Sample Preparation Research Articles

3D Printed Coated Blade Spray-Mass Spectrometry Devices.

Coated blade spray-mass spectrometry (CBS-MS) has emerged as a powerful tool for the rapid screening of target compounds at trace levels in complex biological matrices. Despite its potential, the broader adoption of CBS-MS technology has been hindered by the lack of commercially available, user-friendly MS interfaces and extraction devices. In this work, we present comprehensive CBS-MS solutions developed using 3D printing, including a versatile MS interface and two extraction devices tailored to different analytical needs: one is compatible with LC vials for large-volume samples, while the other is optimized for single-drop blood analysis. The MS interface features a novel design that separates the immobilization station from the blade holder, significantly simplifying the operational workflow and minimizing the contamination risks and hazards associated with manual blade handling. The first extraction cartridge can process 48 samples simultaneously with an average sample preparation time of less than 20 s, while the second extraction device enables extraction from 8 single-drop blood samples via on-blade extraction. The developed devices were successfully tested for the rapid screening of seven drugs in both urine and single-drop blood samples, demonstrating a promising analytical performance. Additionally, potential contamination issues related to the use of 3D-printed materials as extraction phases were examined, emphasizing the importance of ensuring that 3D-printed materials do not leach contaminants into samples or solvents and contaminate the MS.

Innovative sample preparation using alcohol dehydration and high refractive index medium enables acquisition of two-channel super-resolution 3D STED image of an entire oocyte.

Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.

Live Cell Fluorescence Microscopy—An End-to-End Workflow for High-Throughput Image and Data Analysis

Abstract Fluorescence microscopy images of biological samples contain valuable information but require rigorous analysis for accurate and reliable determination of changes in protein localization, fluorescence intensity and morphology of the studied objects. Traditionally, cells for microscopy are immobilized using chemicals, which can introduce stress. Analysis often focuses only on colocalization and involves manual segmentation and measurement, which are time-consuming and can introduce bias. Our new workflow addresses these issues by gently immobilizing cells using a small agarose block on a microscope coverslip. This approach is suitable for cell-walled cells (yeast, fungi, plants, bacteria), facilitates their live imaging under conditions close to their natural environment and enables the addition of chemicals during time-lapse experiments. The primary focus of the protocol is on the presented analysis workflow, which is applicable to virtually any cell type—we describe cell segmentation using the Cellpose software followed by automated analysis of a multitude of parameters using custom-written Fiji (ImageJ) macros. The results can be easily processed using the provided R markdown scripts or available graphing software. Our method facilitates unbiased batch analysis of large datasets, improving the efficiency and accuracy of fluorescence microscopy research. The reported sample preparation protocol and Fiji macros were used in our recent publications: Microbiol Spectr (2022), DOI: 10.1128/spectrum.01961-22; Microbiol Spectr (2022), DOI: 10.1128/spectrum.02489-22; J Cell Sci (2023), DOI: 10.1242/jcs.260554.

Recent Advances in Monitoring Microbial Toxins in Food Samples by HPLC-Based Techniques: A Review

This study examines the significant impact of bacterial, algal, and fungal toxins on foodborne illnesses, and stresses the importance of advanced detection techniques, such as high-performance liquid chromatography (HPLC)-based methodologies. It emphasizes the urgent need for further advancements in these techniques to ensure food safety, as they offer significant benefits, including low detection limits and the ability to be combined with other techniques to detect a wide range of toxins. In this regard, HPLC has emerged as a versatile and sensitive analytical technique for this purpose. Various HPLC methods, often enhanced with detectors such as ultraviolet (UV), fluorescence (FD), and mass spectrometry (MS), have been developed to identify and quantify microbial toxins in a wide variety of food samples. Recent advancements include HPLC-FD methods that utilize the natural fluorescence of certain aflatoxins, improving detection sensitivity. HPLC-MS/MS and UHPLC-MS/MS techniques offer high selectivity and sensitivity, making them suitable for detecting a wide range of toxins in trace quantities. The adaptability of HPLC, combined with innovative detection technologies and sample preparation methods, holds significant potential for enhancing food safety monitoring and reducing the global burden of foodborne diseases.

