Standard Sample Preparation Research Articles

Development of a new HPLC method for wogonin in rat plasma: Compatibility of standard and test samples.

In the current paper, an HPLC/UV method was developed and validated for determination of wogonin in plasma. Considerable attention was paid to the preparation of standard samples and factors affecting drug distribution. A preparation procedure was devised to simulate the conditions the drug is expected to experience in vivo while pointing to the shortcomings of previously published methods. The method was validated according to the FDA regulations and showed to be highly efficient and capable of extracting the drug and IS from the plasma accurately and precisely within the specified range of 50-500 ng mL-1. Further, the standard sample preparation of this method can be used as a guideline for other methods, particularly when highly hydrophobic drugs with considerable protein binding are involved and could be valuable in the field of bioanalysis to improve the reliability of methods.

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells.

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3-diaminobenzidine (DAB) and hydrogen peroxide (H2O2).

MALDI-TOF MS typing enables the classification of brewing yeasts of the genus Saccharomyces to major beer styles.

Brewing yeasts of the genus Saccharomyces are either available from yeast distributor centers or from breweries employing their own “in-house strains”. During the last years, the classification and characterization of yeasts of the genus Saccharomyces was achieved by using biochemical and DNA-based methods. The current lack of fast, cost-effective and simple methods to classify brewing yeasts to a beer type, may be closed by Matrix Assisted Laser Desorption/Ionization–Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) upon establishment of a database based on sub-proteome spectra from reference strains of brewing yeasts. In this study an extendable “brewing yeast” spectra database was established including 52 brewing yeast strains of the most important types of bottom- and top-fermenting strains as well as beer-spoiling S. cerevisiae var. diastaticus strains. 1560 single spectra, prepared with a standardized sample preparation method, were finally compared against the established database and investigated by bioinformatic analyses for similarities and distinctions. A 100% separation between bottom-, top-fermenting and S. cerevisiae var. diastaticus strains was achieved. Differentiation between Alt and Kölsch strains was not achieved because of the high similarity of their protein patterns. Whereas the Ale strains show a high degree of dissimilarity with regard to their sub-proteome. These results were supported by MDS and DAPC analysis of all recorded spectra. Within five clusters of beer types that were distinguished, and the wheat beer (WB) cluster has a clear separation from other groups. With the establishment of this MALDI-TOF MS spectra database proof of concept is provided of the discriminatory power of this technique to classify brewing yeasts into different major beer types in a rapid, easy way, and focus brewing trails accordingly. It can be extended to yeasts for specialty beer types and other applications including wine making or baking.

Improved bacterial identification directly from urine samples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) contributes to rapid identification of pathogens in the clinic but has not yet performed especially well for Gram-positive cocci (GPC) causing complicated urinary tract infection (UTI). The goal of this study was to investigate the possible clinical use of MALDI-TOF MS as a rapid method for bacterial identification directly from urine in complicated UTI. MALDI-TOF MS was applied to urine samples gathered from 142 suspected complicated UTI patients in 2015-2017. We modified the standard procedure (Method 1) for sample preparation by adding an initial 10minutes of ultrasonication followed by centrifugation at 500g for 1minutes to remove debris such as epithelial cells and leukocytes from the urine (Method 2). In 133 urine culture-positive bacteria, the rate of corresponded with urine culture in GPC by MALDI-TOF MS in urine with standard sample preparation (Method 1) was 16.7%, but the modified sample preparation (Method 2) significantly improved that rate to 52.2% (P=.045). Method 2 also improved the identification accuracy for Gram-negative rods (GNR) from 77.1% to 94.2% (P=.022). The modified Method 2 significantly improved the average MALDI score from 1.408±0.153 to 2.166±0.045 (P=.000) for GPC and slightly improved the score from 2.107±0.061 to 2.164±0.037 for GNR. The modified sample preparation for MALDI-TOF MS can improve identification accuracy for complicated UTI causative bacteria. This simple modification offers a rapid and accurate routine diagnosis for UTI, and may possibly be a substitute for urine cultures.

