18 •-GLYCYRRHETINIC ACID 77 + 0.250 O. 000- - 0.250 I I I I I 200 300 qO0 (nm) Figure 5. Second-order derivative UV spectra of: (a) methanoJic analytical solution from a hydrogel sample containing 1% GTP (solid line) (b) methanolic solution from the same hydrogel preparation without GTP. UV detection at 248 nm. Under these conditions, GT and the internal standard (tes- tosterone acetate) were appropriately separated from the other components of the cream (Figure 6). A linear calibration graph was obtained by plotting the ratio of GT area to internal standard area against the corresponding GT concentrations (Table I). All the samples were analyzed by the HPLC method described, and the results, summarized in

78 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS I I I 2 I I I 12 16 20 Figure 6. Representative HPLC chromatogram from a cream sample containing 0.06% glycyrrhetinic acid 1 = internal standard (testosterone acetate) 2 = glycyrrhetinic acid. Column: 5 p•m Hypersil C18, 250 X 4.5 mm I.D. Mobile phase: acetonitrile:0.02 M potassium phosphate buffer (pH = 3.0) 65:35 (v/v) at a flow rate of 1.2 ml min-•. Detection: UV at 248 nm. Table II, proved to be in good agreement with the derivative spectrophotometric methods. The same chromatographic conditions were used to determine the GT content in GTP the result obtained (34.9%) was in agreement with that from the spectropho- tometric assay (34.2%). Table III illustrates the composition of the commercial cosmetic preparations analyzed. Using a modified mobile phase composition (60% acetonitrile) at a flow rate of 1 ml ß --I min , good resolution among the different components of the cream was achieved. Under these chromatographic conditions, the HPLC method was able to identify all the substances isolated in the different stages of the extraction procedure for the sample preparation (Figures 7 and 8). The identification was based on retention times, com- pared with those of the individual components dissolved in the same solvent. The described clean-up procedure, compared with a previously described method (9), was found to be more convenient, providing cleaner analytical solutions, longer column life, and more symmetrical peaks.