Determination of oxysterols in oxidatively modified low-density lipoprotein by semi-micro high-performance liquid chromatography with electrochemical detection.

A simple method for the determination of oxysterols was developed by semi-micro high-performance liquid chromatography with electrochemical detection (semi-micro HPLC-ECD). Semi-micro HPLC-ECD was established using a C30 microbore column, acetonitrile containing 50 mmol/L LiClO(4) as a mobile phase, and an applied potential at +2.8V versus Ag/AgCl. The current peak height was linearly related to the amount of sterol injected from 12.5 to 250 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.9% (n=6). This method was applied to the determination of seven oxysterols in oxidatively modified low-density lipoprotein (Ox-LDL). Oxysterols were determined with a recovery of more than 78.0% and an RSD of less than 2.9% (n=6) except for 7-ketocholesterol. 7-Ketocholesterol was determined as a sum of intact 7-ketocholesterol and its degradation product on saponification, cholesta-3,5-dien-7-one, with a recovery of 98.0% and an RSD of 2.5% (n=6). From these results, the current method enabled the simultaneous determination of seven oxysterols without any derivatization, providing a useful tool for the assessment of oxysterol contents in Ox-LDL.