Sample Preparation Method Research Articles

Multiscale mechanical performance evaluation of L-DED Ti6Al4V by novel ultrasonic burnishing (UB)

Laser-directed energy deposition (L-DED), as a novel technology to produce titanium (Ti) alloys (e.g., Ti6Al4V), has potential impactful applications. However, Ti alloys by L-DED is largely compromised by defects (porosity, cracks, etc.) at or near surfaces, which prevents strength-ductility synergy. In this study, a new method for Ti6Al4V sample preparation using ultrasonic burnishing coupled with heat treatment (UB/HT) after L-DED has been suggested. The results show that UB/HT has a more positive effect than mere UB to overcome turning's disadvantages, when the roughness, porosity, microstructures, and multiscale mechanical performance of L-DED Ti6Al4V are considered. UB/HT transforms residual tensile stress into compressive stress and causes severe plastic deformation (SPD) within a depth of approximately 10 μm on the surface layer. With this, UB/HT improves multiscale mechanical properties, e.g., hardness to 428 HV and strength to 976.7 MPa, and the related failure modes and fracture features induced by the UB/HT crack inhibition effect are confirmed with the fatigue results. With these observations, this research provides a good theoretical and practical reference for multi-scale mechanical analysis and understanding of L-DED Ti6Al4V, which expand the UB/HT applications.

Optimization and validation of a simple extraction method coupled with ICP-MS for the determination of essential and toxic elements in liquid dietary supplement products

This study aimed to develop and validate a fast, easy, cost-effective, and accurate method for determining Mn, Co, Ni, Cr, Cu, Fe, Zn, As, Cd, Hg, Sb, and Pb in liquid dietary supplements using an inductively coupled plasma mass spectrometer (ICP-MS). Two sample preparation methods were compared: microwave acid digestion and direct analysis after dilution. Both methods provided acceptable recovery values (87.7–111 %), with RSD values up to 6.35 %. The microwave digestion method was validated using various certified reference materials (CRMs) and a proficiency test (PT). Additionally, 34 domestic dietary supplements were analyzed using both methods, and the results were similar. Only one sample exceeded the maximum allowable limit for arsenic, which is 150 mg/kg set by the United States Pharmacopeia (USP). Two products had high Fe and Zn levels exceeding the recommended daily allowance (RDA), according to the Food and Drug Administration (FDA). This study demonstrates the effectiveness and advantages of the direct analysis method for measuring essential and toxic elements in liquid dietary supplements. This method promotes green chemistry principles and provides valuable information for ensuring consumer safety and product quality.

Laser desorption mass spectrometry of rubrene and photodissociation of its cation

Laser desorption mass spectrometry was employed to study rubrene using three different sample preparation methods. Pressed-pellet and films drop-cast from solution were investigated with a laser-desorption time-of-flight spectrometer. Jet-cooled rubrene cations were produced in a supersonic molecular beam by laser desorption from a film-coated metal rod and detected with time-of-flight mass spectrometry. The films for this process were produced by vacuum sublimation of powder samples. The mass spectra from each of these samples contained the parent molecular ion and fragments resulting from phenyl ring elimination - a pattern similar to that produced by electron impact ionization. The amount of fragmentation varied with sample preparation and desorption laser wavelength. The rubrene cation was mass selected and studied with UV laser photodissociation at 355 nm. The resulting fragmentation mass spectrum indicated the loss of one or two phenyl groups, but no more than this. Computational studies of the ion energetics were used to investigate the stable fragment ion structures and understand the energetics of the dissociation process.

Rapid and Online Microvolume Flow-Through Dialysis Probe for Sample Preparation in Veterinary Drug Residue Analysis.

A rapid and online microvolume flow-through dialysis probe designed for sample preparation in the analysis of veterinary drug residues is introduced. This study addresses the need for efficient and green sample preparation methods that reduce chemical waste and reagent use. The dialysis probe integrates with liquid chromatography and mass spectrometry (LC-MS) systems, facilitating automated, high-throughput analysis. The dialysis method utilizes minimal reagent volumes per sample, significantly reducing the generation of solvent waste compared to traditional sample preparation techniques. Several veterinary drugs were spiked into tissue homogenates and analyzed to validate the probe's efficacy. A diagnostic sensitivity of >97% and specificity of >95% were obtained for this performance evaluation. The results demonstrated the effective removal of cellular debris and particulates, ensuring sample integrity and preventing instrument clogging. The automated dialysis probe yielded recovery rates between 27 and 77% for multiple analytes, confirming its potential to streamline veterinary drug residue analysis, while adhering to green chemistry principles. The approach highlights substantial improvements in both environmental impact and operational efficiency, presenting a viable alternative to conventional sample preparation methods in regulatory and research applications.

