HPLC ANALYSIS OF VITAMIN E IN COSMETICS 363 statistical difference between the two investigated methods of extraction as indicated by t-test (t=0.215 and p=0.834) and the non-parametric Mann-Whitney test ( p=0.814). Fur- thermore, to further assess the extraction effi ciency of method 2, known quantities (2.5 μg) of T and TA were added to a 500-mg sample of the commercial cosmetic product and the percent recovery of T/TA was estimated. The percent recoveries of T and TA with extrac- tion method 2 were 99.05% and 96.01%, respectively (Table VII). Since both methods were of similar extraction effi ciency, only method 2 was applied throughout the present study. The negative effect of hexane may be avoided by evaporating the solvent mixture and reconstituting with methanol. However, method 2 remains simpler, needs less pro- cessing time, and maintains the column's integrity, ultimately with equal extraction effi ciency. In summary, the advantages of the present method in comparison with the previously described method by Guaratini et al. (8) include the following: (a) there is simultaneous determination of both T and TA, which was achieved by modifying the mobile phase (methanol-water, 97:3 %v/v) instead of pure methanol, (b) the previous method was tested in a gel–cream base that does not resemble the complex nature of commercial products, containing diverse oily, fatty, and waxy ingredients, (c) the use of isopropanol as extraction solvent in the reported method (8) was unsuccessful in extraction of T and TA from the preparations tested in the present study, and (d) other reported methods for T and TA analysis in dosage forms like tablets, capsules, injctions, etc. (9) were unsuc- cessful due to the different nature of the matrices compared with cosmetic products. Table VII Extraction Effi ciency by Methanol (method 2) of Commercial Product (C) after Addition of Known Amount of Tocopherol (T) and Tocopherol Acetate (TA) Tocopherol Tocopherol acetate Nominal conc. (μg/ml) Found conc., mean ± S.D (% CV), n = 3 Extraction effi ciency (%) Nominal conc. (μg/ml) Found conc., mean ± S.D (% CV), n = 3 Extraction effi ciency (%) 10 9.905 ± 0.054 (0.55) 99.05 20 19.201 ± 0.44 (2.34) 96.01 Table VI Mean Extraction Effi ciency of Tocopherol (T) and Tocopherol Acetate (TA) From Experimental Cream Formulations Based on Nominal and Found Concentrations (mean ± %RSD) (n=4) Vitamin Method 1 Method 2 Nominal conc. (%) Found conc. (%) Recovery (%) Nominal conc. (%) Found conc. (%) Recovery (%) T 0.5319 0.5312 99.87 0.5319 0.5172 97.24 (4.71) (4.84) TA 0.5394 0.5315 98.54 0.5394 0.5282 97.89 (1.803) (0.809)

JOURNAL OF COSMETIC SCIENCE 364 CONCLUSIONS 1. The described analytical procedure proved to be accurate, precise, and suitable for simultaneous determination of T and TA in real commercial cosmetic products. 2. Both tested methods of extraction proved to be accurate and precise and statistically indifferent for the extraction of T and TA in cosmetic preparations. 3. Methanol alone (method 2) is equally as effi cient as a mixture of methanol with 10% hexane (method 1) in terms of extraction effi ciency. 4. Method 2 is simpler and less time-consuming. 5. Method 2 maintains column integrity and thus is more suitable for routine determi- nation of T and TA in cosmetic preparations. 6. The use of highly non-polar solvents, e.g., hexane, should be avoided unless the ex- tract is evaporated, reconstituted with methanol, and centrifuged before injection into the HPLC column. ACKNOWLEDGMENTS This work was supported by Kuwait University, Research Grant No. [PP01/05]. The authors appreciate the sincere efforts of Dr. F. Bandarkar, Mrs. E. Abraham, Mr. S. Abraham, and Mrs. D. Nabil, as well as those of Mrs. L. Varghese and Mrs. A. Thomas. REFERENCES (1) A. Chiu and A. B. Kimball, Topical vitamins, minerals and botanical ingredients as modulators of envi- ronmental and chronological skin damage, Br. J. Dermatol., 149, 68–69 (2003). (2) F. Nachbar and H. C. Korting, The role of vitamin E in normal and damaged skin, J. Mol. Med., 73, 7–17 (1995). (3) W. Gehring, J. Fluhr, and M. Gloor, Infl uence of vitamin E acetate on stratum corneum hydration, Arzneimittelforschung, 48, 772–775 (1998). (4) H. L. Gensler and M. Magdaleno, Topical vitamin E inhibition of immunosuppression tumorigenesis induced by ultraviolet irradiation, Nutr. Cancer, 15, 97–106 (1991). (5) J. J. Thiele, C. Schroeter, S. N. Hsieh, M. Podda, and L. Packer, The antioxidant network of the stratum corneum, Curr. Probl. Dermatol., 29, 26–42 (2001). (6) J. Z. Huo, H. J. Nelis, P. Lavens, P. Sorgeoloos, and A. P. De Leenheer, Simultaneous determination of α-tocopheryl acetate and tocopherols in aquatic organisms and fi sh feed, J. Chrom. B, 724, 249–255 (1999). (7) S. Wielinski and A. Olszanowski, Development and validation of HPLC method for simultaneous determination of fat soluble vitamins in capsules, J. Liq. Chrom. Rel. Technol., 24, 201–213 (2001). (8) T. Guaratini, M. D. Gianeti, and P. M. Campos, Stability of cosmetic formulations containing esters of vitamins E and A: Chemical and physical aspects, Int. J. Pharm., 327, 12–16 (2006). (9) F. G. Ruperes, D. Martin, E. Herrera, and C. Barbas, Chromatographic analysis of α-tocopherol and re- lated compounds in various matrices, J. Chrom. A, 935, 45–69 (2001). (10) M. M. Almeida, J. M. P. Alves, D. C. S. Patto, C. R. R. C. Lima, J. S. Qenca-Guillen, M. I. R. M. Santoro, and E. R. M. Kedor-Hackmann, Determination of tocopheryl acetate and ascorbyl tetraisopalmitate in cosmetic formulations by HPLC, Int. J. Cosmet. Sci., 31, 445–450 (2009). (11) A. Dingler, R. P. Blum, H. Niehus, R. H. Muller, and S. Gohla, Solid lipid nanoparticles (SLNTM/ Lipopearls™) a pharmaceutical and cosmetic carrier for the application of vitamin E in dermal products, J. Microencapsul., 16, 751–767 (1999). (12) A. A. Zaghloul, H. H. El-Shattawy, A. A. Kassem, E. A. Ibrahim, I. K. Reddy, and M. A. Khan, Honey, a prospective antibiotic: Extraction, formulation, and stability studies, Pharmazie, 56, 643–647 (2001).