Standard Sample Preparation Research Articles

Combined Analysis of Grade Differences in Lapsang Souchong Black Tea Using Sensory Evaluation, Electronic Nose, and HS-SPME-GC-MS, Based on Chinese National Standards.

Tea standard samples are the benchmark for tea product quality control. Understanding the inherent differences in Chinese national standards for Lapsang Souchong black tea of different grades is crucial for the scientific development of tea standardization work. In this study, Lapsang Souchong black tea of different grades that meet Chinese national standards was selected as the research object. The aroma characteristics were comprehensively analyzed through sensory evaluation, electronic nose, and HS-SPME-GC-MS (headspace solid-phase microextraction gas chromatography-mass spectrometry). The findings indicate that the higher-grade Lapsang Souchong has a higher evaluation score. The results of electronic nose analysis indicate that the volatiles with differences in tea of different grades were mainly terpenoids and nitrogen oxides. The results of HS-SPME-GC-MS analysis show that the odor characteristics of the super-grade samples are mainly floral and fruity, and these substances mainly include D-Limonene, 3,7-dimethyl-1,6-octadien-3-ol and 3-Hydroxymandelic acid, and ethyl ester. The primary aroma characteristics of the first-grade samples are floral, sweet, woody, and green, with key contributing compounds including 2-Furanmethanol, 1-Octen-3-ol, and 5-ethenyltetrahydro-α,α,5-trimethyl-cis-, 4,5-di-epi-aristolochene. The main aroma characteristics of the second-grade samples are green, herbal scent, and fruity, and the main substances include 3,7-dimethyl-1,6-octadien-3-ol, 2,3-dimethylthiophene, Dihydroactinidiolide, and Naphthalene-1-methyl-7-(1-methylethyl)-. It is worth noting that the second-grade samples contain a large amount of phenolic substances, which are related to the smoking process during processing. This study lays a solid foundation for the preparation of tea standard samples and the construction of the tea standard system.

Detection of Salmonella by competitive ELISA of lipopolysaccharide secreted into the culture medium

Detection of Salmonella in food is topical due to known cases of salmonellosis epidemics. Immunochemical methods including ELISA are widely used for Salmonella detection. Traditionally, commercial ELISA kits are based on sandwich technique and detect lipopolysaccharide (LPS), which is considered to be the component of the outer membrane of Gram-negative bacteria. Our aim was elaboration of competitive ELISA test for Salmonella detection in food with improved parameters. It was shown that in the Salmonella culture after the standard sample preparation procedure LPS is present mainly outside cells as a component of outer membrane vesicles. Improved sample preparation procedure includes separation of bacteria from the medium and analysis of the medium, which increases analytical sensitivity. Immobilization of the bovine serum albumin (BSA)-LPS conjugate in microplate wells allows to obtain a more homogeneous coating than immobilization of LPS itself. Thus, we have developed test system for Salmonella detection in food by competitive ELISA of LPS secreted into the culture medium with the immobilized BSA-LPS conjugate and monoclonal antibodies (mAb) to LPS core in the liquid phase. New competitive ELISA test is high sensitive, give reproducible results, allows the detection of any Salmonella serotype and is important for the protection of human health.

Biochemical Properties of CARM1: Impact on Western Blotting and Proteomic Studies.

CARM1 is an arginine methyltransferase that has crucial roles in a number of cellular pathways and is being explored as a therapeutic target in diseases such as cancer and neurodegenerative disorders. Its deregulation at the protein level was found to have potential prognostic value, and as such, its protein levels are regularly assessed through the common practice of western blotting (WB). Our group uncovered that CARM1 has biochemical properties that complicate its analysis by standard WB sample preparation techniques. Here, we show that CARM1 has the ability to form SDS-resistant aggregates that effectively hinder gel migration in SDS-PAGE. CARM1 levels and the temperature at the denaturation step can both influence CARM1 aggregation, which prompts the use of additional measures to ensure representative detection at the protein level. We have demonstrated the formation of CARM1 aggregates in both cell and tissue extracts, making these findings an important consideration for any CARM1-related study. We also show how aggregate formation in models of CARM1 overexpression can hinder proteomic studies. Having identified factors that can induce CARM1 aggregation, we suggest alternative sample preparation techniques that allow for clear resolution of the protein in stringent denaturing conditions while avoiding aggregation.