A Review of Recent Advances in Chromatographic Quantification Methods for Cyanogenic Glycosides

Cyanogenic glycosides are naturally occurring compounds found in numerous plant species, which can release toxic hydrogen cyanide upon hydrolysis. The quantification of cyanogenic glycosides is essential for assessing their potential toxicity and health risks associated with their consumption. Liquid chromatographic techniques coupled with various detectors have been widely used for the quantification of cyanogenic glycosides. In this review, we discuss recent advances in chromatographic quantification methods for cyanogenic glycosides, including the development of new stationary phases, innovative sample preparation methods, and the use of mass spectrometry. We also highlight the combination of chromatographic separation with mass spectrometric detection for the identification and quantification of specific cyanogenic glycosides and their metabolites in complex sample matrices. Lastly, we discuss the current challenges and future perspectives in the development of reliable reference standards, optimization of sample preparation methods, and establishment of robust quality control procedures. This review aims to provide an overview of recent advances in chromatographic quantification methods for cyanogenic glycosides and their applications in various matrices, including food products, biological fluids, and environmental samples.

Affordable and Reliable RP-HPLC Method for Verapamil Hydrochloride Quantification in Rabbit Plasma for Pharmacokinetics

Background: Existing bioanalytical methods for verapamil hydrochloride (VH) are often complex, requiring advanced instrumentation and specialized expertise, which limits their use in resource-constrained laboratories. Aim: The goal of this study is to fill this gap by developing a simplified, robust RP-HPLC-UV approach for the estimation of verapamil hydrochloride in rabbit plasma. Designed to enhance accuracy and precision while minimizing sample preparation challenges, this method addresses existing limitations by providing an affordable and reliable alternative for laboratories lacking sophisticated instrumentation. Methods: The bioanalytical method was implemented on C-18 stationary phase (5 μ, 250 × 4.6 mm) using acetonitrile/0.1% tetrahydrofuran (THF) in water (80:20, in volume) as the liquid system at a 1 mL/min flow speed, employing carvedilol as an internal standard. Results: The reported retention times of verapamil hydrochloride and carvedilol were ~7.64 and 4.69 min, respectively, at sufficiently high system suitability standards. The linearity of the bioanalytical approach can be seen between 0.025 and 5.0 µg/mL (r2 = 0.9991). The findings indicated that there was no matrix influence in terms of accuracy (≥98.96 ± 2.68%), intra- and inter-day precision (≤3.68%), recovery (101.98 ± 2.76%), and procedure efficiency (100.65 ± 1.82%). Benchtop, long-term, and short-term stability investigations all revealed that the verapamil hydrochloride in the bio-samples was stable. The pharmacokinetic parameters (Cmax—3.47 µg/mL; Tmax—1.59 h) were studied from time-dependent plasma concentrations of verapamil hydrochloride estimated after 40 mg oral dosing in New Zealand white rabbits. Conclusions: The developed bioanalytical method provided easier quantitative analysis of verapamil hydrochloride from rabbit plasma and was effectively used in a pharmacokinetic investigation of an oral bolus. The reliable performance of this method under practical conditions positions it as a crucial tool for advancing pharmacokinetic studies across various research environments.

Cross-validation of methods for the quantitative determination of phenazepam and its active metabolite in human blood plasma at various extractions

