312 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS steroids remain in the organic phase, permitting the removal of a great quantity of interfering compounds. At pH 13 the phenolic steroids moved to the aqueous phase. An alternative cleanup procedure is column chromatography, as described by Jones (15). For identification TLC is the most convenient and generally applicable method. Visualization in case of samples with low-level oestrogen is however a problem, as too many interfering compounds of the cream matrix respond to the chromogenic sprays. Only in samples containing DES a more specific method of visualization is available (16), namely by u.v. radiation, which converts DES into a yellow phenanthrene derivative, which fluoresces under long-wave u.v. light. For the steroidal oestrogens we find a GLC confirmation very useful. Gas chromato- graphy alone is however not practicable. GLC-data of a natural mixture of (silyl- ated) steroidal compounds clearly shows that GLC alone cannot make a good identification. Combinations of TLC and GLC generally result in reliable results. Outline of the chemical analysis The outline of the analysis on Table H is mainly based on the information of each product regarding its claimed cosmetic action. Procedures Reagents: Acetic acid, acetonitrile p.a., n-hexane, acetone, pentane, toluene p.a., ethanol p.a. (96•o), chloroform, ethylacetate p.a., benzene p.a., sodium hydroxide (4•a and 0.5M), hydrochloric acid (4ta), potassium hydroxide (0.5•a in ethanol), sulphuric acid (98•o), anhydrous sodium sulphate, sodium chloride, hyflosupercel (filtering aid), alumina for column chromatography (activity II, neutral and activity IV, basic), anisaldehyde, oestradiol-17[•-di- benzoate (Merck), oestradiol-17[• (Organon and Merck 8964), N, O-bis(trimethyl- silyl) acetamide, carbon disulphide, oestrone (Merck 8966), oestriol (Organon and Merck 24609 Lab) and diethylstilbestrol (British Drug Houses). Extraction Three extraction procedures (A, B and C) are described. General extraction procedure (A) (0.5 g sample per ml extract). Weigh 2 g of the sample. Add 10 ml of toluene. Distil off the azeotrope toluene/water, in a rotational evaporator under reduced pressure. Add to the dehydrated residue 10 ml of 96•o ethanol, 10 ml of acetonitrile and 20 ml of pentane. Shake vigorously and wait until the phases have separated well. Discard the pentane fraction. Evaporate the acetonitrile/ethanol fraction and dissolve the residue in 4 ml of chloroform. Extraction for low-level 'free' phenolic oestrogens (B) (4 g sample per ml extract). Use 2 ml of extract A. Evaporate solvent. Dissolve residue in 5 ml sodium hy- droxide 4•a. Shake successively with 2 x 5 ml chloroform and 1 x 5 ml pentane.

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