Optimizing Sample Preparation for Destructive Testing in Resistance Spot Welding

Abstract The integrity and reliability of resistance spot welding, a crucial process in various manufacturing industries, are significantly influenced by the quality of sample preparation. This study focuses on advancing the methodologies and practices in the preparation of samples for resistance spot welding destructive tests, aiming to improve the precision and repeatability of the results. We discuss comprehensive approaches encompassing material selection, surface treatment, dimensional accuracy, and alignment procedures, emphasizing the importance of meticulous handling and preparation techniques, A significant focus is placed on the utilization of sophisticated alignment and measurement tools to enhance the consistency of the welding area subjected to destructive testing. our study demonstrates a significant improvement in the reliability and accuracy of welding machine calibration, ultimately contributing to the overall quality of the welding process. By integrating these refined processes, the study highlights a significant improvement in the reliability of welding tests, contributing to enhanced welding quality and performance in practical applications. Our findings underscore the critical role of detailed and standardized sample preparation in achieving accurate and reliable resistance spot welding destructive test outcomes, providing valuable insights for both industrial applications and further research in welding technology.

Effects of Sample Handling on Visible and Near-Infrared Soil Reflectance

Soil reflectance is a cumulative attribute determined by interactions between light (photons) and the physical, chemical, and biological properties of soil. Soil properties such as organic matter, moisture, mineral oxide contents, soil texture, and surface roughness all influence soil reflectance at unique wavelengths. Many standard sample preparation techniques are designed to alter soil properties, so as to homogenize samples to improve the consistency of reflectance data collected. This study aims to quantify the effects of one standard sample preparation activity, drying, and repeated wetting–drying cycles on visible–near-infrared soil reflectance, by collecting reflectance data from nine samples which were dried then wetted for a total of three times. This study demonstrates the major, permanent effects of a drying and wetting cycle on soil reflectance, and then presents a model which can be used to correct for these effects. These results have direct implications for remote sensing activities, soil libraries, and soil spectral libraries. These results show that without correction, data from soil spectral libraries, spectral data collected from stored samples, and spectral data transferred between studies, have limited utility for characterizing soils as they exist in the field. Soil spectral data collected in the lab requires correction before it may be used to predict soil properties in the field, as standard sample handling procedures change intrinsic soil properties and introduce systematic error into these data.

Separation of microplastics from deep-sea sediment using an affordable, simple to use, and easily accessible density separation device

Microplastics accumulate in the environment but methods to extract particles from sediment for quantification and identification often lack accuracy and reproducibility. Existing methods vary greatly and many do not achieve adequate microplastic separation. During method development for extraction procedures, spike-recovery experiments (positive controls) are essential to ensure accurate and reproducible results from each sample matrix. Furthermore, the large variability in grain size and organic matter can affect the extraction of microplastics from the matrix. Scientists have used density separation to separate microplastics from matrices for decades, but apparatuses are often made of plastic, need to be custom made, and require multiple sample transfers from one apparatus to another. This study presents an affordable, easily accessible, and simple to use Density Separation Device (DSD) to remove plastics from deep-sea sediments. Eight polymers were spiked into replicates of environmental sediment, including six fragments: high density polyethylene (HDPE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), nylon (PA6), and crumb rubber (CR) and two fibers: cellulose acetate (CA) and polyester (PEST). Two size classes of polymers were used: 100 μm to 300 μm and > 300 μm. Using a sodium polytungstate solution at a density of 1.9 g/mL and reflectance FTIR microscopy for particle identification, mean recoveries of all fragments exceeded 78% (CR: 92.7% ± 30.8%, PP: 78.4% ± 34.0%, HDPE: 93.8% ± 13.5%, PS: 86.9% ± 25.7%, PA6: 98.4% ± 63.2%, PVC: 100.0% ± 12.4%). Fiber recovery was much lower (PEST: 28.1% ± 28.1% and CA: 25.9% ± 17.3%) because they aggregated, passed through sieves vertically, or were obscured under other particles. The fragment recovery success, accessibility (available online, all parts under $200) and ease of use of this DSD should facilitate widespread use, thus helping to standardize sample preparation methods for microplastic metrology.

