How Confocal Is Confocal Raman Microspectroscopy on the Skin? Impact of Microscope Configuration and Sample Preparation on Penetration Depth Profiles

Abstract

Background/Aims: The aim of the study was to elucidate the effect of sample preparation and microscope configuration on the results of confocal Raman microspectroscopic evaluation of the penetration of a pharmaceutical active into the skin (depth profiling). Methods: Pig ear skin and a hydrophilic formulation containing procaine HCl were used as a model system. The formulation was either left on the skin during the measurement, or was wiped off or washed off prior to the analysis. The microscope configuration was varied with respect to objectives and pinholes used. Results: Sample preparation and microscope configuration had a tremendous effect on the results of depth profiling. Regarding sample preparation, the best results could be observed when the formulation was washed off the skin prior to the analysis. Concerning microscope configuration, the use of a 40 × 0.6 numerical aperture (NA) objective in combination with a 25-µm pinhole or a 100 × 1.25 NA objective in combination with a 50-µm pinhole was found to be advantageous. Conclusion: Complete removal of the sample from the skin before the analysis was found to be crucial. A thorough analysis of the suitability of the chosen microscope configuration should be performed before acquiring concentration depth profiles.

Determination of skin penetration profiles by confocal Raman microspectroscopy: statistical evaluation of optimal microscope configuration

“…Thereafter, it remains constant. This low value of the normalized procaine signal is similar to the one found in untreated skin and corresponds to a small endogenous peak at 1600–1625 cm −1 . The error bars are similar for the data obtained by lateral scanning and by depth profiling with the 40 × 0.6 NA objective and 25 µm pinhole and higher for the profile obtained with the 100 × 1.25 NA objective and 50 µm pinhole.…”

“…The use of a pinhole with a smaller opening was found to improve the results as it decreased the bias by signals from out‐of‐focus regions. Two microscope configurations evolved as being potentially appropriate for depth profiling: a 40 × 0.6 NA objective in combination with a 25 µm pinhole and a 100 × 1.25 NA (oil immersion) objective in combination with a 50 µm pinhole …”

“…Thereafter, penetration profiles of procaine were established by acquiring spectra from the skin surface up to 20 µm into the depth of the skin at a step size of 1 µm. The penetration and permeation characteristics of procaine HCl from this formulation into the skin and CRM investigation thereof have been reported elsewhere . Statistical analysis of the penetration profiles was carried out using modified Bland–Altman plots to compare the differences between penetration plots obtained by depth profiling and lateral scanning.…”

“…Two microscope configurations evolved as being potentially appropriate for depth profiling: a 40 × 0.6 NA objective in combination with a 25 μm pinhole and a 100 × 1.25 NA (oil immersion) objective in combination with a 50 μm pinhole. [22] The aim of the present study was to statistically evaluate these two microscope configurations with regard to their suitability for depth profiling of the skin. To this end, the results from depth profiling with the two mentioned configurations were compared to the results from lateral scanning of cross sections of skin samples.…”

“…The penetration and permeation characteristics of procaine HCl from this formulation into the skin and CRM investigation thereof have been reported elsewhere. [22,29] Statistical analysis of the penetration profiles was carried out using modified Bland-Altman plots to compare the differences between penetration plots obtained by depth profiling and lateral scanning. Furthermore, mean relative variations and AUC values were calculated.…”