Confocal Raman microscopy is a novel optical method for studies of pro-drug and drug delivery. This method is a promising technique that enables non-destructive measurement of the permeation profile through skin layers. Peaks of compounds are usually normalised to skin peaks (amino-acid and amide I) for semi-quantitative evaluation. The present study seeks to optimise a methodology for complete quantitative measurement of the amount of an active compound at different depths. Caffeine was used as a tracer to evaluate compound's skin penetration using confocal Raman microscopy. A semi-quantitative depth profile of caffeine was obtained with normalisation of the Raman intensities. These ratios of Raman intensities were correlated with the caffeine concentration using an external calibration curve. The calibration curve was carried out with porcine skin incubated in different concentrations of caffeine; afterwards, each skin sample was analysed by confocal Raman microscopy and HPLC to determine the relation between the Raman signal intensity and the caffeine concentration per skin mass and to create a depth profile. These correlation curves allow the full quantification of the caffeine in skin from Raman intensity ratios at different depths.
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