The sensitive and simple measurement of underivatized cholesterol and its oxygen derivatives in biological materials by capillary gas chromatography coupled to a mass-selective detector

A method for determining underivatized cholesterol and its oxygen derivatives (ChlOs) in animal tissue specimens and some food has been developed using capillary gas chromatography (GC) and mass spectrometry (MS) with 5α-cholestane as the internal standard. The analysis preparation procedure for cholesterol and ChlOs consisted of lipid extraction followed by gentle saponification using 2 M KOH in ethanol. The analyzed samples were saponified overnight at room temperature in the dark with shaking. Then, free cholesterol and ChlOs were extracted using diethyl ether. The extracts were dried under argon, and the residues were redissolved in chloroform before GC-MS analysis. Cholesterol was analyzed in biological materials using the short column temperature program. The simultaneous quantification of cholesterol and its oxides in biological materials was performed using the longer column temperature program. Cholesterol was eluted faster than ChlOs. The chromatographic method that used column temperature program A can be successfully used for the simple and rapid quantification of cholesterol in biological materials, while the method using column temperature program B is more suitable for the simultaneous quantification of cholesterol and its oxides in biological materials.