Risk of false conformity decisions due to measurement uncertainty of Active Pharmaceutical Ingredient: A multiparametric evaluation

Abstract Medicines must be subject to physical, chemical, and biological analysis to guarantee their quality, safety, and effectiveness. Despite the efforts to ensure the reliability of analytical results, some uncertainty will always be associated with the measured value, which can lead to false decisions regarding conformity/non-conformity assessment. This work aims to calculate the specific risk of false decisions regarding conformity/non-conformity of acetaminophen oral solution dosage form. The acetaminophen samples from five different manufacturers (A, B, C, D, and E) were subject to an active pharmaceutical ingredient assay, density test, and dose per drop test according to the official compendia. Based on measured values and their respective uncertainties, the risk values were calculated using the Monte Carlo method implemented in an MS Excel spreadsheet. The results for two acetaminophen oral solution samples (C and D) provided an increased total risk value of false acceptance (33.1% and 9.6% for C and D, respectively). On the other hand, the results for the other three acetaminophen samples (A, B, and E) provided a negligible risk of false acceptance (0.004%, 0.025%, and 0.045% for A, B, and E, respectively). This indicates that measurement uncertainty is very relevant when a conformity assessment is carried out, and information on the risks of false decisions is essential to ensure proper decisions.

Multivariate guard-bands and total risk assessment on multiparameter evaluations with correlated and uncorrelated measured values

Abstract The quality, efficacy, and safety of medicines are usually verified by analytical results. Measurement uncertainty is a critical aspect for the reliability of these analytical results. The pharmacopeial compendia usually adopt a simple acceptance rule that does not consider information from measurement uncertainty. In this work, we compared decision-making using simple acceptance and decision rules with the use of guard-band for multiparameter evaluation of ofloxacin ophthalmic solution and acyclovir topical cream. Ciprofloxacin ophthalmic solution and acyclovir topical cream samples were subject to pharmacopeial tests and assays. Multivariate guard-band widths were calculated by multiplying the standard uncertainty (u) by an appropriate multivariate coverage factor (k'). The multivariate coverage factor (k') was obtained by the Monte Carlo method. According to the simple acceptance rule, all the results obtained for ciprofloxacin ophthalmic solution and acyclovir topical cream are within the specification limits. However, the risk of false conformity decisions increases for ciprofloxacin tests. Decisions made using the simple acceptance rule and decision rules with the use of guard-band may differ. The simple acceptance rule may increase the risk of false conformity decisions when the measured value is close to the regulatory specification limits and/or when the measurement uncertainty value is inappropriately high. Nevertheless, the guard-band decision rule will always reduce the risk of false conformity decisions. Therefore, using information on measurement uncertainty in conformity assessment is highly recommended to ensure the proper efficacy, safety, and quality of medicines.

Desirability Function in analytical method development for determination of glitazones and metabolites employing HF-LPME

Abstract Thiazolidinedione, often shortened to TZD or glitazone, helps lower insulin resistance, which is the underlying problem for many people with type 2 diabetes. The two most known glitazones are pioglitazone (PGZ), with the brand name medicine Actos®, and rosiglitazone (RSG), which is Avandia®. This study presented a multivariate optimization in the microextraction procedure employing Fractional Factorial Design (FFD) combined with Desirability Function (DF) to determine TZD and metabolites in biological samples. Microextraction requires several parameters to be optimized; however, most of them still use univariate optimization. Finding optimum conditions by simple response is relatively simple, but the problems, in case of microextractions, are often more complex when it has more responses. For example, changing one factor that promotes one response may suppress the effect of the others. Thus, this multivariate optimization was applied for two bioanalytical methods for determination of TZD and metabolites, one by HPLC and other by CE, both using Hollow Fiber Liquid-Phase Microextraction (HF-LPME). The results establish the optimal values and elucidate how the factors that affect HF-LPME procedure perform in extraction efficiency for TZDs. Additionally, this study demonstrates that DF can be an important tool to optimize microextraction procedures.

