Sample Preparation Research Articles

Modified Complex Electrical Resistivity Technique: Applications to Saturated and Unsaturated Soils

Abstract Direct current (DC) electrical resistivity is a common laboratory soil characterization method to support geotechnical infrastructure design and to supplement site investigations. The complex electrical resistivity method has the potential to provide additional electrical soil properties to enhance electrical soil characterization. Both methods are conventionally performed under fully saturated soil conditions; however, many environments exist where soil is not fully saturated, such as ballast structure supporting railways. In this study, a new experimental setup featuring a current enhancing agent (agar) for complex electrical resistivity testing is evaluated by testing five different soil specimens reconstituted at saturated and unsaturated conditions. Results showed that the new experimental setup is valid and can be used to obtain repeat measurements, particularly for specimens reconstituted in the unsaturated conditions where the traditional DC electrical resistivity setup yields results that are unreliable. This is one of the very few studies where tolerances for triplicate specimens are reported to establish differences in measurements from sample preparation versus discernable variability between different geomaterials. Additionally, all results are supported by a Cole-Cole model. The results show that the additional data collected in a complex electrical resistivity test can be used to differentiate different soil types that are ambiguous with the DC electrical resistivity method. The additional data have the potential to more fully characterize the electrical properties of saturated and unsaturated soils, as well as to help distinguish unique geophysical signatures of various geomaterials to enhance a geophysical site investigation for identifying soil variability in the subsurface.

Reports of tropane alkaloid poisonings and analytical techniques for their determination in food crops and products from 2013 to 2023.

Food safety is crucial to attaining food security and sustainability. Unsafe foods for human and animal consumption lead to product recalls and rejection, negatively impacting the global economy and trade. Similarly, climate change can adversely affect the availability of safe and nutritious food at the table. The changing climatic conditions and global food trade and transport can make the movement of toxic plants possible, resulting in food crops being increasingly invaded by some species of plants that produce toxic secondary metabolites, such as tropane alkaloids (TAs).Datura stramoniumfrom the Solanaceae plant family is an invasive and virulent plant that produces high amounts of two TAs, atropine and scopolamine. Various food poisoning events following accidental or deliberate ingestion of foods contaminated by atropine and scopolamine from seeds ofD. stramoniumhave been recorded in different locations globally. Due to these incidents, regulatory agencies require the development of plant toxin detection methods that can be used in the food chain as early as possible. This systematic review thus focuses on the TA determination techniques in food and feeds published between 2013 and 2023. A particular focus was given to the sample preparation methods, the improvements of each technique claimed, and data to support the performance of each method, especially the ability to measure at or below the maximum level. The review concludes with other technological advancements, including rapid spectroscopy, electrophoresis, and colorimetric methods, as well as the possibility of coupling with smartphones for use in on-farm detection and the challenges in applying them.

Sample preparation of Hedyotis diffusa Willd. for two-dimensional gas chromatography-time of flight mass chromatography analysis enhanced chemical profiling

Sample preparation of Hedyotis diffusa Willd. for two-dimensional gas chromatography-time of flight mass chromatography analysis enhanced chemical profiling

Electrochemical determination of nitazoxanide in environmental and pharmaceutical samples using a boron-doped diamond electrode

Consumption of Nitazoxanide (NTZ), a compound belonging to the class of nitrothiazoles, has increased due to its efficiency in treating viral gastroenteritis caused by rotavirus and norovirus “coronavirus disease”. Thus, developing a method capable of monitoring NTZ in wastewater and pharmaceutical formulations is relevant to human health control. However, most methods for NTZ determination are expensive and time-consuming. Hence, we present a fast, efficient, and low-waste method for NTZ monitoring in drugs and water samples using a boron-doped diamond electrode. A 3D-printed electrochemical cell with a capacity of 2 mL was used for all studies. Besides the simplicity of low waste generation, the method provided good analytical performance with a linear range of 2.0–20.0 µmol/L of NTZ, and a limit of detection (LOD) of 0.57 µmol/L. Recovery percentages between 99 and 107 % were obtained to analyze spiked samples. In addition, we demonstrate that the LOD value can be significantly improved by 50 times after applying a pre-concentration step using solid-phase extraction. Therefore, we believe the described approach is a simple and efficient electrochemical method for monitoring NTZ levels in pharmaceutical and environmental samples without laborious sample preparation procedures. This is the first report to describe the use of BDD electrodes for NTZ monitoring and environmental sample analysis using an eco-friendly approach.