Introduction. In conducting of therapeutic drug monitoring (TDM), often such situation arises where the drug concentration has measured by different methods or in different laboratories. To combine and analyze the data obtained with different methods, it is necessary to perform cross-validation procedure. Insufficient attention is paid to the statistical approaches used for this purpose.Aim. Performing cross-validation of different analytical methods for the quantitative determination of phenazepam (PHEN) and 3-hydroxyphenazepam (3-OH-PHEN) using the Bland – Altman analysis.Materials and methods. PHEN and 3-OH-PHEN concentrations in the blood plasma of patients (n = 100) with alcohol withdrawal syndrome were measured using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The quantification of both analytes in each sample was measure twice by two different methods: solid phase extraction (SPE) and supported liquid extraction (SLE). Both methods have been fully validated before the experiment began. Cross-validation was performed at the end of the experiment using data from study samples. The Bland – Altman analysis was used to evaluate accuracy and precision. Deming regression was also used to identify a systematic error between measurement results.Results and discussion. The regression equations have been obtained between concentrations both analytes measured by different sample preparation methods. 95 % confidence intervals (CI) of the regression coefficients of both equations included one, and 95 % CI of the intercepts included zero. 95 % CI of the geometric mean of the individual SLE/SPE ratios was within the acceptable range (0.87; 1.15). These results confirm the absence of the influence of quantitative methods on the measurement of both analytes concentration. 66.7 % CI of the percent difference between two measurements was within acceptable limits (–0.2; 0.2), not exceeding 20 % of the range of their mean value. This confirms the acceptable precision between the methods. The estimated CIs were displayed in the Bland – Altman plots.Conclusion. The statistical approaches used in the work have confirmed the reproducibility of the results of different sample preparation methods. In addition to cross-validation, the statistical algorithm from this paper using Bland – Altman analysis can be successfully employed to assess accuracy and precision during bioanalytical method validation and evaluation of the acceptance of analytical runs, as well as to determine the level of reproducibility of incurred samples.

Personalization of pharmacotherapy with sirolimus based on volumetric absorptive microsampling (VAMS) in pediatric renal transplant recipients-from LC-MS/MS method validation to clinical application.

The benefits of pharmacotherapy with sirolimus (SIR) in pediatric transplant recipients are well established. Traditionally, whole blood samples have been used to measure SIR concentrations. Volumetric Absorptive Microsampling (VAMS) is an alternative sampling strategy suitable for Therapeutic Drug Monitoring (TDM). In this study, we developed and validated two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for determining SIR concentrations in whole blood (WB) and capillary whole blood samples collected using a VAMS-Mitra™ device. We used protein precipitation during WB sample preparation and dispersive liquid-liquid microextraction (DLLME) with methyl tert-butyl ether for VAMS sample preparation to optimise the analyte extraction process. The described validation protocols were cross-validated, confirming the equivalence of the whole-blood and VAMS-based methods. Furthermore, the developed methods were evaluated in two three-level rounds of an external proficiency-testing scheme. The analytical methods were successfully validated within the calibration range of SIR (0.5-60 ng/ml). The validation parameters met the European Medicines Agency (EMA) and the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDM&CT) acceptance criteria. No hematocrit (tested in the range of 24.3-64.1%), matrix, or carry-over effects were observed. Cross-validation confirmed the interchangeability between VAMS-LC-MS/MS and WB-LC-MS/MS methods. The developed methods were successfully implemented for SIR determination in 140 clinical samples (70 each of WB and VAMS) from pediatric renal transplant recipients, demonstrating their practicality and reliability. The VAMS-based method has been rigorously tested and is clinically equivalent to the reference WB-LC-MS/MS method. Additionally, clinical validation confirmed the utility of the presented methods for TDM of the SIR in the pediatric population after renal transplantation.

Resolving Structures of Paramagnetic Systems in Chemistry and Materials Science by Solid-State NMR: the Revolving Power of Ultra-Fast MAS.

