Comparison of three experimental designs employed in gentamicin microbiological assay through agar diffusion

Abstract

Gentamicin is a broad-spectrum antibiotic complex produced by actinomycetes belonging to Micromonospora genus and classified among aminoglycoside antibiotics, used in the treatment of serious infections derived from Gram-negative microorganisms. Alterations of their antimicrobial activity not shown in chemical assays can be evaluated through microbiological assays. The aim of this work was to compare 5 x 1, 2 x 2 and 3 x 1 experimental designs, evaluating validation parameters of specificity, linearity, range, precision, and accuracy for each experimental design in different levels of concentration, presentation, and lots. It consisted of 81 assays (in 3 replicas) of gentamicin microbiological dosage. The concentrations of the solutions used were employed in a range from 1.0 &#956;g/mL to 5.0 &#956;g/mL, diluted in phosphate buffer 0.1 M pH 8.0. Antibiotic medium number 11 was used, with Staphyloccocus epidermis (ATCC 12228). 21ml of medium were used as base layer and 4 ml of medium inoculated at 1% were used as surface layer. The dishes were incubated for 18 hours at 37 ± 1 ºC. The three designs employed showed adequate specificity for analysis of dermatological cream and injectable solution containing gentamicin sulphate. They also showed accuracy and linearity in the range evaluated, but not a significant difference concerning precision. The results were compared by means of the determination of the rates of measurement system capacity. The statistical analysis demonstrated that there is no significant difference among the results obtained through 5 x 1, 2 x 2, and 3 x 1, being these equivalent and interchangeable.<br>A gentamicina é um complexo antibiótico de largo espectro, produzido por actinomicetos do gênero Micromonospora e classificado entre os antibióticos aminoglicosídeos, utilizado no tratamento de infecções graves, devidas a microrganismos Gram-negativos. Alterações da sua atividade antimicrobiana, não demonstradas pelos ensaios químicos, podem ser avaliadas pelos ensaios microbiológicos. O objetivo deste trabalho foi comparar os delineamentos experimentais 5 x 1, 2 x 2 e 3 x 1, avaliando-se os parâmetros de validação de especificidade, linearidade, faixa ou intervalo, precisão e exatidão para cada delineamento experimental em diferentes níveis de concentração, apresentações e lotes. O plano de trabalho constituiu-se na realização de 81 ensaios (em 3 réplicas) de doseamento microbiológico de gentamicina. As concentrações das soluções empregadas foram preparadas numa faixa de 1,0 &#956;g/mL a 5,0 &#956;g/mL, diluídos em tampão fosfato 0,1 M pH 8,0. O meio utilizado foi o meio antibiótico no. 11, com Staphyloccocus epidermidis (ATCC 12228). Empregou-se 21 mL de meio como camada base e 4 mL de meio inoculado à 1% como camada superfície. As placas foram incubadas por 18 horas à 37 ± 1 °C. Os três delineamentos empregados apresentaram especificidade adequada para análise de creme dermatológico e solução injetável contendo sulfato de gentamicina. Também apresentaram exatidão e linearidade no intervalo ava...

Development and Validation of Microbiological Assay for Ceftriaxone and its Application in Photo-stability Study

“…The United States Pharmacopeia experimental design [7,12] was adopted in the determination of the potencies. Commercial samples were diluted in phosphate buffer pH 6.0 in order to obtain a 4.0 µg/mL concentration solution.…”

Sensitive immunochemical approaches for quantitative (FPIA) and qualitative (lateral flow tests) determination of gentamicin in milk

“…Rapid and fast test methods for determination of gentamicin on-site are representing a very useful and perspective tool for agriculture and diary allowing on-time to remove unsafe livestock and milk from production. For preliminary screening of aminoglycosides in milk some rapid microbiological tests were repeatedly described [13][14][15] and even commercially available (Delvotest SP, SNAP test). The shortcomings of this class of the detection techniques as an extensive incubation time and deficient sensitivity resulted in a progressive replacement of microbiological kits by the rapid and convenient immunochemical rapid tests [Ballyabio (the cut-off is the EU MRL), Quicking (the cut-off is 50 ppb), Ballya combo (the cut-off is the EU MRL)].…”

Simple Estimation of Uncertainty in the Quantification of Cefazolin by HPLC and Bioassay

“…It is highly advisable to adopt an experimental planning which, without further effort, supplies better results and enables an indication respecting the assay validity. The number and nature of the samples, as well as the results reliability level, are among the most important factors to be taken into account, in the selection of a design [13][14][15].…”