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Background: Extracellular vesicles (EV) are enriched with proteins and RNA cargo, promoting cell-to-cell communication. Biofluid derived EV cargo is used for discovering disease specific markers for diagnosis and disease monitoring., Rational: Blood is a complex fluid with an abundance of protiens and thus isolation of EVs is challenging. Therefore, methods for EV isolation, including commercial kits use thromboplastin D (TP-D) for pretreatment of plasma to increase EV purity and yield. This pretreatment can introduce contaminants., Method and Results: We performed a comparative study to evaluate the effect of EV isolation methods focusing on (a) pretreatment of plasma with additives, which include: rabbit TP (rTP) versus human recombinant thromboplastin (huTP), to increase purity and yield (b) an additional centrifugation step prior to freezing plasma and (c) comparison of frozen versus fresh plasma EV isolations. Pretreatment with rTP generated a dynamic range of proteins, however, most of these proteins were contaminants, introduced from the rTP (99.1% purity). As an alternative, huTP was used, which did not introduce any significant contaminants, however, this did not increase yield or purity. Additionally, an extra 10,000 g centrifugation did not improve either EV yield or purity. Finally, comparison of fresh or frozen plasma showed no significant difference, an important factor when sourcing plasma from biobanks., Conclusion: Appropriate controlsare required when adding any additives during EV isolation as even a small percentage of contaminants can have a major effect on results. Furthermore, biobanked plasma can be used with no major changes to processing., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Solid-phase microextraction coupled to gas chromatography-mass spectrometry is a common approach to analyze the volatile profile of cooked meat. The present study aims to investigate the combined effect of sample preparation, including meat presentation (minced and steak) and cooking method (stewed and grilled), and extraction temperature (30, 60 and 80 °C) and time (30 and 50 min) on the volatile composition of cooked deer meat. The statistical results indicated that extraction temperature was the most relevant factor affecting the meat volatile profile of cooked meat followed by the extraction time. Higher extraction temperatures improved the detection of heavy volatile compounds, while sample preparation had little influence on the meat volatile profile, probably due to the accurate control of the parameters used for meat presentation and cooking methods. The results of this work can assist in the standardization of analytical procedures for the characterization of volatile compounds in cooked meat., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

During winemaking a wide variety of processing aids such as albumin, caseinates and lysozyme are often used. These proteins are considered allergenic and could become a human health risk in susceptible individuals. In our knowledge, there are no methods published for the analysis of these three proteins simultaneously by liquid chromatography with tandem mass spectrometry, with electrospray ionization. Therefore, in this work, a sample preparation approach for the analysis of α-casein, β-casein, albumin and lysozyme, in a single run, was performed and compared with published data. Through a pH adjustment, combining the use of cellulose ester membranes, a precipitation with organic solvents and a final concentration/clean-up, we achieved recovery values from 90.7 to 108.6%. The method was validated, showing determination coefficients R 2  ≥ 0.99. This method was able to quantify proteins even at lower levels (limits of quantification from 0.01 to 0.25 mg/L) than the current legal limits., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Tea is a popular drink around the world and contains essential minerals and trace elements for human health. In this study, the analytical capabilities of the total-reflection X-ray fluorescence method (TXRF) were considered for the analysis of tea. Different sample preparation techniques, e.g. suspension, open vessel acid digestion, and microwave acid digestion were examined. The influence of particle size was investigated in the analysis of the suspension of tea samples. Mass-absorption coefficients for the tea matrix and the critical surface density of the specimen were calculated. The data obtained explain the presence of absorption effects in the determination of P, S, Cl, and K in suspensions. The digestion procedure is chosen as an optimal sample preparation technique for the TXRF analysis of tea. Nineteen real tea samples were analyzed using TXRF. The advantages of TXRF have been presented through the comparison of results with the wavelength-dispersive X-ray fluorescence spectrometry method., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Impact of the "Intensification of Vaporization by Decompression to the Vacuum" (IVDV) on extraction of polyphenols from olive leaves was investigated. Using Response Surface Methodology, the effect of three variables were studied: initial water content of leaves, processing time and steam pressure on total phenolic content (TPC). Extractions of TPC from leaves were achieved either using 100% water as a solvent (w 100 ), or 50% (v/v) aqueous ethanol (w 50 ). Following IVDV pretreatment, TPC yields were enhanced with both solvents by approximately 3 times compared to the negative controls. Furthermore, oleuropein and hydroxytyrosol were intensified by up to 600% and 238% respectively. Antioxidant-antiradical assays revealed higher activities, up to 3.5 times, in extracts from IVDV-treated leaves. Calculation of the extraction indices Zp, reflecting cellular damage, confirmed the beneficial effect of IVDV on the extraction yield. Finally, Scanning Electron Microscopy (SEM) permitted the morphological observation of IVDV-treated as compared to untreated leaves., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mass spectrometry imaging (MSI) enables the visualization of molecular distributions on complex surfaces. It has been extensively used in the field of biomedical research to investigate healthy and diseased tissues. Most of the MSI studies are conducted in a 2D fashion where only a single slice of the full sample volume is investigated. However, biological processes occur within a tissue volume and would ideally be investigated as a whole to gain a more comprehensive understanding of the spatial and molecular complexity of biological samples such as tissues and cells. Mass spectrometry imaging has therefore been expanded to the 3D realm whereby molecular distributions within a 3D sample can be visualized. The benefit of investigating volumetric data has led to a quick rise in the application of single-sample 3D-MSI investigations. Several experimental and data analysis aspects need to be considered to perform successful 3D-MSI studies. In this review, we discuss these aspects as well as ongoing developments that enable 3D-MSI to be routinely applied to multi-sample studies.

