Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum.

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR-partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400-1500 cm-1) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R2 = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R2 (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies.

Confocal Raman Spectroscopic Imaging for Evaluation of Distribution of Nano-Formulated Hydrophobic Active Cosmetic Ingredients in Hydrophilic Films.

Film-forming systems are highly relevant to the topical administration of active ingredients (AI) to the body. Enhanced contact with the skin can increase the efficacy of delivery and penetration during prolonged exposure. However, after the evaporation of volatile solvents to form a thin film, the distribution of the ingredient should remain homogenous in order to ensure the effectiveness of the formula. This is especially critical for the use of hydrophobic molecules that have poor solubility in hydrophilic films. In order to address this concern, hydroxyphenethyl esters (PHE) of Punica granatum seed oil were prepared as a nanosuspension stabilised by poloxamers (NanoPHE). NanoPHE was then added to a formulation containing polyvinyl alcohol (PVA) as a film forming agent, Glycerol as a plasticiser and an antimicrobial agent, SepicideTM HB. Despite their reliability, reference methods such as high-performance liquid chromatography are increasingly challenged due to the need for consumables and solvents, which is contrary to current concerns about green industry in the cosmetics field. Moreover, such methods fail to provide spatially resolved chemical information. In order to investigate the distribution of ingredients in the dried film, Confocal Raman imaging (CRI) coupled to Non-negatively Constrained Least Squares (NCLS) analysis was used. The reconstructed heat maps from a range of films containing systematically varying PHE concentrations highlighted the changes in spectral contribution from each of the ingredients. First, using NCLS scores it was demonstrated that the distributions of PVA, Glycerol, SepicideTM HB and PHE were homogenous, with respective relative standard deviations (RSD) of 3.33%, 2.48%, 2.72% and 6.27%. Second, the respective relationships between ingredient concentrations in the films and their Raman responses, and the spectral abundance were established. Finally, a model for absolute quantification for PHE was be constructed using the percentage of spectral abundance. The prepared %w/w concentrations regressed against predicted %w/w concentrations, displaying high correlation (R2 = 0.995), while the Root Mean Squared Error (0.0869% w/w PHE) confirmed the precision of the analysis. The mean percent relative error of 3.75% indicates the accuracy to which the concentration in dried films could be determined, further supporting the suitability of CRI for analysis of composite solid film matrix. Ultimately, it was demonstrated that nanoformulation of hydrophobic PHE provides homogenous distribution in PVA based film-forming systems independent of the concentration of NanoPHE used in the formula.

Nanoscale investigation of human skin and study of skin penetration of Janus nanoparticles.

Janus nanoparticles (JNP) are innovative nanocarriers with an interesting pharmaceutical and cosmetic potential. They are characterized by the presence of a lipid compartment associated with an aqueous compartment delimited by a phospholipid bilayer containing phospholipids and non-ionic surfactants. The hydrodynamic diameter of JNP varies between 150 and 300 nm. The purpose of this study was to answer the following questions: after cutaneous application, are JNP penetrating? If so, how deep? And in which state, intact or degraded? It was essential to understand these phenomena in order to control the rate and kinetics of diffusion of active ingredients, which can be encapsulated in this vehicle for pharmaceutical or cosmetic purposes. An innovative technique called AFM-IR, was used to elucidate the behavior of JNP after cutaneous application. This instrument, coupling atomic force microscopy and IR spectroscopy, allowing to perform chemical analysis at the nanometer scale thanks to local absorption measurements. The identification of organic molecules at the nanoscale is possible without any labelling. Before cutaneous application of JNP, the nano-structure of untreated human skin was investigated with AFM-IR. Then, in vitro human skin penetration of JNP was studied using Franz cells, and AFM-IR allowed us to perform ultra-local information investigations.

ATR-FTIR Characterization of Janus Nanoparticles-Part II: Follow-Up Skin Application.

Attenuated total reflection by Fourier transform infrared (ATR-FTIR) was used to implement reliable infrared descriptors over time of Janus nanoparticles (JNP), to follow their behavior before and after cutaneous application. In the last study, ATR-FTIR spectroscopic analysis allowed us to identify the evolution of intensity ratio of ν(C=O) at 1739 cm-1 and δ(H-O-H) at 1639 cm-1 as a spectroscopic descriptor, for JNP before cutaneous application (on the CaF2 window). This descriptor can be used to follow the physical stability (presence) of nanoparticles over time. The purpose of this study was to understand the behavior of JNP on the surface of the human skin. Therefore, a comparative study with the untreated skin and the skin after cutaneous application of lipophilic phase (Labrafil) of JNP was conducted using Franz cells. The suitability of the ATR-FTIR descriptor of JNP was evaluated, and a research of other descriptors was performed to understand the interaction that may exist between nanoparticles and the skin.

ATR-FTIR Characterization of Janus Nanoparticles. Part I: Implementation of Spectroscopic Descriptors.

The present work deals with original bicompartmental lipid Janus nanoparticles (JNPs), which are characterized by the presence of an oily compartment associated with an aqueous compartment delimited by a phospholipid-based bilayer. The size of JNP varies between 150 and 300 nm. As JNP are promising candidates for cutaneous application, the purpose of this study was to implement reliable infrared descriptors over time of JNP, to follow the physical stability of JNP in open air and over time. Therefore, a comparative study with the nanoemulsion and the physical mixture formulations was conducted by attenuated total reflection by FTIR spectroscopy. We defined herein spectroscopic descriptor reflecting the integrity of the JNP. Principal component analysis and orthogonal partial least square-discriminant analysis were used to validate the relevant descriptor and permitted to extract relevant and useful information from the spectral data. Dynamic light scattering measurements were also carried and gave supporting data for our conclusion on the fate of JNP over time.