Add 1,0 ml of internal standard solution (4.3.1.1) to the flask if sitosterol is to be determined, or (4.3.1.2) if stigmasterol is to be determined. Mix thoroughly. Transfer the contents of the flask quantitatively into a 500 ml separating funnel (3.2) washing the flask in turn with 50 ml water and 250 ml diethyl ether (4.4). Shake the separating funnel vigorously for two minutes and alow the phases to separate. Run of the lower aqueous layer and wash the ether layer by shaking with four successive 100 ml aliquots of water.
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