Analytical and Bioanalytical Chemistry

, Volume 408, Issue 27, pp 7785–7793 | Cite as

A novel chiral stationary phase HPLC-MS/MS method to discriminate between enzymatic oxidation and auto-oxidation of phosphatidylcholine

  • Junya Ito
  • Kiyotaka Nakagawa
  • Shunji Kato
  • Takafumi Hirokawa
  • Shigefumi Kuwahara
  • Toshiharu Nagai
  • Teruo Miyazawa
Research Paper

Abstract

To elucidate the role of enzymatic lipid peroxidation in disease pathogenesis and in food deterioration, we recently achieved stereoselective analysis of phosphatidylcholine hydroperoxide (PCOOH) possessing 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-9Z,11E-HPODE) using HPLC-MS/MS with a CHIRALPAK OP (+) column. Because enzymatic oxidation progresses concurrently with auto-oxidation, we need to distinguish them further. Here, we attempted such an analysis. First, we used lipoxygenase, linoleic acid, and lysophosphatidylcholine (LPC) to synthesize the enzymatic oxidation product 13(S)-9Z,11E-HPODE PC, and the auto-oxidation products 13(RS)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC, which were used as standards to test the ability of various columns to separate the enzymatic oxidation product from auto-oxidation products. Separation was achieved by connecting in series two columns with different properties: CHIRALPAK OP (+) and CHIRALPAK IB-3. The CHIRALPAK OP (+) column separated 13(R)-9Z,11E-HPODE PC and 13(S)-9Z,11E-HPODE PC, whereas CHIRALPAK IB-3 enabled separation of 13(S)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC. The results for the analysis of both enzymatically oxidized and auto-oxidized lecithin (an important phospholipid mixture in vivo and in food) indicate that our method would be useful for distinguishing enzymatic oxidation and auto-oxidation reactions. Such information will be invaluable for elucidating the involvement of PCOOH in disease pathogenesis and in food deterioration.

Keywords

Chiral stationary phase Chiral column Phosphatidylcholine hydroperoxide Lipid oxidation LC-MS/MS 

Abbreviations

16:0 LPC

1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine

16:0/18:2 PC

1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine

C18

octadecylsilane

CD3OD

methanol-d4

CSP

chiral stationary phase

DCC

dicyclohexylcarbodiimide

DMAP

4-dimethylaminopyridine

HPETE

hydroperoxyeicosatetraenoic acid

HPODE

hydroperoxyoctadecadienoic acid

LA

linoleic acid

LOX

lipoxygenase

LPC

lysophosphatidylcholine

MxP

2-methoxypropene

NMR

nuclear magnetic resonance

PC

phosphatidylcholine

PCOOH

phosphatidylcholine hydroperoxide

PPTS

pyridinium p-toluenesulfonate

SRM

selected reaction monitoring.

Notes

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research (B) Grant Number 15H04497.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest associated with this work.

Supplementary material

216_2016_9882_MOESM1_ESM.pdf (542 kb)
ESM 1 (PDF 541 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Junya Ito
    • 1
  • Kiyotaka Nakagawa
    • 1
  • Shunji Kato
    • 1
  • Takafumi Hirokawa
    • 2
  • Shigefumi Kuwahara
    • 2
  • Toshiharu Nagai
    • 3
  • Teruo Miyazawa
    • 1
    • 4
  1. 1.Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural ScienceTohoku UniversitySendaiJapan
  2. 2.Laboratory of Applied Bioorganic Chemistry, Graduate School of Agricultural ScienceTohoku UniversitySendaiJapan
  3. 3.Tsukishima Foods Industry Co., Ltd.TokyoJapan
  4. 4.New Industry Creation Hatchery Center (NICHe)Tohoku UniversitySendaiJapan

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