Apply separately 200 µL of this solution and 20 µL of a solution of -cholestanol in chloroform (1 in 100) to a thin-layer chromatographic plate coated with 0.25-mm silica gel having an application zone that was previously dipped under 3 cm of a solution prepared by dissolving 13 g of potassium hydroxide in 20 mL of water and diluted to 1000 mL with methanol. [note—Allow the plate to dry, and heat it to 100 for 1 hour before use. The plate can be stored in a desiccator containing calcium chloride until the time of use.] Develop with a solvent consisting of a mixture of hexanes and ether (7:3) until the solvent front has moved 17 to 19 cm. Keep the chamber temperature between 15 and 20. Dry the plate in a current of warm air, then spray with an alkaline solution of 2,7-dichlorofluorescein in alcohol (0.2 in 100). Observe the plate under 366-nm wavelength light and identify the bands corresponding to the sterols by referring to the -cholestanol spot. Scrape off these bands and transfer them to a test tube. Add 10 mL of warm chloroform, and shake for 2 minutes with the aid of several glass beads. Filter the chloroform solution, wash the filter with chloroform, and evaporate the combined filtrate and washings to dryness in vacuum. Dissolve the residue with some drops of anhydrous acetone and evaporate in vacuum. Dry the residue in an oven at 105 for 15 minutes. Dissolve the residue in 0.2 mL of Derivatizing reagent, and allow to stand for not less than 15 minutes at room temperature.