Felipe Rebello Lourenço

Measurement uncertainty in pharmaceutical analysis and its application

The measurement uncertainty provides complete information about an analytical result. This is very important because several decisions of compliance or non-compliance are based on analytical results in pharmaceutical industries. The aim of this work was to evaluate and discuss the estimation of uncertainty in pharmaceutical analysis. The uncertainty is a useful tool in the assessment of compliance or non-compliance of in-process and final pharmaceutical products as well as in the assessment of pharmaceutical equivalence and stability study of drug products.

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

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.

Antibiotic microbial assay using kinetic-reading microplate system

O objetivo deste trabalho e determinar as condicoes experimentais ideais para o desenvolvimento de metodologia para a dosagem microbiologica de apramicina empregando microplacas e modo de leitura cinetico e validar o metodo desenvolvido, atraves da avaliacao dos parâmetros de especificidade e seletividade, linearidade, faixa ou intervalo linear, limite de deteccao e quantificacao, exatidao e precisao. O ensaio turbidimetrico e simples: a solucao-teste e adicionada a suspensao de microrganismo-teste em meio de cultura, a mistura e incubada em condicoes apropriadas e o crescimento microbiano e medido por meio de leitura fotometrica. O emprego de metodo de microplacas com leitura cinetica para a dosagem de antibioticos e de interesse consideravel, uma vez que possibilita reduzir quantidade de material e tempo de analise necessarios e permite o ensaio de grande numero de amostras simultaneamente, com leitura e calculo automatizados. As condicoes estabelecidas abrangem curva-padrao de apramicina com concentracoes entre 5 e 35 μg/mL, e emprego de meio de cultura caldo de triptona-soja inoculado com Escherichia coli (ATCC 8739) na proporcao de 5%. Foram obtidos resultados satisfatorios apos 2 horas de incubacao. O metodo desenvolvido apresentou especificidade e seletividade adequadas, linearidade na faixa de 5 a 35 μg/mL, limite de deteccao e quantificacao de 0,1 e 0,4 μg/mL, respectivamente, exatidao (recuperacao = 98,5%) e precisao (DPR = 6,0%) satisfatorias. O ensaio em microplaca agrega caracteristicas dos ensaios microbiologicos (avaliacao da atividade do antibiotico frente a microrganismo-teste sensivel) e fisico-quimicos (facilidade operacional e maior rapidez na obtencao dos resultados).

Rational development and validation of a new microbiological assay for linezolid and its measurement uncertainty.

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.

How pH, Temperature, and Time of Incubation Affect False-Positive Responses and Uncertainty of the LAL Gel-Clot Test

The limulus amebocyte lysate (LAL) test is the simplest and most widely used procedure for detection of endotoxin in parenteral drugs. The LAL test demands optimal pH, ionic strength, temperature, and time of incubation. Slight changes in these parameters may increase the frequency of false-positive responses and the estimated uncertainty of the LAL test. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. LAL tests were performed in nominal conditions (37 °C, 60 min, and pH 7) and in different conditions of temperature (36 °C and 38 °C), time of incubation (58 and 62 min), and pH (6 and 8). Slight differences in pH increase the frequency of false-positive responses 5-fold (relative risk 5.0), resulting in an estimated of uncertainty 7.6%. Temperature and time of incubation affect the LAL test less, showing relative risks of 1.5 and 1.0, respectively. Estimated uncertainties in 36 °C or 38 °C temperatures and 58 or 62 min of incubation were found to be 2.0% and 1.0%, respectively. Simultaneous differences in these parameters significantly increase the frequency of false-positive responses. LAY ABSTRACT: The limulus amebocyte lysate (LAL) gel-clot test is a simple test for detection of endotoxin from Gram-negative bacteria. The test is based on a gel formation when a certain amount of endotoxin is present; it is a pass/fail test. The LAL test requires optimal pH, ionic strength, temperature, and time of incubation. Slight difference in these parameters may increase the frequency of false-positive responses. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. We find that slight differences in pH increase the frequency of false-positive responses 5-fold. Temperature and time of incubation affect the LAL test less. Simultaneous differences in these parameters significantly increase the frequency of false-positive responses.

Determination of apramycin in oral soluble powder by a HPLC method using pre-column derivatization with o-phthalaldehyde and UV detection

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% both for intra and inter-day precision) accuracy (average recuperation of 99.33%) 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% to 105% 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.

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

This work proposes a procedure to estimate the measurement uncertainty for the quantification of cefazolin by high-performance liquid chromatography (HPLC) and microbiological assay (bioassay). HPLC method was performed according to United States Pharmacopeia (USP) and bioassay according to Brazilian Pharmacopeia (FB). The quantification of cefazolin by high-performance liquid chromatography was assess through the calibration curve equation (Y=0.0199 X–0.0323). The HPLC uncertainty was estimated based on the error of the slope and the intercept (between 4.4% and 5.3%). The bioassay uncertainty was estimated based on the standard deviation of inhibition zones of samples and standards solutions (between 6.2 and 6.7%). The uncertainties for bioassay are expected to be higher those obtained by HPLC. Both HPLC method and bioassay estimated uncertainties are reasonable for the scope of each method.

