Multivariate Experimental Design Research Articles

Fundamental investigation of impact of water and TFA additions in peptide sub/supercritical fluid separations

The retention of three peptides was studied under analytical and overloaded conditions at different concentrations of trifluoroacetic acid (TFA) and water added to the co-solvent methanol (MeOH). Four columns with different stationary phase properties, i.e., silica, diol, 2-ethylpyridine and cyanopropyl (CN) columns, were evaluated in this investigation. The overall aim was to get a deeper understanding on how column chemistry as well as water and TFA in the co-solvent affect the analytical and overloaded elution profiles using multivariate design of experiments and adsorption measurements of co-solvent components. Multivariate experimental design modeling indicated that water had on average around five times higher effect on the retention than the addition of TFA. The results also showed that the retention increases with the addition of TFA and water to the co-solvent on all columns except the CN column, on which the retention decreased. When examining the effect of adding water to the co-solvent, evidence of a hydrophilic interaction liquid chromatography (HILIC)-like retention mechanism was found on the three other column with more polar stationary phases. However, on the CN column water acted as an additive, decreasing the retention due to competition with the peptide for available adsorption surface. Adsorption isotherm measurements of the polar solvent MeOH showed that MeOH adsorbs much weaker on the CN column than on the other columns. Addition of TFA and water to the co-solvent substantially sharpened the elution profiles under both overloaded and analytical conditions. Adding a small amount of TFA (from 0% to 0.05%) to the co-solvent substantially improved the peak shape of the elution profiles, while further addition (from 0.05% to 0.15%) had only a minor effect on the elution profile shape. The reduced retention on the CN column could not be explained by TFA adsorption, which was very weak on all studied columns (retention factor, 0.05–0.15). One could therefore speculate that the ion-pairing complex formed between the peptide and TFA in the mobile phase, reduce the retention due to its reduced polarity. On the other columns displaying HILIC-like properties, the TFA probably just decreased the pH of the mobile phase, thereby promoting the partitioning of the peptide into the water-rich layer. Finally, peak deformation due to diluent–eluent mismatch was observed under overloaded conditions. This was most severe in the cases where MeOH adsorption to the stationary phase was strong and the peptides were only mildly retained. Adding 1,4-dioxan to the diluent resolved this issue.

Olaparib Process Development Employing Quality by Design (QbD) Principles.

This study focuses on multivariate experimental design and statistical analysis to optimize the process of Olaparib 1. Quality by design (QbD) methodology was adopted for optimization of the Olaparib process consisting of three reaction steps: (1) amidation, (2) deprotection, and (3) acylation. Every chemical conversion was studied in isolation, employing risk assessment to identify key material attributes and key process parameters that may have the potential to impact the reaction. Thereafter, the screening design of experiment (DoE) was employed to scrutinize the factors that significantly impacted yield. Moving forward, the scrutinized factors which were found to impact the responses, the set of critical material attributes (CMAs) and critical process parameters (CPPs), were considered for optimization by applying I-Optimal design to define design space arriving at a robust setting wherein the predefined targets were supposedly optimal. To our delight, we got 95, 91, and 75% yield with more than 99% purity in amidation, deprotection, and acetylation, respectively, which enabled us to systematically identify design space to meet the desired quality target of the product consistently. More importantly, to distinguish the CMAs and CPPs, these elements ought to be monitored to have control of the quality parameter throughout the active pharmaceutical ingredient (API) value chain until commercial manufacturing followed by marketing. Eventually, we have developed a greener process in comparison to precedented one for Olaparib 1.

Integrated Approach of Life Cycle Assessment and Experimental Design in the Study of a Model Organic Reaction: New Perspectives in Renewable Vanillin-Derived Chemicals.

