Development of an Untargeted Method for the Analysis of the Volatile Profile of Onions via Solid-Phase Microextraction Arrow-Headspace-Gas Chromatography-Mass Spectrometry Using Design of Experiments.

The distinctive aroma of onions, consisting primarily of sulfur-containing compounds, is one of the reasons for the popularity of the vegetable. The rapid enzymatic and chemical reactions that occur after the destruction of onion bulb tissue render the analysis of the volatile profile challenging. Therefore, sample preparation is a crucial step in the analysis of the onion volatilome, but it often does not receive the necessary attention in the literature. In this work, we focused on two aspects: Firstly, we compared different sample preparation approaches to maximize the volatile yield and to enable the description of the onion volatile profile as it would emerge after a solid-phase microextraction (SPME) Arrow sampling procedure. For headspace (HS)-gas chromatography-mass spectrometry analysis, onion juice with the addition of an ammonium sulfate solution proved to be the best option. Secondly, we optimized the HS sampling and desorption parameters (enrichment time, enrichment temperature, agitator speed, desorption time) for onion volatiles using the efficient design of experiments (DoE) approach. The optimal conditions for the analysis with HS-SPME Arrow were an enrichment time of 75 min at 60°C with an agitator speed of 713 rpm and a desorption time of 120 s.

Investigation of impact of siderophore and process variables on production of iron enriched Saccharomyces boulardii by Plackett-Burman design.

The primary cause of anemia worldwide is due to poor diet and iron deficiency. Iron (Fe) enriched yeast can be the most effective way to manage anemia because of the capability for biotransformation of mineral to organic and bioavailable iron. To overcome the low richness of yeast, the use of siderophore as cellular iron carriers is a new approach. In this research, for the first time the potential of siderophore in increasing the Fe enrichment of Saccharomyces boulardii (S. boulardii), which is important because of its probiotic properties and resistance to different stresses, has been investigated to produce of potential iron supplements. For this purpose, siderophore was produced by Pseudomonas aeruginosa (P. aeruginosa). Siderophore impact, along with ten other independent process variables, has been studied on the efficiency of iron biotransformation by the Plackett-Burman design (PBD). The results showed that the highest biotransformation yield was 17.77 mg Fe/g dry cell weight (DCW) in the highest biomass weight of 9 g/l. Iron concentration is the most important variable, with contributions of 46% and 70.79% for biomass weight and biotransformation, respectively, followed by fermentation time, agitation speed, and KH2PO4 concentration. But increasing the level of siderophore and zinc led to a significant negative effect. siderophore inefficiency may be attributed to the absence of membrane receptors for pyoverdine (Pvd) and pyochelin (Pch) siderophores. Also, the steric hindrance of the cell wall mannan, the stickiness and sediment ability of the yeast, can create limitations in the absorption of elements. Such yeast can be used as a potential source of iron even for vegetarians and vegans in the form of medicinal and fortified food products to improve the treatment of anemia. It is recommended that further research be focused on increasing the iron enrichment of yeast by overcoming the structural barrier of the cell wall, investigating factors affecting membrane permeability and iron transport potential of other types of siderophores.

Robustness evaluation of weak anion exchange chromatography method for the purity analysis of therapeutic oligonucleotides.

Therapeutic oligonucleotides are becoming an important class of therapeutics. Their manufacturing processes can result in the formation of impurities, particularly truncated species. To ensure the quality and safety of the product, it is crucial to evaluate the presence of these species. Liquid chromatography analysis enables such purity determination. In this context, a recently described weak anion exchange chromatography method was optimized to allow the effective separation of different impurities. The optimization addressed the complexity and instability of the mobile phases, which contained salts and organic compounds. Adjustments were made to the mobile phase composition and gradient to meet the requirements of QC laboratories. Additionally, to ensure the method's reliability, a robustness study was conducted based on a risk assessment. Five factors were considered potential risks and were assessed experimentally on different chromatographic outputs. This led to the definition of a robust space, ensuring the method's reliability for the purity determination of oligonucleotides.

An artificial bee colony optimization algorithms for solving fuzzy capacitated logistic distribution center problem.