Standardizing Protein Corona Characterization in Nanomedicine: A Multicenter Study to Enhance Reproducibility and Data Homogeneity.

We recently revealed significant variability in protein corona characterization across various proteomics facilities, indicating that data sets are not comparable between independent studies. This heterogeneity mainly arises from differences in sample preparation protocols, mass spectrometry workflows, and raw data processing. To address this issue, we developed standardized protocols and unified sample preparation workflows, distributing uniform protein corona digests to several top-performing proteomics centers from our previous study. We also examined the influence of using similar mass spectrometry instruments on data homogeneity and standardized database search parameters and data processing workflows. Our findings reveal a remarkable stepwise improvement in protein corona data uniformity, increasing overlaps in protein identification from 11% to 40% across facilities using similar instruments and through a uniform database search. We identify the key parameters behind data heterogeneity and provide recommendations for designing experiments. Our findings should significantly advance the robustness of protein corona analysis for diagnostic and therapeutics applications.

Versatile injector for inline renewable solid-phase extraction: Application to cyclodextrin-based bioaccessibility assessment in environmental solids

BackgroundSolid phase extraction (SPE) is a standard sample preparation technique in HPLC workflows. Inline cartridges are high-performance alternatives to manual or robotic systems but at long term, they suffer from irreversible sorption of matrix components and sorbent compaction. Bead injection (BI) is a niche fluidic technique that allows renewing a sorbent bed through the manipulation of its suspension. However, there is a need for a versatile and reliable tool in HPLC that can exchange the inline sorbent automatically, resorting to inexpensive and assorted bulk sorbents. ResultsWe present a new flow path for a liquid chromatographic injector to perform inline micro-solid phase extraction. The sample is processed at real time, trapping the analytes and discarding the matrix. Cleaning the matrix and injecting 10 μL of sample takes 70 s, comparable with the injection in commercial HPLC systems. If the aim is to preconcentrate the analytes, average enrichment factors of 250 have been obtained after processing sample volumes of 3200 μL in 16 min (interleavable with the chromatographic step), keeping the peak position and width independent of the injected volume (compared to large volume direct injection).The desired bed mass is automatically and pressure-driven manipulated in the valve, retained by an inline frit, and optionally, after the analysis, removed by forward flow.The chromatographic performance of the new design is compared to the standard 6-port, 2-position HPLC injector. As a case study, we have monitored the extraction kinetics of a cyclodextrin-based bioaccessibility extraction test of persistent organic contaminants in soil, by extracting several fractions in valve, process them with inline SPE with a balanced hydrophilic-hydrophobic reversed-phase sorbent, and inject the bioaccessible compounds into HPLC. Aiming at avoiding carryover, the sorbent bed (ca. 3 mg) is exchanged before every run. It should be noted that this contribution focuses on HPLC, but other non-separative techniques, such as Flow Injection Analysis, can equally benefit from this injection platform. SignificanceThis contribution reports the first use of inline BI-solid phase extraction in HPLC workflows, without heart-cut eluate injection, in which the sorbent can be exchanged automatically by forward flow. This performance is enabled by prototyping a valve that can autonomously swap sorbents in real-time for diverse samples, as a cartridge exchanger, but using cost-effective and environmentally friendlier bulk sorbents (bed masses from sub-mg to 5 mg) without requiring additional hardware.

