Simplified and Detailed Evaluation of the Uncertainty of the Measurement of Microbiological Contamination of Pharmaceutical Products.

BACKGROUND: The control of the microbial contamination of pharmaceutical products, PP, is crucial to ensure their safety and efficacy. The validity of the monitoring of such contamination depends on the uncertainty of this quantification. Highly uncertain quantifications due to the variability of determinations or the magnitude of systematic effects affecting microbial growth or other analytical operations make analysis unfit for the intended use. The quantification of the measurement uncertainty expressing the combined effects of all random and systematic effects affecting the analysis allows the sound decision about quantification adequacy for their intended use. The complexity of the quantification of microbial analysis uncertainty led to the development of simplified ways of performing this evaluation. OBJECTIVE: This work assesses the adequacy of the simplified quantification of the uncertainty of the determination of the microbial contamination of PP by log transforming microbial count and dilution factor of the test sample whose uncertainty is combined in a log scale using the uncertainty propagation law. METHODS: This assessment is performed by a parallel novel bottom-up and accurate evaluation of microbial analysis uncertainty involving the Monte Carlo Method simulation of the Poisson log-normal distribution of counts and of the normally distributed measured volumes involved in the analysis. Systematic effects are assessed and corrected on results to compensate for their impact on the determinations. Poisson regression is used to predict precision affecting determinations on unknown test samples. RESULTS AND CONCLUSION: This work concludes that triplicate determinations are required to produce results with adequately low uncertainty and that simplified uncertainty quantification underevaluate or overevaluate the uncertainty from determinations based on low or high colonies numbers, respectively. Therefore, detailed uncertainty evaluations are advised for determinations between 50% and 200% of PP's maximum admissible contamination value.

Nonparametric estimation of measurement uncertainty arising from sampling.

BACKGROUND: Increasingly, measurement uncertainty has been used by pure and applied analytical chemistry to ensure decision-making in commercial transactions and technical-scientific applications. Until recently, it was considered that measurement uncertainty boiled down to analytical uncertainty; however, over the last two decades, uncertainty arising from sampling has also been considered. However, the second version of the EURACHEM guide, published in 2019, assumes that the frequency distribution is approximately normal or can be normalized through logarithmic transformations, without treating data that deviate from the normality. RESULTS: Here, six examples (four from Eurachem guide) were treated by classical ANOVA and submitted to an innovative nonparametric approach for estimating the uncertainty contribution arising from sampling. Based on bootstrapping method, confidence intervals were used to guarantee metrological compatibility between the uncertainty ratios arising from the results of the traditional parametric tests and the unprecedented proposed nonparametric methodology. SIGNIFICANCE AND NOVELTY: The present study proposed an innovative methodology for covering this gap in the literature based on nonparametric statistics (NONPANOVA) using the median absolute deviation concepts. Supplementary material based on Excel spreadsheets was developed, assisting users in the statistical treatment of their real examples.

Fatty Acid Profile and Escherichia coli and Salmonella sp. Load of Wild-Caught Seaweed Fly Fucellia maritima (Haliday, 1838) (Diptera: Anthomyiidae).