A standardized protocol for sample preparation for scanning electron microscopy to visualize extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) are circular DNA structures associated with cancer and drug resistance. One specific type, double minute (DM) chromosomes, has been studied since the 1960s using imaging techniques like cytogenetics and fluorescence microscopy. Specialized techniques such as atomic force microscopy (AFM) and scanning electron microscopy (SEM) offer micro to nano-scale visualization, but current sample preparation methods may not optimally preserve ecDNA structure. Our study introduces a systematic protocol using SEM for high-resolution ecDNA visualization. We have optimized the end-to-end procedure, providing a standardized approach to explore the circular architecture of ecDNA and address the urgent need for better understanding in cancer research.

Point-of-need mastitis pathogen biosensing in bovine milk: From academic sample preparation novelty to industry prototype field testing

Bovine mastitis is an inflammation of the mammary gland, and it is the most common infectious disease in dairy cattle. Mastitis reduces milk yield and quality, costing dairy farmers millions of dollars each year. The aim of this study was to develop a point-of-need test for identifying mastitis pathogens that is field portable, cost-effective and can be used with minimal training. Using a proprietary polymer-based milk sample preparation method to rapidly extract pathogen DNA in milk samples, we demonstrated quantitative Polymerase Chain Reaction (qPCR) assays for six common bovine bacterial mastitis pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Mycoplasma bovis and Escherichia coli. We also implemented this sample preparation method on a prototype point-of-need system in a proof-of-concept field trial to evaluate user experience. Importantly, the protype system enabled a sample-to-result turnaround time of within 70 min to quantitatively detect all six target pathogens. The key advantage of our point-of-need prototype system is being culture-independent yet providing automated milk sample preparation for molecular identification of key mastitis pathogens by non-expert users. Our point-of-need prototype system showed a good correlation to laboratory-based qPCR for target pathogen detection outcomes, thus potentially removing the need for milk samples to be transported off-site for laboratory testing. Above all, we successfully achieved our objective of developing a point-of-need biosensor technology for mastitis and increased its readiness level with industry partners towards technology commercialization.

Automated high throughput IgG N-glycosylation sample preparation method development on the Tecan Freedom EVO platform.

Glycomics, focusing on the role of glycans in biological processes, particularly their influence on the folding, stability and receptor interactions of glycoconjugates like antibodies, is vital for our understanding of biology. Changes in immunoglobulin G (IgG) N-glycosylation have been associated with various physiological and pathophysiological conditions. Nevertheless, time-consuming manual sample preparation is one of the limitations in the glycomics diagnostic implementation. The study aimed to develop an automated method for sample preparation on the Tecan Freedom Evo 200 platform and compare its efficiency and precision with the manual counterpart. The initial method development included 32 pooled blood plasma technical replicates. An additional 24 pooled samples were used in the method comparison along with 78 random duplicates of plasma samples collected from 10,001 Dalmatians biobank to compare the manual and automated methods. The development resulted in a new automated method. For the automated method, glycan peaks comprising 91% of the total sample glycan showed a variation of less than 5% while 92% of the total sample showed a variation of less than 5% for the manual method. The results of the Passing-Bablok regression indicated no differences between the automated and manual methods for 12 glycan peaks (GPs). However, for 8 GPs systematic difference was present, while both systematic and proportional differences were present for four GPs. The developed automated sample preparation method for IgG glycan analysis reduced exposure to hazardous chemicals and offered a simplified workflow. Despite slight differences between the methods, the new automated method showed high precision and proved to be highly comparable to its manual counterpart.

Microvolume analysis of 226Ra by inductively coupled plasma mass spectrometry: Environmental applications to high-resolution profile of wetland soil pore waters

