251 J. Cosmet. Sci., 71, 251–262 (September/October 2020) Caffeine in Hair Care and Anticellulite Cosmetics: Sample Preparation, Solid-Phase Extraction and HPLC Determination KRISTINA MLADENOV and SLAVICA SUNARIĆ, Department of Pharmacy, Faculty of Medicine, University of Niš, Niš, Serbia (K.M.), Department of Chemistry, Faculty of Medicine, University of Niš, Niš, Serbia (S.S.) Accepted for publication April 24, 2020. Synopsis Caffeine is extensively used in cellulite and hair growth cosmetic products. Regulations in the field of cosmetics require manufacturers to list caffeine in the ingredient list on product labels, but its exact content in these products is not declared. On the other hand, daily exposure to caffeine from all sources may approach health reference values. For that reason, it is important to know the exact caffeine content in products for skin and hair care. Cosmetics are often viscous or semisolid products of very complex chemical composition. To analyze caffeine in these complex sample matrices by liquid chromatographic methods, an extraction step is often necessary. This article presents the applicability of the solid-phase extraction (SPE) procedure for the caffeine extraction and high-performance liquid chromatography (HPLC) determination in anticellulite gels, shampoos, and hair balsam. The samples of gels were centrifuged after ammonia addition to precipitate carbomer. In cellulite reduction, gel caffeine content was found to be in the range of 0.7–1.7%, whereas in the hair-care products, it was about 1.0%. I NTRODUCTION Caffeine, an organic nitrogenous base, is a naturally occurring purine alkaloid of the methylxanthine class (Figure 1). Caffeine is being increasingly used in cosmetics because of its high biological activity and ability to penetrate the skin barrier (1). Based on the research studies that have been conducted in the last three decades, caffeine is used as an ingredient in hair growth products and cellulite reduction products. Cellulite deposition is one of the most common aesthetic problems associated with the female population throughout the world, especially at the age greater than 35 years. Caf- feine is seen as a potential way to prevent excessive accumulation of fat and promotes the lipolysis process. According to Hexsel et al. (2), it inhibits phosphodiesterases (PDEs), which lead to lipogenesis reduction. Caffeine also stimulates the draining lymph systems Address all correspondence to Kristina Mladenov at kristina_mladenov@yahoo.com.

JOURNAL OF COSMETIC SCIENCE 252 in fatty tissues by removing accumulated fat and toxins, which can improve the microcir- culation of blood vessels (1). The commercially available topical anticellulite formula- tions usually contain 1–2% of caffeine, although some products may have up to 3% of this ingredient (3). In addition, hair products containing caffeine have recently been used to prevent and treat alopecia. 5-α-reductase is an enzyme that converts testosterone into the more active dihydrotestosterone, which is responsible for baldness (1). Earlier inves- tigations have shown that caffeine in concentrations of 0.001% and 0.005% inhibits the activity of 5-α-reductase (4) and phosphodiesterase. This increases the intracellular con- centration of cyclic adenosine monophosphate (5), stimulating microcirculation, cellular metabolism, and delivery of nutrients to the hair follicle, thereby contributing to strengthened and more rapid human hair growth (1,6). It has been documented that the application of caffeine on the skin leads to its fast absorp- tion and occurrence in the blood. Caffeine is detected in blood samples only 5 min after topical application of formulation containing 2.5% caffeine, reaching the highest value after 1 h (7,8). It is known that a high dose of caffeine can cause insomnia, anxiety, elevated blood pressure, and tachycardia (9). Because of this fact, information on the exact caffeine content in a cosmetic product may be important for some consumer groups. Caffeine is also frequently daily administered through beverages (coffee, soft and energy drinks, tea, and cocoa) or certain analgesic and nonsteroidal anti-infl ammatory drug mixtures, which is another reason for increased attention when using cosmetics with caffeine. On the cosmetic product label, caffeine is listed according to the International Nomenclature of Cosmetic Ingredients. The precise content is not declared therefore, the exact concentration of this substance is unknown to the consumers. According to the U.S. Food and Drug Adminis- tration, thigh creams may more appropriately be classifi ed as drugs under the Food, Drug, and Cosmetic Act because removal or reduction of cellulite affects the “structure or function” of the body (10). All of these points increase the need to develop test methods to assess the presence and the content of caffeine in different products for human use. So far, the interest in determining caffeine has been mainly focused on foods and bever- ages. Limited number of articles has described caffeine quantifi cation in cosmetics (11–13). Because cosmetics are complex semisolid or high viscous emulsions, for their analysis, different and lengthy sample pretreatments may be required (14). Sample prep- aration in the cosmetic analysis is a crucial step as the complex matrices may seriously interfere with the determination of target analytes (15). Sample preparation and clean- Figure 1. Chemical structure of caffeine (1,3,7-trimethylxanthine).