The chromatographic procedure may be carried out using:
— a flame-ionisation detector,
maintaining the temperature of the column at 275 °C, that of the injection port at 285 °C and that of the detector at 300 °C.
Inject 1.0 µl of each solution. The assay is not valid unless the resolution between the peaks due to pregnenolone isobutyrate and cholest-5-en-3β-ol in the chromatogram obtained with the reference solution is at least 10.0.
Calculate the percentage content of cholest-5-en-3β-ol, using the declared content of cholest-5-en-3β-ol in cholesterol CRS. Calculate the percentage content of total sterols by adding together the contents of cholest-5-en-3β-ol and other substances with a retention time less than or equal to 1.5 times the retention time of cholest-5-en-3β-ol. Disregard the peaks due to the internal standard and the solvent.