World aquaculture is expected to continue to grow over the next few decades, which amplifies the need for a higher production of sustainable feed ingredients for aquatic animals. Insects are considered good candidates for aquafeed ingredients because of their ability to convert food waste into highly nutritional biomass. However, commercially available terrestrial insect species lack n-3 long-chain polyunsaturated fatty acids (LC-PUFAs), which are essential biomolecules for marine cultured species. Nevertheless, several coastal insect species feature LC-PUFAs in their natural fatty acid (FA) profile. Here, we analysed the lipidic profile of wild-caught seaweed fly Fucellia maritima, with a focus on their FA profile, to evaluate its potential to be used as an aquafeed ingredient, as well as to screen for the presence of pathogenic bacteria. Results showed that the flies had a total lipid content of 13.2% of their total dry weight. The main classes of phospholipids (PLs) recorded were phosphatidylethanolamines (PEs) (60.8%), followed by phosphatidylcholine (PC) (17.1%). The most abundant FA was palmitoleic acid (C16:0) with 34.9% ± 4.3 of total FAs, followed by oleic acid (C18:1) with 30.4% ± 2.3. The FA composition of the flies included essential fatty acids (EFAs) for both freshwater fish, namely linoleic acid (C18:2 n-6) with 3.4% ± 1.3 and alpha-linoleic acid (C18:3 n-3) with 3.4% ± 1.9, and marine fish, namely arachidonic acid (C20:4 n-6) with 1.1% ± 0.3 and eicosapentaenoic acid (C20:5 n-3) with 6.1% ± 1.2. The microbiological analysis found 9.1 colony-forming units per gram (CFU/g) of Enterobacteriaceae and no presence of Salmonella sp. was detected in a sample of 25 g of fresh weight. These findings indicate that Fucellia maritima biomass holds the potential to be used as an additional aquafeed ingredient due to its FA profile and the low count of pathogenic bacteria, which can contribute to the optimal growth of fish and shrimp with a low risk of pathogen transfer during the feed production chain.

How do physicochemical properties contribute to inhibitory activity of promising peptides against Zika Virus NS3 protease?

CONTEXT AND RESULTS: Flavivirus diseases' cycles, especially Dengue and Yellow Fever, can be observed all over Brazilian territory, representing a great health concern. Additionally, there are no drugs available in therapy. In this scenario, in silico methodologies were applied to obtain physicochemical properties, as well as to better understand the ligand-biological target interaction mode of 20 previously reported NS2B/NS3 protease inhibitors of Dengue virus. Since catalytic site of flavivirus hold similarities, such as the same catalytic triad (His51, Asp75 e Ser135), the ability of this series of molecules to fit in Zika NS3 domains can be achieved. We performed an exploratory data analysis, using statistical methodologies, such as PCA (Principal Component Analysis) and HCA (Hierarchical Component Analysis), to assist the comprehension of how physicochemical properties impact the interaction observed by the docking studies, as well as to build a correlation between the respective ranked characteristics. Based on these previous studies, peptides were selected for the dynamics simulations, which were useful to better understand the ligand-protein interactions. Information relating to, for instance, energy, ΔG, average number of hydrogen bonds and distance from Ser135 (one of the main amino acids in the catalytic pocket) were discussed. In this sense, peptides 15 (considering ΔG value and Hbond number), 7 (ΔG and energy) and 1, 6, 7 and 15 (the proximity to Ser135 throughout the dynamics simulation) were highlighted as promising. Those interesting results could contribute to future studies regarding Zika virus drug design, since this infection represents a great concern in neglected populations. METHODS: The models were constructed in the ChemDraw software. The ligand parametrization was performed in the CHEM3D 17.0, UCSF Chimera. Docking simulations were carried out in the GOLD software, after the redocking validation. We used ASP as the function score. Additionally, for dynamics simulations we applied GROMACS software, exploring, mainly, free binding energy calculations. Exploratory analysis was carried out in Minitab 17.3.1 statistical software. Prior to the exploratory analysis, data of quantum chemical properties of the peptides were collected in Microsoft Excel spreadsheet and organized to obtain Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA).

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.

Enhancing analytical development in the pharmaceutical industry: A DoE-QSRR model for virtual Method Operable Design Region assessment.