Pathogen identification in prostate cancer biopsies using transcriptome sequencing.

e16545 Background: Tumor biopsies may frequently be associated with microbial species due to proximity with microbial communities or due to contamination during tissue processing. We examined sequencing data from tumor biopsies to explore the tumor-associated microbiome in prostate cancer. Methods: Patients were enrolled in a prospective Precision Medicine study to evaluate genomic alterations based on freshly obtained tissue biopsies. Total RNA was prepared for RNA sequencing using the standard Illumina mRNA sample preparation protocol. Paired-end RNA-sequencing at read lengths of 50 or 51 bp was performed with the HiSeq 2000 (Illumina). A total of ~268 million paired-end reads were generated, corresponding to 27 billion bases. Kraken and FusionCatcher were used to identify the metagenome that maps to microbial species. R was used to analyze the microbial pathogens and to create a heatmap of microbial abundance. We focused our analysis on prostate cancer. Results: From 1/30/2013 to 12/16/15, 83 patients with urothelial cancers were identified, of which 32 patients with prostate cancer (n = 23 from primary tumor, n = 9 metastatic prostate cancer) were sequenced. Of the 9 metastatic samples, 5 were metastatic prostate cancer to bone, 4 were other metastases. A giant cell tumor of bone was used as a control. The metagenome was identified and mapped to the Kraken pathogen database. 43 unique pathogens characterized the microbiome associated with prostate cancer, including Streptomyces species, Acinetobacter baumannii, Enterobacteriaceae, and Corynebacterium species. The most prevalent species in prostate cancer bone metastases were also the most prevalent in primary prostate tumors. Prostate cancer bone metastases clustered separately from the primary prostate cancer samples, the other metastases, and primary bone cancer. Conclusions: We have identified a putative microbiome signature associated with prostate cancer. Prostate cancer bone metastases appear to cluster separately from prostate cancer, suggesting a non-random association with microbial communities. Ongoing work is looking to (1) validate microbial association using orthogonal analyses and (2) determine the exact origin of the signature.

Molecular weight analysis of water-soluble poly(phenylene ethynylene)s using MALDI-TOF MS

Molecular weights of seven poly(phenylene ethynylene)-based water-soluble conjugated polyelectrolytes (CPEs) obtained through Sonogashira coupling are determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A standard sample preparation protocol is developed to characterize the seven CPEs using 2,5-dihydroxybenzoic acid as the matrix (M) and AgTFA as the cationization reagent (CR). High-quality MALDI mass spectra are obtained at volume mixing ratios (CPE/M/CR) of 5/5/1 for anionic polymers (P1–P4) and 5/50/1 for cationic polymers (P5–P7). Molecular weight, molecular weight distribution, and end-group information are analyzed. The effects of molecular weight of CPEs on optical and quenching properties are also studied. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017

Particle/cell separation on microfluidic platforms based on centrifugation effect: a review

Particle/cell separation in heterogeneous mixtures including biological samples is a standard sample preparation step for various biomedical assays. A wide range of microfluidic-based methods have been proposed for particle/cell sorting and isolation. Two promising microfluidic platforms for this task are microfluidic chips and centrifugal microfluidic disks. In this review, we focus on particle/cell isolation methods that are based on liquid centrifugation phenomena. Under this category, we reviewed particle/cell sorting methods which have been performed on centrifugal microfluidic platforms, and inertial microfluidic platforms that contain spiral channels and multi-orifice channels. All of these platforms implement a form of centrifuge-based particle/cell separation: either physical platform centrifugation in the case of centrifugal microfluidic platforms or liquid centrifugation due to Dean drag force in the case of inertial microfluidics. Centrifugal microfluidic platforms are suitable for cases where the preparation step of a raw sample is required to be integrated on the same platform. However, the limited available space on the platform is the main disadvantage, especially when high sample volume is required. On the other hand, inertial microfluidics (spiral and multi-orifice) showed various advantages such as simple design and fabrication, the ability to process large sample volume, high throughput, high recovery rate, and the ability for multiplexing for improved performance. However, the utilization of syringe pump can reduce the portability options of the platform. In conclusion, the requirement of each application should be carefully considered prior to platform selection.