Fabric phase sorptive extraction (FPSE) is an evolutionary sample preparation approach which was introduced in 2014, meeting all green analytical chemistry (GAC) requirements by implementing a natural or synthetic permeable and flexible fabric substrate to host a chemically coated sol-gel organic-inorganic hybrid sorbent in the form of an ultra-thin coating. This construction results in a versatile, fast, and sensitive micro-extraction device. The user-friendly FPSE membrane allows direct extraction of analytes with no sample modification, thus eliminating/minimizing the sample pre-treatment steps, which are not only time consuming, but are also considered the primary source of major analyte loss. Sol-gel sorbent-coated FPSE membranes possess high chemical, solvent, and thermal stability due to the strong covalent bonding between the fabric substrate and the sol-gel sorbent coating. Subsequent to the extraction on FPSE membrane, a wide range of organic solvents can be used in a small volume to exhaustively back-extract the analytes after FPSE process, leading to a high preconcentration factor. In most cases, no solvent evaporation and sample reconstitution are necessary. In addition to the extensive simplification of the sample preparation workflow, FPSE has also innovatively combined the extraction principle of two major, yet competing sample preparation techniques: solid phase extraction (SPE) with its characteristic exhaustive extraction, and solid phase microextraction (SPME) with its characteristic equilibrium driven extraction mechanism. Furthermore, FPSE has offered the most comprehensive cache of sorbent chemistry by successfully combining almost all of the sorbents traditionally used exclusively in either SPE or in SPME. FPSE is the first sample preparation technique to exploit the substrate surface chemistry that complements the overall selectivity and the extraction efficiency of the device. As such, FPSE indeed represents a paradigm shift approach in analytical/bioanalytical sample preparation. Furthermore, an FPSE membrane can be used as an SPME fiber or as an SPE disk for sample preparation, owing to its special geometric advantage. So far, FPSE has overwhelmingly attracted the interest of the separation scientist community, and many analytical scientists have been developing new methodologies by implementing this cutting-edge technique for the extraction and determination of many analytes at their trace and ultra-trace level concentrations in environmental samples as well as in food, pharmaceutical, and biological samples. FPSE offers a total sample preparation solution by providing neutral, cation exchanger, anion exchanger, mixed mode cation exchanger, mixed mode anion exchanger, zwitterionic, and mixed mode zwitterionic sorbents to deal with any analyte regardless of its polarity, ionic state, or the sample matrix where it resides. Herein we present the theoretical background, synthesis, mechanisms of extraction and desorption, the types of sorbents, and the main applications of FPSE so far according to different sample categories, and to briefly show the progress, advantages, and the main principles of the proposed technique.

The abnormal functionality of mitochondria has been linked to many life-threatening diseases such as cancers, failure of cardiovascular functions, and neurodegenerative disorders. Therefore, in vitro analysis of mitochondria has garnered great interest for understanding the mechanism of mitochondrial dysfunction-related disease development and therapeutics. However, due to the intrinsic heterogeneity of cell membrane stiffness, it remains challenging to standardize the protocols for the extraction of mitochondria and adequate disruption of the cellular membrane while retaining the functionality of mitochondria. We have previously developed a microfluidics-based cell shredder capable of serving the purpose. In this protocol, we describe the step-by-step procedures to empirically identify the threshold shear stress using this microfluidics-based cell shredder for mitochondrial extraction. The optimal shear stress to disrupt human embryonic kidney cell (HEK 293) and mice muscle cell (C2C12) has been characterized at around 16.4 Pa, whereas cell lines with stiffer membrane stiffness, for example, neuroblastoma cells (SH-SY5Y), require 27.4 Pa to effectively lyse the cells. This protocol also provides detailed procedures to determine the quality of extracted mitochondria based on the membrane potential and the integrity of extracted mitochondria. A comparison with the widely employed Dounce homogenizer has shown that the proposed microscale cell shredder can yield at least 40% more functional mitochondria and retain higher integrity regarding extracted mitochondria than the counterparts extracted from Dounce homogenizer, especially for low cell concentrations (5-20 × 10 4  cells/mL) and small sample volume (<200 μL).

Gold nanoparticles (AuNPs) are increasingly being used as diagnostic and therapeutic agents owing to their excellent properties; however, there is not much data available on their dynamics in vivo on a single particle basis in a single mouse. Here, we developed a method for the direct analysis of nanoparticles in trace blood samples based on single particle inductively coupled plasma-mass spectrometry (spICP-MS). A flexible, highly configurable, and precisely controlled sample introduction system was designed by assembling an ultralow-volume autosampler (flow rate in the range of 5-5000 μL/min) and a customized cyclonic spray chamber (transfer efficiency up to 99%). Upon systematic optimization, the detection limit of the nanoparticle size (LOD size ) of AuNPs in ultrapure water was 19 nm, and the detection limit of the nanoparticle number concentration (LOD NP ) was 8 × 10 4 particle/L. Using a retro-orbital blood sampling method and subsequent dilution, the system was successfully applied to track the dynamic changes in size and concentration for AuNPs in the blood of a single mouse, and the recovery for the blood sample was 111.74%. Furthermore, the concentration of AuNPs in mouse blood reached a peak in a short period of time and, then, gradually decreased. This study provides a promising technique for analyzing and monitoring the size and concentration of nanoparticles in ultralow-volume blood samples with low concentrations, making it a powerful tool for analyzing and understanding the fate of nanoparticles in vivo.

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, chemistry, and their potential to enable advanced materials. To effectively understand and regulate the physico-chemical properties and potential adverse effects of nanoparticles, validated measurement procedures for the various properties of nanoparticles need to be developed. While procedures for measuring nanoparticle size and size distribution are already established, standardized methods for analysis of their surface chemistry are not yet in place, although the influence of the surface chemistry on nanoparticle properties is undisputed. In particular, storage and preparation of nanoparticles for surface analysis strongly influences the analytical results from various methods, and in order to obtain consistent results, sample preparation must be both optimized and standardized. In this contribution, we present, in detail, some standard procedures for preparing nanoparticles for surface analytics. In principle, nanoparticles can be deposited on a suitable substrate from suspension or as a powder. Silicon (Si) wafers are commonly used as substrate, however, their cleaning is critical to the process. For sample preparation from suspension, we will discuss drop-casting and spin-coating, where not only the cleanliness of the substrate and purity of the suspension but also its concentration play important roles for the success of the preparation methodology. For nanoparticles with sensitive ligand shells or coatings, deposition as powders is more suitable, although this method requires particular care in fixing the sample.