Recently, the pharmaceutical industry has increasingly adopted the Analytical Quality by Design (AQbD) approach for analytical development. To facilitate AQbD approach implementation in the development of chromatographic methods for determining cephalosporin antibiotics, an in silico tool capable of performing virtual DoEs was developed enabling to obtain virtual operable regions of method. To this end, the drugs cephalexin, cefazolin, cefotaxime and ceftriaxone were analyzed using four experimental designs, deriving a DoE-QSRR model and employing Monte Carlo method. The DoE-QSRR model and virtual DoEs were validated using data not used in model's construction, obtaining coefficients of determination of 84.72 % for DoE-QSRR model and over 77 % for virtual DoEs. Virtual MODRs were constructed using data from the virtual DoEs. The virtual MODRs were validated by comparing them with experimental MODRs under various scenarios, with overlap areas reaching values exceeding 84 %. Therefore, the in silico tool was considered suitable for indicating analyte trends under different analytical conditions, being capable of performing virtual DoEs for cephalosporin drugs with sufficient assertiveness to guide analytical development and allow obtaining a MODR capable of providing results of adequate quality.

Bioaccumulation and Depletion of the Antibiotic Sulfadiazine 14C in Lambari (Astyanax bimaculatus).

Antibiotics are present in the environment, primarily due to their release through wastewater treatment plants, agricultural practices, and improper disposal of unused medications. In the environment, these drugs can be bioaccumulated by organisms and transferred along the food chain. This is a problem when considering the consumption of fish meat. In the United States, legislation stipulates that the maximum residue limit for sulfadiazine (SDZ) should not exceed 100 µg kg-1. Lambari fishes have potential economic importance in aquaculture, as they are relatively easy to breed and can be raised in small-scale operations. Finally, studying the biology and ecology of lambari could provide valuable information about freshwater ecosystems and their inhabitants. The current work aimed to measure the bioaccumulation and depletion of the antibiotic SDZ 14C in lambari (Astyanax bimaculatus). For this purpose, the tests were divided into two stages; seven days of exposure and seven days of depletion, where one fish was randomly selected and sampled every day. In the exposure phase, the fish were fed the medicated feed three times a day at a concentration of 2.5 mg·g-1. The control fish were fed uncontaminated feed. For the depletion phase, the remaining lambari were transferred to clean tanks and fed uncontaminated feed three times a day. The fish samples were burned in the Oxidizer and the reading of radioactivity was performed in a liquid scintillation spectrometer. It is worth noting that on day 7 and day 14, the water in the aquariums was filtered through filter paper to collect the metabolic excrement. SDZ concentrations increased over the days and accumulation occurred in the fish, with day seven presenting the maximum accumulation value of 91.7 ng·g-1 due to feeding uptake. After the depletion phase on day 13, the value found was 0.83 ng·g-1. The bioconcentration factor calculated was 20 L·kg-1. After the bioaccumulation period, the concentrations of SDZ in the water and excreta were 4.5 µg·L-1 and 363.5 ng·g-1, respectively. In the depletion period, the concentrations in the water and excreta were 0.01 µg·L-1 and 5.96 ng·g-1, respectively. These results imply that there was little SDZ bioaccumulation in the fish, but that it was distributed in larger amounts in the water. This is due to the physicochemical properties of the molecule with the low Log P value. Regarding the maximum residue limit, the value was below the established value. This study contributes to understanding SDZ dynamics in an aquatic species native to Brazil.

Measurement uncertainty arising from sampling and analytical steps of dissolution test of prednisone tablets.

Dissolution is used to determine the rate and extent of drug release from the dosage form into a dissolution medium, which allow to assess the batch-to-batch variability. Considering that the dissolution test is used to predict the vivo performance of the drug as well, it is important to guarantee the quality and reliability of dissolution test results. The aim of this work was to evaluate the measurement uncertainty arising from sampling and analytical steps of dissolution test of prednisone tablets. Dissolution test was performed using 900 mL of purified water as dissolution medium and a dissolution apparatus equipped with paddles rotating at 50 rpm for 30 min. Quantification was performed by UV spectrophotometer. Uncertainty arising from sampling was estimated using the duplicate method (empirical approach), using 17-sampling target, two samples for each sampling target, and three replicas for each sample, totalizing 102 analyses. Uncertainty arising from analytical steps considered the uncertainty from dissolution step (estimated using Monte Carlo method and regression equation obtained using DoE) and uncertainty from quantification step. Overall uncertainty value was found to be 2.2%, which is below the target uncertainty value (ut =2.5%). The contributions of uncertainty sources in this study were as follows: 24% from sampling uncertainty, 29% from the dissolution step uncertainty, and 47% from the quantification step uncertainty. The results of dissolution test should be compared to the specification limits (Q). According to the pharmacopeia requirements, the batch of the medicine should be declared compliant if the dissolved amount of prednisone for six tablets are above the specification limits + 5% (Q+5%=85%). Since the measured values for all six tablets (96.5%, 94.0%, 96,4%, 95.3%, 96.0%, and 96.9%) were above the multivariate acceptance limit (90.2%, calculate as the standard uncertainty multiplied by multivariate coverage factor), the batch of the prednisone tablets was declared complaint, with a reduced total risk of false decision (total risk value below 5%).

