tMCR-ALS Method for the Determination of Water Concentration Profiles in the Stratum Corneum of Skin in Vivo

Skin moisturizing is important in dermatology and cosmetics. Raman microspectroscopy is a well-suited method to determine water concentration profiles in the stratum corneum (SC) of untreated and formulation-treated skin in vivo. Until now, the water in the SC was quantified using the ratio of water to proteins, and other SC components were neglected.

In this study, we apply the tailored multivariate curve resolution-alternating least squares (tMCR-ALS)method to improve the quantitative determination of water in the SC in the high wavenumber region.

For this purpose a sophisticated optical measuring system confocal Raman microscopy was used.

Six healthy volunteers aged from 23 to 62 years were included in the study. The petrolatum were evenly applied at an amount of 2mg/cm² upon marked areas of 2*2cm² of the inner forearm and rubbed homogeneously. After one hour oil-treated area was measured at least ten times down to the depth of 40µm in the skin at increments of 2 µm.

From the measured skin Raman spectra (Fig.), two methods based on the water-protein peak ratio(Method 1 and Method 2) and tMCR-ALS method (Method 3) were used to determine the skin water concentration.

Fig. Raman Spectra of human stratum corneum, petrolatum and water

Method 1 is based on the water-protein ratio in the high-wavenumber region, and Method 2 is based on the water-protein ratio in the fingerprint region.

The water concentration in Method 1 is determined by the following equation :

Water concentration(%)=(W/P)/(W/P+R)

Here, P and W are the AUCs of protein(2910–2965 cm^-1) and water-related (3350–3550 cm^-1)bands, respectively. R is a proportionality constant calculated from the calibration curve by using the water and protein mixture.

In Method 2, The water mass percentage is calculated by the ratio of AUCs of the water-related Raman band (3350–3550 cm^-1) and the Raman band of Amide I (1650–1676 cm^-1).

Water concentration(%)=W_HWN/P_FP= W_HWN/(AUC₁₆₅₀-R_WW_HWN)

Here, the AUC₁₆₅₀ is the AUC of the band centered at 1650 cm^-1, WHWN is the AUC of water-related(3350–3550 cm^-1) band and RW is the proportionality constant determined from the Raman spectra of distilled water.

Method3, tMCR-ALS method was derived from the open source code of "MCR-ALS GUI 2.0" and was implemented using Matlab R2015a. The mass percentage of water in Method 3 is determined from the following equation :

Water concentration(%)=m_w/M=m_w/(m_w+m_l+m_p+m_o)

Here, m_w, m_l, m_p, m_o and M are the masses of water, lipids, proteins, oil (or other topically applied substances), and the total mass of the skin, respectively.

Method 1 calculates the incorrect water concentration curve at 0-70% depth of the stratum corneum and Method 2 at 0-10% depth. Method 3 shows results which coincide well with the modern knowledge of the moisturizing effect of cosmetic oils in the stratum corneum.

The results were published in "Journal of Raman Spectroscopy" under the title of "tMCR-ALS method for the determination of water concentration profiles in the stratum corneum of untreated and treated skin in vivo" (https://doi.org/10.1002/jrs.6349).