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The chromatographic procedure may be carried out using:

—  a fused-silica column 30 m long and 0.25 mm in internal diameter coated with poly(dimethyl)siloxane R (film thickness 0.25 µm),

—  helium for chromatography R as the carrier gas with a split ratio of 1:25 at a flow rate of 2 ml/min,

—  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.

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