Update and Revalidation of Ghose's Cellulase Assay Methodology.

New studies on cellulolytic enzymes aiming to improve biofuels production lead to a concern over the assaying methods commonly applied to measure their activity. One of the most used methods is Ghose's cellulase and endoglucanase assay, developed by the International Union of Pure and Applied Chemistry in 1987. Carrying out this method demands high volumes of reagents and generation of high amounts of chemical residues. This work aimed to adapt Ghose's methodology to reduce its application cost and residue generation and validate the adjustments. To do so, International and Brazilian laws were applied to validate methodologies. Method's modifications were successfully validated according to all institutions and were considered linear, accurate, precise, and reproducible. It was possible to reduce the volume of reagents and residues in 12 times. Considering the routine work of most laboratories, it is a great reduction on material costs and residue treatment, which reflects in sustainability and environmental impacts.

Development and validation of a stability indicating high performance liquid chromatography method for trimethobenzamide

A simple, accurate, precise and robust stability indicating RP-HPLC assay method has been developed for the estimation of trimethobenzamide in stress sample. An isocratic separation of trimethobenzamide was achieved on Kromasil 100 C-18 column (250 X 4.6mm, 5µ) with a flow rate of 1.0 ml/min and by using a photodiode array detector to detect the analyte at 213nm. The optimized mobile phase consisted of methanol: ammonium formate (44:56, v/v). The drug was subjected to different forced degradation conditions according to ICH guidelines including acid, base, neutral hydrolysis, oxidation, photolysis and thermal degradation. Degradation products were found only in basic and oxidative degradation conditions. All the degradation products got eluted in an overall analytical run time of 12min. The developed analytical method has been validated according to the ICH guidelines. Response of trimethobenzamide was linear over the concentration range of 0.5-50µg/mL (r2 = 0.999). Accuracy was found to be in between 94.03% to 100.39%. Degradation products resulting from the stress studies did not interfere with the detection of the analyte.

Effect of different storage media on root dentine composition and viability of fibroblasts evaluated by several assay methods.

AIM: To perform multiparametric analysis of the effects of soya milk (SM), whole milk (WM) and Hank's balanced salt solution (HBSS) on the viability of fibroblasts (HGF). The study also aimed to evaluate the influence of these solutions on bovine root dentine according to OH- and PO43- on the surface. METHODOLOGY: The HGF cytotoxicity was determined according to XTT, NR and SRB assays at 1, 3 and 6 h. Root dentine fragments were assessed by Fourier infrared (FTIR) spectrophotometer before and after immersion in the solutions for the same periods. The positive control group included cells and tooth fragments maintained in Dulbecco's modified Eagle's medium (DMEM), and the negative control included tooth fragments that were kept dry. Data were analysed using anova and Tukey's test. RESULTS: No significant difference was found in cell viability evaluated by XTT (P > 0.05). Using the NR assay, WM and HBSS had significantly lower cell viability compared to the positive control group at 6 h (P < 0.05). SM had similar cell viability to the positive control group at all periods evaluated when assessed using all three tests (P > 0.05). A significant difference was found in values of OH- for the negative control group at 1 h (P = 0.002). CONCLUSIONS: Soya milk promoted better cell viability, whereas on dentine composition, the solutions behaved similarly. The association of different assay methods is promising for improving cell viability analysis. The 1-h time-point is a crucial factor in the prognosis of dental replantation because the teeth remain more hydrated and help maintain cell viability.