Antimicrobial Activity of Ethanol Extract of Sea Pandan Leaves (Pandanus odorifer) Against Staphylococcus aureus and Escherichia coli

Sea pandan (Pandanus odorifer) is used in traditional medicine in India. Sea pandan is efficacious in treating pain, parasites, digestive, liver, and cognitive disorders. Sea pandan is found in coastal areas in South Kalimantan. Sea pandan contains flavonoid compounds that have the potential to be antimicrobials. This study aimed to determine the antimicrobial activity of sea pandan leaf extract. The study began with sample preparation, which included washing the leaves, drying them using an oven, refining the particle size, extracting 70% ethanol, and evaporating the solvent until a thick extract was obtained. Total flavonoid levels in sea pandan leaf extract were determined using the colourimetric method using quercetin standards. Antimicrobial testing was conducted using the diffusion method at concentrations of 30%, 40%, and 50% against Staphylococcus aureus and Escherichia coli. The positive control used was chloramphenicol. The determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values ??was carried out using the dilution method. The results showed that sea pandan leaf extract contains phenolics, flavonoids, saponins, steroids, and tannins. The total flavonoid content in sea pandan leaf extract is 10.01% w/w equivalent of quercetin. The antimicrobial activity of sea pandan leaf extract against Staphylococcus aureus bacteria is 10.15 - 25.36 mm, while against Escherichia coli in the range of 8.15 - 23.16 mm. Ethanol extract of sea pandan leaves at a concentration of 50% is included in the very strong category in inhibiting Staphylococcus aureus and Escherichia coli. Ethanol extract of sea pandan leaves has an MIC value of 7.5% on Staphylococcus aureus and 15% on Escherichia coli. MBC of sea pandan extract is 15% on Escherichia coli bacteria. This study concludes that the ethanol extract of sea pandan leaves has antimicrobial activity against Staphylococcus aureus (bacteriostatic) and Escherichia coli (bactericidal).

A Brief Review of Hemp Fiber Length Measurement Techniques

Accurate fiber length measurement is essential for the processing and quality management of textile products. This article reviews the current methods used to measure fiber length, including manual, photoelectric, capacitive, and optical techniques. Existing sample preparation processes for natural fiber characterization have been primarily developed for cotton and wool fibers. However, hemp fibers present unique challenges due to their greater length variability, high strength, and low elongation, making some traditional sample preparation methods less effective. Image processing offers a promising approach for scalable and precise measurement of hemp fiber length. Nevertheless, current image processing techniques are limited by the inability to effectively handle overlapping fibers, which increases both the time and cost of testing. Continued research into developing more advanced segmentation algorithms could lead to more widely adopted commercial methods for fiber measurement.

Mycotoxins in Food: Cancer Risks and Strategies for Control.

Mycotoxins are toxic compounds produced by fungi such as Aspergillus, Penicillium, and Fusarium, contaminating various food crops and posing severe risks to food safety and human health. This review discusses mycotoxins' origins, significance, and impact, particularly in relation to cancer risk. Major mycotoxins like aflatoxins, ochratoxins, fumonisins, zearalenone, and patulin are examined, along with their sources and affected foods. The carcinogenic mechanisms of these toxins, including their biochemical and molecular interactions, are explored, as well as epidemiological evidence linking mycotoxin exposure to cancer in high-risk populations. The review also highlights critical methodologies for mycotoxin detection, including HPLC, GC-MS, MS, and ELISA, and the sample preparation techniques critical for accurate analysis. Strategies for controlling mycotoxin contamination, both pre- and post-harvest, are discussed, along with regulations from organizations like the FAO and WHO. Current challenges in detection sensitivity, cost, and control effectiveness are noted. Future research is needed to develop innovative analytical techniques, improve control strategies, and address the influence of climate change on mycotoxin production. Finally, global collaboration and emerging technologies are essential for advancing mycotoxin control and enhancing food safety.