Rapid identification of microbial contaminants in pharmaceutical products using a PCA/LDA-based FTIR-ATR method

Microbiological quality of pharmaceuticals is fundamental in ensuring efficacy and safety of medicines. Conventional methods for microbial identification in non-sterile drugs are widely used; however they can be time-consuming and laborious. The aim of this paper was to develop a chemometric-based rapid microbiological method (RMM) for identifying contaminants in pharmaceutical products using Fourier transform infrared with attenuated total reflectance spectrometry (FTIR-ATR). Principal components analysis (PCA) and linear discriminant analysis (LDA) were used to obtain a predictive model capable of distinguishing Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), Enterococcus faecium (ATCC 8459), Escherichia coli (ATCC 8739), Micrococcus luteus (ATCC 10240), Pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538), and Staphylococcus epidermidis (ATCC 12228) microbial growth. FTIR-ATR spectra provide data on proteins, DNA/RNA, lipids, and carbohydrates constitution of microbial growth. Microbial identification provided by PCA/LDA based on FTIR-ATR method were compatible with those obtained using traditional microbiological methods. The chemometric-based FTIR-ATR method for rapid identification of microbial contaminants in pharmaceutical products was validated by assessing the sensitivity (93.5%), specificity (83.3%), and limit of detection (17-23 CFU/mL of sample). Therefore, we propose that FTIR-ATR spectroscopy may be used for rapid identification of microbial contaminants in pharmaceutical products and taking into account the samples studied

Intra-articular concentration of gentamicin administered by intravenous regional limb perfusion in healthy horses / Concentração intra-articular de gentamicina administrada por perfusão regional intravenosa em membros de equinos sadios

ABSTRACT: Intravenous regional limb perfusion (IRLP) is an efficient method to treat horses with synovial infections. However, information on the dose, volume, and intervals of administration is lacking. The aim of this study was to evaluate the intra-articular concentration of gentamicin administered by IRLP in horses over 24 h post-administration and evaluate the influence of the total perfused volume. Twenty horses were assigned to two treatment groups. Gentamicin 60 group (G60, n=10) and Gentamicin 250 group (G250, n=10) received IRLP of 6.6 mg/kg of gentamicin diluted in Ringer's lactate for a total volume of 60 ml (G60) and 250 ml (G250), respectively. Synovial fluid harvests were performed in the metacarpophalangeal joint before (0) and 4, 8, 12, 16, 20, and 24 hours after IRLP. Agar diffusion was the assay for measuring gentamicin concentrations. Four hours after IRLP, the intra-articular concentration of gentamicin was 47.58±49.21 μg/ml in G60 and 10.92±6.11 μg/ml in G250. During the 24 h of harvest, the intra-articular concentration of gentamicin remained above the minimum inhibitory concentration for horses (MIC; 2 μg/ml) in both experimental groups. In G60, the concentration of gentamicin in synovial fluid reached the maximal concentration and remained 8 to 10-fold higher than the MIC (Cmax: 16 to 20 μg/ml) during 12 h after IRLP. These findings support that IRLP with 6.6 mg/kg of gentamicin diluted to a volume of 60 ml promotes intra-articular concentrations higher than 250 ml, remaining above the MIC for 24 h and at the maximum concentration for 12 h.