N-linked glycosylation is a post-translational modification that occurs on many proteins during biosynthesis. The profile of different glycans on the protein is a critical quality attribute of some recombinant biopharmaceutical proteins including monoclonal antibodies (mAbs). Methods for profiling glycan should be robust, fast, and sensitive. Isolating glycans from proteins and tagging a label on glycans is the most commonly used technique for glycan profiling. Currently, existing protocols for sample preparation can be complicated, time-consuming, and expensive, which can limit the wide adaptation of glycan profiling methods. As a further barrier to use, an expensive ultra-high-pressure liquid chromatography (UHPLC) system is frequently required for the profile. In this article, a low cost and easily-used workflow of sample preparation is coupled with a standard high-performance liquid chromatography (HPLC) system to achieve comparable results to UHPLC. The number of steps required in the protocol and the time, as well as the cost associated with the sample preparation, is significantly reduced, while maintaining robust analytical performance. We describe the creation and validation of a human serum IgG glycan library to be used as the calibration standard, and successful profiling of glycoforms from a variety of mAbs., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Single-cell proteomics can provide critical biological insight into the cellular heterogeneity that is masked by bulk-scale analysis. We have developed a nanoPOTS (nanodroplet processing in one pot for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample volumes, the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challenge, we have developed a nanoPOTS autosampler allowing fully automated sample injection from nanowells to LC-MS systems. We also developed a sample drying, extraction, and loading workflow to enable reproducible and reliable sample injection. The sequential analysis of 20 samples containing 10 ng tryptic peptides demonstrated high reproducibility with correlation coefficients of >0.995 between any two samples. The nanoPOTS autosampler can provide analysis throughput of 9.6, 16, and 24 single cells per day using 120, 60, and 30 min LC gradients, respectively. As a demonstration for single-cell proteomics, the autosampler was first applied to profiling protein expression in single MCF10A cells using a label-free approach. At a throughput of 24 single cells per day, an average of 256 proteins was identified from each cell and the number was increased to 731 when the Match Between Runs algorithm of MaxQuant was used. Using a multiplexed isobaric labeling approach (TMT-11plex), ∼77 single cells could be analyzed per day. We analyzed 152 cells from three acute myeloid leukemia cell lines, resulting in a total of 2558 identified proteins with 1465 proteins quantifiable (70% valid values) across the 152 cells. These data showed quantitative single-cell proteomics can cluster cells to distinct groups and reveal functionally distinct differences.

Discovery of new protein biomarker candidates has become a major research goal in the areas of clinical chemistry, analytical chemistry, and biomedicine. These important species constitute the molecular target when it comes to diagnosis, prognosis, and further monitoring of disease. However, their analysis requires powerful, selective and high-throughput sample preparation and product (analyte) characterisation approaches. In general, manual sample processing is tedious, complex and time-consuming, especially when large numbers of samples have to be processed (e.g., in clinical studies). Automation via microtiter-plate platforms involving robotics has brought improvements in high-throughput performance while comparable or even better precisions and repeatability (intra-day, inter-day) were achieved. At the same time, waste production and exposure of laboratory personnel to hazards were reduced. In comprehensive protein analysis workflows (e.g., liquid chromatography-tandem mass spectrometry analysis), sample preparation is an unavoidable step. This review surveys the recent achievements in automation of bottom-up and top-down protein and/or proteomics approaches. Emphasis is put on high-end multi-well plate robotic platforms developed for clinical analysis and other biomedical applications. The literature from 2013 to date has been covered., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow 1 . Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as β-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

We report a miniaturized filter aided sample preparation method (micro-FASP) for low-loss preparation of submicrogram proteomic samples. The method employs a filter with ∼0.1 mm 2 surface area, reduces the total volume of reagents to <10 μL, and requires only two sample transfer steps. The method was used to generate 25 883 unique peptides and 3069 protein groups from 1000 MCF-7 cells (∼100 ng protein content), and 13 367 peptides and 1895 protein groups were identified from 100 MCF-7 cells (∼10 ng protein content). Single blastomeres from Xenopus laevis embryos at the 50-cell stage (∼200 ng yolk free protein/blastomere) generated 20 943 unique peptides and 2597 protein groups; the proteomic profile clearly differentiated left and right blastomeres and provides strong support for models in which this asymmetry is established early in the embryo. The parallel processing of 12 samples demonstrates reproducible label free quantitation of 1 μg protein homogenates.

Background: The growth of halal food consumption worldwide has resulted in an increase in the request for halal authentication. DNA-based detection using powerful real-time polymerase chain reaction (PCR) technique has been shown to be highly specific and sensitive authentication tool. The efficient DNA extraction method in terms of quality and quantity is a backbone step to obtain successful real-time PCR assays. In this study, different DNA extraction methods using three lysis buffers were evaluated and developed to recommend a much more efficient method as well as achieve a successful detection using real-time PCR., Results: The lysis buffer 2 (LB2) has been shown to be the best lysis buffer for DNA extraction from both raw and processed meat samples comparing to other lysis buffers tested. Hence, the LB2 has been found to be ideal to detect meat and porcine DNAs by real-time PCR using pairs of porcine specific primers and universal primers which amplified at 119 bp fragment and 93 bp fragment, respectively. This assay allows detection as low as 0.0001 ng of DNA. Higher efficiency and sensitivity of real-time PCR via a simplified DNA extraction method using LB2 have been observed, as well as a reproducible and high correlation coefficient (R 2  = 0.9979) based on the regression analysis of the standard curve have been obtained., Conclusion: This study has established a fast, simple, inexpensive and efficient DNA extraction method that is feasible for raw and processed meat products. This extraction technique allows an accurate DNA detection by real-time PCR and can also be implemented to assist the halal authentication of various meat-based products available in the market. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