Development and optimization of a LC-MS/MS compatible method for quantification of losartan, hydrochlorothiazide and their impurities using AQbD approach and measurement uncertainty evaluation.

The concept of Analytical Quality by Design (AQbD) comes as a more robust, economical, and scientifically based alternative for analytical development, to the detriment of OFAT (one factor at a time). This new understanding applicable to analytical development is recommended since regulatory flexibility can be achieved and ensure more reliable results throughout the life of the product. This new approach was applied to develop an analytical procedure indicative of stability for a pharmaceutical product of association of Losartan Potassium and Hydrochlorothiazide, considered a potential first line for the treatment of hypertension. The first stage of the analytical development consisted of defining analytical target profile (ATP), follow by a bibliographic survey of the physicochemical properties of the molecules in question to define an initial method. After defining the initial analytical conditions, statistical tools for design of experiments (DoE) were used for the screening and optimization steps. In the screening stage, the Plackett-Burman design was chosen, using 11 factors and 2 levels, through which it was possible to evaluate numerous variables and determine their significance in relation to the responses. Next, optimization was carried out with the experimental design of a central composite with 4 factors and 5 levels, which allowed modeling a complex response surface and evaluating the phenomena of interactions between the factors. Thus, the optimized analytical conditions were defined, considering a 0.3% formic acid gradient as eluent A and a mixture of acetonitrile and methanol (80:20) as eluent B, X-Bridge C18 chromatographic column (150 mm × 4 .6 mm × 3.5 µm), column temperature of 40°C, flow rate of 1.3 mL/min, injection volume of 10 µL. Through this methodology, it was possible to identify an unknown degradation product of Hydrochlorothiazide, formed by the reaction with lactose (excipient present in the drug formulation), proving that the method can be applicable both to DAD detectors and to spectrometry and mass detectors. It was also proven through the forced degradation study that the method is indicative of stability, in addition to being validated and robust for its purpose.

Measurement Uncertainty and Conformity Assessment Applied to Drug and Medicine Analyses - A Review.

Analytical results are often used in scientific research, industrial and clinical applications to support decision making. Despite all efforts to ensure the reliability of analytical results (including method validation, internal quality control, use of certified reference materials, proficiency tests, and ISO 17025 accreditation), there will always be an uncertainty associated with the measured value. The measurement uncertainty expresses the quality of the analytical result and allows the comparability between analytical results or between the measured value and the specification limit(s). This work discusses the importance of measurement uncertainty, including the steps involved in the measurement uncertainty evaluation, the bottom-up and top-down approaches used in measurement uncertainty calculation, the measurement uncertainty evaluation in drug and medicine analyses, and the application of measurement uncertainty in conformity assessment for quality control, stability studies, and pharmaceutical equivalence.

Development and optimization of stability-indicating method of ethinylestradiol, levonorgestrel, and their main impurities using quality by design approach.