RESUMO: A perfusão regional intravenosa (PRI) em membros de equinos é um método comprovadamente eficiente para o tratamento de infecções sinoviais, porém ainda existem lacunas quanto à dose, volume e intervalos de administração a serem utilizados. Neste estudo, objetivou-se avaliar a concentração intra-articular da gentamicina administrada por PRI em equinos, ao longo de 24 horas pós-administração, bem como avaliar a influência do volume total perfundido. Vinte equinos foram distribuídos em dois grupos experimentais, Grupo Gentamicina 60 (G60) e Grupo Gentamicina 250 (G250), representados por 10 equinos em cada grupo que receberam, por PRI, 6,6 mg/kg de gentamicina diluída em Ringer lactato para um total de 60 ml (G60) ou 250 ml (G250). As colheitas de líquido sinovial foram realizadas, na articulação metacarpo-falangeana, antes do início do experimento e após 4, 8, 12, 16, 20, e 24 horas. Difusão em ágar foi o método para doseamento das concentrações de gentamicina. Quatro horas após a PRI, a concentração intra-articular da gentamicina era de 47,58±49,21 µg/ml no G60 e 10,92±6,11 µg/ml no G250. Durante as 24 horas de colheita, a concentração intra-articular de gentamicina permaneceu acima da concentração inibitória mínima (CIM) (2 μg/ml) em ambos os grupos experimentais. No G60, as concentrações de gentamicina no líquido sinovial atingiram a concentração máxima e mantiveram-se 8 a 10 vezes mais elevadas que a CIM (Cmáx: 16 a 20 μg/ml) durante 12 horas após a PRI. Conclui-se que a PRI em membros, com 6,6 mg/kg de gentamicina diluída para o volume de 60 ml, promove concentrações intra-articulares mais elevadas que o volume de 250 ml, permanecendo acima da CIM durante 24 horas e em concentração máxima durante 12 horas.

Design of Experiments (DoE) applied to Pharmaceutical and Analytical Quality by Design (QbD)

According to Quality by Design (QbD) concept, quality should be built into product/method during pharmaceutical/analytical development. Usually, there are many input factors that may affect quality of product and methods. Recently, Design of Experiments (DoE) have been widely used to understand the effects of multidimensional and interactions of input factors on the output responses of pharmaceutical products and analytical methods. This paper provides theoretical and practical considerations for implementation of Design of Experiments (DoE) in pharmaceutical and/or analytical Quality by Design (QbD). This review illustrates the principles and applications of the most common screening designs, such as two-level full factorial, fractionate factorial, and Plackett-Burman designs; and optimization designs, such as three-level full factorial, central composite designs (CCD), and Box-Behnken designs. In addition, the main aspects related to multiple regression model adjustment were discussed, including the analysis of variance (ANOVA), regression significance, residuals analysis, determination coefficients (R2, R2-adj, and R2-pred), and lack-of-fit of regression model. Therefore, DoE was presented in detail since it is the main component of pharmaceutical and analytical QbD.

Measurement uncertainty of dissolution test of acetaminophen immediate release tablets using Monte Carlo simulations

ABSTRACT Analytical results are widely used to assess batch-by-batch conformity, pharmaceutical equivalence, as well as in the development of drug products. Despite this, few papers describing the measurement uncertainty estimation associated with these results were found in the literature. Here, we described a simple procedure used for estimating measurement uncertainty associated with the dissolution test of acetaminophen tablets. A fractionate factorial design was used to define a mathematical model that explains the amount of acetaminophen dissolved (%) as a function of time of dissolution (from 20 to 40 minutes), volume of dissolution media (from 800 to 1000 mL), pH of dissolution media (from 2.0 to 6.8), and rotation speed (from 40 to 60 rpm). Using Monte Carlo simulations, we estimated measurement uncertainty for dissolution test of acetaminophen tablets (95.2 ± 1.0%), with a 95% confidence level. Rotation speed was the most important source of uncertainty, contributing about 96.2% of overall uncertainty. Finally, it is important to note that the uncertainty calculated in this paper reflects the expected uncertainty to the dissolution test, and does not consider variations in the content of acetaminophen.