This paper presents a methodological approach to solving the fuzzy capacitated logistic distribution center problem, with a focus on the optimal selection of distribution centers to meet the demands of multiple plants. The distribution centers are characterized by fixed costs and capacities, while plant demands are modeled using fuzzy triangular membership functions. The problem is mathematically formulated by converting fuzzy demands into crisp values, providing a structured framework for addressing uncertainty in logistic planning. To support future research and facilitate comparative analysis, 20 benchmark problems were generated, filling a gap in the existing literature. Three distinct artificial bee colony algorithm variants were hybridized with a heuristic: one using the best solution per iteration, another incorporating chaotic mapping and adaptive procedures, and the third employing convergence and diversity archives. An experimental design based on Taguchi's orthogonal arrays was employed for optimizing the algorithm parameters, ensuring systematic exploration of the solution space. The developed methods offer a comprehensive toolkit for addressing complex, uncertain demands in logistic distribution, with code provided for reproducibility. Key contributions include:•Development of a fuzzy model for the selection of distribution centers with fixed costs and capacities under uncertain plant demands.•Generation of 20 benchmark problems to advance research in the fuzzy capacitated logistic distribution center problem domain.•Integration of a heuristic approach with three distinct ABC algorithm variants, each contributing unique methodological insights.

Evaluation of different techniques for wet granulation and pelletization processes using milk as innovative pharmaceutical excipient for pediatric use.

The present study focused on the use of milk as a novel excipient for the manufacture of pharmaceutical dosage forms specifically designed for the pediatric population. Dairy milks with different fat contents were studied to deliver paracetamol orally. The World Health Organization included milk in the list of GRAS (generally recognized as safe) substances, which together with its taste-masking ability and solubility solving properties, makes it a good candidate as an excipient in formulations containing paracetamol for pediatrics. The influence of the fat content in the milk, the fraction of paracetamol, the type of diluent and drying temperature (considered independent variables) were systematically investigated using a Design of Experiments (DoE) approach for the preparation of granules for oral administration, by wet agglomeration using different processing techniques, enabled the construction of mathematical models reflecting the correlation between the variables. Four different techniques were evaluated: wet granulation by low shear mixer, wet granulation by high shear mixer, wet granulation by fluidized bed, and extrusion and spheronization. The granules and pellets obtained were characterized for size, and size distribution of agglomerates, and complete release of the drug (dependent variables), according to the European Pharmacopoeia. The fraction of fat content in the milk promoted an increase on the dissolution rate of paracetamol. The key finding of the first two process techniques was a migration of paracetamol from powdered agglomerates towards the larger particles, probably due to friction and attrition events, which created a fraction of smaller size granules due to the fragmentation and loss of powder from the larger granules. The study has confirmed the potential of milk to be a novel and efficient excipient that can be used as a liquid binder in various agglomeration techniques to deliver drugs orally.

The importance of factorial design on the optimization of biosensor performance: immobilization of glucose oxidase as a case study.

Conventionally, the optimization of glucose biosensors is achieved by varying the concentrations of the individual reagents used to immobilize the enzyme. In this work, the effect and interaction between glucose oxidase enzyme (GOx), ferrocene methanol (Fc), and multi-walled carbon nanotubes (MWCNTs) at different concentrations were investigated by a design of experiments (DoE). For this analysis, a factorial design with three factors and two levels each was used with the software RStudio for statistical analysis. The data were obtained by electrochemical experiments on the immobilization of GOx-Fc/MWCNT at different concentrations. The results showed that the factorial DoE method was confirmed by the non-normality of the residuals and the outliers of the experiment. When examining the effects of the variables, analyzing the half-normal distribution and the effects and contrasts for GOx-Fc/MWCNT, the factors that showed the greatest influence on the electrochemical response were GOx, MWCNT, Fc, and MWCNT:Fc, and there is a high correlation between the factors GOx, MWCNT, Fc, and MWCNT:Fc, as shown by the analysis of homoscedasticity and multicollinearity. With these statistical analyses and experimental designs, it was possible to find the optimal conditions for different factors: 10 mM mL-1 GOx, 2 mg mL-1 Fc, and 15 mg mL-1 MWCNT show a greater amperometric response in the glucose oxidation. This work contributes to advancing enzyme immobilization strategies for glucose biosensor applications. Systematic investigation of DoE leads to optimized immobilization for GOx, enables better performance as a glucose biosensor, and allows the prediction of some outcomes.

