JOURNAL OF COSMETIC SCIENCE 316 micrometers, depending primarily on the refractive index of the ATR crystal. For exam- ple, for a zinc selenide (ZnSe) crystal at 1000 cm−1 and a sample with a refractive index of 1.5 and an angle of incidence of 45°, the penetration is about 2 μm. As a nondestructive, noninvasive technique, it is ideal for skin measurements and has been used for such measurements in the past (6). However, direct calibration of the technique has proven diffi cult. Application of quantitative standards to the skin has been used (6), but it may not be safe for low levels of analyte because of the solvents needed to dilute the analyte. In addition, the solvents may change the extent of absorption of the applied compounds. Direct application to the ATR crystal of analytes in dilute solution, however, is a tech- nique that yields excellent results. EXPERIMENTAL APPARATUS A BioRad model 575C enhanced intensity FT-IR spectrometer with Pike Technologies’ ATR skin analyzer accessory was used in this work. The ATR crystal was made of zinc selenide with a fl at geometry of 45°, and was 0.40-cm thick and 1.0-cm wide by 10.0-cm long. DigiLab Win-IR Pro software was used to collect the spectra on the PC platform computer. The ATR-FT-IR instrument was aligned prior to the experiments, and all spectra were collected between 4000 and 700 cm−1. Hamilton Gastight® syringes (0.500 ml, #1750) were used to assure accurate delivery of solvents and solutions. REAGENTS Hexane Spectranal® grade (Riedel-de Haën from Sigma Aldrich) was used as solvent in preparing all standards and for rinsing the ATR crystal, glassware, etc. High-purity sol- vent was found to be critical to the method (see below.) PROCEDURES In order to calibrate the instrument, a spectrum of a known amount of analyte must be measured while the skin is in contact with the ATR crystal. Ideally, the standard would be applied to the subject’s arm with the known amount of analyte dissolved in solvent, but solvent interaction with the skin would likely change the skin lipids, the penetration of analyte into the skin, or any number of other important parameters. Depositing the calibration standards directly onto the crystal in dilute solution eliminated these issues. Known volumes of known concentration solutions were carefully delivered by calibrated Gastight syringes, taking care to distribute the solution across the surface of the crystal. The choice of solvent was critical: while solvent must evaporate from the crystal, it must not evaporate too rapidly since the solution may not be delivered quantitatively to the surface. For example, dichloromethane did not pass this requirement. Furthermore, the solvent must be one that evaporates fully from the crystal, leaving no residue. Only high- purity solvents are acceptable early attempts with regular laboratory grade hexane left a residue that produced absorption peaks in the same area, 3200–2600 cm−1 (the C–H

LOTION TRANSFER TO SKIN 317 region), as the lotion we were measuring. Most lotion substrate products, including lotioned facial tissues, have as primary lotion components petrolatum and mineral oil, with key absorbance peaks in this region. Calibration method. The ATR FT-IR apparatus was calibrated for spectral response versus the amount of lotion deposited onto the ATR crystal, with and without the presence of hu- man skin on the crystal. The lotion used for calibration and analysis was similar to those used most commonly in consumer substrate products such as facial tissues. The main com- ponents in these lotions were mineral oil, petroleum wax, cetyl, and stearyl alcohols. A standard stock solution of the lotion in hexane was prepared by weight at 5071.97 μg lotion per gram of solution. From the standard stock solution ten calibration standards were pre- pared by dilution with hexane, ranging from 9 to 1230 μg/g. The spectra of each standard solution were obtained, with and without skin contact, using 0.250 ml of the given stan- dard solution, spread over the entire surface (10 cm2) of the ATR crystal. The human skin portion used in these studies was on the subjects’ volar forearms subjects’ selection criteria, prerequisites, and skin preparation methods are detailed in the next section. Human skin study design. The following study protocol was adhered to throughout this study. Participation in the study was completely voluntary. At the initial visit, subjects were screened for any conditions that would exclude them from the study. Subjects were screened for study eligibility based on the following criteria. Excluded from the study was anyone who: (a) was participating in another clinical trial (b) had an excess amount of hair on the volar forearm (c) was perspiring excessively due to laborious work or exercise, thus resulting in increased skin moisture (d) had allergies to soap, detergent, perfume, cosmetics, toiletries, and/or lotioned tissue products (e) had eczema or psoriasis on the forearms within the past six months (f) had been diagnosed with skin cancer within the past twelve months (g) had any cuts, scratches, rashes or any condition on their inner forearms (e.g., damaged skin, extensive scarring, birth marks, port wine stains, tattoos, moles) that may pre- vent a clear assessment of their skin (h) was using or had used, within the past two weeks, any topical prescription or over- the-counter (OTC) medications (e.g., hydrocortisone cream, antibiotic ointment) on their forearms (i) was a smoker The entire volar forearm was submerged in running water (37°C ± 5°C), washed with a liquid detergent (e.g., 2 ml Dawn® dishwashing detergent), rinsed with running water until all of the soapy water was removed, and then blotted dry with a lotion-free paper towel (e.g., Bounty® paper towel). Subsequently, a timer was set and the forearm was al- lowed to dry for at least two minutes. After drying, the washed area of the volar forearm was positioned on the ATR prism and the FT-IR spectrum was collected. While the spec- trum was being acquired, the subject’s arm remained motionless. The scan was completed within one minute. Prior to each measurement, the prism was cleaned with Kimwipes® and solvent. A blank spectrum was collected to be certain the crystal was clean. This initial scan on cleaned skin became the baseline scan of the volar forearm. Use of this clean arm as “control” helped negate differences in arm pressure on the ATR surface.