sports, Science and Technology, Japan, and by a Grant-in-Aid from the Japan Society for the Promotion of Science.


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Shu-Ping Hui
    • 1
  • Yudai Taguchi
    • 1
  • Seiji Takeda
    • 1
  • Futaba Ohkawa
    • 1
  • Toshihiro Sakurai
    • 1
    • 2
  • Shinobu Yamaki
    • 1
  • Shigeki Jin
    • 1
  • Hirotoshi Fuda
    • 1
  • Takao Kurosawa
    • 3
  • Hitoshi Chiba
    • 1
    Email author
  1. 1.Faculty of Health SciencesHokkaido UniversitySapporoJapan
  2. 2.Research Fellow of the Japan Society for the Promotion of ScienceTokyoJapan
  3. 3.Faculty of Pharmaceutical SciencesHealth Sciences University of HokkaidoHokkaidoJapan

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