End-To-End Automated Intact Protein Mass Spectrometry for High-Throughput Screening and Characterization of Bispecific and Multispecific Antibodies.

Bispecific antibodies (bsAbs) and multispecific antibodies (msAbs) represent a promising frontier in therapeutic antibody development, offering unique capabilities not achievable with traditional monoclonal antibodies. Despite their potential, significant challenges remain due to their increased molecular complexity. One prominent challenge is the correct assembly of light and heavy chains, as improper pairing leads to mispaired or incompletely assembled species that lack therapeutic efficacy and possess undesired properties, impairing the developability, manufacturability, and safety. There is a critical need for rapid, sensitive analytical tools to monitor and control these undesired species and ensure the quality assessment of bsAbs and msAbs. To address this need, we present a novel high-throughput, format-agnostic intact mass workflow that significantly enhances the efficiency of detecting and quantifying biotherapeutic products and related impurities. This workflow integrates automated sample preparation, novel high-resolution rapid mass detection powered by SampleStream-MS, and an advanced data analysis pipeline. It offers increased throughput and data quality while substantially reducing analysis turnover time and labor. This was demonstrated in a pilot program where ∼800 multispecific antibodies were processed in 10 working days. The article details the evaluation and validation of our method, demonstrating its repeatability and intermediate precision in terms of measurement accuracy and relative quantification of various product-related species. We underscore the transformative potential of this end-to-end high-throughput workflow in expediting bispecific and multispecific antibody discovery, optimizing production processes, and ensuring high-quality development and manufacturing for therapeutic antibodies.

Antioxidant and Antibactery Activity of Ethanol Extract of Selutui Puka Leaves (Tabernaemontana macrocarpa Jack.) Against Propionibacterium acnes

The selutui puka plant (Tabernaemontana macrocarpa Jack.) is a plant that has many benefits for the Dayak community in the West Kutai area, Karangan Village, Mook Manaar Bulatn District, East Kalimantan. Empirically, the Dayak tribe uses selutui puka to treat cancer, herpes, scabies, itchy skin, acne and blisters. Metabolites contained in selutui puka leaves such as alkaloids, flavonoids, tannins, saponins, and steroids have potential as antioxidants and antibacterials. Selutui puka leaf can be used as an alternative to natural acne treatment, because it is known that the use of natural ingredients has lower side effects. The purpose of this study was to determine the antioxidant and antibacterial activity of the ethanol extract of selutui puka leaves against Propionibacterium acnes. This research was conducted by experimental method. The research sample was selutui puka leaf. The research phase includes sample preparation, plant determination, simplicia manufacture, extraction, testing of antioxidant activity using UV/VIS Spectrophotometer at extract concentrations of 300 ppm, 325 ppm, 350 ppm, 375 ppm and 400 ppm. Quercetin as a positive control and comparison. The antibacterial activity test consisted of making media, culturing Propionibacterium acnes, and testing antibacterial activity using the disc diffusion method at extract concentrations of 5%, 10%, and 15%. Data were analyzed descriptively. The data from the results of this study are presented in the form of tables and figures. IC50 of ethanol extract of selutui puka leaves was 333.60 ppm with very weak category. The diameter of the inhibition zone of the ethanolic extract of selutui puka leaves against Propionibacterium acnes was 7.81 mm (5%), 7.44 mm (10%) and 8.63 mm (15%) in the medium category.

Evaluating MIR and NIR Spectroscopy Coupled with Multivariate Analysis for Detection and Quantification of Additives in Tobacco Products.