This work is focused on performing a quantitative assessment of the environmental impacts associated with an organic synthesis reaction, optimized using an experimental design approach. A nucleophilic substitution reaction was selected, employing vanillin as the substrate, a phenolic compound widely used in the food industry and of pharmaceutical interest, considering its antioxidant and antitumoral potential. To carry out the reaction, three different solvents have been chosen, namely acetonitrile (ACN), acetone (Ace), and dimethylformamide (DMF). The syntheses were planned with the aid of a multivariate experimental design to estimate the best reaction conditions, which simultaneously allow a high product yield and a reduced environmental impact as computed by Life Cycle Assessment (LCA) methodology. The experimental results highlighted that the reactions carried out in DMF resulted in higher yields with respect to ACN and Ace; these reactions were also the ones with lower environmental impacts. The multilinear regression models allowed us to identify the optimal experimental conditions able to guarantee the highest reaction yields and lowest environmental impacts for the studied reaction. The identified optimal experimental conditions were also validated by experimentally conducting the reaction in those conditions, which indeed led to the highest yield (i.e., 93%) and the lowest environmental impacts among the performed experiments. This work proposes, for the first time, an integrated approach of DoE and LCA applied to an organic reaction with the aim of considering both conventional metrics, such as reaction yield, and unconventional ones, such as environmental impacts, during its lab-scale optimization.

Ultrasonic-assisted extraction and UHPLC determination of ascorbic acid, polyphenols, and half-maximum effective concentration in Citrus medica and Ziziphus spina-christi fruits using multivariate experimental design

This study aimed to determine the concentrations of ascorbic acid and polyphenols in fruits and peels of Citrus medica and Ziziphus spina-christi grown in Ethiopia. Conditions of ultrasound-assisted extraction (UAE) and ultra-high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) were optimized, using a multivariate experimental design. The optimum conditions of UAE were 15 min extraction time at 35 ℃, with 75 % aqueous methanol as solvent, and a fruit powder-to-solvent ratio (m/v) of 1:15. Among the different drying conditions investigated, freeze-drying was found to be appropriate for analyzing ascorbic acid, polyphenols, and antioxidant potential. The overall ranges, across the fruits and peels, of ascorbic acid, total polyphenols, and antioxidant potentials (EC50) obtained were 8.7 ± 1.4–91.2 ± 2.6 mg/100 g, 253.0 ± 6.3–764.1 ± 25.8 mg GAE/100 g and 2.4 ± 0.1–26.1 ± 2.9 mg/mL, respectively. This indicates that the fruits and peels of the studied plants are advantageous as sources of ascorbic acid and polyphenols.

An integrated method for rapid extraction of fluorescent whitening agents from selected cosmetics by in situ formation of deep eutectic solvent

A novel extraction methodology was developed based on deep eutectic solvent (DES) in situ formation. The solvent was synthesized from a binary mixture of L-cysteine, functioning as a hydrogen bond acceptor, and 2-methylphenol, serving as a hydrogen bond donor. The DES’s inherent molecular structures and extensive hydrogen bonding networks were characterized using the Fourier-transform infrared spectroscopy (FT-IR), while its hydrophobic nature was assessed by determining molecular polarity. Its extraction efficiency was demonstrated through the extraction of fluorescent whitening agents (FWAs) in cosmetic products, acting as a model analytical application. This was accomplished using vortex-assisted emulsification solid–liquid extraction (SLE) method, followed by high-performance liquid chromatography-fluorescence detection. Response surface methodology was employed to optimize extraction conditions in a multivariate experimental design. This approach streamlined the multi-operation extraction process via ingeniously integrating extraction and clean-up into a single step, resulting in significant reductions in extraction time required and high sample throughputs of approximately 40 samples per hour. The methodology was validated and exhibited excellent linearity in concentration ranges of 0.01–24 mg/L for five FWAs and 0.01–40 mg/L for other three FWAs, with correlation coefficients (r) ranging from 0.9985 to 1.000. The limits of detection and quantification were in the range of 0.03–0.26 mg kg−1 and 0.10–0.87 mg kg−1, respectively. The reliability and practicality of the proposed method were affirmed by quantifying eight FWAs in ninety-four commercially available facial masks and whitening creams, yielding recoveries values between 90.0 % and 106.0 % with relative standard deviations (RSDs) of 1.0–4.3 %. Inorganic ions and organic compounds present in the samples had minimal impact on FWAs detection following sample pre-treatment. This procedure showcased exceptional extraction capabilities towards a diverse array of FWAs with the aid of multiple interactions, suggesting significant potential for increasing sample throughput while reducing organic solvent and material consumptions compared with conventional SLE methods. The low-toxicity DES is an excellent alternative to traditional extractants typically applied to SLE techniques.