BackgroundThe release to the environment of natural radionuclides like 226Ra due to human activities have raised concerns about human and ecosystem exposure. 226Ra’s resemblance to calcium and involvement in biological processes accentuate the importance of understanding its geochemical behavior and migration pathways. Soil and sediment pore waters provide valuable information but have proved to be challenging to analyze, requiring to push the limits of current sample preparation and detection methods often not scaled for low concentrated microvolumes of samples. A more efficient and accurate analytical method is therefore needed to get reliable data to support hydro-geochemical modeling. ResultsWe describe here a novel comprehensive approach for accurate 226Ra quantification in pore waters of a wetland located downstream of the former Rophin uranium mine. In order to highlight the migration of 226Ra between the different soil layers, microvolumes of soil pore waters were in situ sampled up to 50 cm depth employing Diffusive Equilibrium in Thin-Films (DET) probes. The workflow included spiking DET extracts with an in-house 228Ra tracer, specific solid-phase extraction with 50 mg of AnaLig Ra-01® resin, and elution fraction calcination. This process eliminated interferences and simplify the analysis matrix. Quantification relied on isotope dilution after measurement by ICP-MS hyphenated to a desolvator module and to a microsampler (150 µL injection). The resulting 1 cm-high-resolution profile revealed unusual 226Ra enrichment with depth, signs of Ra mobility from the solid contamination source. These elements are essential for assessing 226Ra distribution coefficients across soil horizons and subsequent wetland remobilization considerations. Significance and noveltyThis first methodological advance for 226Ra constitutes a significant step to understand 226Ra behavior i.e., to quantify its activity level, fluxes inside of wetland soils or at the interface overlying waters/sediments. Its potential extension to studies involving other elements underscores its broader applicability in environmental research. The findings contribute to evidence-based decision-making for environmental protection and management, aiding in the preservation of ecological integrity and human health.

Cyanotoxin Monitoring and Detection Using Passive Sampling Application

Cyanobacterial blooms in water have been extensively studied as they produce bioactive and toxic metabolites, commonly known as cyanotoxins. Additionally, the presence of cyanobacteria and, consequently, the production of cyanotoxins, have increased in extent and frequency worldwide. Therefore, the risk associated with the presence of these microorganisms and their toxins has become a matter of great concern. On the other hand, conventional processes for water treatment are inefficient for their elimination and/or degradation, so their presence in water persists at trace and ultra-trace concentrations. In this regard, it is important to develop alternatives to monitor cyanotoxins and allow their detection at low levels in water supply and purification systems, in order to ensure water of good quality for human consumption. In this work, different methodologies, implemented both at laboratory scale and in situ in aqueous bodies, are described. Among these methodologies, traditional and passive techniques are highlighted. Appropriate analytical and sample preparation methods used in the detection and quantification of cyanotoxins are also addressed. It was found that the use of passive samplers is a convenient and a cost-effective method of identifying the presence of these toxins in water at concentrations in the order of µg/L and ng/L. Moreover, studying the by-products generated from the degradation of natural toxins in aquatic environments and evaluating their possible adverse effects is crucial in terms of the management and control of cyanobacteria and cyanotoxin pollution in water.

Determination of silicon isotopic composition in soil using gas-source isotope ratio mass spectrometry by thermal decomposition of barium hexafluorosilicate

Determining the Si isotopic compositions of samples of the Earth’s surface is crucial to understanding the implications of the biogeochemistry cycling of Si. This study established an alternative method for high-precision Si isotopic measurement in soil samples using gas-source isotope ratio mass spectrometry, which involved the conversion of Si in the samples into SiF4 gas, which was purified cryogenically and collected for isotopic analysis. The proposed method was applied to determine the Si isotopic composition of two reference materials (GBW04421 and GBW04422), and the results were found to be in good agreement with the certified values. Moreover, the proposed method exhibited a long-term measurement precision of 0.05 ‰–0.08 ‰ (2 s) for the δ30Si values. The δ30Si values obtained from ten soil reference materials ranged from −1.84 ‰ to −0.17 ‰, which were within the range of the values reported in the literature (−1.82 ‰ to −0.15 ‰). The sample preparation and analysis method in this study provided an easy and effective approach to determining the Si isotopic composition of solid samples and offers to the promise of being applicable to biogeochemical analyses of the Earth’s surface processes.

Improved Mass Spectrometry–Based Methods Reveal Abundant Propionylation and Tissue-Specific Histone Propionylation Profiles

Histone posttranslational modifications (PTMs) have crucial roles in a multitude of cellular processes, and their aberrant levels have been linked with numerous diseases, including cancer. Although histone PTM investigations have focused so far on methylations and acetylations, alternative long-chain acylations emerged as new dimension, as they are linked to cellular metabolic states and affect gene expression through mechanisms distinct from those regulated by acetylation. Mass spectrometry is the most powerful, comprehensive, and unbiased method to study histone PTMs. However, typical mass spectrometry–based protocols for histone PTM analysis do not allow the identification of naturally occurring propionylation and butyrylation. Here, we present improved state-of-the-art sample preparation and analysis protocols to quantitate these classes of modifications. After testing different derivatization methods coupled to protease digestion, we profiled common histone PTMs and histone acylations in seven mouse tissues and human normal and tumor breast clinical samples, obtaining a map of propionylations and butyrylations found in different tissue contexts. A quantitative histone PTM analysis also revealed a contribution of histone acylations in discriminating different tissues, also upon perturbation with antibiotics, and breast cancer samples from the normal counterpart. Our results show that profiling only classical modifications is limiting and highlight the importance of using sample preparation methods that allow the analysis of the widest possible spectrum of histone modifications, paving the way for deeper insights into their functional significance in cellular processes and disease states.