Whiteness, redness, blueness and greenness profile assessment and design of experiments approach to microwave-aided sensitive and green spectrofluorophotometric determination of baclofen.

As a gamma amino butyric acid-ergic agonist, Baclofen is often prescribed to adults and children for the treatment of severe spasticity that originates in the brain or spinal cord. Even after reviewing the literature extensively, no one has documented a method for estimating baclofen using microwave-assisted stability-indicating spectrofluorimetric techniques, despite the abundance of options for baclofen stability, assay, and bioanalysis. Organic solvents, which are typically necessary for current procedures but may be costly and toxic, have a severe effect on aquatic life and the environment. Using green solvents and 4-chloro-7-nitrobezofuran as a fluorescent probe, this work conducted a stability-indicating spectrofluorimetric estimate of baclofen. Through the use of a design-of-experiments technique, a reliable microwave-aided spectrofluorimetric method was developed, with little solvent consumption and time for sample analysis. Prior to conducting response surface analysis and optimizing important variables and responses, a fractional factorial design was used to screen method variables and responses. A central composite design was then employed for these purposes. This flexible spectrofluorimetric technique was used to assess baclofen concentrations in forced degraded samples and marketed formulations. For baclofen determination, the suggested spectrofluorimetric approach was found to be green, quick, easy to use, economical, and user-friendly.

Influence of articular cartilage sample geometry on mechanical response and properties using finite element simulation.

Mechanical testing of articular cartilage yields highly variable results, posing challenges for tissue characterization. Many factors cause variability, one is sample geometry. Using in-situ phase-contrast enhanced synchrotron micro-tomographs of cartilage samples while tested in unconfined compression (stress relaxation) our group found high variability in the mechanical response. Since all samples originated from a single bovine knee, they were assumed to share mechanical properties. Microscale tomography images showed geometric irregularities in samples that were not accounted for in the often assumed intended cylindrical shape. We aimed to determine the influence of sample shape on mechanical response in unconfined compression and how sample geometry affects identified mechanical properties. Using a parametric FE model incorporating geometric irregularities in a Design of Experiments approach, results were analysed with 2-way ANOVA. Furthermore, a material parameter fitting was done with multiple segmented sample-specific finite element models simultaneously to assess the influence of sample geometry on material parameters. Results revealed that the average inclined sample surface (4°) caused a 15 % decrease in reaction forces compared to the intended cylinder. Fitting multiple sample-specific geometries simultaneously altered material parameters between -70 to +159 % compared to the average model. Strikingly, initial fibril stiffness and permeability increased by 137 % and 159 %, while the root-mean-square error of the fit was reduced by ∼2/3 compared to using parameters from a cylindrical shape model. In conclusion, minor variability in sample geometry affects property characterization and can account for some of the inter-sample variability in the mechanical data for cartilage.

Development and integration of a continuous horizontal belt filter into drug production procedure.

In the pharmaceutical industry, filtration is traditionally carried out in batch mode. However, with the spread of continuous technologies, there is an increasing demand for robust continuous filtration strategies suitable for processing suspensions produced in continuous crystallizers. Accordingly, this study aimed to investigate a lab-scale horizontal conveyor belt filtration approach for pharmaceutical separation purposes for the first time. The newly developed continuous horizontal belt filter (CHBF) was tested under different systems (microcrystalline cellulose (MCC)/water, lactose/ethanol and acetylsalicylic acid (ASA)/water) and diverse conditions. Filtration was robust using a well-defined unimodal particle size distribution MCC in water system, where the residual moisture content varied within narrow limits of 45-52% independently from the process conditions. Besides, the residual moisture content highly depended on the applied solvent and particle size. It could be reduced to below 2% by processing the suspensions of either a volatile solvent (lactose in ethanol) or an aqueous slurry of a large particle size ASA. Finally, the CHBF was connected to a mixed suspension mixed product removal (MSMPR) or a plug flow crystallizer (PFC). The residual moisture content of the CHBF-filtered ASA product and operation characteristics (onset of steady-state) were evaluated in both continuous crystallizer-filter systems. The MSMPR-CHBF system operated with a longer startup period. The size of the in situ-produced crystals was of a similar order magnitude in both systems, resulting in a similar residual moisture content (around 20%). Overall, the tested continuous filter was robust, did not modify the crystal morphology in the examined experimental range, and could be effectively integrated with continuous crystallizers.

