Preliminary Protocol Development of a HPLC-TBARS-EVSC (Ex Vivo Stratum Corneum) Assay for Skin Research: Application in a Sunscreen System

Abstract

Considering the importance of the cutaneous tissue investigation and the need for the development of new protocols to non-invasively establish the safety and efficacy of dermocosmetics and topical products, we aimed at developing an HPLC-TBARS-EVSC (high performance liquid chromatography–thiobarbituric acid reactive species–ex vivo stratum corneum) assay for the lipid peroxidation measurement on subjects’ stratum corneum (SC) obtained by tape stripping; additionally, we applied the HPLC-TBARS-EVSC assay in an emulsified sunscreen system containing ethylhexyl triazone and bemotrizinol as UV filters. HPLC analysis was performed in isocratic mode with 35% methanol/65% phosphate buffer (pH 7.0) as the mobile phase. The diode detector was set at 532 nm to quantify the malondialdehyde (MDA)-TBA adduct. An ex vivo tape stripping method was applied in 10 volunteers in three pre-defined regions of the volar forearms: the control; the irradiated; and the site containing the sunscreen (2.0 mg·cm−2). Ten adhesive tapes per region were used for SC removal. An exclusive ex vivo protocol to measure SC lipid peroxidation was preliminarily developed with linearity and selectivity. The protocol suggested the use of an artificial irradiation dose (5506 KJ·m−2) to improve the assay response from the SC. The sunscreen system had a significative decrease in SC lipoperoxidative damage compared to the control. Our protocol can aid in the efficacy establishment of anti-UV and antioxidant agents, for instance, in studies that aim at elucidating the level of SC lipid peroxidation and even in carrying out baseline investigations characterizing different ethnicities and genders.