Analyzing Alkyl Bromide Genotoxic Impurities in Febuxostat Based on Static Headspace Sampling and GC-ECD.

Herein, a sensitive and selective gas chromatography-electron capture detector (GC-ECD) method was developed and validated for the quantification of trace levels of five bromo-containing genotoxic impurities in Febuxostat active pharmaceutical ingredient (API) after headspace sampling (HS). Multivariate experimental designs for the optimization of static headspace parameters were conducted in two stages using fractional factorial design (FFD) and central composite design (CCD). The optimum headspace conditions were 5 min of extraction time and a 120 °C extraction temperature. Baseline separation on the analytes against halogenated solvents was carried out using an Agilent DB-624 (30 m × 0.32 mm I.D., 1.8 μm film thickness) stationary phase under isothermal conditions. The method was validated according to ICH guidelines in terms of specificity, linearity, the limits of detection and quantification, precision and accuracy. The linearity was assessed in the range of 5-150% with respect to the specification limit. The achieved LOD and LOQ values ranged between 0.003 and 0.009 and 0.01 and 0.03 μg mL-1, respectively. The accuracy of the method (expressed as relative recovery) was in the range of 81.5-118.2%, while the precision (repeatability, inter-day) was less than 9.9% in all cases. The validated analytical protocol has been successfully applied to the determination of the impurities in various Febuxostat API batch samples.

APLICATIVO GAMMA-GUI: UMA INTERFACE GRÁFICA AMIGÁVEL PARA PLANEJAMENTO DE EXPERIMENTOS NO MATLAB

GAMMA-GUI APP: A FRIENDLY GRAPHICAL USER INTERFACE FOR THE MULTIVARIATE DESIGN OF EXPERIMENTS IN MATLAB. We present to users unfamiliar with the programming language a user-friendly interface for the multivariate design of experiments (DoE) in Matlab®. The interface of the GAMMA app - Grupo de Análise Multivariada em Matrizes Alimentares, in Portuguese-BR, is available for free download on GitHub https://github.com/appGAMMA, and has a YouTube channel https://www.youtube.com/@appGAMMA with a series of tutorial videos. In this tutorial, some practical demonstrations are made using the DoE interface with real applications: (i) screening design for variable selection; (ii) optimization by Box-Behnken design; (iii) optimization by central composite design; (iv) factorial design for variable selection following by optimization with Doehlert design; (v) optimization by mixture design; (vi) optimization by mixture design with pseudocomponents; (vii) optimization by mixture design with process variable; and (viii) multiple-response optimization; also reproduced in the software Statistica v.13, StatSoft® for comparison. The GAMMA-GUI is constantly developing; suggestions for improvements or new ideas are welcome and can be directed to the developers.

Optimization and evaluation of the distribution of Fischer-Tropsch products over a cobalt-based catalyst utilising design expert software

Modelling biomass to liquid via the Fischer-Tropsch synthesis (FTS) system allows researchers to investigate the most efficient parameters while running the system under optimal conditions. As part of the design of experiments (DOE) procedure, a special data simulation method based on response surface methodology (RSM) is utilized to thoroughly analyse the impact of operating circumstances. The objective of this study was to examine the factors that affect the production of C1, C2–C4, and C5+ in FTS process, and then optimize the critical factors utilising factorial design and response surface techniques. The parameters evaluated were reaction temperature, reaction pressure and the crystallite size of cobalt. The effects of these factors and their potential for synergy were explored simultaneously using multivariate DOE, with the yield of different hydrocarbon composition selectivity's as the measured responses. In the concept generation phase, optimization was based on the literature consulted, which proved to be an effective method for determining the optimization parameters. The detailed conceptual design included the generation of models using statistical methods and response surface models. Finally, the optimized design was validated using catalysts and parameters obtained during the optimization process, and this were compared to the output recorded in the theoretical modelling. The optimized parameters resulted in performance consistency, with the theoretical model for each group of hydrocarbons being validated by actual experiments. The established models were seen to characterize hydrocarbon distributions accurately and repeatedly over a wide range of reaction conditions (200–270 °C, 5–20 Bar, and 3–26 nm) using a cobalt-based catalyst. According to the detailed quantitative models developed, for higher C5+ production, 220 °C, 10 barg and 11 nm (cobalt crystallite) benchmark parameters were set to produce 19.3 % C1, 11.4 % C2–C4 and 69 % C5+ selectivity's. Comparative analysis showed a 1.9 %, 3.9 % and 0.3 % percentage difference between the theoretical output and the actual output of C1, C2–C4 and C5+, respectively.