A Lab-Scale Evaluation of Parameters Influencing the Mechanical Activation of Kaolin Using the Design of Experiments.

This research investigates the mechanical activation of kaolin as a supplementary cementitious material at the laboratory scale, aiming to optimize milling parameters using the response surface methodology. The study evaluated the effects of rotation speed and milling time on the amorphous phase content, the reduction in crystalline kaolinite, and impurity incorporation into the activated clay through the Rietveld method. The results demonstrated that adjusting milling parameters effectively enhanced clay activation, which is crucial for its use in low-carbon cements. High rotation speeds (300/350 rpm) and prolonged grinding times (90/120 min) in a planetary ball mill increased the pozzolanic activity by boosting the formation of amorphous phases from kaolinite and illite and reducing the particle size. However, the results evidenced that intermediate milling parameters are sufficient for reaching substantial degrees of amorphization and pozzolanic activity, avoiding the need for intensive grinding. Exceedingly aggressive milling introduced impurities like ZrO2 from the milling equipment wear, underscoring the need for a balanced approach to optimizing reactivity while minimizing impurities, energy consumption, and equipment wear. Achieving this balance is essential for efficient mechanical activation, ensuring the prepared clay's suitability as supplementary cementitious materials without excessive costs or compromised equipment integrity.

Chondroitin Sulfate for Cartilage Regeneration, Administered Topically Using a Nanostructured Formulation.

In the pharmaceutical sector, solid lipid nanoparticles (SLN) are vital for drug delivery incorporating a lipid core. Chondroitin sulfate (CHON) is crucial for cartilage health. It is often used in osteoarthritis (OA) treatment. Due to conflicting results from clinical trials on CHON's efficacy in OA treatment, there has been a shift toward exploring effective topical systems utilizing nanotechnology. This study aimed to optimize a solid lipid nanoparticle formulation aiming to enhance CHON permeation for OA therapy. A 3 × 3 × 2 Design of these experiments determined the ideal parameters: a CHON concentration of 0.4 mg/mL, operating at 20,000 rpm speed, and processing for 10 min for SLN production. Transmission electron microscopy analysis confirmed the nanoparticles' spherical morphology, ensuring crucial uniformity for efficient drug delivery. Cell viability assessments showed no significant cytotoxicity within the tested parameters, indicating a safe profile for potential clinical application. The cell internalization assay indicates successful internalization at 1.5 h and 24 h post-treatment. Biopharmaceutical studies supported SLNs, indicating them to be effective CHON carriers through the skin, showcasing improved skin permeation and CHON retention compared to conventional methods. In summary, this study successfully optimized SLN formulation for efficient CHON transport through pig ear skin with no cellular toxicity, highlighting SLNs' potential as promising carriers to enhance CHON delivery in OA treatment and advance nanotechnology-based therapeutic strategies in pharmaceutical formulations.

Can We Simplify Liposome Manufacturing Using a Complex DoE Approach?

Microfluidic liposome production presents a streamlined pathway for expediting the translation of liposomal formulations from the laboratory setting to clinical applications. Using this production method, resultant liposome characteristics can be tuned through the control of both the formulation parameters (including the lipids and solvents used) and production parameters (including the production speed and mixing ratio). Therefore, the aim of this study was to investigate the relationship between not only total flow rate (TFR), the fraction of the aqueous flow rate over the organic flow rate (flow rate ratio (FRR)), and the lipid concentration, but also the solvent selection, aqueous buffer, and production temperature. To achieve this, we used temperature, applying a design of experiment (DoE) combined with machine learning. This study demonstrated that liposome size and polydispersity were influenced by manipulation of not only the total flow rate and flow rate ratio but also through the lipids, lipid concentration, and solvent selection, such that liposome attributes can be in-process controlled, and all factors should be considered within a manufacturing process as impacting on liposome critical quality attributes.