Serial block-face scanning electron microscopy of adherent cells on thin plastic substrate

Abstract Serial block-face (SBF) scanning electron microscopy (SEM) is used for imaging the entire internal ultrastructure of cells, tissue samples or small organisms. Here, we present a workflow for SBF SEM of adherent cells, such as Giardia parasites and HeLa cells, attached to the surface of a plastic culture dish, which preserves the interface between cells and plastic substrate. Cells were embedded in situ on their substrate using silicone microwells and were mounted for cross-sectioning which allowed SBF imaging of large volumes and many cells. A standard sample preparation and embedding protocol for thin section electron microscopy provided already sufficient resolution and image quality to visualize larger structures. To improve resolution and image quality of SBF imaging, we stepwise tested modifications of the protocol, such as the moderate increase of the heavy metal content of the sample. Modifications of the embedding by either the reduction of the resin layer (minimal embedding) or the addition of silver colloid to the resin were evaluated at high and low vacuum imaging conditions. The optimized sample preparation protocol is very similar to the standard preparation protocol for thin section electron microscopy, so that the samples can also be used for this application. The protocol applies a higher concentration of osmium tetroxide, a higher temperature for heavy metal incubation and an additional lead en bloc staining. In summary, the presented workflow provides a generic and adaptable solution for studying adherent cells by SBF SEM.

Mass Spectrometry Imaging and Histology for the Analysis of Budding Intestinal Organoids.

Three-dimensional (3D) organoids have been at the forefront of regenerative medicine and cancer biology fields for the past decade. However, the fragile nature of organoids makes their spatial analysis challenging due to their budding structures and composition of single layer of cells. The standard sample preparation approaches can collapse the organoid morphology. Therefore, in this study, we evaluated several approaches to optimize a method compatible with both mass spectrometry imaging (MSI) and immunohistological techniques. Murine intestinal organoids were used to evaluate embedding in gelatin, carboxymethylcellulose (CMC)-gelatin-CMC-sucrose, or hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) solutions. Organoids were assessed with and without aldehyde fixation and analyzed for lipid distributions by MSI coupled with hematoxylin and eosin (H&E) staining and immunofluorescence (IF) in consecutive sections from the same sample. While chemical fixation preserves morphology for better histological outcomes, it can lead to suppression of the matrix-assisted laser desorption/ionization (MALDI) lipid signal. By contrast, leaving organoid samples unfixed enhanced MALDI lipid signal. The method that performed best for both MALDI and histological analysis was embedding unfixed samples in HPMC and PVP. This approach allowed assessment of cell proliferation by Ki67 while also identifying putative phosphatidylethanolamine (PE(18:0/18:1)), which was confirmed further by tandem MS approaches. Overall, these protocols will be amenable to multiplexing imaging mass spectrometry analysis with several histological assessments and help advance our understanding of the biological processes that take place in district subsets of cells in budding organoid structures.

Degradation processes in polyolefins with phenolic stabilizers subjected to ionizing or non-ionizing radiation

We investigated degradation processes in polymer plaques made of polyolefin (HDPE or UHMWPE or COC) prepared by melt-mixing with or without phenolic stabilizer (natural α-tocopherol or synthetic Irganox®1010) and spin trapping agent (TTBNB; 2,4,6-Tri‑tert-butylnitrosobenzene). The degradation was initiated either by low-energy, non-ionizing radiation (wavelengths corresponding to terrestrial range of solar UV radiation) or high-energy, ionizing radiation (electrons accelerated at 1.5 MeV). The motivation for the study was our recent finding that some phenolic stabilizers exhibited a surprising pro-oxidant activity in polyolefins subjected to non-ionizing radiation. Consequently, we asked if the phenolic stabilizers in polyolefins behave in the same way during long-term, low-energy, non-ionizing UV irradiation and after short-term, high-energy, ionizing e-beam irradiation. All samples were characterized thoroughly by means of electron spin resonance (ESR; the information about radiation-induced radicals), infrared microspectroscopy (IR; the detection of oxidation products and structural changes), instrumented microindentation (MHI; local mechanical properties) and light and electron microscopy (LM and SEM; surface morphology). The results proved that (i) the degradation processes in polyolefins subjected to non-ionizing or ionizing radiation were different, (ii) the phenolic stabilizers exhibited mostly their expected antioxidant activity, while pro-oxidant activity was detected only for specific conditions and/or systems subjected to non-ionizing radiation, and (iii) the selected spin trapping agent, TTBNB, was stable enough to survive standard sample preparation by melt-mixing and catch short-living unstable radicals.