Equivalência Farmacêutica de produtos oncológicos comercializados no Brasil / Pharmaceutical equivalence of oncology products marketed in Brazil

The aim of this work was to assess pharmaceutical equivalence among medicinal products containing cisplatin, doxorubicin hydrochloride and paclitaxel that are marketed in Brazil by various manufacturers. We analyzed 14 lots of cisplatin injectable solution from 4 manufacturers (Labs B, C, H and I), 15 lots of doxorubicin hydrochloride injectable lyophilized powder from 5 manufacturers (Labs C, F, G, H and J) and 38 lots of paclitaxel injectable solution from 8 manufacturers (Labs A, B, C, D, E, F, G and H). All products complied with the criteria established in the Brazilian and American pharmacopoeias. The assay results for contents of cisplatin, doxorubicin hydrochloride and paclitaxel were 94.3-105.9%, 97.1-106.6% and 90.2-109.4%, respectively. Statistical analysis showed that the same products from the various manufacturers were equivalent.

O objetivo deste trabalho foi avaliar a equivalência farmacêutica entre medicamentos de diferentes fabricantes contendo cisplatina, doxorrubicina cloridrato e paclitaxel comercializados no Brasil. Foram analisados 14 lotes de cisplatina solução injetável de 4 fabricantes (laboratórios B, C, H e I), 15 lotes de doxorrubicina cloridrato pó liófilo injetável de 5 fabricantes (laboratórios C, F, G, H e J) e 38 lotes de paclitaxel solução injetável de 8 fabricantes (laboratórios A, B, C, D, E, F, G e H). Todos os produtos apresentaram resultados em conformidade com os critérios estabelecidos nas farmacopeias brasileira e americana. Os resultados de teores obtidos para cisplatina, doxorrubicina cloridrato e paclitaxel foram 94,3-105,9 %, 97,1-106,6 % e 90,2-109,4%, respectivamente. A análise estatística demonstrou que há equivalência entre produtos dos diferentes fabricantes avaliados.

Comparison of dissolution profile of extended-release oral dosage forms - two one-sided equivalence test

The aim of this work is to present the two one-sided test (TOST) as an alternative approach to compare dissolution profiles of extended-release dosage forms. The dissolution profiles of oxycodone extended-release tablets containing 10 mg, 20 mg and 40 mg (reference and generic) were evaluated according to the requirements described in United States Pharmacopeia. These dissolution profiles were compared using the conventional similarity factor (f2) and the proposed TOST as an equivalence test. TOST is a simple and alternative approach to compare dissolution profiles of extended-release dosage forms. It allows us to identify the time-point (or time-points) that did not show similarity. We concluded that the two one-sided test performed at a significance level of 5% and defined as D = 10 showed results comparable to those obtained by the conventional similarity factor (f2).

O objetivo deste trabalho é apresentar o teste uni-caudal duplo (TOST) como uma abordagem alternativa na comparação do perfil de dissolução de formas farmacêuticas de liberação prolongada. Os perfis de dissolução de comprimidos de liberação prolongada de oxicodona contendo 10 mg, 20 mg e 40 mg (genérico e referência) foram avaliados de acordo com os requisitos descritos na Farmacopeia Americana. Estes perfis de dissolução foram comparados empregando-se o fator de semelhança convencional (f2) e o método TOST como teste de equivalência. TOST é uma abordagem simples e alternativa para a comparação de perfis de dissolução de formas farmacêuticas de liberação prolongada. Este permite identificar o ponto (ou pontos) que não apresentou semelhança. Considerando-se D = 10, concluímos que o teste uni-caudal duplo num nível de significância de 5% apresenta resultados comparáveis àqueles obtidos com o fator de semelhança convencional (f2).

Using fluid bed granulation to improve the dissolution of poorly water-soluble drugs

In this study, fluid bed granulation was applied to improve the dissolution of nimodipine and spironolactone, two very poorly water-soluble drugs. Granules were obtained with different amounts of sodium dodecyl sulfate and croscarmellose sodium and then compressed into tablets. The dissolution behavior of the tablets was studied by comparing their dissolution profiles and dissolution efficiency with those obtained from physical mixtures of the drug and excipients subjected to similar conditions. Statistical analysis of the results demonstrated that the fluid bed granulation process improves the dissolution efficiency of both nimodipine and spironolactone tablets. The addition of either the surfactant or the disintegrant employed in the study proved to have a lower impact on this improvement in dissolution than the fluid bed granulation process.