The association of Ethinylestradiol 0.03 mg and Levonorgestrel 0.15 mg is a hormonal contraceptive that combines estrogen and progestogen. According to a bibliographic survey, these combined drugs present at least 18 known degradation products, which are required to control the potential impurities harmful to human health. The high number of impurities and the low concentrations of the active pharmaceutical ingredients (APIs) and their respective degradation products increase the complexity of the stability-indicating method development for this medicine. Thus, this work aimed to develop and optimize the stability-indicating method using the quality by design (QbD) approach and in-silico tools for application in samples of oral contraceptives sold in Brazil. The analysis samples were initially subjected to a forced degradation study through 7 days of exposure under acid and alkali hydrolysis, oxidative condition, and oxidation by metal ions. In addition to the chemical exposure, the sample was subjected to physical stress through 10 days of exposure under dry heat, moisture, and photolytic degradation. These exposure samples were analyzed in the development and optimization of chromatographic conditions. As a result, the developed method was able to separate 20 known substances, including the two APIs and their respective 18 degradation products, as well as unknown degradation products obtained by the forced degradation study. Finally, this stability-indicating method was successfully applied for comparative analysis of contraceptive drugs marketed in Brazil, newly purchased and subjected to accelerated stability condition at 40 °C and 75% RH over the 6-month period.

Definition of multivariate acceptance limits (guard-bands) applied to pharmaceutical equivalence assessment.

The quality assessment of medicines involves multiple compliance parameters, such as identity, dosage, purity, potency, content uniformity, disintegration time, dissolution rate, among others. The measurement uncertainty associated with a measured value can affect the conformity assessment and, consequently, it impacts decision-making. Even if the particular risks are acceptable, the total risk may be significantly high. Thus, the aim of this work was to develop a procedure for the definition of acceptance (or rejection) limits applied to multiple compliance assessments, that ensure acceptable particular and total risks. The multiple compliance assessments were performed and applied in the pharmaceutical equivalence studies for cisplatin injectable solution, carboplatin injectable solution, ranitidine tablets, and acetaminophen oral solution from several manufacturers. Pharmaceutical equivalence studies were performed adopting pharmacopeial analytical procedures. All chromatographic system suitability results complied with the requirements regarding the resolution between peaks, the capacity factor, the tailing factor, the theoretical plates, and the relative standard deviation for replicate injections. Univariate and multivariate guard-bands (g and g', respectively) were calculated by multiplying the standard uncertainty (u) by an appropriate univariate and multivariate coverage factor (k and k', respectively).The values of multivariate guard band (g') were higher than the values of univariate guard bands (g), which leads to more restrictive acceptance intervals. Measured values between the conventional and the multivariate acceptance limits will ensure particular risk values below the maximum acceptable value, however, the total risk may be significantly high. On the other hand, measured values within the multivariate acceptance limits ensure that particular risk values and total risk value are below the maximum acceptable value The application of multivariate guard bands is a simple way to ensure reduced particular and total risks of false conformity decisions, which is of great interest to regulatory agencies and the manufactures of the medicines.

Conformity assessment of medicines containing antibiotics - A multivariate assessment.

Pharmaceutical products must meet quality requirements to ensure the efficacy and safety of pharmacological treatment. Non-compliance of medicines can cause economic losses and compromise the patient's health. In this work, the risks of false compliance/non-compliance decisions for parenteral antibiotics (cephalothin, ciprofloxacin and metronidazole) were evaluated on the basis of analytical results, measurement uncertainties and specification limits. Physicochemical and biological quality assays were performed according to pharmacopeial procedures. Measurement uncertainties were determined using the bottom-up approach or the probability of false-positive/false-negative results. The risks of false compliance/non-compliance decisions were estimated using the frequentist approach and Monte Carlo simulations. Guard-bands were determined through a validated spreadsheet for calculating univariate and multivariate acceptance limits. All risk values (particular risks and total risk, consumer's or producer's risk) were below the maximum permissible risk value. The univariate and multivariate guard-bands defined more restrictive specification values, reducing the risks of false compliance/non-compliance decisions. One antibiotic presented unsatisfactory results regarding the drug content and was classified as out of specification product. The application of risk management tools in the pharmaceutical area contributes to improving the quality and safety of products and supports decision-making.