Easy Access to Super-Resolution Applications with DeepSIM

Abstract Super-resolution microscopy is becoming more and more relevant and widespread for solving complex biological questions. However, many super-resolution techniques require high-level expertise and specific sample preparation protocols limiting the kind of fluorophore that can be used and consequently the applications. CrestOptics DeepSIM supports researchers deploying standard sample preparation protocols used for confocal microscopy while offering staggering resolution, the possibility of doing live cell imaging, and the ability to image deep into tissues. DeepSIM also allows staining with commonly used fluorescent markers, making it compatible with a large variety of samples and applications.

Optimization of therapeutic drug monitoring of vancomycin in newborns using “Dried Blood Spot” method

Therapeutic drug monitoring (TDM) is used to increase the individualization of pharmacotherapy, especially in patient groups with a high interindividual variability in pharmacokinetic (PK) parameters. One of these groups of patients is newborn children, for whom drug therapy, especially drugs with a narrow therapeutic range, causes a few difficulties or cannot be used in principle.The aim of the work was to develop and validate quantitative HPLC-MS/MS methods for the determination of vancomycin in “dried blood spot” samples using new protocols and comparison of the results obtained with the results in plasma samples using standard sample preparation methods.Materials and methods. To prepare stock and standard solutions of vancomycin and norvancomycin as an internal standard, dry portions of the corresponding certified standards of vancomycin (Servier, France) and norvancomycin (Augsburg, Germany, purity grade >95.0%) were used. A chromatographic separation of the components was carried out on a Poroshell 120 C18 column (4.6×50 mm, 2.7 µm). When developing conditions for a mass spectrometric detection of the desired substances using the multiple reaction monitoring (MRM) method, precursor ions and their corresponding product ions were determined.Results. A quantitative HPLC-MS/MS method for the determination of vancomycin in «dried blood spot» samples was developed and validated. A comparison was made between vancomycin concentrations in «dried blood spot» samples and plasma samples. Moreover, more than 95% of the calculated average concentrations are within the limits of d-2s and d+2s, which correspond to the values of –10.2 and 12.2. That confirms the suitability of the developed method for the analysis of patient samples.Conclusion. The results obtained make it possible for us to recommend the “dried blood spot” method for therapeutic monitoring of vancomycin, additional studies of PK in this group of patients with subsequent use of this drug in newborns and pediatric patients.

Catalytic and biocatalytic degradation of microplastics

AbstractIn recent years, there has been a surge in annual plastic production, which has contributed to growing environmental challenges, particularly in the form of microplastics. Effective management of plastic and microplastic waste has become a critical concern, necessitating innovative strategies to address its impact on ecosystems and human health. In this context, catalytic degradation of microplastics emerges as a pivotal approach that holds significant promise for mitigating the persistent effects of plastic pollution. In this article, we critically explored the current state of catalytic degradation of microplastics and discussed the definition of degradation, characterization methods for degradation products, and the criteria for standard sample preparation. Moreover, the significance and effectiveness of various catalytic entities, including enzymes, transition metal ions (for the Fenton reaction), nanozymes, and microorganisms are summarized. Finally, a few key issues and future perspectives regarding the catalytic degradation of microplastics are proposed.

Ultrasensitive determination of selenium in food samples and its speciation in water and beverages using thiosemicarbazide-incorporated graphene and total-reflection X-ray fluorescence spectrometry

The paper presents a new analytical procedure for the determination and speciation of trace and ultratrace selenium in water, beverages, seafood, milk, and vegetables. The developed method is based on the dispersive micro-solid phase extraction with the use of new thiosemicarbazide-incorporated graphene as a solid sorbent, in combination of the total-reflection X-ray fluorescence spectrometry (TXRF). As a result, we have created an auspicious analytical tool for fast and sensitive analysis of samples with a complex matrix. Regardless of the specimen type, the method is characterized by a very low detection limit of 1.7 pg mL−1 and high precision. The developed strategy allowed us to solve common problems associated with selenium loss during the sample preparation for the TXRF measurement and also improve its performance toward the analysis of beverages and high saline/solid samples, which may even be impossible to perform using standard sample preparation procedures for a TXRF measurement.