Lignin extraction from acacia wood: Crafting deep eutectic solvents with a systematic D-optimal mixture-process experimental design.

Lignin is a complex biopolymer whose efficient extraction from biomass is crucial for various applications. Deep eutectic solvents (DES), particularly natural-origin DES (NADES), have emerged as promising systems for lignin fractionation and separation from other biomass components. While ternary DES offer enhanced fractionation performance, the role of each component in these mixtures remains unclear. In this study, the effects of adding tartaric acid (Tart) or citric acid (Cit) to a common binary DES mixture composed of lactic acid (Lact) and choline chloride (ChCl) were investigated for lignin extraction from acacia wood. Ternary Cit-based DES showed superior performance compared to Tart-based DES. Using a combined mixture-process D-Optimal experimental design, the Lact:Cit:ChCl DES composition and extraction temperature were optimized targeting maximum lignin yield and purity. The optimal conditions (i.e., Lact:Cit:ChCl, 0.6:0.3:0.1 molar ratio, 140 °C) resulted in a lignin extraction yield of 99.63 ± 1.24 % and a lignin purity of 91.45 ± 1.03 %. Furthermore, this DES exhibited feasible recyclability and reusability without sacrificing efficiency.

Development, evaluation of critical method variables and stability assessment using a Box-Behnken design for the determination of organic impurities in a pharmaceutical dosage form of a centrally acting muscle relaxant drug chlorzoxazone.

This study validates a stability-indicating LC method for detecting organic impurities in the chlorzoxazone dosage form. Using a Waters X-Select R HSS T3 analytical column, mobile phase of it was made by mixing of water, methanol, and glacial acetic acid in the ratio of 700:300:10 (v/v/v). The drug product and drug substance were subjected to the stress conditions such as acid, base, oxidation, heat, and photolysis as per the recommendations of the International Conference on Harmonization (Q2) methodology. The study revealed the susceptibility of 4-chloro-2-aminophenol to alkaline environments, emphasizing peak homogeneity and stability. The method verification, per ICH guidelines and USP<1225>, established precision, specificity, linearity, accuracy, and robustness for quality control. The mean impurity recovery ranged from 95.5% to 105.2%, the correlation coefficient (r) was greater than 1.000, and the RSD values (n = 6) ranged from 0.6% to 5.1% across the LOQ-150% ranges. Full-factorial design tested final method conditions, evaluating multiple parameters concurrently. Graphical optimization within the design space defined strong method requirements, ensuring consistent and reliable outcomes. The study develops and validates chlorzoxazone stability-indicating methods, employing advanced statistical approaches like design of experiments and factorial design, with resilient conditions established through graphical optimization of the design space.

Quality by Design for Preclinical In Vitro Assay Development.

Quality by Design (QbD) is an approach to assay development to determine the design space, which is the range of assay variable settings that should result in satisfactory assay quality. Typically, QbD is applied in manufacturing, but it works just as well in the preclinical space. Through three examples, we illustrate the QbD approach with experimental design and associated data analysis to determine the design space for preclinical assays.

Design and evaluation of magnetic-targeted bilosomal gel for rheumatoid arthritis: flurbiprofen delivery using superparamagnetic iron oxide nanoparticles.

