[F15. PROCEDURE U.K.
Using a 500 μL micro-syringe (3.4) introduce into the 250 mL flask (3.1) a volume of the α-cholestanol internal standard solution (4.10) and a volume of 1-eicosanol (4.12) containing an amount of cholestanol and eicosanol corresponding to approximately 10 % of the sterol and alcohol content of the sample. For example, for 5 g of olive oil sample add 500 μL of the α-cholestanol solution (4.10) and 250 μL of 1-eicosanol solution (4.12). For pomace olive oils add 1500 μL of both α-cholestanol solution (4.10) and 1-eicosanol (4.12). Evaporate until dryness with a gentle current of nitrogen in a warm water bath. After cooling the flask, weigh 5,00 ± 0,01 g of the dry filtered sample into the same flask.
Note 1: Animal or vegetable oils and fats containing appreciable quantities of cholesterol may show a peak having a retention time identical to cholestanol. If this case occurs, the sterol fraction will have to be analysed in duplicate with and without internal standard. U.K.
Add 50 mL of 2M ethanolic potassium hydroxide solution (4.2) and some pumice, fit the reflux condenser and heat to gentle boiling until saponification takes place (the solution becomes clear). Continue heating for a further 20 minutes, then add 50 mL of distilled water from the top of the condenser, detach the condenser and cool the flask to approximately 30 °C.
Transfer the contents of the flask quantitatively into a 500 mL separating funnel (3.2) using several portions of distilled water (50 mL). Add approximately 80 ml of ethyl ether (4.6), shake vigorously for approximately 60 seconds, periodically releasing the pressure by inverting the separating funnel and opening the stopcock. Allow standing until there is complete separation of the two phases (Note 2). Then draw off the soap solution as completely as possible into a second separating funnel. Perform two further extractions on the water-alcohol phase in the same way using 60 to 70 mL of ethyl ether (4.6).
Note 2: Any emulsion can be destroyed by adding small quantities of ethanol (4.7). U.K.
Combine the three ether extracts in one separating funnel containing 50 mL of water. Continue to wash with water (50 mL) until the wash water no longer gives a pink colour on the addition of a drop of phenolphthalein solution (4.11). When the wash water has been removed, filter on anhydrous sodium sulphate (4.4) into a previously weighed 250 mL flask, washing the funnel and filter with small quantities of ethyl ether (4.6).
Evaporate the solvent by distillation in a rotary evaporator at 30 °C under vacuum. Add 5mL of acetone (4.5) and remove the volatile solvent completely in a gentle current of nitrogen. Dry the residue in the oven at 103 ± 2 °C for 15 min. Cool in desiccators and weigh to the nearest 0,1 mg.]