Alessandro Morais Saviano

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.

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.

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.

Kinetic microplate bioassays for relative potency of antibiotics improved by partial Least Square (PLS) regression

Microbiological assays are widely used to estimate the relative potencies of antibiotics in order to guarantee the efficacy, safety, and quality of drug products. Despite of the advantages of turbidimetric bioassays when compared to other methods, it has limitations concerning the linearity and range of the dose-response curve determination. Here, we proposed to use partial least squares (PLS) regression to solve these limitations and to improve the prediction of relative potencies of antibiotics. Kinetic-reading microplate turbidimetric bioassays for apramacyin and vancomycin were performed using Escherichia coli (ATCC 8739) and Bacillus subtilis (ATCC 6633), respectively. Microbial growths were measured as absorbance up to 180 and 300min for apramycin and vancomycin turbidimetric bioassays, respectively. Conventional dose-response curves (absorbances or area under the microbial growth curve vs. log of antibiotic concentration) showed significant regression, however there were significant deviation of linearity. Thus, they could not be used for relative potency estimations. PLS regression allowed us to construct a predictive model for estimating the relative potencies of apramycin and vancomycin without over-fitting and it improved the linear range of turbidimetric bioassay. In addition, PLS regression provided predictions of relative potencies equivalent to those obtained from agar diffusion official methods. Therefore, we conclude that PLS regression may be used to estimate the relative potencies of antibiotics with significant advantages when compared to conventional dose-response curve determination.

Reduced incubation time for inhibition zone formation based on diffusion and growth mechanism elucidation

Microbiological agar diffusion methods are widely employed for clinical, pharmacological and industrial purposes. Usually, inhibition zone sizes are defined after a few hours of incubation; however, they can only be visualized after overnight microbial growth. Thus, the aim of this work was to determine the critical parameters and optimize them using the tool Solver (Microsoft Excel®), in order to elucidate the mechanism of inhibition zone formation and develop a microbiological assay for linezolid with a reduced incubation time. A Box–Behnken design was used to determine the critical parameters, such as the critical concentration (C′), critical population (N′), critical time (T′), diffusion coefficient (D), lag phase time (L), and generation time (G). Optimized critical parameters were coherent with the mean values of experimental parameters and were contained within the confidence interval 95%, validating the results obtained with the tool Solver. Furthermore the formation of inhibition zones was elucidated with 97.7% certainty. Increased inoculum sizes reduce critical time, which allow us to use TTC to visualize inhibition zones after 6 hours. The value of the critical concentration obtained with reduced incubation time (8.90 mg L−1) was consistent with the experimental value with standard incubation time (8.62 ± 0.53 mg L−1; CI95% 7.77 to 9.45 mg L−1). Therefore our method was able to reduce incubation from 18 to 24 hours to approximately 6 hours.

Rapid microbiological methods (RMMs) for evaluating the activity of cephalosporin antibiotics employing triphenyltetrazolium chloride

Agar diffusion method has been used to evaluate antimicrobial activity since the discovery of penicillin. Nevertheless, little progress has occurred in reducing the time required to determine growth inhibition zones. The aim of this work was to develop, optimize and validate rapid microbiological methods (RMMs) for cephalosporin antibiotics using triphenyltetrazolium chloride (TTC) to reduce the incubation time of the assays. Through a factorial design in which the inoculum suspension, incubation time, and percentage of the TTC solution were varied, it was possible to validate the RMMs for cefazolin, cefuroxime, ceftriaxone, and cefepime. The validated conditions employed 5 mL of MHA medium inoculated with 2% of Staphylococcus aureus suspension, incubation time of 5 h and 30 min at 37 ± 1 °C and the addition of 0.3% TTC solution in 1% agar, cefazolin and cefuroxime in concentrations from 15 to 60 µg mL-1, ceftriaxone and cefepime in concentrations from 20 to 80 µg mL-1. The methods were selective, linear (for standard and sample curves, respectively), accurate, precise, robust and rugged. The results found by the RMMs were statistically similar to those found by conventional microbiological methods, but the advantages of the former decreased the incubation time from 22 h to 5 h and 30 min. Therefore, RMMs can be used in the evaluation and quantification of cephalosporin antibiotics, ensuring their quality, safety, and therapeutic efficacy.