Antibiotic microbial assay using kinetic-reading microplate system / Ensaio microbiano antibiótico utilizando cinética de leitura de microplacas sistema

The aim of this study was to determine the optimal experimental conditions to develop a methodology for microbiological assay of apramycin employing microplate and kinetic reading mode, and to validate the developed method, through evaluation of parameters of selectivity, linearity, linear range, limits of detection and quantification, accuracy and precision. The turbidimetric assay principle is simple: the test solution is added to a suspension of test microorganism in culture media, the mixture is incubated under appropriate conditions and the microbial growth is measured by photometric reading. Microplate with kinetic reading mode employed in antibiotic assay is of considerable interest since it allows reduction of material and analysis time and enables a large number of samples to be analyzed simultaneously, with automated reading and calculating. Established conditions considered the standard-curve of apramycin at concentrations from 5.0 to 35.0 μg mL-1, and tryptic soy broth inoculated with 5 percent Escherichia coli (ATCC 8739) suspension. Satisfactory results were obtained with 2 hours of incubation. The developed method showed appropriate selectivity, linearity in the range from 5.0 to 35.0 μg mL-1, limits of detection and quantification of 0.1 and 0.4 μg mL-1, respectively, as well as satisfactory accuracy (recuperation = 98.5 percent) and precision (RSD = 6.0 percent). Microplate assay combined the characteristics of microbiological (evaluation of antibiotic activity against sensitive test microorganism) and physico-chemical (operationally straightforward and faster results) assays.

O objetivo deste trabalho é determinar as condições experimentais ideais para o desenvolvimento de metodologia para a dosagem microbiológica de apramicina empregando microplacas e modo de leitura cinético e validar o método desenvolvido, através da avaliação dos parâmetros de especificidade e seletividade, linearidade, faixa ou intervalo linear, limite de detecção e quantificação, exatidão e precisão. O ensaio turbidimétrico é simples: a solução-teste é adicionada à suspensão de microrganismo-teste em meio de cultura, a mistura é incubada em condições apropriadas e o crescimento microbiano é medido por meio de leitura fotométrica. O emprego de método de microplacas com leitura cínética para a dosagem de antibióticos é de interesse considerável, uma vez que possibilita reduzir quantidade de material e tempo de análise necessários e permite o ensaio de grande número de amostras simultaneamente, com leitura e cálculo automatizados. As condições estabelecidas abrangem curva-padrão de apramicina com concentrações entre 5 e 35 μg/mL, e emprego de meio de cultura caldo de triptona-soja inoculado com Escherichia coli (ATCC 8739) na proporção de 5 por cento. Foram obtidos resultados satisfatórios após 2 horas de incubação. O método desenvolvido apresentou especificidade e seletividade adequadas, linearidade na faixa de 5 a 35 μg/mL, limite de detecção e quantificação de 0,1 e 0,4 μg/mL, respectivamente, exatidão (recuperação = 98,5 por cento) e precisão (DPR = 6,0 por cento) satisfatórias. O ensaio em microplaca agrega características dos ensaios microbiológicos (avaliação da atividade do antibiótico frente a microrganismo-teste sensível) e físico-químicos (facilidade operacional e maior rapidez na obtenção dos resultados).