The detection and quantification of additives in tobacco products are critical for ensuring consumer safety and compliance with regulatory standards. Traditional analytical techniques, like gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and others, although effective, suffer from drawbacks, including complex sample preparation, high costs, lengthy analysis times, and the requirement for skilled operators. This study addresses these challenges by evaluating the efficacy of mid-infrared (MIR) spectroscopy and near-IR (NIR) spectroscopy, coupled with multivariate analysis, as potential solutions for the detection and quantification of additives in tobacco products. So, a representative set of tobacco products was selected and spiked with the targeted additives, namely caffeine, menthol, glycerol, and cocoa. Multivariate analysis of MIR and NIR spectra consisted of principal component analysis (PCA), hierarchical clustering analysis (HCA), partial least squares-discriminant analysis (PLS-DA) and soft independent modeling of class analogy (SIMCA) to classify samples based on targeted additives. Based on the unsupervised techniques (PCA and HCA), a distinction could be made between spiked and non-spiked samples for all four targeted additives based on both MIR and NIR spectral data. During supervised analysis, SIMCA achieved 87-100% classification accuracy for the different additives and for both spectroscopic techniques. PLS-DA models showed classification rates of 80% to 100%, also demonstrating robust performance. Regression studies, using PLS, showed that it is possible to effectively estimate the concentration levels of the targeted molecules. The results also highlight the necessity of optimizing data pretreatment for accurate quantification of the target additives. Overall, NIR spectroscopy combined with SIMCA provided the most accurate and robust classification models for all target molecules, indicating that it is the most effective single technique for this type of analysis. MIR, on the other hand, showed the overall best performance for quantitative estimation.

Automatic Algorithm Applied for Calculating Thermal Conductivity by Transient Plane Source Method

As a thermal conductivity measurement method, Transient Plane Source (TPS) method has gained much popularity because of its broad applicability, short measurement times, high precision and simple sample preparation. However, the accuracy of thermal conductivity calculations based on temperature rise data is often hindered by factors such as probe thickness, contact thermal resistance, and input power. Currently, there is no standardized criteria for selecting effective temperature rise data for thermal conductivity calculation. Consequently, the accuracy of results are limited by the operator's understanding of the TPS methods, and repeatability of the results is often poor. To address this issue, an automatic algorithm based on the international standard (ISO22007-2:2008) is proposed in this paper. By applying this algorithm to the measurement of different materials, it has been demonstrated that the proposed algorithm can produce more precise and consistent results than the conventional method. Additionally, the integration of the time window function , typically utilized solely for result validation in conventional methods, further enhances the objectivity and reproducibility of the results obtained by the automatic algorithm.

Spatial proteomics of single cells and organelles on tissue slides using filter-aided expansion proteomics.

Hydrogel-based tissue expansion combined with mass spectrometry (MS) offers an emerging spatial proteomics approach. Here, we present a filter-aided expansion proteomics (FAXP) strategy for spatial proteomics analysis of archived formalin-fixed paraffin-embedded (FFPE) specimens. Compared to our previous ProteomEx method, FAXP employed a customized tip device to enhance both the stability and throughput of sample preparation, thus guaranteeing the reproducibility and robustness of the workflow. FAXP achieved a 14.5-fold increase in volumetric resolution. It generated over 8 times higher peptide yield and a 255% rise in protein identifications while reducing sample preparation time by 50%. We also demonstrated the applicability of FAXP using human colorectal FFPE tissue samples. Furthermore, for the first time, we achieved bona fide single-subcellular proteomics under image guidance by integrating FAXP with laser capture microdissection.

Development of a Novel Label-Free Subunit HILIC-MS Method for Domain-Specific Free Thiol Identification and Quantitation in Therapeutic Monoclonal Antibodies.