The decision on the fitness of a measurement for its intended use and the interpretation of an analytical result requires the assessment of the measurement uncertainty. Frequently, the determination of analytes in complex matrices involves demanding sample preparations in which analyte losses are observed. These losses should be considered when reporting the results, which can be corrected for low recovery by taking the mean recovery observed in the analysis of reference items (e.g. spiked samples) or, alternatively, by subjecting calibrators to the same pre-treatment performed on the samples. In these cases, neat (NC) or adjusted (AC) calibrators are used, respectively. The way analyte losses are handled impacts on the measurement uncertainty. The top-down evaluation of the measurement uncertainty involves combining precision, trueness and additional uncertainty components. The trueness component is quantified by pooling various analyte recovery determinations. This work assesses and compares the uncertainty of polycyclic aromatic hydrocarbons (PAHs) measurements in water based on HPLC-FD calibrations with NC or AC. The trueness component is estimated by pooling mean recoveries observed from the analysis of different spiked samples to which mean recovery uncertainty and degrees of freedom are used to estimate a weighted mean recovery and respective uncertainty. The performance of measurements based on NC and AC are associated with equivalent uncertainty except when large analyte losses are observed, namely in the determination of Naphtalene. In this case, the processing of AC reduces the expanded relative uncertainty from 9.9% to 3.5%. The evaluated expanded uncertainty ranged from 3.5% to 12% of the measured value., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Metabolomics tactics have been applied in the research associated with embryonic zebrafish. However, the report regarding the evaluation of impacts of sample pretreatment on metabolomics results from zebrafish embryos is limited. In the present study, different data normalization approaches, extraction solvents, and extraction strategies for off-line derivatization gas chromatography coupled with mass spectrometry-based metabolomics analysis of zebrafish eleutheroembryos were evaluated and optimized. The results showed that, when 4-chlorophenylalanine normalization, sample homogenization and pure methanol combined with ultrasonic extraction were conducted, better repeatabilities, higher signals and broader coverages of detected metabolites can be achieved. The recovery and standard deviation of most standards were in the range of 82%-121% and 6.6%-12%, respectively, while the relative standard deviation of major detected metabolites ranged from 5.4% to 19%, indicating good extraction efficiencies and method precision. Under the developed method, 87 important endogenous metabolites such as citric acid and hypoxanthine were identified by universal databases or standards among 270 extracted metabolites, which consisted of sugars, amines, amino acids, nucleotides, fatty acids, and sterols. Therefore, the results could provide a proper pretreatment protocol for the analysis of wide-coverage metabolome in embryonic zebrafish. In addition, this study highlights the impact of normalization and extraction methods on the data quality of metabolomics analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The effect of high-temperature and mild-pressure (HTMP) pretreatment on the enzymatic hydrolysis of phosvitin and the structural characteristics of the phosphopeptides produced were analyzed using tandem mass spectrometry. The HTMP pretreatment hydrolyzed phosvitin at random sites and helped the subsequent enzyme hydrolysis of the peptides produced. With the HTMP pretreatment alone, 154 peptides were produced, while the use of trypsin, Protex 6L, and Multifect 14L in combination with the pretreatment produced 252, 280, and 164 peptides, respectively. The use of two enzyme combinations (trypsin + Protex 6L and trypsin + Multifect 14L) helped the hydrolysis further. The number of phosphopeptides produced increased when the modifications within the same amino acid sequences were considered. This study indicated that HTMP pretreatment was a breakthrough method to improve the enzymatic hydrolysis of phosvitin that enabled an easy production of phosvitin phosphopeptides for their subsequent functional characterizations.

Rapid sample preparation is one of the leading bottlenecks to low-cost and efficient sample component detection. To overcome this setback, a technology known as Lyse-It has been developed to rapidly (less than 60 seconds) lyse Gram-positive and-negative bacteria alike, while simultaneously fragmenting DNA/RNA and proteins into tunable sizes. This technology has been used with a variety of organisms, but the underlying mechanism behind how the technology actually works to fragment DNA/RNA and proteins has hitherto been studied. It is generally understood how temperature affects cellular lysing, but for DNA/RNA and protein degradation, the temperature and amount of energy introduced by microwave irradiation of the sample, cannot explain the degradation of the biomolecules to the extent that was being observed. Thus, an investigation into the microwave generation of reactive oxygen species, in particular singlet oxygen, hydroxyl radicals, and superoxide anion radicals, was undertaken. Herein, we probe one aspect, the generation of reactive oxygen species (ROS), which is thought to contribute to a non-thermal mechanism behind biomolecule fragmentation with the Lyse-It technology. By utilizing off/on (Photoinduced electron transfer) PET fluorescent-based probes highly specific for reactive oxygen species, it was found that as oxygen concentration in the sample and/or microwave irradiation power increases, more reactive oxygen species are generated and ultimately, more oxidation and biomolecule fragmentation occurs within the microwave cavity., Competing Interests: The University of Maryland, Baltimore County (UMBC) has filed and has had issued several patents related to microwave-based lysing and DNA fragmentation. Those patents are licensed to Lyse-It LLC, a Maryland-based biotechnology company, which Professor Geddes currently holds stock. Patent #: US US9500590B2, “Assays for pathogen detection using microwaves for lysing and accelerating metal-enhanced fluorescence.” Patent #: US10294451B2, “Flow and static lysing systems and methods for ultra-rapid isolation and fragmentation of biological materials by microwave irradiation”, Chris D. Geddes. Lyse-It LLC was not involved in the study design of the material within this paper, nor do any of the authors receive any salaries or consultancies from Lyse-It LLC. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The effects of four different extraction methods (Folch, Soxhlet, two-step, and enzyme-assisted aqueous extraction) on the yields, lipid class, fatty acids (FAs) composition, minor components (including carotenoid, cholesterol), and thiobarbituric acid reactive substances values of lipids in the hepatopancreas of Chinese mitten crab (Eriocheir sinensis) were investigated. The C16:0, C18:1, and C18:2 were identified to be the dominant FAs in crab lipids, and the FAs were present in the form of triglycerides. The Soxhlet and enzyme-assisted extraction were more suitable for crab lipids extraction, showing higher extraction rates and oxidative stability. Especially, the lipid extracted by enzyme-assisted extraction has high carotenoids content. The components of crab lipids extracted by enzyme-assisted aqueous extraction were further identified using untargeted metabolomics methods. The polyunsaturated fatty acid, sterols, amino acids, products of lipid β-oxidation and ATP degradation, phosphatidyl ethanolamine, and astaxanthin were founded in crab oil. PRACTICAL APPLICATION: The Chinese mitten crab (Eriocheir sinensis) is a popular aquatic food in China. The hepatopancreas is the major lipid storage organ of crab, and the distinctive flavor of crab is mainly from it. To compare the different extraction methods on yield, composition and properties of crab lipids can be helpful for lipids production from crab hepatopancreas. Meanwhile, the crab hepatopancreas lipids are rich in polyunsaturated fatty acids and astaxanthin, and have potential to be as a functional component and a crab flavor additive in food industry., (© 2019 Institute of Food Technologists®.)

