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

Abstract

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 CuSO(4) 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.

Mass Spectrometric Discrimination of Squalene Monohydroperoxide Isomers

“…choline, m/z 184 20,21 , but the MRM 790/184 could not distinguish the hydroperoxide positional isomers e.g. 1-palmitoyl-2-9-hydroperoxy-octadecadienoyl -sn-glycero-3-phosphocholine 16:0/9-HpODE PC and 1-palmitoyl-2-1 3 -h y d r o p e r o x y -o c t a d e c a d i e n o y l -s n -g l y c e r o -3-phosphocholine 16:0/13-HpODE PC .…”

Oxidative Lipidomics Coming of Age: Advances in Analysis of Oxidized Phospholipids in Physiology and Pathology

“…Uchikata et al used this approach to measure oxidized PCs in liver from mice fed on a high-fat diet: As an example, the monoperoxide of SAPC was monitored by transitions from m/z 842.6 to 184.1, 808.4 (loss of H 2 O 2 ), and 824.4 (loss of H 2 O) (136). It is important to note that the loss of water is not a very specific transition, as it occurs for hydroxides and hydroperoxides, although it has been used in an SRM approach (50 -adducts, respectively (82,85), and an advantage is that two transitions of comparable intensity, one for each fatty acyl chain, should be detectable. The corresponding neutral loss of fatty acyl fragments is observed in the positive ion mode, and facilitates structural determination, though these signals are generally weak and very dependent on lipid structure.…”

Identification of Organic Hydroperoxides and Peroxy Acids Using Atmospheric Pressure Chemical Ionization – Tandem Mass Spectrometry (APCI-MS/MS): Application to Secondary Organic Aerosol

“…There are limited studies on direct analysis of ROOH in the literature. Using ESI-MS, Hui et al identified ROOH formed from the oxidation of cholesteryl ester, reporting ammonium and sodium adducts of the ROOH molecular ions under positive ion mode and acetate adducts of ROOH molecular ions under negative ion mode (Hui et al, 2012a;2012b). Reinnig et al (2009) identified ROOH using on-line ion trap mass spectrometry, proposing that neutral loss of 34 Da (loss of H 2 O 2 ) from the protonated 20 molecular ions is a characteristic fragmentation route for ROOH.…”