Ultra-fast magic-angle spinning (100+ kHz) has revolutionized solid-state NMR of biomolecular systems but has so far failed to gain ground for the analysis of paramagnetic organic and inorganic powders, despite the potential rewards from substantially improved spectral resolution. The principal blockages are that the smaller fast-spinning rotors present significant barriers for sample preparation, particularly for air/moisture-sensitive systems, and are associated with lower sensitivity from the reduced sample volumes. Here these difficulties are overcome by improvements in rotor ceramics and handling methods that enable the routine use of this setup for repetitive, high-throughput analysis of sensitive samples. Furthermore, we demonstrate that the sensitivity penalty is less severe than expected for highly paramagnetic solids and is more than offset by the associated improved resolution. While previous approaches employing slower MAS are often unsuccessful in providing sufficient resolution, we show that ultra-fast 100+ kHz MAS allows site-specific assignments of all resonances from complex paramagnetic solids. This opens the way to routine characterisation of geometry and electronic structures of functional paramagnetic systems in chemistry, including catalysts and battery materials. We benchmark this approach on a hygroscopic luminescent Tb3+ complex, an air-sensitive homogeneous high-spin Fe2+ catalyst, and a series of mixed Fe2+/Mn2+/Mg2+ olivine-type cathode materials.

A Review of the Application of the Laser-Light Backscattering Imaging Technique to Agricultural Products

Growing concerns about food safety and waste have increased consumer demand for high-quality agricultural products, particularly at the postharvest stage. This demand has prompted the development of non-destructive methods to assess or inspect the internal quality of fruits and vegetables. The backscattering imaging technique, also known as diffuse reflectance imaging, is considered a highly promising approach. Numerous studies have focused on practical applications, using laser light at selected wavelengths to develop quick multispectral methods. Due to the rapid interaction of photons with biological tissue, together with the highly computational performance of machine vision, backscattering imaging can offer a valuable alternative to traditional methods. Its primary benefits include quick measurements without chemical sample preparation, easy integration with high-throughput automatic quality control, and reduced waste, since this non-destructive technique does not damage samples. This review presents a comprehensive overview of backscattering imaging, including the measurement geometry, data analysis, and design considerations for vision systems. Additionally, it explores this technique’s advantages, challenges, and accuracy, as demonstrated using various case studies.

Features of laboratory study of the core of weakly cemented reservoir rocks

Background. Currently, there is a problem of studying intervals represented by weakly cemented core, although such rocks can be the main reservoirs. Lack of reliable petrophysical information leads to underestimation of the main parameters of porosity and permeability, which in the future is likely to lead to incorrect estimation of hydrocarbon reserves. The relevance of the work is due to the lack of unified regulatory and methodological approaches to the research of weakly cemented core material. Objective. To develop our own methodological approach by selecting technologies and techniques, which allows to carry out primary preparation, sampling, profile measurements and petrophysical studies of weakly cemented core. Materials and methods. We used weakly cemented core from the Volga-Ural oil and gas province and conducted the research in accordance with the requirements specified in the guidelines for laboratory tests using necessary supplies. The methods considered in the article cover the time interval from the early 2000s. Results. In the course of the work, standard methods of core investigation were considered, and the possibility of their application to the samples of weakly cemented rock was evaluated. The peculiarities of sample preparation and research on weakly cemented core were also revealed. Conclusions. A methodological approach was developed that allows primary preparation, sampling, profile measurements and petrophysical studies of weakly cemented core.

Development of a Versatile High-through-put Oligonucleotide LC-MS Method to Accelerate Drug Discovery.

Liquid chromatography-mass spectrometry (LC-MS) is an effective tool for high-throughput quantification of oligonucleotides that is crucial for understanding their biological roles and developing diagnostic tests. This paper presents a high-throughput LC-MS/MS method that may be versatilely applied for a wide range of oligonucleotides, making it a valuable tool for rapid screening and discovery. The method is demonstrated using an in-house synthesized MALAT-1 Antisense oligonucleotide (ASO) as a test case. Biological samples were purified using a reversed liquid-liquid extraction process automated by a liquid handling workstation and analyzed with ion-pairing LC-MS/MS. The assay was evaluated for sensitivity (LLOQ = 2nM), specificity, precision, accuracy, recovery, matrix effect, and stability in rat cerebrospinal fluid (CSF) and plasma. Besides some existing considerations such as column selection, ion-pairing reagent, and sample purification, our work focused on the following four subtopics: 1) selecting the appropriate Multiple Reaction Monitoring (MRM) transition to maximize sensitivity for trace-level ASO in biological samples; 2) utilizing a generic risk-free internal standard (tenofovir) to avoid crosstalk interference from the oligo internal standard commonly utilized in the LC-MS assay; 3) automating the sample preparation process to increase precision and throughput; and 4) comparing liquid-liquid extraction (LLE) and solid-phase extraction (SPE) as sample purification methods in oligo method development. The study quantified the concentration of MALAT-1 ASO in rat CSF and plasma after intrathecal injection and used the difference between the two matrices to evaluate the injection technique. The results provide a solid foundation for further internal oligonucleotide discovery and development.