In HPLC analyses of soluble dietary fiber, desalting processes using open, mixed-bed ion-exchange columns are time-consuming and labor-intensive. We developed and validated a simple desalting method using tandem cation/anion exchange SPE cartridges. We found that combining Bond Elut Jr SCX (upstream) and Bond Elut PSA (downstream) cartridges provided adequate desalting of test solutions. The developed method was then validated in an inter-laboratory study. Five test samples were prepared by mixing food matrixes with purified soluble dietary fiber and treated to generate solutions to test the desalting process. These solutions were then analyzed by eight different laboratories. The results demonstrated that the developed method is simple and reliable for desalting samples containing 140 to 945 mg/100 mL of soluble dietary fiber in preparation for HPLC analysis of soluble dietary fiber.

Sample preparation is a highly important and integral part of bioanalysis for cleaning up the complex biological matrices and thereby minimizing matrix effect. Matrix effect can jeopardize the precise quantification and adversely affect the reliability of liquid chromatography-mass spectrometry-based analytical results by alteration of analyte ionization. Matrix components result in suppression or enhancement of the intensity of analyte response. In spite of the high specificity and selectivity of tandem mass spectrometry, a relatively higher concentration of coeluted matrix elements present in biofluids may alter the efficiency of quantification of a bioanalytical method. Numerous literature reports different types of sample preparation techniques employed in bioanalysis. In this review, the strategies for selection of the appropriate sample clean-up technique in bioanalysis are discussed extensively. A paradigm shift in the arena of sample preparation and bioanalytical approaches involving the liquid chromatography-mass spectroscopic technique has been scrutinized. Current trends and possible future advancements in the field of biological sample extraction methods, including instrumental techniques are analyzed in detail.

Reactive oxygen species (ROS) and chronic inflammation contribute to DNA damage of many organs, including the prostate. ROS cause oxidative damage to biomolecules, such as lipids, proteins, and nucleic acids, resulting in the formation of toxic and mutagenic intermediates. Lipid peroxidation (LPO) products covalently adduct to DNA and can lead to mutations. The levels of LPO DNA adducts reported in humans range widely. However, a large proportion of the DNA adducts may be attributed to artifact formation during the steps of isolation and nuclease digestion of DNA. We established a method that mitigates artifacts for most LPO adducts during the processing of DNA. We have applied this methodology to measure LPO DNA adducts in the genome of prostate cancer patients, employing ultrahigh-performance liquid chromatography electrospray ionization ion trap multistage mass spectrometry. Our preliminary data show that DNA adducts of acrolein, 6-hydroxy-1, N 2 -propano-2'-deoxyguanosine (6-OH-PdG) and 8-hydroxy-1, N 2 -propano-2'-deoxyguanosine (8-OH-PdG) (4-20 adducts per 10 7 nucleotides) are more prominent than etheno (ε) adducts (<0.5 adducts per 10 8 nucleotides). This analytical methodology will be used to examine the correlation between oxidative stress, inflammation, and LPO adduct levels in patients with benign prostatic hyperplasia and prostate cancer.

Sample pretreatment is an important process in liquid chromatography-mass spectrometry-based quantitative lipidomics. Reversed-phase solid phase extraction (RP-SPE) has been widely used for analyzing various types of samples, including aqueous samples such as cell culture media, plasma, serum, urine, and other biological fluids. Because lipid mediators are often protein-bound, prior deproteinization is necessary for their effective recovery. Deproteinization is typically performed by the addition of organic solvents, which requires time-consuming evaporation-reconstitution, or dilution with aqueous solvents before RP-SPE; however, both of these approaches compromise the analytical performance. As a potential alternative, we attempted to utilize supported liquid extraction (SLE), an automation-compatible variant of liquid-liquid extraction, for the determination of eicosanoids and related metabolites in aqueous samples. We screened 81 different sample diluent-eluent conditions and found that the use of 0.1% formic acid-water as the diluent and 0.1% formic acid-methyl acetate as the eluent enabled the optimum recovery of a variety of eicosanoids, except for peptide leukotrienes. The optimized SLE method efficiently removed protein from human plasma, while phospholipids and neutral lipids were modestly recovered. Moreover, the proposed method exhibited a quantitative performance comparable to that of typical ordinary RP-SPE method in the analysis of human platelets stimulated with thrombin receptor-activating peptide 6. Thus, we propose SLE as an attractive option for rapid lipid mediator extraction from aqueous samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Protein analysis of potential disease markers in blood is complicated by the fact that proteins in plasma show very different abundances. As a result, high-abundance proteins dominate the analysis, which often render the analysis of low-abundance proteins impossible. Depleting high-abundance proteins is one strategy to solve this problem. Here, we present, for the first time, a very simple approach based on selective binding of serum proteins to the surface of nanodiamonds. In our first proof-of-principle experiments, we were able to detect, on average, eight proteins that are present at a concentration of 1 ng/mL (instead of 0.5 ng/mL in the control without sample preparation). Remarkably, we detect proteins down to a concentration of 400 pg/mL after only one simple depletion step. Among the proteins we could analyze are also numerous disease biomarkers, including markers for multiple cancer forms, cardiovascular diseases, or Alzheimer's disease. Remarkably, many of the biomarkers we find also could not be detected with a state-of-the-art ultrahigh-performance liquid chromatography column (which depletes the 64 most-abundant serum proteins).

In the past decade, mass spectrometry studies of skeletal muscles have become common. In this tissue, the abundance of several contractile proteins significantly limits the depth of the panoramic proteome analysis. The use of isobaric labels allows improving assessment of the changes in the protein content, while analyzing up to 10 samples in a single run. Here we present the results of a comparative study of various methods for the fractionation of skeletal muscle peptides labeled with an isobaric label iTRAQ. Samples from m. vastus lateralis of eight young males were collected with a needle biopsy. After digestion into peptides and labeling, the preparations were carried out according to three different protocols: (1) peptide purification, HPLC-MS/MS; (2) peptide purification, isoelectric focusing, HPLC-MS/MS; (3) high pH reverse-phase LC fractionation, HPLC-MS/MS. Fractionation of labeled peptides by high pH reverse-phase LC was the optimal strategy for increasing the depth of the proteome analysis. This approach, in addition to contractile and mitochondrial proteins, allowed us to detect a variety of regulatory molecules, including the nucleic acids binding the proteins, chaperones, receptors, and transcription factors.

