High-performance liquid chromatography/electrospray ionization ion-trap tandem mass spectrometric analysis and quantification of phosphatidylcholine molecular species in the serum of cystic fibrosis subjects supplemented with docosahexaenoic acid.

Since phosphatidylcholine (PC) is the most abundant phospholipid (PL) class in human serum, its concentration represents an important marker for the evaluation of lipid absorption and metabolism. High-performance liquid chromatography coupled on-line with electrospray ionization ion-trap tandem mass spectrometry (HPLC/ESI-MS/MS) was successfully applied to the quantitative analysis of PC molecular species from serum of cystic fibrosis (CF) subjects before and after supplementation with docosahexaenoic acid (DHA). Seven molecular species of PC (containing C16:0/C20:4, C16:0/C22:6, C18:0/C20:4, C18:0/C22:6, C16:0/C18:1, C16:0/C18:2 and C18:0/C18:2, respectively) were quantified using MS in the negative scan mode with 1,2-diundecanoyl-sn-glycero-phosphocholine as the internal standard. The molecular species containing DHA, C16:0/C22:6 and C18:0/C22:6, increased from 41.3 +/- 31.7 and 33.1 +/- 18.2 to 85.4 +/- 20.4 and 52.1 +/- 20.7 microg/mL serum, respectively, after a 3-month supplementation. Interestingly, the species containing arachidonic acid (C18:0/C20:4 and C16:0/C20:4) decreased from 115 +/- 55 and 139 +/- 57 to 58.1 +/- 22.5 and 70.5 +/- 28.1, respectively. HPLC/ESI-MS/MS allowed the direct analysis of the lipid extract without previous purification of PLs, thus it is a useful analytical support in CF research in order to understand the extent of lipid dysfunctions typical of CF or other diseases. The present method might also be used for quantitative analysis of each serum phospholipid class molecular species. However, the instrument response was found to be very dependent on the phospholipid class considered, and thus the use of appropriate standards for each class of PLs is recommended.

Normal phase liquid chromatography-electrospray ionization tandem mass spectrometry analysis of phospholipid molecular species in blood mononuclear cells: application to cystic fibrosis.

The use of HPLC coupled on-line with a mass spectrometer is a very powerful tool in order to analyze intact PLs molecular species (PMS) without the need of derivatization, thus decreasing the risk of artifacts formation. A normal-phase HPLC-ESI-MS-MS method has been developed in order to study the human blood mononuclear cell PMS composition. This method was applied to characterize PMS from seven CF subjects and from seven age-matched healthy subjects. More than 140 phospholipid molecular species from phosphatidylethanolamine (PE), plasmalogen phosphatidylethanolamine (pPE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC) and sphingomyelin (Sph) were identified and compared. Differences between the two groups were found in pPE (p16:0/22:6), pPE (p18:0/22:6), PE (16:0/20:4) and PC (16:0/18:2) which were significantly lower in CF subjects and in PC (16:0/16:1) which was significantly higher in CF subjects.