Using Analytical Quality by Design to improve analytical method development in vaccines quality control: Application to an optimized quantitative high-performance anion-exchange chromatographic method

Analytical quality by design (AQbD) is an enhanced approach for the development of analytical methods. AQbD has received much industrial interest, being the subject of several recently published draft guidelines. This article demonstrates the application of AQbD to determine the quantity of non-adsorbed polysaccharide polyribosyl ribitol phosphate (PRP) and percentage of depolymerized PRP in a commercial hexavalent liquid vaccine, and establishment of an analytical control strategy (ACS). The quantification method developed is high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection, preceded by ultracentrifugation (sample preparation) for separation of the depolymerized polysaccharide from the native adsorbed polysaccharide. The first step was to develop the analytical target profile (ATP) which defines the purpose of the analytical measurement as well as the development scope. As a second step, risk assessment tools were used for identification and ranking of the critical method variables (CMVs) which have a potential impact on method performance if not controlled.Based on a multivariate Design of Experiments (DoE) approach, a proposed method operational design region (MODR) was determined for seven CMVs. Finally, the ACS was established from the understanding of the analytical method and the robustness study. This article focuses on robust and operational ranges of critical parameters linked to the ultracentrifugation and chromatographic steps for depolymerized polysaccharide content control. The design space proposed for CMVs corresponds to the ranges that ensure a product that complies with the previously established precision criteria (±2% equivalent to ± 10 % around the product criterion, which is 20 % for depolymerized polysaccharide control limit).The following design space was established from the DoE statistical modeling for ultracentrifugation critical parameters: [483,000–520,000] g for speed, [11–19]°C for temperature, [29–34] minutes for duration, and from extemporaneous to 8 min for holding time before supernatant recuperation after the ultracentrifugation. For chromatographic critical parameters, the MODR is [2–6] psi for mobile phase helium pressure, [0–7] days for mobile phase storage time, and [0–3] days for samples storage time in the autosampler at 5 °C.Methods optimized using the AQbD approach provide strong justifications during regulatory filing for the selection of analytical CMVs, and for the ACS to be applied during the lifecycle management of the method.

Preparation of red seaweed extract for use as a flocculant agent in sugarcane juice and comparison between two experimental designs

The sugar-energy sector is seeking to reduce its negative environment impact through productivity gains and process savings as a result of research and development of new technologies. For example, during the processing of sugarcane and the extraction of the juice, unwanted compounds are removed, requiring treatments such as clarification, which uses acrylamide compounds in the processing. However, there is a need to reduce the presence of these compounds in the process as they present carcinogenic potential. In this context, the present study aimed to develop a natural flocculant from red seaweed, based on carrageenan extraction. The best conditions for its preparation, dosage, and application in the treatment of sugarcane juice were determined by comparing two experimental designs. The experiment was carried out at the Universidade do Estado de Minas Gerais, Frutal, Brazil, using sugarcane variety CTC072361 (2022/2023 Harvest). Seaweed samples were purchased from commercial centers. Regarding the red seaweed extract, the study was carried out in two stages. First, a univariate experimental design was used to determine the relevant variables that could influence the preparation of the seaweed extract. There was no direct correlation between the parameters. Subsequently, a multivariate design was carried out to verify the interaction between the assessed parameters. Thus, the seaweed samples were previously dried in an oven at 65 ºC, and the standardization and characterization procedures of the seaweed extracts were evaluated, considering the analysis parameters: drying time, granulometry, concentration, pH, temperature, and mixing time, in triplicate. Then, the dosage tests were carried out using a decanter, performing 12 different tests with doses of red seaweed extract (0, 5, 10, 35, 50, 75, 100, 150, 250, 400, 500 and 1000 mg.L-1) for univariate experimental design, and (0, 10, 100, 125, 275, 350, 425, 500, 800, 900, 1000 and 1200 mg.L-1) for the multivariate experimental design. After the retention time, the clarified juice was recovered by siphoning determining its Brix, Total Reducing Sugars, pH, Acidity, and Total Phenolic Compounds. This was used for checking whether the treatments differed from each other. Therefore, by comparing the experimental designs, significant differences were verified between the treatments applied, with emphasis on dosages of 500 mg.L-1 (multivariate design) and 1000 mg.L-1 (univariate design). For both experimental designs, the results obtained were satisfactory; however, considering all the interactions between the variables, the multivariate design was more suitable for the assessed data.