Effect of Polydextrose on the Growth of Pediococcus pentosaceus as Well as Lactic Acid and Bacteriocin-like Inhibitory Substances (BLIS) Production.

Pediococcus pentosaceus was cultivated in MRS medium supplemented or not with polydextrose under different conditions in order to evaluate its effect on cell growth, lactic acid and bacteriocin-like inhibitory substance (BLIS) production. Independent variables were pH (4.0, 5.0, 6.0), rotational speed (50, 100, 150 rpm), polydextrose concentration (0.5, 1.0, 1.5%) and temperature (25, 30, 35 °C), while cell concentration and productivity after 24 h, maximum specific growth rate, specific rate of substrate (glucose) consumption, volumetric and specific lactic acid productivities, yields of biomass and lactic acid on consumed substrate were the dependent. The maximum cell concentration (10.24 ± 0.16 gX L-1) and productivity (0.42 ± 0.01 gX L-1 h-1) were achieved at pH 6.0, 35 °C, 150 rpm using 1.5% polydextrose, while the maximum specific growth rate (0.99 ± 0.01 h-1) and yield of biomass (2.96 ± 0.34 gX gS-1) were achieved at the same pH and polydextrose concentration, but at 25 °C and 50 rpm. The specific substrate consumption rate (0.09 ± 0.02 gS gX-1 h-1) and the volumetric lactic acid productivity (0.44 ± 0.02 gP L-1 h-1) were maximized at pH 6.0, 35 °C, 50 rpm and 0.5% polydextrose. BLIS produced in this last run displayed the highest antibacterial activity against Escherichia coli, while the same activity was displayed against Enterococcus faecium using 1.5% polydextrose. These results appear to be quite promising in view of possible production of this BLIS as an antibacterial agent in the food industry.

Besifloxacin Nanocrystal: Towards an Innovative Ophthalmic Preparation.

Bacterial conjunctivitis significantly impacts public health, including more than one-third of eye diseases reported worldwide. It is an infection caused by various aerobic and anaerobic bacteria and is highly contagious. Therefore, it has a high incidence of bacterial resistance to the antibiotics commonly used for treatment. Among the most recent antibiotics, besifloxacin is a fourth-generation fluoroquinolone antibiotic indicated exclusively for topical ophthalmic use. Due to its importance in treating bacterial conjunctivitis and its low solubility in water, limiting its efficacy, a nanotechnology-based drug delivery preparation was developed to overcome this hurdle. Besifloxacin nanocrystals were prepared by small-scale wet milling and response surface methodology, using Povacoat® as a stabilizer. The particle's average hydrodynamic diameter (Z-ave) was approximately 550 nm (17 times smaller than raw material), with a polydispersity index (PdI) of less than 0.2. The saturation solubility increased about two times compared to the raw material, making it possible to increase the dissolution rate of this drug substance, potentially improving its bioavailability and safety. The optimized preparation was stable under an accelerated stability study (90 days). The Z-ave, PZ, PdI, and content did not alter significantly during this period. Furthermore, the 0.6% m/m besifloxacin nanocrystals at the maximum dose and the Povacoat® stabilizer did not show toxicity in Galleria mellonella larvae. The innovative ophthalmic preparation minimum inhibitory concentration (MIC) was 0.0960 µg/mL and 1.60 µg/mL against Staphylococcus aureus and Pseudomonas aeruginosa, respectively, confirming in vitro efficacy. Therefore, besifloxacin nanocrystals revealed the potential for reduced dosing of the drug substance, with a minor occurrence of adverse effects and greater patient adherence to treatment.

Antibiotic-loaded lipid-based nanocarrier: A promising strategy to overcome bacterial infection.

