CAFFEINE IN HAIR-CARE AND ANTICELLULITE COSMETICS 259 sis of a seven-point calibration curve was used to calculate analytical parameters such as slope, intercept, linearity, limit of detection (LOD), and limit of quantifi cation (LOQ). A regression curve was obtained for the caffeine concentration of 0.01–0.2 mg/mL in fi nal extracted solutions, which corresponded to assumed caffeine amounts of 0.08–1.6% in cosmetic products (Table I). High sensitivity of the method was enabled by a high value of the slope. A good linear relationship with a regression coeffi cient R2 = 0.9997 was found. The real LOD and LOQ were estimated by injecting different volumes of caffeine- free shampoo spiked to 0.08% caffeine until the signal-to-noise (S/N) ratio reached three for LOD and 10 for LOQ (Table I). ACCURACY AND PRECISION OF THE METHOD The overall accuracy and precision of the developed HPLC-SPE procedure were estimated by using the standard addition method. Caffeine-free shampoo (from the same producer as one of the shampoos with caffeine) was used as a blank. Ten millilitre of the dissolved blank shampoo was spiked at four concentration levels (0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, and 0.6 mg/mL) which corresponded to caffeine content of 0.1%, 0.2%, 0.4%, and 1.2% in the product, and three replicate measurements were conducted. The mean recoveries of the extraction, calculated as the ratio of found and added caffeine, were 78%, 85%, 90%, and 97% respectively, and relative SDs were from 3.5% to 10.7%. These results indicate satisfactory accuracy and precision of the method, having in mind the sample complexity and the necessity of performing the extraction step. APPLICATION OF THE METHOD TO THE COSMETIC SAMPLES After SPE treatment of th e cosmetics, clear and nonviscous extracts were injected into the HPLC column. The chromatograms of shampoo 1 and anticellulite gel 1 are shown in Figures 2 and 4. Very clean chromatograms with no interfering peaks were obtained, in- dicating a good selectivity of the method. Identifi cation of caffeine was achieved based on retention times and by UV spectrum of the chromatographic peaks obtained by using the DAD detector (Figures 3 and 5). The caffeine peaks exhibit a purity factor of 989.6 for shampoos and 999.2 for gels. The average retention time was 3.34 ± 0.007 min, with a relative SD of 0.21%. The caffeine content in the examined samples (Table III) was deter- mined by using the calibration curve. Table III Content of Caffeine in Cosmetic Products Cosmetic product Caffeine (g/100 g ± SD)a RSDb (%) Caffeine percent in products (%) Anticellulite gel 1 1.7 ± 0.05 2.9 1.7 Anticellulite gel 2 0.73 ± 0.05 6.9 0.73 Shampoo 1 1.08 ± 0.1 9.3 1.08 Shampoo 2 1.04 ± 0.08 7.7 1.04 Hair balsam 1.0 ± 0.07 7.0 1.0 a The average concentration of caffeine from three measurements expressed per 100 g of the products . b The relative SD of three measurements .

JOURNAL OF COSMETIC SCIENCE 260 Because of the lack of refer ence methods for caffeine determination in cosmetics, we could only compare our results with the expected recommended maximum values and with the results reported in other studies. Thus, the found contents in cellulite reduc- tion cosmetics were in agreement within the recommended values of 1–3% of caffeine (3). In addition, the obtained results are comparable with those given by Injac et al. (1.2–1.6% of caffeine) (11) and Marchei et al. (0.03–5.6% of caffeine) (12). Also, the examined hair-care products contained caffeine in accordance with the usual amounts of 1.0–2.5% (7,8). Because caffeine is considered as a stimulating bioactive compound that can be readily absorbed through the skin (7,8), its exact content in cosmetics is important, especially for caffeine-sensitive consumers. According to the European Food Safety Authority, daily intake of up to 400 mg of caffeine from all sources does not raise safety concerns for adults. On the other hand, the estimated proportion of the adult population exceeding daily intakes of 400 mg ranges from about 6% to almost 33% (23). All the examined cosmetics contained a signifi cant amount of caffeine however, none of them listed its precise content. Moreover, the anticellulite gel packaging does not indicate the presence of caffeine on the product`s principal display panel, whereas hair-care products have this information on the front panel. CONCLUSIONS Methods to monitor and control the caffeine content in cosmetics are of great importance to the cosmetic industry. Caffeine is dispersed in the complex cosmetic matrices there- fore, its extraction is diffi cult and depends on physicochemical properties of the sample and other excipients. Solid-phase extraction was an effective and useful method for pre- treatment and clean-up of cosmetic formulations before HPLC analysis. By appropriate selection of cartridge sorbent, very good extraction of caffeine can be achieved. After SPE Figure 5. UV spectrum of the caffeine peak from anticellulite gel 1 extract on the HR-X cartri d ge.