Design of experiments for micellar electrokinetic chromatography method development for the monitoring of water-soluble vitamins in cell culture medium.

Biopharmaceutical production takes place in complex processes which should be thoroughly understood. Therefore, the iConsensus project focuses on developing a monitoring platform integrating several process analytical technology tools for integrated, automated monitoring of the biopharmaceutical process. Water-soluble vitamin monitoring using (microchip) capillary electrophoresis (CE) is part of this platform. This work comprises the development of conventional CE methods as the first part towards integrated vitamin monitoring. The vitamins were divided based on their physical-chemical properties to develop two robust methods. Previously, a method for the analysis of cationic vitamins (pyridoxine, pyridoxal, pyridoxamine, thiamine and nicotinamide) in cell culture medium was developed. This work focused on the development of a micellar electrokinetic chromatography method for anionic and neutral vitamins (riboflavin, d-calcium pantothenate, biotin, folic acid, cyanocobalamin and ascorbic acid). By employing multivariate design of experiments, the background electrolyte (BGE) could be optimised within one experiment testing only 11 BGEs. The optimised BGE conditions were 200mM borate with 77mM sodium dodecyl sulphate at a pH of 8.6. Using this BGE, all above-mentioned cationic, anionic and neutral vitamins could be separated in clean samples. In cell culture medium, most anionic and neutral vitamins could be separated. Combining the two methods allows for analysis of cationic, anionic and neutral vitamins in cell culture medium samples. The next step towards integrated vitamin monitoring includes transfer to microchip CE. Due to the lack of fast and reliable methods for vitamin monitoring, the developed capillary methods could be valuable as stand-alone at-line process analytical technology solutions as well.

Direct determination of tyrosine and tryptophane enantiomers in pumpkin (Cucurbita moschata) by HPLC-UV/Vis: Effect of cooking treatment on enantiomers profile

An analytical method to determine the content of tryptophan (Trp) and tyrosine (Tyr) enantiomers in pumpkin (Cucurbita moschata) is developed and validated. The method consists of a sample preparation procedure with a direct liquid chromatographic separation of enantiomers on a Whelk-O2 chiral stationary phase. The chromatographic separation was performed under gradient reversed-phase conditions in which the effect of pH and organic modifier were studied. A multivariate design of experiments was applied in the optimization of the extraction procedure. After optimization the complete analytical process was validated. Great accuracy was achieved with recovery between 92–109% with a relative standard deviation of less than 10%. Limits of detection obtained ranged between 0.04 and 0.07 mg L-1. The resulting analytical method is applied with success to dose the content of Trp and Tyr and their enantiomers in several pumpkin cultivars. The impact of several traditional cooking methods on the content of these amino acids was also studied. The obtained data showed that studied pumpkin is a good source of free Trp and Tyr, mainly L-Trp. The results confirm the natural presence of D-Trp in this vegetable. Finally, the results demonstrate that the levels of all analytes are modified during traditional cooking treatments.

