18 [3-GLYCYRRHETINIC ACID 7 ! standard solutions, respectively. The amplitude values were then plotted against the corresponding GT and GTP concentrations to obtain the calibration graphs. Second derivative method. The GT (7.2-36 •g ml -•) and GTP (30-150 •g ml -•) analytical solutions were prepared in 1% methanolic potassium hydroxyde. The zero- order UV spectra of the solutions were recorded against the solvent blank, and the second-order UV spectra were then derived. The amplitude of the positive peak at 275.2 nm (2D275.2) to the zero line was measured for each standard solution and was plotted against the corresponding concentration to obtain the calibration graph. HPLC method. Standard solutions of GT (6.0-15.0 •g ml -•) in the mobile phase, containing 4.3 •g ml- • of testosterone acetate as internal standard, were injected into the chromatograph. The ratios of analyte area to internal standard area were plotted against the corresponding analyte concentrations to obtain the calibration graph. ANALYSES OF COMMERCIAL FORMULATIONS Commercially available creams and hydrophilic gels containing GT and GTP at different concentrations (see Table II) were analyzed. First derivative method. Quantities of 300 mg of commercial sample 3 (1% GT) and 1 g of gels 4 and 5 (1% GTP) were transferred into a separator containing 50 ml of chloroform and extracted with 3 x 15 ml of aqueous hydrochloric acid solution (pH 2.0). The chloroform solution was then adjusted to the volume of 50 ml in a volumetric flask. An aliquot of 10 ml of this solution was extracted with 1 x 10 ml and 3 x 5 ml of 1% ammonium hydroxide solution. The combined ammoniacal extracts were adjusted to the volume of 25 ml in a volumetric flask. The filtered resulting solutions were then analyzed as described under Calibration Graphs (first derivative method), and the GT and GTP content was calculated by comparison with an appropriate standard solution according to the formula Cs(Du/Ds), in which Cs is the concentration, }xg per ml, of GT in the standard solution and Du and Ds are the amplitudes 1D248 in the first derivative UV spectrum of the sample solution and standard solution, respectively. The same sample preparation procedure was applied when the blanks of the cosmetic samples, which contained all the components except GT and GTP, were analyzed. Second derivative method. This method was applied to the analysis of the samples 4 and 5 (hydrogels, 1% GTP content). A 1.0-g quantity of the product was dissolved in chlo- roform and washed three times with a solution of hydrochloric acid in water (pH = 2.0). The chloroform extract was adjusted to the volume of 50 ml in a volumetric flask, and an aliquot of 10 ml of the chloroform solution was evaporated to dryness. The residue was dissolved with 1% methanolic potassium hydroxide, diluted to 25 ml in a volumetric flask. The filtered solution was then subjected to the second derivative method, and the amplitude 2D275. 2 was used to calculate the GTP content in each sample by comparison with the appropriate standard solution, according to the formula Cs(Du/Ds), in which Cs is the concentration, in }xg per ml, of GT in the standard solution and Du and Ds are the amplitude 2D275. 2 in the second derivative UV spectrum of the sample solution and standard solution, respectively. The same sample preparation procedure was applied to the GTP lacking hydrogels. HPLC method. A suitable amount of sample was dissolved in a mixture of chloroform- methanol 1:1 to obtain a solution containing 13 }xg ml- • of GT. An aliquot of 10 ml

72 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS of this solution was evaporated to dryness and the residue suspended with the mobile phase containing 4.3 •xg ml- • of testosterone acetate as an internal standard. The volume was adjusted to 10 ml in a volumetric flask. The resulting solution was filtered and then injected into the chromatograph in triplicate. The unknown samples were run concurrently with a standard, and the peak area ratio of GT to internal standard was used to calculate the GT content according to the formula Cs(Ru/Rs) in which Cs is the concentration, in •xg per ml, of GT in the standard solution and Ru and Rs are the ratios of the peak responses of the GT peak to the internal standard peak obtained from the sample solution and the standard solution, respectively. HPLC IDENTIFICATION OF THE COSMETIC FORMULATION COMPONENTS The sample preparation procedure described under First Derivative Method was applied to all the blanks of the commercial samples, i.e. preparations containing all the ingre- dients, with the exception of the glycyrrhetinic acid. The aqueous acidic extract was directly injected into the chromatograph. Aliquots of the chloroform solutions before and after the extraction with ammonium hydroxide were evaporated to dryness the residue was suspended in acetonitrile:phosphate buffer (0.02 M pH = 3) 60:40 v/v, and the volume adjusted to 10 ml in a volumetric flask. The filtered solutions were then injected into the chromatograph. A 5-ml aliquot of the ammoniacal solutions was acidified with 0.2 ml of 17% phosphoric acid solution in water, diluted to the volume of 10 ml with acetonitrile, and injected into the chromatograph. The components of the analyzed cosmetics (preservatives and antioxidants) were iden- tified through their retention times, by comparison with the pure standards. DETERMINATION OF THE GT CONTENT IN GTP HPLC method. A solution of 28.5 •xg ml- • of GT in the mobile phase, containing 4.3 •g ml- • of testosterone acetate as internal standard, was injected into the chromato- graph in triplicate and run concurrently with a solution containing 120 •xg ml-• of GTP (solvent and concentration of internal standard being the same). The peak area ratios of GTP to internal standard were then calculated and the amount of GT in GTP determined by comparison with the GT standard solution. The GT content in GTP was found to be 34.9%. Spectrophotometric second derivative method. The zero order spectra of a solution of GT (c = 30.7 •xg ml- •) and GTP (c = 74.9 •xg ml- •) in methanolic potassium hydroxide were recorded. The second derivative spectra were then derived, and the GT content in GTP was calculated by comparing the GTP 2D277 value to the GT one (GT in GTP = 34.2%). RESULTS AND DISCUSSION SPECTROPHOTOMETRY The zero-order UV spectra of GT and GTP in neutral medium show the same absorption maximum at 248 nm, which is slightly shifted (to 258 nm) at basic pHs. On the other