Evaluation of air oxidized PAPC: A multi laboratory study by LC-MS/MS

Zhixu Ni, Bebiana C. Sousa, Simone Colombo, Catarina B. Afonso, Tania Melo, Andrew R. Pitt, Corinne M. Spickett, Pedro Domingues, M. Rosário Domingues, Maria Fedorova, Angela Criscuolo

Research output: Contribution to journalArticlepeer-review


Oxidized LDL (oxLDL) has been shown to play a crucial role in the onset and development of cardiovascular disorders. The study of oxLDL, as an initiator of inflammatory cascades, led to the discovery of a variety of oxidized phospholipids (oxPLs) responsible for pro-inflammatory actions. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is frequently used by the scientific community as a representative oxPL mixture to study the biological effects of oxidized lipids, due to the high abundance of PAPC in human tissues and the biological activities of oxidized arachidonic acids derivatives. Most studies focusing on oxPAPC effects rely on in-house prepared mixtures of oxidized species obtained by exposing PAPC to air oxidation. Here, we described a multi-laboratory evaluation of the compounds in oxPAPC by LC-MS/MS, focusing on the identification and relative quantification of the lipid peroxidation products (LPPs) formed. PAPC was air-oxidized in four laboratories using the same protocol for 0, 48, and 72 h. It was possible to identify 55 different LPPs with unique elemental composition and characterize different structural isomeric species within these. The study showed good intra-sample reproducibility and similar qualitative patterns of oxidation, as the most abundant LPPs were essentially the same between the four laboratories. However, there were substantial differences in the extent of oxidation, i.e. the amount of LPPs relative to unmodified PAPC, at specific time points. This shows the importance of characterizing air-oxidized PAPC preparations before using them for testing biological effects of oxidized lipids, and may explain some variability of effects reported in the literature.
Original languageEnglish
Pages (from-to)156-166
Number of pages11
JournalFree Radical Biology and Medicine
Early online date15 Jun 2019
Publication statusPublished - 20 Nov 2019

Bibliographical note

© 2019 The Authors. Published by Elsevier Inc. CC BY-NC-ND.

Funding: EU H2020 funded project MASSTRPLAN (Grant number 675132; to all authors except TM) and German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept for SysMedOS project (to MF) are gratefully acknowledged. Thanks are due to University of Aveiro, Marine Lipidomics Laboratory, Fundação para a Ciência e a Tecnologia (FCT, MECPortugal), European Union, QREN, Programa Operacional Factores de Competitividade (COMPETE) and FEDER for the financial support to QOPNA (FCT UID/QUI/00062/2019) and CESAM (UID/AMB/50017/2019) research units; Portuguese Mass Spectrometry Network (LISBOA-01-0145-FEDER-402-022125), FCT/MEC through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.


  • Multi-laboratory study
  • Oxidized phospholipids
  • PAPC
  • Reverse phase chromatography
  • Tandem mass spectrometry


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