Introduction: The study aimed to systematically enhance the fabrication process of flurbiprofen-loaded bilosomes (FSB) using Quality by Design (QbD) principles and Design of Experiments (DOE). The objective was to develop an optimized formulation with improved entrapment efficiency and targeted drug delivery capabilities. Methods: The optimization process involved applying QbD principles and DOE to achieve the desired formulation characteristics. Superparamagnetic iron oxide nanoparticles (SPIONs) were incorporated to impart magnetic responsiveness. The size, entrapment efficiency, morphology, and in vitro release patterns of the FSB formulation were evaluated. Additionally, an in situ forming hydrogel incorporating FSB was developed, with its gelation time and drug release kinetics assessed. In vivo studies were conducted on osteoarthritic rats to evaluate the efficacy of the FSB-loaded hydrogel. Results: The optimized FSB formulation yielded particles with a size of 453.60 nm and an entrapment efficiency of 91.57%. The incorporation of SPIONs enhanced magnetic responsiveness. Morphological evaluations and in vitro release studies confirmed the structural integrity and sustained release characteristics of the FSB formulation. The in situ forming hydrogel exhibited a rapid gelation time of approximately 40 ± 1.8 s and controlled drug release kinetics. In vivo studies demonstrated a 27.83% reduction in joint inflammation and an 85% improvement in locomotor activity in osteoarthritic rats treated with FSB-loaded hydrogel. Discussion: This comprehensive investigation highlights the potential of FSB as a promising targeted drug delivery system for the effective management of osteoarthritis. The use of QbD and DOE in the formulation process, along with the integration of SPIONs, resulted in an optimized FSB formulation with enhanced entrapment efficiency and targeted delivery capabilities. The in situ forming hydrogel further supported the formulation's applicability for injectable applications, providing rapid gelation and sustained drug release. The in vivo results corroborate the formulation's efficacy, underscoring its potential for improving the treatment of osteoarthritis.

Evaluation of analytical greenness metric for an eco-friendly method developed through the integration of green chemistry and quality-by-design for the simultaneous determination of Nebivolol hydrochloride, Telmisartan, Valsartan, and Amlodipine besylate.

In recent years, the field of analytical chemistry has witnessed a notable shift towards the adoption of greener chromatographic methods, aiming to minimize the environmental impact. An effective strategy involves substituting conventional harmful organic solvents with environmentally friendly alternatives, reducing the use of hazardous chemicals that contribute to environmental concerns. However, separating drug substances without the use of buffers and organic solvents presence is a big challenge. To overcome this challenge, a combination of quality-by-design (QbD) and green analytical chemistry (GAC) was employed in this study for method development. A high-performance liquid chromatography (HPLC) method was successfully developed and validated for the simultaneous determination of Nebivolol hydrochloride, Telmisartan, Valsartan, and Amlodipine besylate. The method utilized a mobile phase composed of a mixture of 0.1 % formic acid in water (pH: 2.5) and ethanol. A regular octadecyl silica (ODS) column was employed, and UV detection at 220 nm was utilized. The method exhibited linearity within the concentration range of 25-75 µg/mL for Telmisartan and 150-450 µg/mL for Nebivolol Hydrochloride, Valsartan, and Amlodipine besylate and the correlation coefficient was greater than 0.999 for all the analytes. Limits of detection (LOD) and quantification (LOQ) were determined as 0.01 and 0.04 µg/mL for Telmisartan, 0.06 and 0.20 µg/mL for Nebivolol Hydrochloride, 0.08 and 0.25 µg/mL for Amlodipine besylate, and 0.14 and 0.46 µg/mL for Valsartan, respectively. The developed method underwent thorough validation, encompassing various parameters such as linearity, accuracy, precision, LOD, LOQ, robustness, and ruggedness. The mean recovery values were observed to range between 98.86 % and 99.89 %. The accuracy demonstrated was consistently above 98.98 % for both intra-day and inter-day precisions were with the relative standard deviations less than 2 %. To establish its robustness, a quality-by-design-based experimental design (DoE) approach was implemented. Additionally, the method's environmental friendliness was evaluated using the Analytical Greenness metric (AGREE) an analytical eco scale, both confirming its alignment with sustainable practices and reduced ecological impact. The sustainability of the solvent used in the current study was evaluated by Green Solvents Selecting Tool (GSST) Further, the developed method greenness was evaluated with the green analytical tools such as Analytical method greenness score (AMGS) and using the recently released White Analytical Chemistry (WAC) using RGB assessment tool. By employing this greener approach to chromatography method, this study contributes to the ongoing efforts in analytical chemistry to promote sustainable practices and minimize the environmental footprint of analytical methods.

Unraveling the complexities of antifungal susceptibility testing in Candida spp.: Insights from design of experiments.