Epigallocatechin-3-gallate (EGCG) reduced expression of Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) and Transforming Growth Factor-β1 (TGF-β1) in visceral pleural tissue cultures of patients with empyema

Abstract* Background Increased expression of signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) and Transforming Growth Factor-β1 (TGF-β1) has been thought to influence the fibrosis process in many tissues. However, increased expression of these two factors has never been assessed in pleural fibrosis. Pleural fibrosis is a disease that usually results from various infectious processes, such as empyema. Fibrosis formation is recently known to be prevented by Epigallocatechin-3-gallate (EGCG), which is the most potent active substance found in Gambir (Uncaria gambir Roxb) leaves. Thus, further research is needed to determine the potential of EGCG to inhibit the process of pleural fibrosis caused by empyema. Methods An in vitro experimental study with post test-only controlled group was conducted on the pleural tissue of all patients who underwent decortication surgery due to empyema at Dr. M. Djamil Hospital, Padang, Indonesia between March 1st and April 30, 2024. Case samples were obtained by consecutive sampling, and three patients who met the inclusion criteria were obtained. Pleural tissue in each patient was then divided into several groups based on the treatment, namely the control group, 50 μg EGCG administration group, and 100 μg EGCG administration group. We used to determine SCUBE3 and TGF-β1 genes expression. Data were tested using ANOVA and Least Significant Difference (LSD) tests. Results There were significant differences in the SCUBE3 and TGF-β1 genes expression of 50 μg and 100 μg EGCG administration groups and the control groups in the 2 × 2 cm sample preparation (p=0.002) (p=0.014, respectively). Significant differences in TGF-β1 expression were also found between the groups treated with EGCG 50 μg and 100 μg and the control group in the 1 × 1 cm preparation (p=0.019). Conclusion EGCG can potentially decrease SCUBE3 and TGF-β1 expression in patients with pleural empyema.

The Addition of Transcriptomics to the Bead-Enabled Accelerated Monophasic Multi-Omics Method: A Step toward Universal Sample Preparation.

Omics-based measurements enable the study of biomolecules in a high-throughput fashion, leading to the characterization and quantification of biological systems. Multi-omics methods aim to incorporate several omics measurements for a more holistic approach, which is crucial for advancing our understanding of the diversity and redundancy of biological systems. Current multi-omics sample preparation methods have achieved proteomics, lipidomics, and metabolomics from individual samples; however, the bioinformatic tools currently available for interpreting data generated from these omics are limited. Alternately, transcriptomics has a wide arsenal of available bioinformatic tools offering intensive sample characterization but has yet to be incorporated into a unified, multi-omics sample preparation technique. Herein we describe the modified bead-enabled accelerated monophasic multi-omics (mBAMM) method, which incorporates RNA extraction for transcriptomics analysis. mBAMM was shown to enable RNA-seq without compromising the isolation of biomolecules for proteomics, lipidomics, and metabolomics. This methodology greatly improves sample characterization and represents a major innovation toward cohesive insights into biological systems.

Method development and optimization for dispersive liquid-liquid microextraction factors using the response surface methodology with desirability function for the ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry determination of organic contaminants in water samples: risk and greenness assessment.

