Fluorometric quantification of protoporphyrin IX in biological skin samples from in vitro penetration/permeation studies

Abstract

A fluorometric analytical method was developed for quantification of protoporphyrin IX (PpIX) in skin samples and receptor phase solution after in vitro cutaneous penetration/permeation studies. Analytical conditions used were: excitation and emission wavelengths: 400 nm and 632 nm; bandwidth: 0.5 nm; excitation and emission slits: 10/10. PpIX was recovered from two different layers of skin, the stratum corneum (SC) and the epidermis plus dermis ([E+D]), by vortex homogenization, probe and bath sonication, using DMSO as an extraction solvent. The detection and quantification limits were 0.002 and 0.005 µg/mL, respectively. The assay was linear from 0.005 -0.5 µg/mL. The within-day and betweenday assay precision and accuracy in DMSO and receptor phase solution were each studied at the two concentration levels 0.04 and 0.2 µg/mL, and 0.01 and 0.08 µg/mL, respectively. The coefficients of variation and deviation from the theoretical values were lower than 5%. The skin recovery of PpIX from SC and [E+D] layers using two different concentrations (0.5 and 1.0 µg/mL) were all above 90.0%. The method described has potential application to in vitro penetration/permeation studies of PpIX using porcine skin as a biological membrane model. Uniterms: Fluorometric quantification. Protoporphyrin IX/determination. Photodynamic therapy. Porcine skin. Cutaneous penetration/in vitro study. Cutaneous permeation/in vitro study.Um método analítico por espectrofluorimetria foi desenvolvido para quantificar a protoporfirina IX (Pp IX) em amostras de pele e fase receptora após a realização de testes in vitro de penetração/permeação cutâneas. As condições analíticas utilizadas foram: comprimentos de onda de excitação e emissão: 400 nm e 632 nm; largura de banda: 0,5 nm; fendas de excitação e emissão: 10/10. A PpIX foi extraída de amostras de estrato córneo (EC) e da epiderme sem estrato córneo + derme ([E+D]) através da agitação em vórtex e sonicação por haste e banho, utilizando-se o DMSO como solvente extrator. O limite de detecção e quantificação foram, respectivamente, de 0,002 e 0,005 µg/mL. O método mostrou-se linear da faixa de 0,005 -0,5 µg/mL. A precisão e exatidão intra e inter-ensaio em DMSO e na fase receptora foram validadas utilizando-se duas concentrações distintas, respectivamente, de 0,004 e 0,2 µg/mL, e 0,01 e 0,08 µg/mL. Os valores de coeficiente de variação e o desvio do valor teórico foram inferiores a 5%. A recuperação da PpIX das camadas da pele (EC e [E+D]) utilizando-se duas concentrações distintas (0,5 e 1,0 µg/mL) foram todas acima de 90,0%. O método descrito pode ser utilizado para determinação da PpIX após estudos de penetração/permeação cutânea in vitro utilizando pele de porco como modelo de membrana.Unitermos: Espectrofluorimetria/análise quantitativa. Quantificação fluorimétrica. Protoporfirina IX/ determinação. Terapia fotodinâmica. Pele porcina. Penetração cutânea/estudo in vitro. Permeação cutânea/estudo in vitro.

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