This paper describes the development of an on-chip nucleic acid (NA) extraction assay from whole blood using a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation. The combination of pneumatic and centrifugal forces makes it possible to perform fluidic operations without the need for integrating active control elements on the microfluidic cartridge. The cartridge is fabricated from thermoplastic polymers (e.g., Zeonor 1060R) and features a simple design that is compatible with injection molding. In addition, the cartridge is interfaced with two external vials for off-chip storage of the blood sample and retrieval of the eluted NA solution, respectively. On-chip capture of NAs is performed using an embedded solid-phase extraction matrix composed of commercial glass microfiber filters (Whatman GF/D and GF/F). The yield of the automated, on-chip extraction protocol, determined by measuring absorbance at 260 nm, is comparable to some of the best manually operated kits (e.g., Qiagen QIAamp DNA Mini Kit) while providing low assay-to-assay variability due to the high level of control provided by the platform for each processing step. The A260/A280 and A260/A230 ratios of the absorbance spectra also reveal that protein contamination of the sample is negligible. The capability of the pneumatic platform to circulate air flux through the microfluidic conduit was used to dry leftover ethanol residues retained in the capture matrix during washing. This method, applied in combination with localized heating, proved effective for reducing ethanol contamination in eluted samples from ∼12% to 1% (v/v).

Mass spectrometry-based urinary proteomics is one of the most attractive strategies to discover proteins for diagnosis, prognosis, monitoring, or prediction of therapeutic responses of urological diseases involving the kidney, prostate, and bladder; however, interfering compounds found in urine necessitate sample preparation strategies that are currently not suitable for urinary proteomics in the clinical setting. Herein, we describe the C4-tip method, comprising a simple, automated strategy utilizing a reverse-phase resin tip-based format and "on-tip" digestion to examine the urine proteome. We first determined the optimal conditions for protein isolation and protease digestion on the C4-tip using the standard protein bovine fetuin. Next, we applied the C4-tip method to urinary proteomics, identifying a total of 813 protein groups using LC-MS/MS, with identified proteins from the C4-tip method displaying a similar distribution of gene ontology (GO) cellular component assignments compared to identified proteins from an ultrafiltration preparation method. Finally, we assessed the reproducibility of the C4-tip method, revealing a high Spearman correlation R-value for shared proteins identified across all tips. Together, we have shown the C4-tip method to be a simple, robust method for high-throughput analysis of the urinary proteome by mass spectrometry in the clinical setting.

Lichens are useful biomonitors for atmospheric polycyclic aromatic hydrocarbons (PAHs). Different sample preparation techniques were explored in this regard, including ultrasound-assisted solvent extraction, microwave-assisted extraction, Soxhlet, and the quick, easy, cheap, effective, rugged, and safe (QuEChERS) technique. It was found that a QuEChERS technique using hexane:acetone (1:1, v/v), never reported before for application to lichens, provided the best recoveries of internal standards, the highest total peak area for all PAHs of interest, and %RSDs comparable with the other preparation techniques tested. The optimized sample preparation technique was found to be a comparatively fast method (45 min), with good recoveries (96%), using less solvents and minimal energy consumption. Strong matrix effects were found: both strong enhancement (for the lighter PAHs) and strong suppression (for the heavier PAHs). The use of matrix-matched standards is thus imperative for the accurate determination of PAH concentrations in the lichen samples. Graphical abstract "Note: This data is mandatory. Please provide."

In this work, we present a method for the clean-up, preconcentration and quantification of the four most widely found estrogens (estrone E1, estradiol E2, estriol E3 and ethynyl estradiol EE2) in seawater samples. A sequential injection analysis-lab on valve system (SIA-LOV) has been developed to perform the microsolid phase extraction (µSPE) of the analytes in a fully automated way. After testing different resins and solvents, C18 resin with acetonitrile (ACN) as eluent have been chosen as they provided the best results. Several parameters affecting the extraction have been studied and optimized. Besides, extraction column lifetime has also been checked as it is indicative of the number of consecutive analysis that the column is able to perform before replacing it. Results showed that the same column can be used up to 50 times. Then, the derivatization of the extracts has been performed unattended by exploiting an in-port derivatization of the analytes with N-methyltrimethylsilyltrifluoroacetamide (BSTFA) prior their quantification using large volume injection with programmable temperature vaporization gas chromatography (LVI-PTV-GC-MS)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims/introduction: It is reported that interfering substances in the blood might influence the value for measurement of active glucagon-like peptide-1 (GLP-1) in human plasma. Solid phase extraction (SPE) pretreatment is recommended to reduce their influence, but it requires a lot of cost and time. However, there is little investigation about causative inhibitory substances and about methods that can replace solid phase extraction. In the present study, we aimed to seek the candidate of the substances that might interfere with an active GLP-1 enzyme-linked immunosorbent assay (ELISA)., Materials and Methods: Two kinds of active GLP-1 ELISA kits using different antibodies, plural extraction carriers and elution solutions were used to evaluate the SPE method. Active GLP-1 concentration was compared with or without SPE, and with or without a heterophilic blocking tube., Results: Active GLP-1 values were often higher without SPE compared with those with SPE pretreatment. This difference was eliminated by pretreatment with a heterophilic blocking tube or ELISA kits that did not use a mouse monoclonal antibody, and was independent of SPE., Conclusions: Substances absorbed to a heterophilic blocking tube carrier might interfere with an active GLP-1 immunoassay. Solid-phase extraction treatment is required for measurement of active GLP-1 by an ELISA kit affected by heterophilic antibodies., (© 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Batch recalls for valsartan containing pharmaceutical products in July 2018 initiated a discussion on possible contaminations with N-nitrosodimethylamine (NDMA). It appeared that NDMA was generated during synthesis of the active pharmaceutical ingredient (API) from the solvent dimethylformamide (DMF) and the reagent nitrite. Discussion on NDMA as API impurity is extended to other drugs since then. Already several years before scientific literature reported NDMA as impurity of several other drugs, thus underlining the apparent risk. At present none of the pharmacopoeias tests for NDMA and only very limited publications of methods for its determination in pharmaceuticals are published so far. This review summarizes aspects for the analyses of nitrosamines (NAs) with special focus on NDMA and discusses their potential applicability for drug analyses. The majority of recent publications utilize GC-MS or GC-MS/MS due to its high selectivity and low detection levels. GC-TEA also provides high selectivity for nitrosamines. However, current availability of this combination is very limited. Alternatively, LC-MS/MS is also performed in NA analysis. An integration of a general test in future pharmacopoeias is suggested due to the toxicological relevance and broader spectrum of possible APIs that may be affected., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Metabolomics is a well-established field in fundamental clinical research with applications in different human body fluids. However, metabolomic investigations in feces are currently an emerging field. Fecal sample preparation is a demanding task due to high complexity and heterogeneity of the matrix. To gain access to the information enclosed in human feces it is necessary to extract the metabolites and make them accessible to analytical platforms like NMR or LC-MS. In this study different pre-analytical parameters and factors were investigated i.e. water content, different extraction solvents, influence of freeze-drying and homogenization, ratios of sample weight to extraction solvent, and their respective impact on metabolite profiles acquired by NMR and LC-MS. The results indicate that profiles are strongly biased by selection of extraction solvent or drying of samples, which causes different metabolites to be lost, under- or overstated. Additionally signal intensity and reproducibility of the measurement were found to be strongly dependent on sample pre-treatment steps: freeze-drying and homogenization lead to improved release of metabolites and thus increased signals, but at the same time induced variations and thus deteriorated reproducibility. We established the first protocol for extraction of human fecal samples and subsequent measurement with both complementary techniques NMR and LC-MS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The demand for understanding the roles genes play in biological systems has steered the biosciences into the direction the metabolome, as it closely reflects the metabolic activities within a cell. The importance of the metabolome is further highlighted by its ability to influence the genome, transcriptome, and proteome. Consequently, metabolomic information is being used to understand microbial metabolic networks. At the forefront of this work is mass spectrometry, the most popular metabolomics measurement technique. Mass spectrometry-based metabolomic analyses have made significant contributions to microbiological research in the environment and human disease. In this chapter, we break down the technical aspects of mass spectrometry-based metabolomics and discuss its application to microbiological research.

