Rapid analysis of oxidized cholesterol derivatives by high-performance liquid chromatography combined with diode-array ultraviolet and evaporative laser light-scattering detection

Extensive evidence of the deleterious biological effects of oxidized 5-cholesten-3 beta-ol (cholesterol) derivatives has led to great interest in their detection. We observed that known oxidized cholesterol derivatives can be rapidly quantitated by combining reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorption and evaporative laser light-scattering (ELSD) detection. Using a 20 X 0.46 cm C18 HPLC column and methanol/acetonitrile (60:40, vol/vol) as the mobile phase at 1.0 mL/min, 10 species of oxidized cholesterol derivative were measured by UV (205, 234, and 280 nm) while 5-cholestan-5 alpha,6 alpha-epoxy-3 beta-ol (5 alpha-epoxycholesterol), 5-cholestan-5 beta,6 beta-epoxy-3 beta-ol (5 beta-epoxycholesterol), and 5-cholestan-3 beta,5 alpha,6 beta-triol (cholestanetriol) were detected by only ELSD. The minimal limits of detection ranged from 100 to 500 ng depending on sterol and detector. The response was linear in the range 0-1000 or 0-2000 ng depending on detector. These oxidized cholesterol derivatives were also identified by HPLC/mass spectrometry analysis combined with UV detector. Heated tallow contained cholestanetriol, 5-cholesten-3 beta,7 alpha-diol (7 alpha-hydroxycholesterol), 5-cholesten-3 beta,7 beta-diol (7 beta-hydroxycholesterol), 5-cholesten-3 beta-ol-7-one (7-ketocholesterol) 5 alpha- and 5 beta-epoxycholesterols under the developed analysis condition. Photooxidized cholesterol had cholestanetriol, 7 alpha- and 7 beta-hydroxycholesterols and 3,5-cholestadien-7-one. On the other hand, 7 alpha- and 7 beta-hydroxycholesterols, 7-ketocholesterol, 5 alpha- and 5 beta-epoxycholesterols and 3,5-cholestadien-7-one were observed in copper-oxidized low-density lipoprotein. Thus, this developed HPLC analysis method could be applied to identification of oxidized cholesterol derivatives in food and biological specimen.