Application of directly coupled HPLC MMR to separation and characterization of lipoproteins from human serum

Disorders in lipoprotein metabolism are critical in the etiology of several disease states such as coronary heart disease and atherosclerosis, Thus, t here is considerable interest in the development of novel methods for the a nalysis of lipoprotein complexes. We report here a simple chromatographic m ethod for the separation of highdensity lipoprotein, low-density lipoprotei n, and very low-density lipoprotein from intact serum or plasma. The separa tion was achieved using a hydroxyapatite column and elution with pH 7.4 pho sphate buffer with 100-muL injections of whole plasma. Coelution of HDL wit h plasma proteins such as albumin occurred, and this clearly limits quantit ation of that species by HPLC peak integration. We also show, for the first time, the application of directly coupled HPLC H-1 NMR spectroscopy to con firm the identification of the three major lipoproteins, The full chromatog raphic run time was 90 min with stopped-now 600-MHz NMR spectra of each lip oprotein being collected using 128 scans, in 7 min. The H-1 NMR chemical sh ifts of lipid signals were identical to conventional NMR spectra of freshly prepared lipoprotein standards, confirming that the lipoproteins were not degraded by the HPLC separation and that their gross supramolecular organiz ation was intact.