Cysteine residues are crucial for the formation of conserved disulfide bonds in therapeutic monoclonal antibodies (mAbs), which are essential for their folding and structural stability. The presence of free thiols in mAbs can indicate incomplete disulfide bond formation, potentially impacting the molecule's conformational stability. Free thiol quantitation has been achieved using labeling-based strategies such as maleimide and haloalkyl derivatives at both intact and peptide levels. However, intact-level measurement only provides total free thiol levels, while peptide-level measurement is time-consuming and more prone to assay-induced artifacts. In this study, we present a novel label-free HILIC-MS method that separates free thiol species at the subunit level, followed by free thiol localization by the MS2 fragmentation pattern. This allows for facile identification and quantitation of intrachain free thiols at domain-specific resolution. Compared to bottom-up approaches, this subunit HILIC-MS method excels in simpler sample preparation and higher throughput and enables chain-specific free thiol analysis for bispecific mAbs. This method can be readily applied for screening mAb candidates with elevated levels of free thiols in early-stage developability assessment and facilitating an effective comparability evaluation of mAb samples during process development.

Development and Validation of an optimized HPLC-UV analytical method for the quantification of 5-Methyltetrahydrofolate (Active Folate) in dietary Supplements: Application to commercial tablets and capsules

5-MTHF is the active form of folic acid, and it is provided as a dietary supplement in the market. Therefore, in this study we provide a new, simple and economic HPLC-UV analysis method to quantify 5-MTHF in dietary supplement. A new HPLC-UV method was developed and validated for the quantification of 5-methyltetrahydrofolate in dietary supplements. The method was optimized in terms of chromatographic conditions, including the mobile phase composition, chromatographic column, and the sample preparation diluent using ascorbic acid. A combination of ammonium acetate buffer with methanol (90:10) was the optimal mobile phase, achieving a retention time of approximately 11.4 min on C18 column with good column performance (N = 6786, Asymmetry = 1.03). The method was validated for selectivity, linearity, precision, accuracy, and robustness. The calibration curve was linear (r2 > 0.999) over the concentration range of 1.20 to 4.80 µg/mL. Precision was demonstrated with RSD% values ranging from 1.38 % to 2.90 % for intra-day precision and 0.96 % to 2.61 % for inter-day precision. Accuracy was confirmed with recovery values between 96.5 % and 110.6 % for spiked samples. To demonstrate the method’s applicability, ten brands of 5-MTHF dietary supplements were collected from local pharmacies. Assay results showed significant variability, with four brands having contents below the acceptable range (90–110 % of labeled contents) and two brands exceeding this range. The stability of 5-MTHF in these products appeared to be formula-dependent, emphasizing the need for regulatory revisions to mandate stability testing for dietary supplements.

Cryo-X-Ray Phase Contrast Imaging Enables Combined 3D Structural Quantification and Nucleic Acid Analysis of Myocardial Biopsies.

Snap-frozen biopsies serve as a valuable clinical resource of archival material for disease research, as they enable a comprehensive array of downstream analyses to be performed, including extraction and sequencing of nucleic acids. Obtaining three-dimensional (3D) structural information before multi-omics is more challenging but can potentially allow for better characterization of tissues and targeting of clinically relevant cells. Conventional histological techniques are limited in this regard due to their destructive nature and the reconstruction artifacts produced by sectioning, dehydration, and chemical processing. These limitations are particularly notable in soft tissues such as the heart. In this study, the feasibility of using synchrotron-based cryo-X-ray phase contrast imaging (cryo-X-PCI) of snap-frozen myocardial biopsies is assessed and 3D structure tensor analysis of aggregated myocytes, followed by nucleic acid (DNA and RNA) extraction and analysis. It is shown that optimal sample preparation is the key driver for successful structural and nucleic acid preservation which is unaffected by the process of cryo-X-PCI. It is proposed that cryo-X-PCI has clinical value for 3D tissue analysis of cardiac and potentially non-cardiac soft tissue biopsies before nucleic acid investigation.

Rapid and Effective Determination of Ethyl Glucuronide in Hair by Micro Extraction by Packed Sorbent (MEPS) and LC-MS/MS.