Morphological peculiarities of regeneration phases and dynamics of growth factor levels at using 2-ethyl-6-methyl-3-hydroxypyridinium n-acetyl-6-aminohexanoate for rat skin burns repairation

BACKGROUND: It was found that new derivatives of acexamic acid — N-acetyl-6-aminohexanoate of silver and 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate stimulate regeneration and mineralization of bone tissues in osteoporosis. However, to date, the effect of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate on the features of reparative histogenesis and the relationship of its stages with the levels of growth factors in regenerating tissues during skin restoration after thermal burn has not been studied. AIM: To estimate the morphology dynamics of skin burn healing and its relationship with the levels of vascular endothelial growth factor and basic fibroblast growth factor growth factors in rat regenerating tissues to characterize the reparative properties of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate. MATERIALS AND METHODS: 63 nonlinear rats were used to establish thermal burn wound model according to standard methodics. Burn wounds of the experimental group animals were treated with 2% ointment of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate daily, control group 1 — the burns were not treated, control group 2 — ointment base was applied daily. Morphological examinations of regenerating tissues from the burn area were carried out on days 7, 14 and 21 of the experiment after standard sample preparation. The concentrations of growth factors in homogenates of regenerating tissues were determined by Enzyme Immunoassay. RESULTS: The inflammation phase is characterized by vascular endothelial growth factor active production and release from cells and morphologically accompanied by early neovasculogenesis in granulation tissue and significant differences in its thickness in rats of the experimental group. During the proliferation phase, activation of fibroblast reproduction and differentiation was associated with an increase in basic fibroblast growth factor levels and the maintenance of elevated vascular endothelial growth factor levels in all groups, especially high in experimental group rats. The epithelialization phase was accompanied by a statistically significant decrease in the content of basic fibroblast growth factor and vascular endothelial growth factor in regenerating skin tissues by 1.9 and 1.8 times, respectively, in comparison with the control groups. CONCLUSIONS: The obtained experimental data indicate 2% ointment with 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate has a reparative potential, as evidenced by the accelerated reduction both the area of burn defects and the average time for their healing in the experimental group compared to the control groups.

MassSpecPreppy-An end-to-end solution for automated protein concentration determination and flexible sample digestion for proteomics applications.

In proteomics, fast, efficient, and highly reproducible sample preparation is of utmost importance, particularly in view of fast scanning mass spectrometers enabling analyses of large sample series. To address this need, we have developed the web application MassSpecPreppy that operates on the open science OT-2 liquid handling robot from Opentrons. This platform can prepare up to 96 samples at once, performing tasks like BCA protein concentration determination, sample digestion with normalization, reduction/alkylation and peptide elution into vials or loading specified peptide amounts onto Evotips in an automated and flexible manner. The performance of the developed workflows using MassSpecPreppy was compared with standard manual sample preparation workflows. The BCA assay experiments revealed an average recovery of 101.3% (SD:±7.82%) for the MassSpecPreppy workflow, while the manual workflow had a recovery of 96.3% (SD:±9.73%). The species mix used in the evaluation experiments showed that 94.5% of protein groups for OT-2 digestion and 95% for manual digestion passed the significance thresholds with comparable peptide level coefficient of variations. These results demonstrate that MassSpecPreppy is a versatile and scalable platform for automated sample preparation, producing injection-ready samples for proteomics research.

Development of a sample preparation method for micro-proteomics analysis of the formaldehyde-fixed paraffin-embedded liver tissue samples