Enhanced surface flatness of vitrinite particles by broad ion beam polishing and implications for reflectance measurements

Standardized measurements of vitrinite reflectance vary significantly between different measuring institutions. The only systematically adjustable influence factor on these differences is the polishing method. We present a recently developed preparation method using broad ion beam polishing, which, applied as additional polishing step or replacement of the standardized sample preparation, provides a dramatically improved surface flatness, and allows to remove scratches and debris of traditional polishing. Mechanical, near-surface damage of the sample, as known from traditional polishing, is not appearing in broad ion beam polished samples. Moreover, its water-free polishing allows excellent preparation of swelling clay-rich samples or granular solid bitumen surfaces.Using broad ion beam polishing the vitrinite reflectance increases by 0.18–0.24% VRr (relative increase: 12–28%) in coal samples (including an ICCP standard sample) and by 4.76% VRmax (relative increase: 90%) at a measured graphite without causing thermal overprinting of the sample based on our preliminary results. A subsequent traditional polishing step after broad ion beam polishing reverses this increase in reflectivity. The presented method has the potential to decrease the range of reflectance-based calculated maximum burial temperatures by up to 50°C if a reduction of 0.2 in the range of the measured vitrinite reflectance standard deviation can be achieved.

Transmission electron microscopy artifacts in characterization of the nanomaterial-cell interactions.

We investigated transmission electron microscopy artifacts obtained using standard sample preparation protocols applied to the investigation of Escherichia coli cells exposed to common nanomaterials, such as TiO2, Ag, ZnO, and MgO. While the common protocols for some nanomaterials result only in known issues of nanomaterial-independent generation of anomalous deposits due to fixation and staining, for others, there are reactions between the nanomaterial and chemicals used for post-fixation or staining. Only in the case of TiO2 do we observe only the known issues of nanomaterial-independent generation of anomalous deposits due to exceptional chemical stability of this material. For the other three nanomaterials, different artifacts are observed. For each of those, we identify causes of the observed problems and suggest alternative sample preparation protocols to avoid artifacts arising from the sample preparation, which is essential for correct interpretation of the obtained images and drawing correct conclusions on cell-nanomaterial interactions. Finally, we propose modified sample preparation and characterization protocols for comprehensive and conclusive investigations of nanomaterial-cell interactions using electron microscopy and for obtaining clear and unambiguous revelation whether the nanomaterials studied penetrate the cells or accumulate at the cell membranes. In only the case of MgO and ZnO, the unambiguous presence of Zn and Mg could be observed inside the cells.

Application and Evaluation of 3D Printed Materials with PALS

In this study, the use of a 3D printer for sample holder fabrication and polymer sample preparation for positron analysis was explored. Custom printed 3D holders may be rapidly made and modified for a variety of thin-film, crystalline, or other diversely-shaped samples. For positron studies a 3D printer allows for the preparation of standard and unique polymer samples. In an initial study, a mesoporous-patterned ABS sample was attempted, without success. Various polymers (ABS, PLA, and PETG) and the same polymers with varied additives (carbon fiber or carbon nanotubes) were studied before and after printing. The different polymers and those with additives are distinguishable via PALS. Samples show a consistently lower I3 value after printing, suggesting a decrease in defect quantity for the printed polymer versus the as-received polymer filament.

