Plasma low-density lipoprotein immobilized silica as stationary phase in nano-liquid chromatography.

ABSTRACT

The feasibility to attach plasma low-density lipoprotein (LDL) particles covalently onto 5 µm silica particles and use them as stationary phase in nano-liquid chromatography (nano-LC) for interaction and oxidation studies was clarified. Before the immobilization, both epoxy silica and aldehyde-activated particles were synthesized, LDL was immobilized to the particles via its protein component and capillary columns were packed with LDL-modified silica materials. The performance of the capillary columns was tested with neutral steroids, and the column with LDL immobilized to aldehyde-activated silica was selected for further studies due to its stronger retention toward steroids. The retention factors of the steroids were used as indicators of the column stability, and the RSDs from 0.8 to 5.7% (n=12) for 168 successive runs within 14 days carried out in the same capillary column and from 0.8 to 3.6% (n=6) in three different capillaries demonstrated that the capillary column was stable and that the capillary column-to-capillary column reproducibility was good. The lifetime of LDL-modified silica stationary phase was around 14 days. The applicability of the column for the separation of steroids and ß-blockers was based mainly on the hydrophobic interactions with lipids of LDL in the stationary phase. The LDL immobilized silica column was successfully exploited also in the copper-mediated in situ oxidation of LDL. The results achieved demonstrated that nano-LC with plasma LDL immobilized silica phase can be exploited as nano-biomimicking tool for interaction studies and as a microreactor for oxidation studies with low consumption of reagents and human materials.