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 (R 2 , R 2-adj, and R 2-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.

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% 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% 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...

Grape pomace retains polyphenols in the peels and in the seeds after winemaking, which is indicative of the high valorization potential of this industrial waste. There is strong evidence that phenolics are robust antioxidants and confer photoprotection; thus, it is rational to apply these active compounds from winemaking waste to sunscreens, in order to increase UV protection. Despite the importance of this class of cosmetics to public health, more efficacious strategies are still needed to overcome the problems caused by the photoinstability of some UV filters. The hydroethanolic extract of Vitis vinifera L. grapes was obtained by percolation and then lyophilized. Six formulations were developed: Type I—cosmetic base and UV filters; Type II—cosmetic base and extract; and Type III—cosmetic base, extract and UV filters. Each formulation was prepared in the pHs 5 and 7. The antioxidant activities of the samples were measured by DPPH• and expressed in Trolox® equivalents (TE), and their photostability and in vitro sun protection factor (SPF) were analyzed by diffuse reflectance spectrophotometry. The anti-radical efficiencies observed in the formulations with grape extract were: (II) 590.12 ± 0.01 μmol TE g−1 at pH 5 and 424.51 ± 0.32 μmol TE g−1 at pH 7; (III) 550.88 ± 0.00 μmol TE g−1 at pH 5 and 429.66 ± 0.10 μmol TE g−1, at pH 7, demonstrating that the UV filters, butylmethoxydibenzoyl methane, ethylhexyl methoxycinnamate and ethylhexyl dimethyl 4-aminobenzoic acid had no influence on this effect. The photoprotective efficacy and the photostability of formulation III containing the extract and UV filters at pH 5 suggested that a synergism between the active molecules provided an 81% increase in SPF. Additionally, this was the only sample that maintained a broad spectrum of protection after irradiation. These results confirmed that the grape pomace extract has multifunctional potential for cosmetic use, mainly in sunscreens, granting them superior performance.

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The aim of this work was to develop and validate a new microbiological assay to determine potency of linezolid in injectable solution. 2(4) factorial and central composite designs were used to optimize the microbiological assay conditions. In addition, we estimated the measurement uncertainty based on residual error of analysis of variance of inhibition zone diameters. Optimized conditions employed 4 mL of antibiotic 1 medium inoculated with 1% of Staphylococcus aureus suspension, and linezolid in concentrations from 25 to 100 µg mL(-1). The method was specific, linear (Y=10.03X+5.00 and Y=9.20X+6.53, r(2)=0.9950 and 0.9987, for standard and sample curves, respectively), accurate (mean recovery=102.7%), precise (repeatability=2.0% and intermediate precision=1.9%) and robust. Microbiological assay׳s overall uncertainty (3.1%) was comparable to those obtained for other microbiological assays (1.7-7.1%) and for determination of linezolid by spectrophotometry (2.1%) and reverse-phase ultra-performance liquid chromatography (RP-UPLC) (2.5%). Therefore, it is an acceptable alternative method for the routine quality control of linezolid in injectable solution.

Microbiological agar diffusion methods are widely employed for clinical, pharmacological and industrial purposes.

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 mg mL -1 , and tryptic soy broth inoculated with 5% 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 mg mL , respectively, as well as satisfactory accuracy (recuperation = 98.5%) and precision (RSD = 6.0%). Microplate assay combined the characteristics of microbiological (evaluation of antibiotic activity against sensitive test microorganism) and physico-chemical (operationally straightforward and faster results) assays.Uniterms: Antibiotics/microbial assay. Apramycin/microbial assay. Apramycin sulfate. Turbidimetric assay/medicines analysis.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 mg/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 mg/mL, limite de detecção e quantificação de 0,1 e 0,4 mg/mL, respectivament...

Ayahuasca tea is a hallucinogenic beverage used for religious purposes in Brazil and many other countries that has therapeutic potential in the treatment of some mental health disorders. In the context of psychedelic research, quantification of the tea’s main alkaloids prior to its administration in animal or human studies is essential. For this reason, this study aims to provide information regarding the stability of the main ayahuasca alkaloids (dimethyltryptamine, DMT; harmine, HRM; tetrahydroharmine, THH; harmaline, HRL) in three different conditions: (1) A year stored in a refrigerator either in plastic or glass containers, (2) seven days at 37 °C to reproduce usual mail transportation, and (3) after three freeze–thaw cycles. Samples were quantified after a dilute-and-shoot procedure using liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). There was no significant degradation of DMT concentration over time in all tested conditions. Harmala alkaloids (THH, HRL, and HRM) showed important variations after long-term and high-temperature storages. Although DMT has proven to be stable in all studied conditions, the harmala alkaloids revealed intense degradation and even concentration increment. This may be caused by degradation, alkaloid inter-conversion, and leaching from tea precipitate material. Therefore, ayahuasca quantification before administration in controlled sets is mandatory.