An optimized fast-performance liquid chromatography method for analyzing lipoprotein profiles using microliter volumes of serum.

Plasma or serum lipoprotein analysis is commonly carried out with a conventional size-exclusion fast-performance liquid chromatography method that requires large sample volumes (1-2 ml). To determine lipoprotein profiles of mice with this method, plasma or serum samples have to be pooled from a group of animals, which often requires sacrificing animals. Here we report an optimized anion-exchange chromatography method with simplified cholesterol collection and detection system. After 5-10 microl serum was injected for anion-exchange chromatography, a stepwise gradient was applied and fractions were collected on a 96-well plate. Cholesterol content in each well was measured using a fluorescence-based detection method. With this method, distinct lipoprotein peaks corresponding to high-density lipoprotein, low-density lipoprotein, and very-low-density lipoprotein, can be easily separated and identified with excellent resolution. The entire high-performance liquid chromatography run takes about 30min and the results are reproducible with a low variability. The small sample size allows analyzing the lipoprotein profile in a given mouse at a given time point with nonterminal bleeding. The method is simple to set up with commercially available parts and convenient to run.