Design Space Approach for Preservative System Optimization of an Anti-Aging Eye Fluid Emulsion

The use of preservatives must be optimized in order to ensure the efficacy of an antimicrobial system as well as the product safety. Despite the wide variety of preservatives, the synergistic or antagonistic effects of their combinations are not well established and it is still an issue in the development of pharmaceutical and cosmetic products. The purpose of this paper was to establish a space design using a simplex-centroid approach to achieve the lowest effective concentration of 3 preservatives (methylparaben, propylparaben, and imidazolidinyl urea) and EDTA for an emulsion cosmetic product. Twenty-two formulae of emulsion differing only by imidazolidinyl urea (A: 0.00 to 0.30% w/w), methylparaben (B: 0.00 to 0.20% w/w), propylparaben (C: 0.00 to 0.10% w/w) and EDTA (D: 0.00 to 0.10% w/w) concentrations were prepared. They were tested alone and in binary, ternary and quaternary combinations. Aliquots of these formulae were inoculated with several microorganisms. An electrochemical method was used to determine microbial burden immediately after inoculation and after 2, 4, 8, 12, 24, 48, and 168 h. An optimization strategy was used to obtain the concentrations of preservatives and EDTA resulting in a most effective preservative system of all microorganisms simultaneously. The use of preservatives and EDTA in combination has the advantage of exhibiting a potential synergistic effect against a wider spectrum of microorganisms. Based on graphic and optimization strategies, we proposed a new formula containing a quaternary combination (A: 55%; B: 30%; C: 5% and D: 10% w/w), which complies with the specification of a conventional challenge test. A design space approach was successfully employed in the optimization of concentrations of preservatives and EDTA in an emulsion cosmetic product.

Development, Optimization, and Validation of a Microplate Bioassay for Relative Potency Determination of Linezolid Using a Design Space Concept, and its Measurement Uncertainty.

The aim of this study was to develop, optimize, and validate a microplate bioassay for relative potency determination of linezolid in pharmaceutical samples using quality-by-design and design space approaches. In addition, a procedure is described for estimating relative potency uncertainty based on microbiological response variability. The influence of culture media composition was studied using a factorial design and a central composite design was adopted to study the influence of inoculum proportion and triphenyltetrazolium chloride in microbial growth. The microplate bioassay was optimized regarding the responses of low, medium, and high doses of linezolid, negative and positive controls, and the slope, intercept, and correlation coefficient of dose-response curves. According to optimization results, design space ranges were established using: (a) low (1.0 μg/mL), medium (2.0 μg/mL), and high (4.0 μg/mL) doses of pharmaceutical samples and linezolid chemical reference substance; (b) Staphylococcus aureus ATCC 653 in an inoculum proportion of 10%; (c) antibiotic No. 3 culture medium pH 7.0±0.1; (d) 6 h incubation at 37.0±0.1ºC; and (e) addition of 50 μL of 0.5% (w/v) triphenyltetrazolium chloride solution. The microplate bioassay was linear (r2=0.992), specific, precise (repeatability RSD=2.3% and intermediate precision RSD=4.3%), accurate (mean recovery=101.4%), and robust. The overall measurement uncertainty was reasonable considering the increased variability inherent in microbiological response. Final uncertainty was comparable with those obtained with other microbiological assays, as well as chemical methods.

Development, optimization and validation of a rapid colorimetric microplate bioassay for neomycin sulfate in pharmaceutical drug products

Microbiological assays have been used to evaluate antimicrobial activity since the discovery of the first antibiotics. Despite their limitations, microbiological assays are widely employed to determine antibiotic potency of pharmaceutical dosage forms, since they provide a measure of biological activity. The aim of this work is to develop, optimize and validate a rapid colorimetric microplate bioassay for the potency of neomycin in pharmaceutical drug products. Factorial and response surface methodologies were used in the development and optimization of the choice of microorganism, culture medium composition, amount of inoculum, triphenyltetrazolium chloride (TTC) concentration and neomycin concentration. The optimized bioassay method was validated by the assessment of linearity (range 3.0 to 5.0μg/mL, r=0.998 and 0.994 for standard and sample curves, respectively), precision (relative standard deviation (RSD) of 2.8% and 4.0 for repeatability and intermediate precision, respectively), accuracy (mean recovery=100.2%) and robustness. Statistical analysis showed equivalency between agar diffusion microbiological assay and rapid colorimetric microplate bioassay. In addition, microplate bioassay had advantages concerning the sensitivity of response, time of incubation, and amount of culture medium and solutions required.