Cleaner production of iron-coated quartz sand composites for efficient phosphorus adsorption in sanitary wastewater: A design of experiments (DoE) approach

Phosphorus (P) adsorption and recovery from sanitary wastewater is a promising route to produce more sustainable fertilizers. Multivariate Design of Experiments (DoE) techniques were used to develop a cleaner process to produce Fe-coated quartz sand composites with applications in Phosphorus (P) adsorption from sanitary effluents. Nine different process parameters were investigated and correlations between process inputs and physicochemical characteristics of adsorbents were elucidated. It was found that Goethite and Magnetite phases with different degrees of crystallinity were obtained depending on the conditions applied during the synthesis by coprecipitation. Statistical models fitted to an experimental multivariate database were used to predict the best conditions for synthesizing an efficient P adsorbent. In addition, a zero-waste process for the production of the adsorbents is reported. The best adsorbent produced, an aqueous suspension containing particles of a SiO2@FeOOH composite, exhibited a P (KH2PO4) adsorption capacity of 12.4 mg P/g (C0 = 10 mg P/L, pH 6.0) and robust performance when applied in simulated sanitary effluent, reaching 97% of P removal efficiency (C0 = 5 mg P/L). Considering the value of the green star area index (GSAI), the optimized method (suspension) was 65% green, reaching the maximum score for four of the ten principles involved, denoting the greenness of the proposed method. The optimized adsorbent has appropriate characteristics to be applied in sanitary effluent treatment processes for capturing and recovering residual P, contributing to a more sustainable management of natural resources.

Modeling and optimization of trivalent arsenic removal from wastewater using activated carbon produced from maize plant biomass: a multivariate experimental design approach

Modeling and optimization of trivalent arsenic removal from wastewater using activated carbon produced from maize plant biomass: a multivariate experimental design approach

Compressive Strength Behavior of Briquettes Produced from Manganese Ore Fines and Metal Dust: Optimization Using Response Surface Methodology

ABSTRACT Electric reduction furnaces cannot be fed fines in the production of manganese ferroalloy as they hinder gas permeability. Thus, agglomeration techniques can be used to minimize the environmental and economic impacts of storing fines. Among these techniques, briquetting is advantageous because it allows for cold agglomeration of the particles and is flexible in terms of particle sizes. Therefore, this work aimed to develop self-reductor briquettes composed of ore manganese fines, dust ferromanganese, charcoal fines, and three different binders. The compaction pressure, curing time, and % binder were optimized to enhance briquette strength through a multivariate experimental design consisting of a screening study using a 23 full factorial design followed by response surface methodology with a spherical central composite design. The results were assessed by analysis of variance, and a regression model was established. The study found that briquettes elaborated with bentonite exhibited an optimized compressive strength of 4.02 MPa, achieved with a compaction pressure of 67.55 MPa and 12.71% bentonite. For briquettes elaborated with pregelatinized starch, a compressive strength of 7.04 MPa was obtained with a compaction pressure of 67.52 MPa, a curing time of 19.51 days, and 8.07% pregelatinized starch. For briquettes agglomerated using slaked lime and molasses, an optimized compaction pressure of 27.09 MPa and a curing time of 23.27 days resulted in a compressive strength of 1.63 MPa. These results suggest that briquettes containing bentonite and pregelatinized starch are feasible substitutes for lump ore in electric furnaces.

Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification.

In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log10 reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65-3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.

Single Molecule with a Large Transistor – SiMoT cytokine IL‐6 Detection Benchmarked against a Chemiluminescent Ultrasensitive Immunoassay Array

AbstractEarly diagnosis and efficient treatments of oncological, neurological, inflammatory, and infectious diseases rely more and more on ultrasensitive detection of protein markers; the ultimate limit is a reliable immunoassay capable of single‐protein detection. Among protein biomarkers, cytokines play a key role in clinical diagnosis as they are involved in developing many complex diseases and disorders, such as chronic inflammatory diseases including metabolic syndrome, neurodegenerative diseases, and cardiovascular diseases, along with autoimmune diseases and cancer. Herein, the improvement of a Single Molecule with Transistor (SiMoT) is reported based on an electrolyte‐gated organic field‐effect transistor applied for the detection of cytokine IL‐6 in blood serum, reaching a limit‐of‐detection (LOD) of 1 ± 1 protein in a sample of 0.1 mL. The analytical performance levels are benchmarked against the Simoa Planar Array SP‐X technology, a benchtop chemiluminescent array. After comprehensive optimization, Simoa SP‐X by using a multivariate experimental design approach exhibits a LOD 103 higher than SiMoT. The proposed SiMoT electronic assay is label‐free, fast (30 min), and selective, paving the way for an ultra‐sensitive point‐of‐care immunoassay platform enabling pre‐symptomatic disease diagnosis.

