A novel, simplified and stability-indicating high-throughput ultra-fast liquid chromatography method for the determination of rosmarinic acid in nanoemulsions, porcine skin and nasal mucosa.

Currently, there is an increasing interest on the development of topical formulations containing rosmarinic acid (RA) due to its well-documented antioxidant activity. This study aimed to develop and validate a stability-indicating ultra-fast liquid chromatography (UFLC) method for the determination of RA in nanoemulsions, porcine skin and nasal mucosa intended to be applied in permeation/retention studies and for development of topical nanoemulsions. Chromatographic separation was carried out using a C18 column packed with 2.6 µm particle size in isocratic conditions using as mobile phase water:acetonitrile (83:17, v/v), acidified with 0.1% trifluoracetic acid (v/v), with a total time of analysis of 3.5 min and detection at 330 nm. RA analysis was specific in the presence of both non-biological (blank nanoemulsion and receptor fluid) and biological matrices (porcine ear skin and porcine nasal mucosa). No interference of degradation products of RA was verified after different stress conditions such as acidic, alkaline, oxidative, light exposure (UV-A and UV-C) and thermal demonstrating the method stability-indicating property. The analytical (0.1-10.0 µg·mL-1) and bioanalytical (0.5-10.0 µg·mL-1) linearity was proved by analysis of the calibration curves of RA and no matrix effect was observed. The method was sensitive, precise and accurate, and showed recovery higher than 85%. The method was considered robust as evaluated by a Plackett-Burman experimental design. In the validated conditions, the RA was determined in the nanoemulsions obtained by spontaneous emulsification procedure (1.007 ±â€¯0.040 mg·mL-1), porcine ear skin (1.13 ±â€¯0.19 µg·cm-2) and nasal mucosa (22.46 ±â€¯3.99 µg·cm-2) after retention/permeation studies. Thus, a highly sensitive, simple, fast and stability-indicating method was developed for RA analysis during the development of topical nanoemulsions and bioanalytical assays in complex matrices.

Determination of the main impurities formed after acid hydrolysis of soybean extracts and the in vitro mutagenicity and genotoxicity studies of 5-ethoxymethyl-2-furfural.

Soybean acid hydrolyzed extracts are raw-materials widely used for manufacturing of pharmaceuticals and cosmetics products due to their high content of isoflavone aglycones. In the present study, the main sugar degradation products 5-hydroxymethyl-2-furfural (HMF) and 5-ethoxymethyl-2-furfural (EMF) were quantitatively determined after acid hydrolysis of extracts from different soybean cultivars by a validated liquid chromatography method. The furanic compounds determined in samples cover the range of 0.16-0.21mg/mL and 0.22-0.33mg/mL for HMF and EMF, respectively. Complementarily, due to the scarce literature regarding the EMF toxicology, this study also assessed the EMF mutagenicity by the Salmonella/microsome test and genotoxicity by the comet assay. The results revealed that EMF did not show mutagenicity at the range of 50-5000µg/plate in S. typhimurium strains TA98, TA97a, TA100, TA102 and TA1535, but induced DNA damage in HepG2 cells at non-cytotoxic doses of 0.1-1.3mg/mL, mainly by oxidative stress mechanisms. Based on literature of HMF genotoxicity, and considering the EMF genotoxicity results herein shown, purification procedures to remove these impurities from extracts are recommended during healthcare products development to ensure the security of the products.

A versatile, stability-indicating and high-throughput ultra-fast liquid chromatography method for the determination of isoflavone aglycones in soybeans, topical formulations, and permeation assays.

There is a growing interest in the pharmaceutical field concerning isoflavones topical delivery systems, especially with regard to their skin care properties and antiherpetic activity. In this context, the present work describes an ultra-fast liquid chromatography method (UFLC) for determining daidzein, glycitein, and genistein in different matrices during the development of topical systems containing isoflavone aglycones (IA) obtained from soybeans. The method showed to be specific, precise, accurate, and linear (0.1 to 5 µg mL(-1)) for IA determination in soybean acid extract, IA-rich fraction obtained after the purification process, IA loaded-nanoemulsions, and topical hydrogel, as well as for permeation/retention assays in porcine skin and porcine esophageal mucosa. The matrix effect was determined for all complex matrices, demonstrating low effect during the analysis. The stability indicating UFLC method was verified by submitting IA to acidic, alkaline, oxidative, and thermal stress conditions, and no interference of degradation products was detected during analysis. Mass spectrometry was performed to show the main compounds produced after acid hydrolysis of soybeans, as well as suggest the main degradation products formed after stress conditions. Besides the IA, hydroxymethylfurfural and ethoxymethylfurfural were produced and identified after acid hydrolysis of the soybean extract and well separated by the UFLC method. The method's robustness was confirmed using the Plackett-Burman experimental design. Therefore, the new method affords fast IA analysis during routine processes, extract purification, products development, and bioanalytical assays.