Nuclear magnetic resonance (NMR) spectroscopy is widely considered to be one of the most robust and reproducible analytical platforms for conducting metabolomic experiments. As a metabolomic platform, NMR is not particularly sensitive, but it is nondestructive and requires no prior derivatization or chromatographic separation. It is also very automatable, easy to perform, and highly reproducible and can be used to accurately quantify dozens of metabolites in complex mixtures. To perform a successful NMR metabolomic experiment, it is important to follow good practices in sample preparation, data acquisition, and data analysis. In this chapter, we will describe, step-by-step, the preparation of different livestock samples, including both biofluids (whole blood, serum, urine, rumen content, and fecal water) and tissues (liver, muscle, testis). We will also describe the protocols for acquiring optimal NMR spectra and the techniques used to identify and quantify water-soluble metabolites by NMR spectroscopy.

In this study, a novel strategy based on acetone stable-isotope derivatization coupled with HPLC-MS for profiling and accurate quantification of aminophospholipids (phosphatidylethanolamine and phosphatidylserine) in biological samples was developed. Acetone derivatization leads to alkylation of the primary amino groups of aminophospholipids with an isopropyl moiety; the use of deuterium-labeled acetone (d6-acetone) introduced a 6 Da mass shift that was ideally suited for profiling and quantification analysis with high selectivity and accuracy. After derivatization, significantly increased column efficiency for chromatographic separation and detection sensitivity for MS analysis of aminophospholipids was observed. Furthermore, an accuracy quantification method was developed. Aminophospholipids in biological samples were derivatized with d0-acetone; while more than two aminophospholipid standards were selected for each class of aminophospholipid and derivatized with d6-acetone, which were then used as the internal standards to typically construct a calibration curve for each class to normalize the nonuniformity response caused by the differential fragmentation kinetics resulting from the distinct chemical constitution of individual aminophospholipid species in the biological samples. The excellent applicability of the developed method was validated by profiling and quantification of aminophospholipids presented in liver samples from rats fed with different diets., (Copyright © 2019 Ma et al.)

The efficacy of faecal microbiota transplantation (FMT) as a therapeutic intervention may depend on the viability of the microorganisms in faecal slurries (FS) prepared from donor stool. However, determining the viability of these organisms is challenging. Most microorganisms in stool are refractory to culture using standard techniques, and culture-independent PCR-based methods derive signal from both viable and non-viable cells. Propidium monoazide (PMA) treatment has been shown to be effective in preventing PCR amplification of DNA from non-viable bacteria in a range of contexts. However, this methodology can be sensitive to factors such as bacterial load and sample turbidity. We describe the optimisation of a PMA treatment methodology for FS that restricts quantitative PCR-based bacterial enumeration to viable cells. When applied to concentrated FS (10-25% stool content), PMA treatment at 100 μM concentration was ineffective in preventing DNA amplification from heat-killed cells. Efficacy was not significantly improved by doubling the PMA concentration. However, PMA treatment efficacy was improved markedly following 10-fold sample dilution, and was found to be optimal at 100-fold dilution. Substantial reductions in viable bacterial load could be observed following both freeze-thaw and heat-treatment of FS. This method successfully prevented DNA amplification of heat-killed Pseudomonas and Staphylococcus spiked into stool and could reliably determine the proportion of live bacteria and viable E. coli counts present in fresh and heat-treated stool. With appropriate sample dilution, PMA treatment excluded >97% of non-viable cells from amplification in all assays, without significantly affecting the amplification of DNA from viable cells. This method can be applied to optimise sample processing of FMT donor material, and to characterise bacterial viability within faecal samples more widely., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The molecular composition of soil organic matter (SOM) sets the foundation for terrestrial microbial community structures and carbon cycling dynamics. However, the specific chemical constituents of SOM are underexplored. In this chapter we present a protocol for the extraction of small molecule metabolites from soil followed by compound detection and identification using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. There are options within the protocol to assess either the extracellular pool of metabolites or the total pool (including intracellular) and either polar or nonpolar metabolites, depending on the reader's research interests. These methods can be followed individually for a more targeted analysis or all methods can be combined to obtain a more comprehensive understanding of SOM metabolite composition (such as amino acids, nucleobases, organic acids, fatty acids, carbohydrates, secondary metabolites, and antibiotics).

