Major human plasma lipid classes determined by quantitative high-performance liquid chromatography, their variation and associations with phospholipid fatty acids

Abstract

An HPLC method with evaporative light-scattering detection (ELSD) was optimized and validated for the simultaneous quantitation of cholesteryl esters (CEs), triacylglycerols (TGs), free cholesterol (FC) and phosphatidylcholine (PC) in human plasma. The separation of CEs from TGs, the most variable plasma lipid class, was improved by speeding up the gradient steps and by increasing the re-equilibration time between runs. The calibrations were made at levels of 0.14–14 μg lipid/injection. The intra- and inter-day precision values of the method ranged between 1.9 and 4.5 and 2.3–7.2% (RSD, n=6), respectively, including determinations at two concentration levels. In comparison to other lipid classes, quantitation of PC proved to be equally repeatable despite its lowest detector response. The relative recoveries varied from 97.0 to 110.3%, showing good accuracy of the method. The methodological variation of the lipid classes covered 0.6–3.1% of their total variation in the study population ( n=48). The CE/FC ratio showed an even closer relationship with phospholipid linoleic acid (18:2 n−6; r=0.65, P<0.001) than with serum cholesterol levels, while eicosapentaenoic acid (20:5 n−3) was significantly associated with PC ( r=0.41, P<0.01). The CE/FC ratio increased ( P<0.01) during soyabean oil substitution and the level of PC increased ( P<0.01) during cold-pressed rapeseed oil substitution.