Immortalized keratinocytes cells generates an effective model of Epidermal Human Equivalent for irritation and corrosion tests.

Three-dimensional skin models, also named 3D skin models, human skin equivalents (HSEs), or Human Epidermal Equivalents (HEEs), have been increasingly used for chemical assessments in terms of efficacy and safety. Considering this, we developed an HEE model using immortalized HaCaT cells, aiming to overcome the limitation of primary tissue source. Our 3D model (HaCaT-HEE) exhibited important markers of cell differentiation (CK10, CK14, involucrin, and filaggrin), although the stratum corneum was shown to be modest. Besides, the model showed a good prediction potential considering membrane permeability, sensitivity, specificity, and accuracy in distinguishing irritant and corrosive effects after exposure to selected chemicals recommended by the OECD protocols. We also validated the formazan determination for the MTT method using High-Performance Liquid Chromatography (HPLC). For that, we considered carry over, linearity, reproducibility/robustness, accuracy, precision, selectivity, and matrix effect, according to the Food and Drug Administration (FDA) guideline. Based on our results, we can conclude that our model has an acceptable predictive value for the safety evaluation of compounds after skin exposure, with the great advantage of being constructed using immortalized cells.

Development and validation of a method for the determination of folic acid in different pharmaceutical formulations using derivative spectrophotometry

ABSTRACT Folic acid is a B complex water-soluble vitamin that is essential to humans, and its deficiency can cause problems including congenital malformations in the fetus as well as heart disease. Most countries affected by diseases associated with a lack of folic acid now supplement foods with the vitamin. There is therefore a need for the development of new analytical procedures able to determine folic acid present in different matrices. This work describes the development of zero order and first order derivative spectrophotometric methods for the determination of folic acid in different pharmaceutical formulations, using 0.1 mol L-1 NaOH as solvent. The methods are shown to be simple, selective, and robust. Good linearity was achieved, with correction coefficients ≥0.9996 and limits of detection and quantification ranging from 0.64 to 0.75 and from 1.80 to 2.85 mg L-1, respectively. Recoveries of 98-104% were obtained in accuracy tests, and precision (as RSD) was between 0.2 and 4.8%. The methods can be used in routine analyses for quality control purposes, offering an alternative to the procedures already reported in the literature

Simultaneous determination of abamectin homologs H 2 B 1a and H 2 B 1b in gel formulation by high performance liquid chromatography

ABSTRACT Abamectin is a drug with antiparasitic properties used in several pharmaceutical formulations. The objective of this research was to develop and validate a high performance liquid chromatographic (HPLC) method for quantification of the two abamectin homologs (H2B1a and H2B1b) in gel formulation. This HPLC method was validated using a LichroCart(r) 100 RP-18 (125 x 4 mm, 5 µm) column. The mobile phase contained of acetonitrile and water (95:5 v/v) with 1% acetic acid. The flow rate was 1.0 mL min-1 and UV detection was performed at 245 nm. Mobile phase solutions were prepared containing a nominal concentration 185.2 µg mL-1 H2B1a and 9.6 µg mL-1 H2B1b. The method displayed good linearity in the concentration range of 148.1 - 222.3 µg mL-1 and 7.7 - 11.5 µg mL-1, for H2B1a and H2B1b, respectively, with a correlation coefficient of (r)> 0.99 for both compounds, calculated by the least mean squares method. Detection limits (DLs) were 2.8 µg mL-1 and 1.2 µg mL-1 and quantitation limits (QLs) were 8.6 µg mL-1 and 3.8 µg mL-1, for H2B1a and H2B1b, respectively. The method is simple, economical and efficient for the quantitative determination of abamectin H2B1a and H2B1b homologs in pharmaceutical preparations.

Rapid and sensitive ultra-high-pressure liquid chromatography method for quantification of antichagasic benznidazole in plasma: application in a preclinical pharmacokinetic study.

Benznidazole (BNZ) and nifurtimox are the only drugs available for treating Chagas disease. In this work, we validated a bioanalytical method for the quantification of BNZ in plasma aimed at improving sensitivity and time of analysis compared with the assays already published. Furthermore, we demonstrated the application of the method in a preclinical pharmacokinetic study after administration of a single oral dose of BNZ in Wistar rats. A Waters® Acquity UHPLC system equipped with a UV-vis detector was employed. The method was established using an Acquity® UHPLC HSS SB C18 protected by an Acquity® UHPLC HSS SB C18 VanGuard guard column and detection at 324 nm. The mobile phase consisted of ultrapure water-acetonitrile (65:35), and elution was isocratic. The mobile phase flow rate was 0.55 mL/min, the volume of injection was 1 µL, and the run time was just 2 min. The samples were kept at 25°C until injection and the column at 45°C for the chromatographic separation. The sample preparation was performed by a rapid protein precipitation with acetonitrile. The linear concentration range was 0.15-20 µg/mL. The pharmacokinetic parameters of BNZ in rats were determined and the method was considered sensitive, fast and suitable for application in pharmacokinetic studies.

Celecoxib determination in different layers of skin by a newly developed and validated HPLC-UV method.

A simple, rapid and sensitive analytical procedure for the measurement of celecoxib (CXB) levels in skin samples after in vitro penetration studies was developed and validated. In vitro permeability studies in porcine skin were performed for quantification of CXB at different layers of skin, the stratum corneum (SC) and epidermis plus dermis (EP + D) as well as in the acceptor solution (AS) to assess CXB permeation through skin. CXB was quantified by HPLC using a C18 column and UV detection at 251 nm. The mobile phase was methanol-water 72:28 (v/v) and the flow-rate was 0.8 mL/min. The CXB retention time was 5 min. The assay was linear for CBX in the concentration range of 0.1-3.0 µg/mL in the AS (drug permeated through skin) and 5.0-50.0 µg/mL for drug retained in SC and [EP + D] in vitro. The linear correlation coefficients for the different calibration curves were equal or greater than 0.99. Intra- and inter-assay variabilities were below 8.0%. Extraction of CXB from skin samples showed recoveries higher than 95.0% after 15 min of ultrasonic sound and centrifugation at 2500 rpm for 3 min. The method was considered appropriate for the assay of CXB in skin samples, after in vitro cutaneous penetration studies.