Assessing the matrix effects on MALDI-MS in the positive and negative ion mode detection for protein-protected metal nanoclusters

Protein-protected metal nanoclusters (MNCs) represent a new class of highly photoluminescent nanomaterials that have wide applications. Suitable reaction conditions combining protein and metal precursors can produce a vast range of different NC sizes (i.e. different number of metal atoms). The average number of metal atoms per protein can be determined by mass spectrometry (MS). MS coupled with matrix-assisted laser desorption ionization (MALDI) presents a number of advantages such as detection with high sensitivity of nanoclusters with high molecular weights. Although many protein-protected MNCs have been characterized by MALDI-MS, a large dispersion in the number of metal atoms has been reported mainly due to sample preparation. In this work, we optimized the protocols for negative and positive ion detection mode as a general MALDI-MS sample preparation method for protein-protected MNCs (bovine serum albumin and lysozyme and with gold and silver). Negative and positive ion mode detection was compared, showing that negative ion mode detection in MALDI-MS can also be used with acidic matrices. Obvious matrix effects on ion signals and peak positions by MALDI-MS were observed. The average metal numbers of MNCs embedded in proteins are different depending on the MALDI matrix. The matrix effects give a warning for more serious consideration on MALDI-MS measurement and spectra analysis of MNCs.

Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens

Effective pathogen detection is essential for plant disease control. However, plant sample preparation for downstream assays, such as quantitative polymerase chain reaction (qPCR), is challenging to perform outside of a laboratory. This paper reports two sample preparation methods featuring chemical and mechanical lysis and nucleic acid extraction using a micro-homogenizer, followed by serial dilution or nucleic acid purification with a paper disk before assay. Five minutes of lysis and extraction resulted in DNA and RNA yields of up to 76.5% and 63.3%, respectively, compared to mortar and pestle controls. Crude lysates were unsuitable for direct use in qPCR assays; however, serial dilution or quick wash using chromatography paper rendered samples ready for such assays. Additionally, the nucleic acids stored on paper disks under various storage conditions remained stable for one month. These methods can facilitate the in-field preparation of citrus samples and allow for both onsite and mail-in diagnostics for growers.

Preparing Retinal Organoid Samples for Transmission Electron Microscopy.

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

EMA beamline at Sirius: A versatile platform to probe glass and glass ceramics under extreme thermodynamic conditions

AbstractGlass and glass ceramics are very functional materials, albeit their structural complexity. Their relevance ranges from fundamental science problems in the fields of physics, chemistry, and geoscience, to applications in health areas, engineering, or technological matters that require high performance. Enhancing our understanding of these materials' performance and refining sample preparation methods remains paramount in this field. Synchrotron facilities offer a suite of powerful techniques for the detailed characterization of glasses and glass ceramics. These methods provide valuable insights into their atomic and molecular structure, phase transformations, mechanical behavior, and thermal properties, ultimately contributing to the development of improved materials for a wide range of applications. In‐depth investigations conducted under extreme conditions of pressure and temperature have yielded pivotal insights into densification mechanisms, phase transitions, crystallization kinetics, and their consequential macroscopic properties. The emergence of fourth‐generation synchrotrons brings in a wave of novel experimental possibilities that may exert a profound influence on this field in the coming decade. In this study, we unveil a selection of the remarkable capabilities now accessible to researchers at the Brazilian Synchrotron Light Source—Sirius, within the realm of extreme methods of analysis (EMA) beamline for investigating vitreous systems under extreme conditions.

Generality of Enhancing the Dissolution Rates of Free Acid Amorphous Solid Dispersions by the Incorporation of Sodium Hydroxide.