Liver micro-proteomics based on the routinely used formaldehyde-fixed paraffin-embedded (FFPE) samples is valuable for innovative research, but the technical approach for sample preparation is often challenging. In this study, we aimed to develop a method for sample preparation for micro-proteomics on using the FFPE liver samples. We collected 2000 individual cells per batch from FFPE liver slices with laser capture microdissection and used them as test samples. We used the microscale fresh-frozen liver samples or HepG2 cells as control samples. For the FFPE samples, we first established a procedure for protein extraction. 2 h incubation at 95 °C in alkaline amine buffer supplemented with 4% sodium dodecyl sulfate allows improved production, efficiency, and quality of protein extraction. Then, we developed a dedicated protocol HDMSP for the micro-concentrated (<0.05 μg/μL) protein preparation for mass spectrometry (MS) based analysis, in which 2 μg/μL carboxyl magnetic beads and 70% acetonitrile are used to induce protein precipitation. For the 0.01 μg/μL protein control samples, protein recovery rate (PRR) by HDMSP is 72.1%, while the PRR is 5.9% if using a standard method solid phase-enhanced sample preparation. For the FFPE samples, the HDMSP PRR is 88.8%, and the subsequent MS analysis demonstrates increased depth, robustness, and quantitation accuracy for HDMSP relative to the control of in-gel digestion. Moreover, the physicochemical properties and subcellular location of the FFPE liver micro-proteome are comparable to those of the fresh-frozen control samples processed with filter-aided sample preparation (FASP). HDMSP is also comparable to FASP in terms of reproducibility and physicochemical properties in liver subcellular proteomes, and meanwhile reduces the sample preparation time by 15.9% and the experimental cost by 30.8%. Overall, the new method is simple and highly effective for preparing the microscale FFPE liver protein samples for MS analysis. This study provides a useful solution for FFPE liver micro-proteomics.

Pharmacokinetic evaluation of 24 representative components of Ling-Gui-Zhu-Gan decoction in acute myocardial infarction model rats via a validated ultrahigh-performance liquid chromatography-tandem mass spectrometry method.

Ling-Gui-Zhu-Gan decoction (LGZGD), one of the 100 herbal classic formulas, is clinically used to treat chronic heart failure with remarkable curative effect. However, LGZGD pharmacokinetic parameters in pathological model rats are poorly understood, in particular for special components. As physicochemical properties are specific to each representative component, no standard sample preparation is available for absolute quantification of representative components of LGZGD in rat plasma. A specific, sensitive and high-throughput ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method capturing 24 representative components was developed and applied to evaluate the pharmacokinetic parameters of LGZGD in acute myocardial infarction (AMI) rat plasma after intragastric administration (2.4, 4.8 and 9.6g/kg). Precipitation and extraction were selected and optimized for plasma preparation, and isopropanol precipitation could offer higher recovery and broader coverage. It was expected that AMI could cause less absorption and slower elimination of most of active components of LGZGD. Most of newly reported special components absorbed quickly and eliminated slowly. The average elimination half-life of the 24 representative components was 10.09 h, which is consistent with the dosage of LGZGD (twice daily). The specificity, linearity, precision and accuracy, recovery, matrix effect and stability were validated according to Food and Drug Administration guidance. The validation results demonstrated that the method could be applied to evaluate the pharmacokinetic parameters of LGZGD in AMI rats. The pharmacokinetic parameters showed substantial improvement in quality research of LGZGD, thereby laying the groundwork for preclinical and clinical trials in chronic heart failure clinical efficacy.

Biological function of Extracellular Vesicles (EVs): a review of the field.

Extracellular vesicles (EVs) theranostic potential is under intense investigation. There is a wealth of information highlighting the role that EVs and the secretome play in disease and how these are being utilized for clinical trials and novel therapeutic possibilities. However, understanding of the physiological and pathological roles of EVs remain incomplete. The challenge lies in reaching a consensus concerning standardized quality-controlled isolation, storage, and sample preparation parameters. Interest in circulating EV cargo as diagnostic and prognostic biomarkers is steadily growing. Though promising, various limitations need to be addressed before there can be successful, full-scale therapeutic use of approved EVs. These limitations include obtaining or manufacturing from the appropriate medium (e.g., from bodily fluid or cell culture), loading and isolating EVs, stability, and storage, standardization of processing, and determining potency. This review highlights specific topics, including circulation of abnormal EVs contribute to human disease and the theranostic potential of EVs. Theranostics is defined as a combination of the word's therapeutics and diagnostics and describes how a specific medicine or technique can function as both. Key findings include, (1) EVs and the secretome are future theranostics which will be utilized as both biomarkers for diagnosis and as therapeutics, (2) basic and translational research supports clinical trials utilizing EVs/secretome, and (3) additional investigation is required to fully unmask the theranostic potential of EVs/secretome in specific diseases and injuries.