Determination of apramycin in oral soluble powder by a HPLC method using pre-column derivatization with o-phthalaldehyde and UV detection / Determinação da apramicina em pó solúvel oral por um método de HPLC utilizando pré-coluna de derivação com o de detecção de ftalaldeído e UV

A high-performance liquid chromatographic method employing pre-column derivatization with o-phthalaldehyde (OPA) and 2-mercaptoacetic acid was developed for the determination of apramycin, an aminoglycoside antibiotic used in veterinary medicine, in the oral soluble powder form. The chromatographic separation was done by ion-pair HPLC using a C18 reversed-phase column, Synergy Hydro (150 mm x 4.6 mm x 4 µm) and mobile phase composed of 0.005 mol/L sodium octanosulfonate in a mixture of acetonitrile: water: acetic acid (45:55:2) (v/v/v) with a flow rate of 1.0 mL/min; the UV detector was operated at 332 nm. The developed method was validated according to official compendia guidelines, having demonstrated robustness, selectivity and linearity for the concentration range of 0.02 to 0.05 mg/mL, precision (with RSD < 2.0 percent both for intra and inter-day precision) accuracy (average recuperation of 99.33 percent) and detectivity (quantification and detection limits of 0.08 and 0.02 µg/mL, respectively). Three batches of commercial apramycin oral soluble powder were analyzed by both the proposed method and the official microbiological method, where all the results obtained were in the acceptable range (95 percent to 105 percent of labeled value of apramycin). Both methods were statistically compared by the t test, which yielded no significant differences (α = 0.05) thereby confirming the equivalence of the methods.

Foi desenvolvido um método por cromatografia líquida alta eficiência empregando derivatização pré-coluna com o-ftalaldeído (OPA) e ácido mercaptoacético para determinação de apramicina, um antibiótico aminoglicosídeo de uso veterinário, em pó oral solúvel. A separação cromatográfica foi feita por fase reversa com pareamento iônico utilizando-se coluna Synergy Hydro C18 (150 x 4,6 mm x 4 µm) e fase móvel composta por octanossulfonato de sódio 0,005 mol/L em mistura de acetonitrila:água:ácido acético nas proporções 45:55:2 (v/v/v), numa vazão de 1.0 mL/min; efetuou-se detecção por UV a 332 nm. O método foi validado de acordo com os compêndios oficiais e demonstrou robustez, seletividade, linearidade na faixa de 0,02 a 0,05 mg/mL, precisão (com DPR < 2,0 por cento tanto para a precisão intra-dia quanto para a precisão inter-dia), exatidão (recuperação média de 99,33 por cento) e detectabilidade (limite de quantificação e de detecção iguais a 0,08 e 0,02 µg/mL, respectivamente). Analisaram-se 3 lotes de apramicina pó oral solúvel pelo método proposto e pelo método microbiológico oficial e todos os resultados obtidos estavam dentro do limite de aceitação (95 por cento-105 por cento valor rotulado de apramicina). Ambos os métodos foram comparados estatisticamente pelo teste t, não sendo encontradas diferenças significativas entre eles para α=0,05, sendo os dois equivalentes.

Comparison of three experimental designs employed in gentamicin microbiological assay through agar diffusion / Comparação de três modelos experimentais empregados no ensaio microbiológico de gentamicina por difusão em ágar

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 μg/mL to 5.0 μg/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 percent 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.

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 μg/mL a 5,0 μg/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 por cento como camada superfície. As placas foram incubadas por 18 horas à 37 ± 1 ºC. 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 avaliado. Os delineamentos não apresentaram diferença significativa quanto a precisão. Os resultados foram comparados através da determinação de índices de capacidade do sistema de medição. A analise estatística demonstrou que não há diferença significativa entre os resultados obtidos pelos delineamentos 5 x 1, 2 x 2 e 3 x 1, sendo equivalentes e intercambiáveis.