The incorporation of a counterion into an amorphous solid dispersion (ASD) has been proven to be an attractive strategy to improve the drug dissolution rate. In this work, the generality of enhancing the dissolution rates of free acid ASDs by incorporating sodium hydroxide (NaOH) was studied by surface-area-normalized dissolution. A set of diverse drug molecules, two common polymer carriers (copovidone or PVPVA and hydroxypropyl methylcellulose acetate succinate or HPMCAS), and two sample preparation methods (rotary evaporation and spray drying) were investigated. When PVPVA was used as the polymer carrier for the drugs in this study, enhancements of dissolution rates from 7 to 78 times were observed by the incorporation of NaOH into the ASDs at a 1:1 molar ratio with respect to the drug. The drugs having lower amorphous solubilities showed greater enhancement ratios, providing a promising path to improve the drug release performance from their ASDs. Samples generated by rotary evaporation and spray drying demonstrated comparable dissolution rates and enhancements when NaOH was added, establishing a theoretical foundation to bridge the ASD dissolution performance for samples prepared by different solvent-removal processes. In the comparison of polymer carriers, when HPMCAS was applied in the selected system (indomethacin ASD), a dissolution rate enhancement of 2.7 times by the incorporated NaOH was observed, significantly lower than the enhancement of 53 times from the PVPVA-based ASD. This was attributed to the combination of a lower dissolution rate of HPMCAS and the competition for NaOH between IMC and HPMCAS. By studying the generality of enhancing ASD dissolution rates by the incorporation of counterions, this study provides valuable insights into further improving drug release from ASD formulations of poorly water-soluble drugs.

High-throughput Proteomics-Guided Biomarker Discovery of Hepatocellular Carcinoma

Liver cancer stands as the fifth leading cause of cancer-related deaths globally. Hepatocellular carcinoma (HCC) comprises approximately 85%-90% of all primary liver malignancies. However, only 20-30% of HCC patients qualify for curative therapy, primarily due to the absence of reliable tools for early detection and prognosis of HCC. This underscores the critical need for molecular biomarkers for HCC management. Since proteins reflect disease status directly, proteomics has been utilized in biomarker developments for HCC. In particular, proteomics coupled with liquid chromatography-mass spectrometer (LC-MS) methods facilitate the process of discovering biomarker candidates for diagnosis, prognosis, and therapeutic strategies.In this work, we investigated LC-MS-based proteomics methods through recent reference reviews, with a particular focus on sample preparation and LC-MS methods appropriate for the discovery of HCC biomarkers and their clinical applications.We classified proteomics studies of HCC according to sample types, and we examined the coverage of protein biomarker candidates based on LC-MS methods in relation to study scales and goals. Comprehensively, we proposed protein biomarker candidates categorized by sample types and biomarker types for appropriate clinical use.In this review, we summarized recent LC-MS-based proteomics studies on HCC and proposed potential protein biomarkers. Our findings are expected to expand the understanding of HCC pathogenesis and enhance the efficiency of HCC diagnosis and prognosis, thereby contributing to improved patient outcomes.

Selection of Suitable Methods for Sample Preparation and Desorbing Agents for Determination of Nutrient Contents in Scots Pine Roots

Selection of Suitable Methods for Sample Preparation and Desorbing Agents for Determination of Nutrient Contents in Scots Pine Roots

Intact quantitation of cysteine-conjugated antibody-drug conjugates using native mass spectrometry.

A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach. A linear dynamic range of 5-100 μg/mL was achieved using 20 μL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement. We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo.

A Review on Sample Preparation for Bio analytical Method Development by HPLC

This article compares the benefits and drawbacks of each approach while reviewing recent advancements in bioanalysis sample preparation methods. It also provides an update on fundamental concepts, theories, applications, and automation opportunities. Because every biological matrix has its own special difficulties and complexity, sample preparation is thought to be the bottleneck stage in bioanalysis. In the last ten years, new sample preparation methods for bioanalysis have developed quickly. In the preclinical and clinical phases of drug development, it is crucial to create reliable bioanalytical method(s). Consequently, it is widely acknowledged that sample preparation A number of excellent review papers have recently been published in the literature that address various scientific and technical facets of bioanalysis. The use of bioanalysis in the pharmacokinetic/pharmacodynamic characterization of novel chemical entities, beginning with their finding and continuing through their market authorization, is now generally acknowledged. Physico-chemical and biological technologies used in bioanalysis, including mass spectrometry, immunoassay, and chromatography. Liquid chromatography-mass spectrometry is a method used in labs for the qualitative and quantitative study of drug substances and metabolites. It makes use of liquid chromatography/RP HPLC. The current review concentrated on different extraction methods, including protein precipitation, solid phase extraction, and liquid-liquid extraction, all of which are crucial for sample preparation and RP HPLC sample detection.