Ethyl glucuronide (EtG) in hair is a reliable biomarker of alcohol consumption habits. Due to its small concentration incorporated into hair, analytical methods sensitive enough to reliably quantify EtG in this matrix are required. Sample preparation is critical in hair analysis, especially for EtG, for which extraction efficiency and matrix effect can strongly influence the results; furthermore, miniaturized methods are sought, to reduce solvent use and times of sample preparation. A micro extraction by packed sorbent (MEPS) procedure coupled to a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of EtG in human hair samples. Fifty milligrams of hair samples were cut into snippets and extracted in water. The cleanup of the extract was carried out by using a MEPS syringe packed with anion exchange sorbent (SAX); all parameters for conditioning, washing, loading and eluting steps were optimized and the eluted aqueous volume was directly injected in the LC-MS/MS system operating in the negative ionization mode. The method was fully validated assessing LOD, LOQ, calibration curve, repeatability, accuracy, matrix effect and carryover. The method was subsequently applied to QCs and authentic hair samples. The developed MEPS method is quick and effective, with low solvent purchase and discard costs, allowing the differentiation between social drinkers and chronic excessive alcohol consumers, according to the cut-offs established by the Society of Hair Testing (SoHT).

A systematic evaluation of quenching, extraction and analysis procedures for metabolomics study of the mechanism of QYSLD intervention in A549 cells.

The preparation of cellular metabolomics samples and how to achieve comprehensive coverage of different polar metabolites in cell samples in the analysis pose a challenge for cellular metabolomics. In this study, we optimized a metabolomics protocol based on ultra-high-performance liquid chromatography high-resolution mass spectrometry (UPLC/HRMS) for theextraction and detection of metabolites in A549 cells and exploration of the intervention effect of Qi-Yu-San-Long decoction (QYSLD) on A549 cells. The results indicate that the lowest level of ATP leakage was observed when A549 cells were quenched under liquid nitrogen. MeOH/chloroform/H2O (1:2:1) extraction yielded more chromatographic peaks and excellent reproducibility, and the relative extraction efficiency of most target metabolites was also high. And we optimized thechromatographic separation conditions in both HILIC and RPLC modes, enabling comprehensive detection and analysis of metabolites with varying polarities. Then, we applied the optimized method to UPLC-Q-TOF/MS-based metabolomics of A549 cells to study the mechanism of QYSLD intervention in non-small cell lung cancer (NSCLC). The CCK-8, EdU staining, and cell cycle assay showed that QYSLD inhibited the proliferation of A549 cells by interfering with the cell cycle and blocking them in theG1 phase. A total of 36 differential metabolites associated with the antitumor effects of QYSLD on NSCLC were identified, mainly involving nicotinate and nicotinamide metabolism, sphingolipid metabolism, and glycerophospholipid metabolism. And western blotting confirmed that thechange in 1-methylnicotinamide levels after QYSLD intervention was associated with theinhibition of nicotinamide N-methyltransferase expression in A549 cells.

Electroextraction of Large Volume Samples Using Paper Points Coupled With Hollow Fiber Membranes: Study of Parameters and Strategies to Enhance Analytical Performance.

Electroextraction (EE) encompasses a range of sample preparation methods whose effectiveness, selectivity, and efficiency are significantly influenced by the physical-chemical characteristics of analytes, samples, and instrumental conditions. This article explores, for the first time, various strategies aimed at enhancing the extraction efficiency of a recent approach of EE utilizing a paper point (PP) combined with a hollow fiber (HF) (abbreviated as PP-HF-EE) to extract various cationic and anionic model compounds from water samples. The study also explores, experimentally, the impact of agitation, organic filter composition, PP diameter, and PP brand on extraction performance, and proves that all these factors are quite important, especially when digital image analysis is utilized for determination. Furthermore, this work demonstrates the ease and feasibility of simultaneously extracting cations and anions using PP-HF-EE and proposes a straightforward method to enhance analyte concentration on the vertex of the PP through a base-to-vertex focusing. Lastly, it is demonstrated, using capillary electrophoresis coupled to a UV-Vis detector, that for PP-HF-EE, the extraction efficiency and pre-concentration factor are less dependent on other parameters when multiple PPs per sample are utilized, with signal enhancement values of up to 111 and 339 for nortriptyline and haloperidol, respectively. All the findings and strategies presented herein constitute significant contributions that can facilitate future research in method development, particularly in the utilization of PP-HF-EE and similar EE approaches.

