Simultaneous determination of serum lathosterol and cholesterol by semi-micro high-performance liquid chromatography with electrochemical detection

Abstract

A simple method has been developed for the simultaneous determination of lathosterol and cholesterol by high-performance liquid chromatography with electrochemical detection (HPLC–ECD). Lathosterol was found to be electrochemically oxidized and its current peak height was linearly related to the amount of lathosterol injected, ranging from 0.15 μmol/L to 300 μmol/L ( r = 0.995). Similar results were obtained with cholesterol from 15 μmol/L to 600 μmol/L ( r = 0.995). The separation was carried out with an ODS column, acetonitrile containing 30 mmol/L lithium perchlorate as a mobile phase, and an applied potential at +2.8 V vs. Ag/AgCl. The detection limit ( S/ N = 3) of lathosterol as well as cholesterol was 0.03 μmol/L (0.15 pmol). Total lathosterol in control human and rat serum was determined by the present method with a recovery of more than 95.8% and an RSD ( n = 5) of less than 7.3%. The present method was applied to an experiment with rats to examine the effect of lathosterol feeding. There were no significant changes in serum lathosterol or cholesterol levels in rats fed with a high-lathosterol diet for six days. Therefore, we found this method to be both simple and useful for the simultaneous determination of lathosterol and cholesterol in serum.