Analysis of Oxidized 1-Palmitoyl-2-Arachidonoyl-Sn-Glycero-3 Phosphocholine Products in Uremic Patients by LC-ESI/MS

A simple liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI/MS) method has been developed to analyze oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products on the lipoproteins of uremic patients. The native PAPC standard was in vitro oxidized by the Fenton reaction, and the ox-PAPC products were analyzed by LC- ESI/MS. For LC, a C-8 column and a mobile phase (acetonitrile-isopropanol containing 0.1% formic acid (70:30, v/v)) were selected. For ESI/MS, the optimal conditions included sheath gas pressure (10 psi), capillary temperature (270 degrees C), and injection time (1000 ms). The identification of ox-PAPC products on human lipoproteins was based on the extracted ion chromatograms (EICs) and the ESI-MS spectra of the in vitro oxidation products of PAPC standard. The EICs and ESI-MS spectra showed good repeatability and sensitivity. A total of 21 ox-PAPC products was determined. Linear analysis has been performed for the phospholipid standard, 1, 2-Di-O-hexadecyl-sn-glycero-3-phosphocholine (PC(O-16:0/O-16:0)). The linear range was 5.0-100.0 mu g/mL, and the coefficient of determination (R-2) was 0.989. The concentration limit of detection (LOD) was 1.50 mu g/mL, and the concentration limit of quantitation (LOQ) was 4.54 mu g/mL. The selected 21 ox-PAPC products have been identified and quantified in very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) of uremic and healthy subjects. Interestingly, the results showed that the levels of 18 products in VLDL, one product in LDL, and 19 products in HDL were significantly higher for uremic patients than healthy controls. This simple LC-ESI/MS method might accelerate the searching for biomarkers of uremia in the future.