Polyamide-coated paper-based sorptive phase applied in high-throughput thin film microextraction designed by 3D printing

A new method was developed for the determination of endocrine disrupting compounds (phthalates, alkylphenols and bisphenols) in water samples by using thin film microextraction technique (TFME) with high-performance liquid chromatography and diode-array detection (HPLC-DAD). This paper reports for the first time the use of a paper-based sorptive phase (PSP) in a high-throughput semi-automated system. This system designed by 3D printing technology allowed us to integrate the PSP with a multi-well plate format. Multivariate design of experiments and one-factor-at-a-time approaches were employed for optimization. The optimized PSP preparation required two dips of the filter paper into the nylon 66 solution followed by the evaporation of the solvent. The best conditions for the extraction step were NaCl 30 % (w/v) added to the sample at pH 2 for 180 min. The desorption showed best results with acetonitrile for 20 min. The calibration curves exhibited coefficients of determination higher than 0.9837. The limits of detection varied from 1.5 to 7.6 μg L-1. Six of ten analytes showed promising results with accuracy ranging from 82 to 117 % and precision (RSD) equal or inferior to 24 % for mineral water sample. We also evaluated a coconut water sample that obtained higher deviations than mineral water sample. The time consumed for each sample was about 8.3 min.

Qualitative imaging of elemental spatial distribution in stalagmites through laser ablation inductively coupled plasma mass spectrometry analysis

The laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) technique is considered versatile for multi-elemental analysis and imaging because it is easy to handle, compatible with different types of solid samples, requires minimal sample preparation, and provides high spatial resolution and sensitivity. One of the challenges of imaging analysis is to obtain accurate and precise spatial information of elements distribution in the sample, so the optimization of the laser ablation (LA) parameters is essential. In this context, this study aimed to optimize the LA parameters for direct analysis of speleothem samples. Laser intensity, frequency, and spot diameter were evaluated through multivariate experimental design and multi-response data, the influence of ablation scan speed and the use of 44Ca as an internal standard (IS) for the qualitative image of 66Zn, 137Ba, 55Mn, 57Fe, 88Sr, 60Ni, and 26Mg distribution in the sample were also evaluated. The multivariate optimization revealed positive interactions between the parameters evaluated, i.e., the greater the laser intensity, LA frequency, and spot diameter, the greater the analyte signal and, thus the sensitivity. Therefore, 90% laser ablation intensity, 20 Hz repetition rate, and 100 µm spot diameter were selected. In the scan speed evaluation, the images obtained with 40 and 20 µm s-1 were very similar for all isotopes. The use of 44Ca as IS did not impact the resolution of the images. The use of 44Ca can provide important information about the speleothem formation and paleoclimate changes.

Multivariate optimization of large-volume sample stacking with polarity switching by capillary electrophoresis for determination of gold nanoparticle size

Gold nanoparticles (AuNPs) are widely studied and applied due to their unique optical characteristics, such as surface plasmon resonance and extinction coefficient. In this study, large sample stacking combined with polarity switching (LVSS-PS) by capillary electrophoresis was established to determine AuNP mixtures with different particle sizes simultaneously. The multivariate experimental design containing fractional factorial design (fFD) and central composite design (CCD) was combined with LVSS-PS to estimate the optimal condition. The influence of factors on the response was evaluated by the use of the chromatography resolution statistic function to simultaneously investigate the resolution and separation under twenty-eight sets of capillary electrophoresis conditions. The results showed that the optimal condition of the separation buffer was 100 mM CAPS (pH 9.5) with 20 mM SDBS and 35 mM SDS. The separation temperature was set at 20 °C. Under this condition, four different diameters of AuNPs (5, 20, 50, and 80 nm) were successfully separated in the same run. The linear relationship between the electrophoretic mobility of AuNPs and their diameters was established, and the correlation coefficient was 0.99. Sodium citrate and gallic acid (GA) were capping agents to synthesize AuNPs. The established method was applied to determine the size of gold nanoparticles synthesized by sodium citrate and gallic acid as capping agents, respectively. The diameters of the AuNPs obtained by LVSS-PS are consistent with transmission electron microscopy.