TRETINOIN ASSAY BY CARBON PHASE EXTRACTION 3 3 3 24O 160 - 120 - 80- 40- I I I I I I _I 60 120 0 I I I 0 180 240 300 360 Eluate fractions (mL) Figure 4. TRT recovery of sequential 10-ml eluate fractions from 1-g carbon column, loaded continuously with a TRT 208 •xg/ml solution. The adsorbance capacity of the chosen carbon to TRT was calculated eluting a 1-g car- bon column with a TRT 208-•g/ml solution in THF and collecting sequential portions of 10 ml. Figure 4, where TRT recovery of the eluate fractions versus volumes are plot- ted, shows the complete adsorption for about the first 80 ml, corresponding to --16.5 mg, and the carbon saturation after --270 mi. Considering that pharmaceuticals con- tain at most 0.5 mg/ml, this capacity value assures complete TRT adsorption when ap- plying the proposed procedure. For some sun-protective formulations the carbon extraction was not able to eliminate completely the signal interferences. In fact, in these preparations the sun filters are present in high amounts (5-10%), and they are not at all adsorbed on the carbon phase. Among the sun filters examined, the 2,2'-dihydroxhy-4,4'-dimethoxy-benzophenone and the 1-(4-methoxyphenyl)-3-tert-buthylphenyl) propan-l,3-dione presented UV curves overlapping TRT absorbance signals, whereas the other ones did not absorb over 350 nm. For these cases, a further separation by HPTLC was defined. To remove any suspended particles, the pyridinic eluate was filtered through a 0.45-•m-pore-size membrane, acidified with HC1, and then extracted with methylene chloride. The extracts were evaporated under N2, and the residue, dissolved in ethanol, was loaded on the plate. A good separation of TRT (Rf 0.72) was obtained with an eluent acetone/hexane mixture (6:4), and its quantitative evaluation was carried out by correlating the analyte concen- tration with the spot area through the regression equation reported in Table II.

334 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS In any case, the presence of the TRT signals in the second- and third-order derivative spectra allows one to quantify the analyte through the relative equations reported in Table II. When these signals are absent or they are influenced by whatever interference, the analyte presence can be assured by applying the HPTLC method. VALIDATION The accuracy of the methods was determined by applying recovery studies on cream bases to which known amounts of TRT over the range of 0.05 to 200 mg/100g had been added. The precision of the methods was determined by assaying five replicated analyses of the samples, obtaining RSD% values from 1.0 to 5.3%. The results, shown in Table III, demonstrate good recovery, with mean values of 99.3 +- 2.3% for the di- Table III TRT Assay Results in Cream Bases and Commercial Cosmetics, Spiked With Varying Amounts of Analyte, by UV-Derivative and HPTLC Methods UV-Derivative HPTLC Added (mg/100g) Found s RSD% Found RSD% Cream bases Restiva© cream base Resriva© ointment base Schering-Plough Essex© base 2.03 1.95 4.87 1.90 3.23 20.32 19.71 3.10 19.31 5.09 203.24 201.21 1.56 192.05 4.63 0.51 0.49 5.23 0.46 5.10 5.08 4.89 2.89 4.72 4.23 50.82 48.39 3.67 47.77 4.84 0.10 0.10 3.52 0.09 5.48 1.02 0.99 4.56 0.95 4.69 10.24 10.04 2.89 9.62 6.23 Commercial samples Korff "Antia 45 ©" N.E b -- N.E -- 0.55 0.53 3.25 0.51 5.23 Vichy "Day cream©" N.F. -- N.E -- 1.06 1.02 4.52 0.98 4.98 Medestea "S. Angelica©" N.E -- N.E -- 5.46 5.24 3.69 4.99 5.64 Rydelle "Ricerca Derm©" -- N.E -- N.E -- 10.65 10.33 2.89 10.12 3.89 Restiva "Angstrom©" -- N.E -- N.E -- 50.10 48.01 2.57 47.30 4.28 Phas "Exigence©" -- N.F. -- N.E -- 100.20 97.20 3.74 95.23 4.34 ROC "Myosphere ©" -- N. E -- N.E -- 2.73 2.62 5.65 2.51 5.82 L' O real "Plenitude ©" -- N.F. -- N. E -- 20.50 19.65 4.12 19.35 5.71 SAverage of five determinations. bN.E: not found. Manufacturers: Resriva (Milan, Italy) Schering-Plough (Milan, Italy) Korff (Vicenza, Italy) Vichy (Cosme- tique Active France, Levallois-Perret, France) Medestea (Turin, Italy) Rydelle (Johnson Wax, Racine, WI) Phas (Phas C.A.I., Paris, France) ROC (Colombes, France).