A simple, cost effective, and efficient dispersive liquid-liquid microextraction method was developed and optimized for the determination of organic contaminants in different environmental water matrices followed by UHPLC-QTOF-MS analysis. In the preliminary experiments, the univariate optimization approach was used to select tetrachloroethylene and acetonitrile as extraction and disperser solvents, respectively. The significant factors influencing DLLME were screened using full factorial design, and the optimal values for each variable were then derived through further optimization using central composite design with desirability function. The optimal conditions were achieved with 195 μL of tetrachloroethylene as the extraction solvent, 1439 μL of acetonitrile as the disperser solvent, and a sample pH of 5.8. Under these conditions, the method provided detection limits ranging from 0.11-0.48 μg L-1 and recoveries ranging from 23.32-145.43% across all samples. The enrichment factors obtained ranged from 11.66-72.72. The proposed method was then successfully applied in real water samples. Only benzophenone was detected in the concentration range of 0.79-0.88 μg L-1 across all the water samples. The calculated risk quotient resulting from benzophenone exposure in water samples showed a low potential risk to human health and the aquatic ecosystem. The method was also evaluated for its environmental friendliness using various metrics tools such as Analytical Eco-Scale (AES), Green Analytical Procedure Index (GAPI), Analytical GREEnness (AGREE), Analytical Greenness for Sample Preparation (AGREEprep), and Sample Preparation Metric of Sustainability (SPMS). Only AES qualified the method as green while it was considered acceptable and sustainable when assessed using SPMS.

Hierarchical Biclustering of Mouse Pancreas Mass Spectrometry Imaging Data Using Recursive Rank-2 Non-negative Matrix Factorization.

One of the main challenges in mass spectrometry imaging data analysis remains the analysis of m/z-spectra displaying a low signal-to-noise ratio caused by their low abundance, sample preparation, matrix effects, fragmentation, and other artifacts. Additionally, we observe that molecules with a high abundance suppress those with lower intensities and misdirect classical tools for MSI data analysis, such as principal component analysis. As a result, the observed significance of a molecule may not always be directly related to its abundance. In this work, we present a recursive rank-2 non-negative matrix factorization (rr2-NMF) algorithm that automatically returns spectral and spatial visualization of colocalized molecules, both highly and lowly abundant. Using this hierarchical decomposition, our method finds spatial and spectral correlations on different levels of abundances. The quality of the analysis is evaluated on MALDI-TOF data of healthy mouse pancreatic tissue for the annotation of molecules of interest in the lower abundances. The results show interesting findings regarding the functioning and colocalization of certain molecules.

Composite Materials Used for Dental Fillings

This article explores the properties of composite materials employed in dental fillings. A traditional nano-hybrid composite containing nanofiller particles exceeding 82% by weight served as a benchmark. The remaining samples were fabricated from ormocer resin, maintaining an identical nanofiller content of 84%. In all specimens, the nanoparticles were dispersed randomly within the matrix. This study presents findings from investigations into surface geometry, hardness, wettability, and tribological behavior. The microscopic observations revealed that ormocer-based samples exhibited greater surface roughness than those composed of the traditional composite. Hardness testing indicated that both ceramic addition and sample preparation significantly influenced mechanical properties. Ceramic-enhanced samples demonstrated superior hardness, surpassing the reference composite by 30% and 43%, respectively. Contact angle measurements revealed hydrophilic characteristics in the classic composite, contrasting with the hydrophobic nature of ceramic-containing samples. Tribological evaluations revealed the superiority of the classic composite in terms of friction coefficients and volumetric wear compared to ormocer-based materials.

Quantifying allergenic proteins using antibody-based methods or liquid chromatography-mass spectrometry/mass spectrometry: A review about the influence of food matrix, extraction, and sample preparation.

