j. Soc. Cosmet. Chem., 44, 249-261 (September/October 1993) Direct measurement of moisture in skin by NIR spectroscopy KATHLEEN A. MARTIN, Helene Curtis, Inc., 4401 W. North Avenue, Chicago, IL 60639. Received March 23, 1993. Synopsis A method to directly determine water content in skin as well as the nature of water binding using near-infrared reflectance spectroscopy is introduced. The method is able to quantirate water on a relative basis and to distinguish free, bulk, and bound water. In addition, scattering of near-infrared radiation off the skin surface can be used as a measure of skin smoothness. As examples of the method's potential, effects of both humidity and moisturizer application on moisture content, water type, and scattering effects have been studied on dry legs. INTRODUCTION According to Kligman (1), skin characterized as dry is not necessarily lacking in mois- ture but is more often considered to have a rough, uneven surface that scatters light efficiently, leading to a dry, matte appearance. Normal dry skin can be caused by climate, cleansing, age, or heredity. The function of a moisturizing product is to retard water loss by creating a barrier to surface evaporation, to create a smoother, softer feel to the skin, and to improve the appearance of the skin. The outermost layer of skin is the stratum corneum (SC), about 10-20 microns thick and composed of partially dehydrated cells in a lipid matrix. It is this layer that is considered to be responsible for the barrier function of skin. Below the SC is the epidermis, about 100-200 microns thick, and below that the dermis, about 2-4 mm thick. Skin becomes increasingly more hydrated at the deeper layers. Obata and Tagami indicate that the main role of water in the stratum corneum is to control softness and pliability (2). Several studies on the effect of humidity on both the strength and number of water-binding sites in the SC have been performed and are summarized by Potts (3). Generally, increasing relative humidity causes an increase in tissue hydration, the rate of hydration being greater at higher relative humidity. The nature of water in the SC was concluded to be different at different relative humidities. Using NMR and IR, Hansen and Yellin (4) determined that at a water content of below 10% the water present was tightly bound, presumably to the polar sites of the proteins. At water contents between 10% and 40%, they found less tightly bound water, which 249

250 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS they suggested was hydrogen-bonded to the protein-bound water. Above 50% water content, the water resembled the bulk liquid. The efficacy of skin moisturizers in vivo is usually determined by indirect measurements of hydration. Tests include high frequency electrical conductivity, TEWL (transepider- mal water loss), biomechanical measurements, and subjective clinical evaluations. All suffer from low precision and no well-understood relationship to water content. Spectroscopic measurements, on the other hand, are related to water content in a straightforward manner through a classical Beer's Law type relationship via absorption of the hydroxyl moieties. The absorbance of a sample is proportional to both concen- tration and the path length that light travels. In the mid-infrared, ATR (attenuated total reflectance) has been used to measure water content of skin in vivo. The ratio of the amide I to the amide II band of skin protein has been used as a measure of water content by exploiting the overlap of the water-bending mode with the amide I band (5). This method assumes that neither the amide I nor amide II band is affected by water content, an unproven assumption. Potts et al. (6) have used the 2100 cm- 1 combination band of water, which has the advantage of being far removed from bands due to skin or products. ATR measurements, however, require occlusion of the skin, which can affect water content. In addition, the degree of contact between the skin and the internal reflectance element is not constant, particularly after skin is treated with a lotion, and the refractive index of skin may change upon hydration, thus affecting the depth of penetration of radiation. In this paper, near-infrared reflectance (NIR) spectroscopy is used to determine skin water content in vivo. NIR has several advantages over mid-infrared spectroscopy. The near-infrared region is more sensitive to hydrogen-bonding differences and thus can distinguish different types of water scattering effects can be used to determine changes in the character of the skin surface, and non-occlusive measurements can be made. The possibility of using NIR for this purpose was previously demonstrated in our laboratory by Walling and Dabney (7). In those experiments, a 10-foot fiber optic cable was coupled to the spectrometer to collect spectra on the skin of legs treated with moisturizer. In situ experiments on pigskin distinguished free and bound water, al- though in vivo experiments did not. This paper presents in vivo results determined on skin without a fiber optic cable. The improved resolution allows several types of water, including free, bulk, and protein-bound, to be distinguished. EXPERIMENTAL Dry leg studies were conducted on 16-22 female subjects in successive clinical trials. Four to five sites on the outer lower legs, each about 2 inches square, were used. An untreated control site was included and used to study the effects of ambient humidity on skin water content. The effects of three formulations used in a single trial were compared by NIR. These formulations were 1) a plasticizer (the exact nature of which is proprietary) in an aqueous gel base containing hydroxyethyl cellulose and preserva- tives, 2) propylene glycol (a penetration enhancer) in the same gel base, and 3) a non-ionic oil-in-water emulsion containing 22% oil phase and a water phase that includes the plasticizer, dipropylene glycol, glycerol, and emulsifiers. Product appli- cations were made twice a day for two to four weeks, followed by a regression period of