Our study delved into the intricate dynamics of antifungal susceptibility testing for Candida spp., employing a Design of Experiments approach. We systematically investigated the influence of pH, temperature, inoculum size, and glucose concentration on both growth patterns and inhibitory concentrations of Candida spp. Our findings underscore the nuanced interplay between these factors, revealing significant impacts on susceptibility outcomes. Notably, even minor adjustments in these parameters yielded substantial variations in growth and inhibitory concentrations, underscoring the critical importance of meticulous control over growth conditions in antifungal susceptibility testing protocols. Each Candida isolates exhibited unique susceptibility profiles, necessitating tailored culture conditions for accurate testing. Our study sheds light on the variability inherent in Candida spp. growth patterns and emphasizes the need for standardized protocols to ensure consistency across laboratories. By leveraging the design of experiments, our research provides a systematic framework for unraveling the complexities of antifungal susceptibility testing, offering valuable insights for optimizing testing protocols and informing clinical decision-making in antifungal treatment. These findings represent a significant step towards enhancing the efficacy and reliability of antifungal susceptibility testing in clinical practice.

Sustainable application of modified Luffa cylindrica biomass for removal of trimethoprim in water by adsorption with process optimization.

The present study describes a set of methodological procedures (seldom applied together), including (i) development of an alternative adsorbent derived from abundant low-cost plant biomass; (ii) use of simple low-cost biomass modification techniques based on physical processing and chemical activation; (iii) design of experiments (DoE) applied to optimize the removal of a pharmaceutical contaminant from water; (iv) at environmentally relevant concentrations, (v) that due to initial low concentrations required determination by ultra-performance liquid phase chromatography coupled to mass spectrometry (UPLC-MS/MS). A central composite rotational design (CCRD) was employed to investigate the performance of vegetable sponge biomass (Luffa cylindrica), physically processed (crushing and sieving) and chemically activated with phosphoric acid, in the adsorption of the antibiotic trimethoprim (TMP) from water. The optimized model identified pH as the most significant variable, with maximum drug removal (91.1 ± 5.7%) achieved at pH 7.5, a temperature of 22.5 °C, and an adsorbent/adsorbate ratio of 18.6 mg µg-1. The adsorption mechanisms and surface properties of the adsorbent were examined through characterization techniques such as scanning electron microscopy (SEM), point of zero charge (pHpzc) measurement, thermogravimetric analysis (TGA), specific surface area, and Fourier-transform infrared spectroscopy (FTIR). The best kinetic fit was obtained by the Avrami fractional-order model. The hypothesis of a hybrid behavior of the adsorbent was suggested by the equilibrium results presented by the Langmuir and Freundlich models and reinforced by the Redlich-Peterson model, which achieved the best fit (R2 = 0.982). The thermodynamic study indicated an exothermic, spontaneous, and favorable process. The maximum adsorption capacity of the material was 2.32 × 102 µg g-1 at an equilibrium time of 120 min. Finally, a sustainable and promising adsorbent for the polishing of aqueous matrices contaminated by contaminants of emerging concern (CECs) at environmentally relevant concentrations is available for future investigations.

Unlocking DOE potential by selecting the most appropriate design for rAAV optimization.

Producing recombinant adeno-associated virus (rAAV) for gene therapy via triple transfection is an intricate process involving many cellular interactions. Each of the different elements encoded in the three required plasmids-pHelper, pRepCap, and pGOI-plays a distinct role, affecting different cellular pathways when producing rAAVs. The required expression balance emphasizes the critical need to fine-tune the concentration of all these different elements. The use of design of experiments (DOE) to find optimal ratios is a powerful method to streamline the process. However, the choice of the DOE method and design construction is crucial to avoid misleading results. In this work, we examined and compared four distinct DOE approaches: rotatable central composite design (RCCD), Box-Behnken design (BBD), face-centered central composite design (FCCD), and mixture design (MD). We compared the abilities of the different models to predict optimal ratios and interactions among the plasmids and the transfection reagent. Our findings revealed that blocking is essential to reduce the variability caused by uncontrolled random effects and that MD coupled with FCCD outperformed all other approaches, improving volumetric productivity 109-fold. These outcomes underscore the importance of selecting a model that can effectively account for the biological context, ultimately yielding superior results in optimizing rAAV production.