Genistein (GEN) has potential advantages for topical skin delivery, but no literature data are available for its quantitation in different skin layers, such as the stratum corneum (SC). Therefore, a simple, rapid, selective and sensitive bioanalytical method was developed and validated for GEN quantitation in porcine skin samples following in vitro permeation studies. GEN was assayed by HPLC with UV-Vis detection (270 nm) using 0.5% acetic acid in water-n-propanol-acetonitrile (50 : 2 : 48, v/v/v) as mobile phase (flowrate of 1.0 mL/min). Specificity was demonstrated since endogenous skin components did not interfere with GEN peak. Standard analytical curve was linear over the concentration range (0.1-60 µg/mL) and the lower limit of quantitation was determined for different skin layers (100 ng/mL). GEN recovery from skin layers ranged from 95.57 to 97.57%. Permeation studies were carried out using an automated vertical diffusion cell apparatus. No fluctuation on the amount of GEN retained in the SC was observed over time, but increasing amounts of the drug were found in deeper layers of the skin. The method was reliable and reproducible for the quantitation GEN in skin samples enabling the determination of the cutaneous penetration profile of this drug in permeation experiments.Key words genistein; HPLC quantitation; bioanalytical validation; skin layer Genistein (GEN) (Fig. 1) is the main isoflavone found in soybeans with several beneficial effects in cardiovascular diseases, osteoporosis and postmenopausal syndrome. Potential advantages of GEN have been reported in different types of cancer, such as breast and prostate, as well as skin cancer. 1) GEN acts in carcinogenesis through different mechanisms such as induction of differentiation, inhibition of topoisomerase II, protein tyrosine kinase activity and angiogenesis.
1,2)Skin cancer arises predominantly from cells located within the epidermis.3) Thus, to reach skin malignancies and achieve therapeutic benefits, antitumoral drugs, such as GEN, have to penetrate the stratum corneum and reach deeper layers of the epidermis. 4) In general, topical administration is a challenge in pharmaceutics. 5) Regardless, previous studies have demonstrated GEN's viability for topical 6) and transdermal permeation from different kinds of vehicles.7) Thus, with the purpose of conducting percutaneous permeation studies, the quantitative determination of the drug in the skin is necessary in order to evaluate the efficiency of the proposed formulations.
8)Due to the complexity of the skin matrices and the need to quantify low levels of the drug in the epidermis, 9) a highly selective analytical tool is required, since most samples usually contain several endogenous components of the skin. In addition, the analytical method must present enough sensitivity, due to possible very low concentrations of the drug present in each skin layer. 10) Considering the complexity and heterogeneity of the skin tissue, an effective extraction procedure to completely recover the analyte from the ...