Straightforward, rapid and high-throughput pretreatment for single nucleotide polymorphisms (SNP) genotyping is critically needed in clinical practice. However, all existing SNP genotyping methods require DNA purification step, which is labor-intensive and time-consuming. We develop a protocol for SNP genotyping by combining whole blood lysis (WBL) with qPCR and justify the practicality of our method in blood samples from 140 donors, including 40 samples from healthy donors, and 100 samples from donors with either low white blood cell counts, high level of serum uric acid or triglyceride. When compared with Sanger sequencing, the gold standard for SNP genotyping, our method exhibits a 100% consistency in the aspect of sensitivity and specificity. In addition, our method can obtain amplifiable DNA within 25 mins (which is the fastest to the best of our knowledge) from 48 samples. The blood samples, even with low white blood cell counts, high level of serum uric acid or triglyceride could not affect the sensitivity and specificity of our method. Our study demonstrates that the combination of WBL and qPCR genotyping can serve as a high-throughput and robust approach for routine clinical SNP genotyping., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Freeze-thaw DNA extraction methods and PCR primers were compared to optimize detection of Cryptosporidium parvum and Toxoplasma gondii oocysts in different matrices. Increasing FT cycles did not increase parasite DNA detection, and primers targeting the 18S ssrRNA gene yielded the most sensitive detection of C. parvum oocysts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aim: Transdermal analysis is proposed for vitamin D 3 and its hydroxylated metabolite to overcome problems associated with blood analysis., Methods: Vitamin D 3 was extracted directly from skin with solid patches and liquid phases. Deuterium-labeled vitamin D 3 was added to the extraction solutions to compensate for variability and accurately determine the rate of transdermal transfer. Of the different extraction solvents tested, 50:50 octanol:isopropanol showed the best results, with an accuracy of 115% and reproducibility better than 30%., Conclusion: The research shows that transdermal route can be used for analysis of vitamin D 3 in porcine skin. When microneedles are used, accurate measurements were obtained in 1 h. With intact skin, the highest accuracy was obtained when extraction was done for 2 h.

The extraction of metabolites turns out to be one of the most important key factors for nontargeted metabolomics approaches as this step can significantly affects the informative value of the successive measurements. Compared to metabolomics experiments of various matrices of bacterial or mammalian origins, there are only few studies, which focus on different extraction methods for plant metabolomics analyses. In this study, various solvent extraction compositions were compared and assessed using an UPLC-ESI-QTOF-MS strategy. Exemplary, white asparagus ( Asparagus officinalis) were employed as a low-fat-, low-protein-, high-water-content model commodity with the objective of designing an optimal nontargeted extraction protocol for polar and nonpolar metabolites. Furthermore, the influence of acid addition, mechanical cell disruption methods (ball mill, ultrasonic bath, vortex mixer), and extract stability have been systematically scrutinized too. The different extraction protocols were compared based on sum of features, sum of peak intensities, sum of peak areas, as well as by analyzing individual signals of as many different substance groups as possible to obtain a maximum overview.

Rapid detection and identification of anaerobic bacteria from blood is important to adjust antimicrobial therapy by including antibiotics with activity against anaerobic bacteria. Limited data is available about direct identification of anaerobes from positive blood culture bottles using MALDI-TOF mass spectrometry (MS). In this study, we evaluated the performance of two sample preparation protocols for direct identification of anaerobes from positive blood culture bottles, the MALDI Sepsityper kit (Sepsityper) and the in-house saponin (saponin) method. Additionally, we compared two blood culture bottle types designed to support the growth of anaerobic bacteria, the BacT/ALERT-FN Plus (FN Plus) and the BACTEC-Lytic (Lytic), and their influence on direct identification. A selection of 30 anaerobe strains belonging to 22 different anaerobic species (11 reference strains and 19 clinical isolates) were inoculated to 2 blood culture bottle types in duplicate. In total, 120 bottles were inoculated and 99.2% (n = 119) signalled growth within 5 days of incubation. The Sepsityper method correctly identified 56.3% (n = 67) of anaerobes, while the saponin method correctly identified 84.9% (n = 101) of anaerobes with at least log(score) ≥1.6 (low confidence correct identification), (p < 0.001). Gram negative anaerobes were better identified with the saponin method (100% vs. 46.5%; p < 0.001), while Gram positive anaerobes were better identified with the Sepsityper method (70.8% vs. 62.5%; p = 0.454). Average log(score) values among only those isolates that were correctly identified simultaneously by both sample preparation methods were 2.119 and 2.029 in favour of the Sepsityper method, (p = 0.019). The inoculated bottle type didn't influence the performance of the two sample preparation methods. We confirmed that direct identification from positive blood culture bottles with MALDI-TOF MS is reliable for anaerobic bacteria. However, the results are influenced by the sample preparation method used., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The purpose of this study was to establish a chemical profiling method to compare the chemical composition of herbal products by using extracts of Belamcandae Rhizoma(EBR) extracted with different polarity solvent as an example, and evaluate the quality of EBR based on the analysis of chemical profiling, efficacy evaluation and pharmacokinetics. As seen from the results of chemical profiling, the PCA and PLS-DA score plot indicated that the dots of Belamcandae Rhizoma water extracts were separated from ethanol extracts obviously, which suggested significant differences of chemical profiling existing in the different solvent extracts. The PCA and PLS-DA loading plot illustrated that the main compounds contributing to chemical profiling differences were tectoridin(TD), iristectorin B(IT B), iridin(ID), tectorigenin(TG), irigenin(IG), iristectorigein A(IG A), dichotomitin(DT) and irisflorentin(IF). Furthermore, the results of HPLC analysis demonstrated that the contents of these main compounds in ethanol extracts were significantly higher than that in water extracts (P < 0.01). Both the pharmacological and hematoxylin-eosin staining studies indicated that the ethanol extracts of Belamcandae Rhizoma had a better therapeutic effect than water extracts in oral ulcer model rats (P<0.01). It is suggested that the ethanol extracts were beneficial to the absorption and bioavailability of TG which was one of the most important bioactive compounds of Belamcandae Rhizoma in pharmacokinetic study in rats. This work provided a novel method to optimize the extraction process of EBR and related herbal products. Compared with the conventional chemical fingerprint methodology, the approach proposed above is not only a powerful tool to identify efficacy-related components for the quality evaluation, but also can be used to predict the therapeutic efficacy of herbal products., (Copyright © 2018 Elsevier B.V. All rights reserved.)