In this study, an extended calculation method for the determination of the water profiles in oil‐treated skin is proposed, which is based on the calculation of the ratio between the Raman band intensities of water (3350‐3550 cm−1) and keratin Amide I at 1650 cm−1. The proposed method is compared with the conventional method based on the ratio of the Raman band intensities of water (3350‐3550 cm−1) and keratin at 2930 cm−1. The conventional method creates artifacts in the depth profiles of the water concentration in oil‐treated skin, showing a lower amount of water in the upper and intermediate layers of the stratum corneum, which is due to the superposition of oil‐ and keratin‐related Raman bands at 2930 cm−1. The proposed extended method shows no artifacts and has the potential to determine the water depth profiles after topical application of formulations on the skin.

The quantitative determination of substance concentration in the stratum corneum (SC) is important for profiling of the skin barrier function-related parameters and for investigating drug delivery through the SC. Hereby, confocal Raman microscopy (CRM) is a widely used method, which can be applied noninvasively and in vivo. Most analysis methods are based on the assumption that keratin is distributed homogeneously throughout the SC. In this study, the depth profiles of keratin-related Raman peaks (1,003, 1,450, 1,650, and 2,935 cm −1 ) are investigated in detail. It was shown that each of these Raman peaks can be used for the precise determination of the skin surface position, which is important in case of overlapping of these with Raman bands associated to a formulation for treated skin. For correction of the depth-dependent signal attenuation, that is, normalization, the 1,450, 1,650, and 2,935 cm −1 peaks can be used, whereas the 1,003 cm −1 peak cannot. Further analysis shows that the keratin concentration is nonhomogeneous throughout the SC and decreases towards its bottom due to water increase. Thus, the concentration of certain substances in the SC, such as urea, lipids, water, or natural moisturizing factor, determined using normalization on the keratin peak intensity, is overestimated in the deep SC layers starting from 30% SC depth. The correction coefficients were calculated for all four Raman peaks and for all SC depths with 10% increments. It was determined that at the bottom of the SC, the values obtained using keratin normalization should be multiplied by 0.88 (for 1,003 or 1,450 cm −1 ), 0.94 (for 1,650 cm −1 ), and 0.92 (for 2,935 cm −1 ) in order to compensate nonhomogeneous distribution of keratin in the SC. Thus, original values are overestimated at the SC bottom by 6-12%. The presented correction mechanism is proposed to be used for further correction.

Objective It is conventionally understood that occlusive effects are the retention of excessive water in the stratum corneum (SC), the increase of SC thickness (swelling) and a decrease of the transepidermal water loss. However, the influence of occlusion on water binding properties in the SC is unknown. Methods The action of plant‐derived jojoba and almond oils, as well as mineral‐derived paraffin oil and petrolatum topically applied on human skin, is investigated in vivo using confocal Raman microspectroscopy. To understand the oils’ influence on the SC on the molecular level, the depth‐dependent hydrogen bonding states of water in the SC and their relationship to the conformation of keratin, concentration of natural moisturizing factor (NMF) molecules and lipid organization were investigated. Results A significant SC swelling was observed only in petrolatum‐treated skin. The water concentration was increased in oil‐treated skin in the intermediate SC region (40–70% SC depth). Meanwhile, the amount of free, weakly and tightly bound water increased, and strongly bound water decreased in the uppermost SC region (0–30% SC depth). The NMF concentration of oil‐treated skin was significantly lower at 50–70% SC depth. The lateral organization of lipids in oil‐treated skin was lower at 0–30% SC depth. The secondary structure of keratin was changed towards an increase of β‐sheet content in mineral‐derived oil‐treated skin and changed towards an increase of α‐helix content in plant‐derived oil‐treated skin. Conclusion The occlusive properties can be summarized as the increase of free water and the transformation of water from a more strongly to a more weakly hydrogen bonding state in the uppermost SC, although some oils cause insignificant changes of the SC thickness. The accompanied changes in the keratin conformation at the intermediate swelling region of the SC also emphasize the role of keratin in the SC’s water‐transporting system, that is the water in the SC transports intercellularly and intracellularly in the intermediate swelling region and only intercellularly in the uppermost non‐swelling region. Bearing this in mind, almond, jojoba and paraffin oils, which are not occlusive from the conventional viewpoint, have an occlusion effect similar to petrolatum on the SC.

The main components of the stratum corneum (SC), water, lipids, and proteins, are non-homogeneously distributed throughout the depth. The quantitative determination of their concentration profiles and penetration depth of topically applied substances are urgent topics of dermatological and cosmetic research. Confocal Raman micro-spectroscopy has distinct advantages when determining semi-quantitative concentrations of SC components and topically applied substances non-invasively and in vivo. In this work, we applied a tailored multivariate curve resolution-alternating least squares (tMCR-ALS) method to analyze Raman spectra of the SC in the 2000-4000 cm À1 region for quantitatively determining the concentrations of water, lipids, proteins, and topically applied oils using substance-related spectral loadings which were allowed to change depth-dependently from the SC's surface toward its bottom. tMCR-ALS makes matching of depth-dependent signal attenuation, that is, the normalization on keratin, unnecessary and requires only a few additional experiments for calibration -Raman spectra of the pure materials and their densities.

No abstract