Ensuring Quality and Consistency in OPC-53 Cement Testing: A Comprehensive Proficiency Testing Evaluation

The proficiency testing (PT) program is a critical part of quality assurance in laboratories, especially within industries like construction where cement testing is essential. This study provides a comprehensive evaluation of the fifth PT scheme for Ordinary Portland Cement (OPC) grade 53, conducted by the Nodal Laboratory. The key parameters assessed in this program were standard consistency, compressive strength, and soundness using the Le-Chatelier method. Twenty-four laboratories participated, and their performance was analyzed using advanced statistical methods, adhering to ISO 17043:2010 and ISO 13528:2015 standards. The results showed that most laboratories performed satisfactorily, with only one outlier identified in the soundness test. This paper covers all aspects of the PT program, from sample preparation to the distribution of materials, as well as the homogeneity and stability tests conducted on the samples. Statistical evaluation was carried out using Z-scores, which allowed the laboratories to be ranked according to their performance in each parameter. This method highlights discrepancies and provides a clear framework for participating laboratories to improve their testing procedures. Proficiency testing is essential for maintaining high standards in laboratory performance. It helps standardize testing methods across multiple laboratories, ensuring that OPC-53 cement meets strict quality and performance specifications. The program enables laboratories to identify performance gaps and take corrective actions, ensuring the reliability and accuracy of results. Participation in PT programs is a step toward continuous improvement, keeping laboratories aligned with international standards, ultimately benefiting the broader construction industry by ensuring the structural integrity of buildings and infrastructure. This study underscores the importance of regular PT participation. It not only provides a benchmark for laboratory performance but also ensures compliance with ISO standards. This fosters the consistent quality of cement used in large-scale construction, reducing the risk of structural failures and contributing to long-term safety and durability. The findings demonstrate the positive impact of PT programs on overall testing accuracy and laboratory reliability, emphasizing their role in advancing best practices in the construction industry.

2D-CEX-FcRn-MS to Study Structure/Function Relation of mAb Charge Variants.

The automated elucidation of the interplay between monoclonal antibody (mAb) structure and function using two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) is reported. Charge variants, induced through forced degradation, are resolved by first-dimension (1D) cation-exchange chromatography (CEX) and subsequently collected in loops installed on a multiple heart-cutting valve prior to transfer to second-dimension (2D) neonatal crystallizable fragment receptor (FcRn) affinity chromatography coupled with MS. As such, binding affinity of the latter mAb variants can elegantly be assessed and a first glimpse of identity provided. To maximize MS sensitivity, charge variants are unfolded upon eluting from the 2D affinity column by postcolumn addition of a denaturing solution. Further structural details, i.e., modification sites and chain distribution, are unraveled by a multidimensional LC-MS (mD-LC-MS) setup incorporating 1D CEX and parallel online middle-up and bottom-up LC-MS analysis in the subsequent dimensions. Identified charge variants could be ranked according to their affinity for FcRn. Binding is predominantly impacted by heavy chain (HC) M253 oxidation and to a lesser extend, M429 oxidation. Oxidation of both HCs more drastically affects FcRn interaction compared to single-chain oxidation, and the more oxidation, the less binding. Other modifications, such as HC glycosylation, HC N385/390, and N326 deamidation or HC C-terminal processing, are not shown to affect binding. The streamlined platform is challenged against the established workflow involving offline collection of charge variants and structural and functional assessment by, respectively, LC-MS and enzyme-linked immunosorbent assay (ELISA). A decent correlation is demonstrated between the binding affinity measured with ELISA and 2D FcRn affinity chromatography. In addition, throughput is improved (7-fold), material requirements are substantially reduced (2 orders of magnitude), and sample preparation artifacts and loss are minimized. With the simultaneous determination of mAb structure and function, the current study takes the concept of multiattribute analysis to the next level, thereby contributing to the future development of safer and more effective antibody therapeutics.