Doseamento microbiológico de apramicina - desenvolvimento e validação de método empregando leitura cinética em microplacas / Apramycin microbiological assay - method development and validation using kinetic microplate reading

O princípio do ensaio turbidimétrico é simples: a solução-teste é adicionada a suspensão do microrganismo-teste em meio de cultura, a mistura é incubada em condições adequadas e o crescimento microbiano é medido através da leitura fotométrica. O emprego de método de microplacas com leitura cínética para a dosagem de antibióticos é de interesse considerável, uma vez que possibilita reduzir quantidade de material e tempo de análise necessários e permite o ensaio de grande número de amostras simultaneamente, com leitura e cálculo automatizados. O objetivo deste trabalho é determinar as condições experimentais ideais para o desenvolvimento de metodologia para a dosagem microbiológica de apramicina empregando microplacas e modo de leitura cinético, e validar o método desenvolvido, através da avaliação dos parâmetros de especificidade e seletividade, linearidade, faixa ou intervalo linear, limite de detecção e quantificação, exatidão e precisão. As condições estabelecidas abrangem curva-padrão de apramicina com concentrações entre 5 e 35 μg/ml, e emprego de meio de cultura caldo de triptona-soja inoculado com Escherichia coli (ATCC 8739) na proporção de 5%. Foram obtidos resultados satisfatórios após 2 horas de incubação. O método desenvolvido apresentou especificidade e seletividade adequadas, linearidade na faixa de 5 a 35 1.19/ml, limite de deteção e quantificação de 0,1 e 0,4 μg/ml, respectivamente, exatidão (recuperação = 98,5%) e precisao (DPR = 6,0%) satisfatórias. O ensaio em microplaca agrega características dos ensaios microbiológicos (avaliação da atividade do antibiótico frente a microrganismo-teste sensível) e físico-químicos (facilidade operacional e major rapidez na obtenção dos resultdos).

The turbidimetric assay principle is simple: the test-solution is added to a suspension of test-microorganism in culture media, the mixture is incubated in appropriate conditions and the microbial growth is measured by photometric reading. Microplate with kinetic reading mode employed in antibiotic assay is considerable interesting, once it allows reduction of material and analysis time and it permits that a great numbers of samples could be analyzed simultaneously, with automated reading and calculating. The aim of this work is to determinate best experimental conditions to development of methodology for microbiological assay of apramycin employing microplate and kinetic reading mode, and to validate the developed method, through evaluation of parameters of specificity and selectivity, linearity, linear range, limit of detection and quantification, accuracy and precision. Established conditions considered standard-curve of apramycin in concentrations from 5 to 35 μg/ml, and tryptic soy broth inoculated with 5% of Escherichia coli (ATCC 8739) suspension. Satisfactory results were obtained with 2 hours incubation. The developed method showed appropriate specificity and selectivity, linearity in the range from 5 to 35 μg/ml, limit of detection and quantification of 0,1 and 0,4 μg/ml, respectively, satisfactory accuracy (recuperation = 98,5%) and precision (RSD = 6,0%). Microplate assay considered characteristics of microbiological assay (evaluation of antibiotic activity against sensible test-microorganism) and physical-chemistry (operational facility and quicker results).

Estimativa da incerteza em ensaio de detecção de endotoxina bacteriana pelo método de gelificação / Estimation of uncertainty in the detection of bacterial endotoxin by gel-clot method

Desde a publicação da ISO 17025:1999, o interesse em métodos para estimativa da incerteza em ensaios qualitativos do tipo passa/não passa, têm ganho grande importância. Uma forma de estimar e informar a incerteza deste tipo de ensaio é o uso das probabilidades de respostas-falsas, particularmente falsos-positivos e falsos-negativos, determinados a partir do teorema de Bayes. O objetivo deste artigo é estabelecer um método para a estimativa de incerteza em ensaios de detecção de endotoxina bacteriana pelo método in vitro Limulus Amebocyte Lysate (LAL). Considerando a confirmação da sensibilidade do LAL e a validação do teste, a probabilidade de uma resposta falsa corresponde à soma da probabilidade dos resultados falso-negativos e falso-positivos. A partir dos resultados obtidos foi verificado que a etapa da confirmação da sensibilidade...