Accurate quantification of allergens in food is crucial for ensuring consumer safety. Pretreatment steps directly affect accuracy and efficiency of allergen quantification. We systematically reviewed the latest advances in pretreatment steps for antibody-based methods and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) protein quantification methods in food. For antibody-based methods, the effects induced by food matrix like decreased allergen solubility, epitope masking, and nonspecific binding are of the upmost importance. To mitigate interference from the matrix, effective and proper extraction can be used to obtain the target allergens with a high protein concentration and necessary epitope exposure. Removal of interfering substances, extraction systems (buffers and additives), assistive technologies, and commercial kits were discussed. About LC-MS/MS quantification, the preparation of the target peptides is the crucial step that significantly affects the efficiency and results obtained from the MS detector. The advantages and limitations of each method for pre-purification, enzymatic digestion, and peptide desalting were compared. Additionally, the application characteristics of microfluidic-based pretreatment devices were illustrated to improve the convenience and efficiency of quantification. A promising research direction is the targeted development of pretreatment methods for complex food matrices, such as lipid-based and carbohydrate-based matrices.

Label-free quantitative phase imaging and analysis of airborne pollen

Abstract Pollen morphology, involving the physical characteristics of pollen grains from seed plants during reproduction, plays an important role in plant biology, ecology, and evolution. High pollen concentrations in the air can degrade air quality and exacerbate respiratory conditions such as asthma. Understanding pollen morphology and its implications for air quality is significant for mitigating respiratory health risks. Conventionally, fluorescence microscopy (FM) is used for pollen imaging, but photobleaching, quenching, and phototoxicity affect the surface morphology and do not provide quantitative data on the pollen grains. For this study, we used bright field (BF) imaging and quantitative phase imaging (QPI), a label-free interferometric microscopy method, to look at differences in the shape of pollen. BF imaging provides information about the shape and size of the different pollens but has a limitation of low contrast. To obtain high-contrast images and quantitative data on the pollen grains without any exogenous agents, we employed QPI and BF imaging in the present study. QPI enables the extraction of detailed information regarding the cell wall, aperture, and thickness of pollen while also maintaining their natural state without the need for chemical treatments. In the present work, we sampled the ambient air from May 2023 to January 2024 on the IIT Delhi campus. Subsequently, QPI and BF imaging have been done for pollen identification and phase analysis of arboreal and non-arboreal pollen. Further, by utilizing the information obtained from BF microscopy and QPI, different species of pollen have been identified, and a pollen calendar has been prepared for exhibiting pollen season throughout the year. To the best of the authors' knowledge, they have conducted the QPI of airborne pollen grains for the first time; this technique holds great potential for characterizing airborne pollens without the need for staining or sample preparation.

MISSMARPLE: MIlan Small-SaMple Automated Radiocarbon Preparation LinE for atmospheric aerosol

Abstract Radiocarbon measurements on the carbonaceous aerosol fractions are an effective tool for aerosol source apportionment. For these measurements, a new sample preparation line (MISSMARPLE: MIlan Small-SaMple Automated Radiocarbon Preparation LinE for atmospheric aerosol) was built in Milan (Italy). MISSMARPLE can separate different carbon fractions (i.e. total carbon (TC), elemental carbon (EC)), automates the sample combustion processes and the CO2 isolation in the “combustion line”, and was designed to handle small samples, of about 50 μg carbon. The CO2 obtained in the combustion line is then reduced to graphite in the graphitization line for subsequent accelerator mass spectrometry (AMS) analysis at the INFN-LABEC in Sesto Fiorentino (Italy). MISSMARPLE was tested for reproducibility of 14C/12C ratio in primary standard samples, for background contamination by the analysis of blank samples (graphite with zero percent Modern Carbon (pMC)), and for accuracy by the analysis of IAEA-C7 for pMC(TC) and NIST RM8785 for pMC(EC) used as secondary standards. Measurements were carried out in different AMS runs. Reproducibility of 14C/12C was within 1.2%; blank values were down to 2.2 ± 0.2 pMC in the latest AMS run, and both IAEA-C7 and NIST RM8785 measurements were within 1σ with the reference value (but for one IAEA-C7 sample within 2.3σ). These results point to MISSMARPLE as a new, valuable tool for aerosol sample preparation for radiocarbon measurements to be exploited not only on traditional 24-h samples but also when small carbon quantities are available (e.g. collected at remote sites or with high temporal resolution).