Determination of cholesterol and triglycerides in serum lipoproteins using flow field-flow fractionation coupled to gas chromatography-mass spectrometry.

Asymmetric flow field flow fractionation (AsFlFFF) was combined with pyrolysis-gas chromatography mass spectrometry for a sized based fractionation and a detailed compositional study of the triglycerides and cholesterol associated with the various lipoprotein subclasses present in human serum. Serum samples were injected in the AsFlFFF instrument and fractionated with a time-delayed exponential decay cross flow program. The fractions collected after AsFlFFF elution were injected into a programmable temperature vaporizer (PTV) GC-injector, containing a fritted liner. A temperature and split-flow program for the PTV injector was optimized for the thermally assisted hydrolysis and methylation of the compounds of interest. The resulting fatty acid and cholesterol methyl esters were separated by GC and characteristic fragment ions were detected by MS. The system was optimized and calibrated with triglyceride and cholesterol standards for quantitative analysis. The possible interference by phospholipids with the quantitative results was investigated and found to be of minor importance. The concentrations and lipoprotein profiles of triglycerides and cholesterol were determined in a pooled serum sample of healthy volunteers and a serum sample of a sepsis patient. The results obtained with the GC-MS approach were compared with those of a previously developed method based on AsFlFFF with a dual enzymatic reaction detection system. A good agreement of the profiles was found, for cholesterol as well as for the triglycerides, even when the GC-MS method quantifies the fatty acids while with the enzymatic reaction method the glycerol concentrations are determined. Total cholesterol and triglyceride concentration values for the serum samples showed good agreement with the results of the standard enzymatic method as used in practice in the university hospital.