Validation of a UV-VIS Spectrophotometric method for the determination of usnic acid /collagen-based membranes

Abstract

O ácido úsnico (AU) é um metabólito secundário de líquens que tem demonstrado potenciais efeitos farmacológicos, tais como atividade antimicrobiana, antiviral, antiproliferativa e anti-inflamatória. O colágeno, por sua vez, é um dos biomateriais mais utilizados visando o desenvolvimento de sistema de liberação de fármacos. Sendo assim, o objetivo deste estudo foi desenvolver e validar um método analítico por espectrofotometria na região do ultravioleta (UV) para determinação de concentrações de AU em membranas de colágeno. Os parâmetros de validação foram determinados de acordo com a Agência Nacional de Vigilância Sanitária (ANVISA). A especificidade revelou que os excipientes na formulação não interferiram na análise. A linearidade nas concentrações de 2-10 µg/mL apresentou um coeficiente de correlação de 0,9994. O método mostrou excelente repetibilidade (desvio-padrão relativo < 1,0%). A precisão revelou uma recuperação média percentual de 100,43%. O método foi robusto para a variação de temperatura e solvente. Os limites de detecção e de quantificação foram 0,109 e 0,364 g/mL, respectivamente. A taxa total de recuperação das membranas analisados apresentaram valores entre 100,4 e 83,2%.Palavras-chave: ácido úsnico, membranas de colágeno, espectrofotometria UV The usnic acid (UA) is a secondary metabolite of lichen that has shown potential pharmacological effects such as antimicrobial, antiviral, antiproliferative and anti-inflammatory activities. Collagen is one of the most useful biomaterials with various applications as drug delivery systems. The aim of this study was to develop and validate a quantitative UV spectrophotometric method for the determination of UA levels in collagen membranes. The validation parameters were assessed according to the Brazilian Health Surveillance Agency (ANVISA). The specificity revealed that the excipients in the formulation did not interfere with the analysis. The linearity in the range of 2-10 μg/mL presented a correlation coefficient of 0.9994. The method showed excellent repeatability (Relative Standard Deviation (RSD) < 1.0%). The accuracy revealed a mean percentage recovery of 100.43%. The method was robust for the variation of temperature and solvent. The detection and quantization limits were found at 0.109 and 0.364 µg/mL. The total rate recovery from the analyzed membranes showed values between 100.4 and 83.2%.

Short-Term Effects of Critical Metal Concentrations on Usnic Acid Production in Cladonia rangiferina

“…Nickel production. Any p-value less than 0.05 in ANOVA testing shows a significant difference between two treatments (Nunes et al, 2015). The large p-values for all three combinations in Figure 6 indicate that there were not any notable changes in usnic acid production within the metal-free, low nickel, and high nickel treatment groups.…”

“…Previous studies on usnic acid synthesis in lichens have identified several of the chemical reactions needed to make this molecule, but certain steps in the metabolic pathway remain unknown (Nunes et al, 2015). As a result, the majority of usnic acid's properties cannot be predicted, and they are generally discovered through sampling or experimental designs instead.…”

“…A machine called a spectrophotometer calculates color intensity of the media and represents this quantity as an absorbance value. According to the Beer-Lambert equation, these absorbance values are proportional to the concentration of usnic acid excreted by the lichens into the media (Nunes et al, 2015). This means that a 50% decrease in the absorbance reading would indicate a 50% decrease in usnic acid production.…”

“…The ANOVA test with Tukey Test Intervals was used to determine where there were significant changes in usnic acid production between treatment groups. This procedure was chosen because the ANOVA test has been used in several other published studies regarding usnic acid production (Bjerke et al, 2005;Larsson, 2009;Nunes et al, 2015). Also, the accuracy of an ANOVA procedure does not depend on the number of replicates in an experiment, making it ideal for use in a method with only 6 lichens (Nash, 2008).…”

Development and validation of a gas chromatography with a flame ionization detector method for quantitative analysis of four major fatty acids extracted shark liver oil

“…ANOVA test (one-way) was performed based on the absorbance values observed for each ascorbic acid concentration during the replicate measurement of the standard solutions. The linearity of the calibration process was investigated by means of the Lack-offit test (Akritas and Papadatos, 2004;de Souza and Junqueira, 2005;Hsieh and Liu, 2008), the quality coefficient (De Beer et al, 2007;Tellinghuisen, 2008), correlation coefficient (Van Loco et al, 2002;de Souza and Junqueira, 2005;Asuero et al, 2006;Hsieh and Liu, 2008), determination coefficient (Van Loco et al, 2002), ANOVA (Nunes et al, 2015;de Haro Moreno et al, 2018).…”