According to the World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC), bacterial infections are one of the greatest threats to global health, food production, and life expectancy. In this sense, the development of innovative formulations aiming at greater therapeutic efficacy, safety, and shorter treatment duration compared to conventional products is urgently needed. Lipid-based nanocarriers (LBNs) have demonstrated the potential to enhance the effectiveness of available antibiotics. Among them, liposome, nanoemulsion, solid lipid nanoparticle (SLN), and nanostructured lipid carrier (NLC) are the most promising due to their solid technical background for laboratory and industrial production. This review describes recent advances in developing antibiotic-loaded LBNs against susceptible and resistant bacterial strains and biofilm. LBNs revealed to be a promising alternative to deliver antibiotics due to their superior characteristics compared to conventional preparations, including their modified drug release, improved bioavailability, drug protection against chemical or enzymatic degradation, greater drug loading capacity, and biocompatibility. Antibiotic-loaded LBNs can improve current clinical drug therapy, bring innovative products and rescue discarded antibiotics. Thus, antibiotic-loaded LBNs have potential to open a window of opportunities to continue saving millions of lives and prevent the devastating impact of bacterial infection.

Hot-melt extrudability of amorphous solid dispersions of flubendazole-copovidone: An exploratory study of the effect of drug loading and the balance of adjuvants on extrudability and dissolution.

The FDA-approved anthelmintic flubendazole has shown potential to be repositioned to treat cancer and dry macular degeneration; however, its poor water solubility limits its use. Amorphous solid dispersions may overcome this challenge, but the balance of excipients may impact the preparation method and drug release. The purpose of this study was to evaluate the influence of adjuvants and drug loading on the development of an amorphous solid dispersion of flubendazole-copovidone by hot-melt extrusion. The drug, copovidone, and adjuvants (magnesium stearate and hydroxypropyl cellulose) mixtures were statistically designed, and the process was performed in a twin-screw extruder. The study showed that flubendazole and copovidone mixtures were highly extrudable, except when drug loading was high (>40%). Furthermore, magnesium stearate positively impacted the extrusion and was more effective than hydroxypropyl cellulose. The extruded materials were evaluated by modulated differential scanning calorimetry and X-ray powder diffraction, obtaining positive amorphization and physical stability results. Pair distribution function analysis indicated the presence of drug-rich domains with medium-range order structure and no evidence of polymer-drug interaction. All extrudates presented faster dissolution (HCl, pH 1.2) than pure flubendazole, and both adjuvants had a notable influence on the dissolution rate. In conclusion, hot-melt extrusion may be a viable option to obtain stable flubendazole:copovidone amorphous dispersions.

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.

Measurement uncertainty evaluation and risk of false conformity assessment for microbial enumeration tests.

Microbial enumeration tests are widely used to assess the microbiological quality of non-sterile pharmaceutical products. Despite of all efforts to guarantee the reliability of microbial enumeration tests, there will always be an uncertainty associated with the measured values, which can lead to false conformity/non-conformity decisions. In this work, we evaluated the measurement uncertainty using a bottom-up approach and estimate the consumer's or producer's risk due to the measurement uncertainty. Three main sources of uncertainty were identified and quantified: dilution factor, plated volume, and microbial plate counts. The contribution of these sources of uncertainty depends on the measured value of microbial load in pharmaceutical products. The contribution of dilution factor and plated volume uncertainties increase with an increase of measured value, while the contribution of microbial plate count uncertainty decreases with an increase of measured value. The overall uncertainty values were expressed as uncertainty factors, which provide an asymmetric 95% level confidence level of microbial load in pharmaceutical products. In addition, the risk of false conformity/non-conformity decisions due to measurement uncertainty was assess using Monte Carlo method. When the measured value is close to the upper specification limit and/or the measurement uncertainty is large, the risk of false conformity/non-conformity decisions may be significantly high. Thus, we conclude that the use of uncertainty factor in the conformity/non-conformity assessment is important to guarantee the reliability of microbial enumeration test results and to support decision-making.