Development of agar diffusion method for dosage of gramicidin

Abstract

Gramicidin, an antimicrobial peptide active against Gram positive bacteria, is commonly used in pharmaceutical preparations for topical use. Considering that only the turbidimetric method has been described in the literature, the present study sought to develop and validate an agar diffusion method for the dosage of gramicidin. The method was developed and validated using the Kocuria rhizophila ATCC 9341 as a test microorganism. Two designs were used: a 3x3 parallel-line model, and a 5x1 standard curve. The validation demonstrated that the method follows the linear model (r 2 = 0.994), presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 5 to 25.3 μg/mL. The results obtained for both designs were precise, having a relative standard deviation (R.S.D.) for intra-day precision of 0.81 for the 3x3 assay and 1.90 for the 5x1 assay. For the inter-day precision, the R.S.D. was 1.35 for the 3x3 and 2.64 for the 5x1. The accuracy was verified and results confirmed to be accurate, having a tolerance interval of 95%, which lay within permitted limits and appropriate trueness. In addition, the method was considered selective, with limit of detection and upper and lower limits of quantification of 2.00, 5.00 and 25.3 μg/mL, respectively. No difference in precision between the designs used in the agar diffusion method was evident (p>0.05). The method proved to be appropriate for the microbiological dosage of the raw material gramicidin.Uniterms: Gramicidin/microbiological assay. Microbiological assay/method validation. Agar diffusion. Cylinder-plate.A gramicidina, um peptídeo antimicrobiano ativo contra bactérias Gram positivo, é utilizada em preparações farmacêuticas de uso tópico. Neste trabalho procurou-se desenvolver e validar outro método para o doseamento de gramicidina tendo em vista que somente o método turbidimétrico é descrito. O método de difusão em ágar foi desenvolvido e validado utilizando como microrganismo teste Kocuria rhizophila ATCC 9341. Foram utilizados dois delineamentos: retas paralelas 3x3 e curva padrão 5x1. A validação demonstrou que o método segue o modelo linear (r 2 = 0,994) havendo regressão significativa entre o diâmetro dos halos de inibição e o logaritmo da concentração na faixa de 5,00 a 25,3 µg/mL. Os resultados obtidos por ambos os delineamentos foram precisos apresentando desvio padrão relativo (DPR) para precisão intra-dia de 0,81 para ensaio 3x3 e de 1,90 para ensaio 5x1. Para a precisão interdias o DPR foi de 1,35 para 3x3 e de 2,64 para 5x1. A exatidão foi verificada e os resultados foram exatos apresentando intervalo de tolerância a 95% dentro dos limites permitidos e veracidade adequada. O método foi seletivo com limites de detecção e quantificação inferior e superior iguais a 2,00, 5,00 e 25,3 µg/mL, respectivamente. Não foi observada diferença entre a precisão dos delineamentos empregados no método de difusão em ágar (p>0.05). O método se mostrou adequado para a dosagem microbiológica de gramic...

Microbiological Antibiotic Assay Validation of Gentamicin Sulfate Using Two-Dose Parallel Line Model (PLM)

“…The investigated design did not show any fixed or absolute tendency and relative tendency, nor being necessary to employ any kind of correction or adjustment of the results obtained [24]. The method had appropriate accuracy, as could be confirmed by the values calculated for the β-tolerance interval (Figure 3) for each concentration level, which showed a maximum variation of ± 17.8% for the current two-dose symmetrical design [39,[49][50][51].…”

“…While the analysis criteria for individual compounds may show wide variations in the commercial products that could reach 20% in the range, it would be necessary to use a sort of biological test to estimate the net resultant true activity with this complex mixture of microbiologically active entities [3,6,16]. Moreover, Intra-laboratory and inter-laboratory variation mitigation in the microbiological assay might be acquired through the implementation of the international guidelines for the antibiotic assay that include media composition, reagents and assay conditions [39].…”

“…Accuracy is represented by the combination of the random (precision) and systematic (trueness) errors, which were considered in the β-tolerance interval calculation. This represents the interval in which β percentage of the future individual results would be expected [39,50]. According to the trueness parameter, there was no evidence indicating systematic errors in either experimental design [39,49,52].…”

Validation of Symmetrical Two-Dose Parallel Line Assay Model for Nystatin Potency Determination in Pharmaceutical Product

“…The original four calibration curves for nystatin were previously constructed (Table 3) from which the pooled standard curve in Figure 1 was charted. All presented a coefficient of correlation (r) greater than 0.98 according to the Brazilian reference (2003) and FDA (2001) and when compared, no significant difference was found between the intercepts and slopes 39 . This will ensure that the high and low doses -viz 10 and 20 μg/ml -will fall within linear ranges covering overage or decline in the target concentration.…”

Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution

“…However, in the literature, there are a large number of reports describing the use of microbiological assays to assess the potency of many antibiotics (Marona, Schapoval, 1998;Mendez et al, 2005;Gomes, Salgado, 2006;Salgado, Lopes, Lucchesi, 2006;Vaugher, Breier, Schapoval, 2006;Souza et al, 2007;Moreno, Salgado, 2007;Salgado, Tozo, 2007;Schmidt et al, 2008;Lopes, Salgado, 2010;Cazedey, Salgado, 2011;Paim et al, 2011;Solano et al, 2011).…”