Impact of sample preparation on physical quantification of filling fats and oils in fresh and stored chocolate

Filling fat and oil migration in filled chocolate products often cause quality loss, such as fat bloom or changes in texture. Therefore, and due to legal restrictions, the detection and quantification of filling fats and oils in chocolate is of interest. Common methods are fatty acid or triacylglycerol (TAG) analysis by GC or HPLC. Additionally, NMR and DSC are used to evaluate migration especially in stored products. Using DSC, the melting and crystallization peak of a filling oil can be detected directly, while NMR turns the reduction of solid fat content (SFC) caused by filling fats and oils to account. However, fat and oil migration is not the only effect during storage, which might affect NMR and DSC measurements. Post‐crystallization is also of interest. Therefore, DSC and NMR methods were used to observe and to evaluate the detectable amount over time. Dark chocolate was mixed with hazelnut oil, butterfat, and coconut oil. Samples were stored and analyzed using NMR and DSC. Additionally, methods to destroy thermal history of the sample were investigated. We found that post‐crystallization, which occurs during the first 2 wk after production, has a major impact on detectability of filling fats and oils using physical methods. However, standardized sample preparation was successfully applied and amounts below 1 g per 100 g sample were detectable and amounts above 1–2 g were quantifiable with slender time and effort.Practical applications: Filling fat and oil migration during production and storage is a major problem with regards to product stability and shelf life. Especially filling migration during storage is reported to be an activator for fat bloom formation. Physical methods are a cost and time efficient way to control and adjust processing. Our results report sample preparation techniques to detect filling fats and oils in fresh and stored chocolate. This provides the possibility of large scale storage tests with focus on fat and oil migration.Fresh and stored dark chocolate was analyzed with regards to filling fat content using DSC and NMR. Impact of sample preparation on decision and determination limit was evaluated.

A Liquid Handling Robot for Robust and Reproducible Preparation of Standard and Quality Control Samples in Bioanalysis

We describe a unique liquid handling platform, based on a Tecan EVO, specifically designed for the preparation of analytic calibrators and quality control samples according to the requirements of the Good Laboratory Practice (GLP). The platform utilizes a combination of off-the-shelf software (Tecan Gemini 4.2) and custom-programmed SPIKE 1.0. The system convinces with robust and reproducibly quality and a very easy, intuitive user interface. All security requirements as per FDA 21CFRpart 11 were considered, when programming the software bundle.

Rapid tin-mediated access to a lysophosphatidylethanolamine (LPE) library: Application to positional LC/MS analysis for hepatic LPEs in non-alcoholic steatohepatitis model mice

Even though lysophospholipids have attracted much interest in recent years on account of their unique bioactivity, research related to lysophospholipids is usually hampered by problems associated with standard sample preparation and discrimination of regioisomers. Herein, we demonstrate a quick tin-chemistry-based synthetic route to lysophosphatidylethanolamines (LPEs) and its application in the positional analysis of hepatic LPEs in non-alcoholic steatohepatitis (NASH) model mice. We found that the preference of hepatic LPE regioisomer largely depends on the unsaturation of acyl chain in both control and NASH model mice. In addition, hepatic C18:2-LPE and C20:5-LPE levels were significantly lower in the NASH model mice than those in the control. The LC/MS technique based on the library of LPE regioisomers allows an accurate observation of hepatic LPE metabolism and might provide useful information to elucidate yet ambiguous pathogenesis of NASH.

Optimisation of sample preparation and analysis conditions for atom probe tomography characterisation of low concentration surface species

The practicalities for atom probe tomography (APT) analysis of near-surface chemistry, particularly the distribution of low concentration elements, are presented in detail. Specifically, the challenges of surface analysis using APT are described through the characterisation of near-surface implantation profiles of low concentration phosphorus into single crystal silicon. This material system was chosen to illustrate this surface specific approach as low concentration phosphorus has significant mass spectra overlaps with silicon species and the near surface location requires particular attention to focused ion beam specimen preparation and deposition of various capping layers. Required changes to standard sample preparation procedure are described and the effects of changes in APT analysis parameters are discussed with regards to this specific material system. Implantation profiles of 14 kV phosphorus ions with a predicted peak concentration of 0.2 at .% were successfully analysed using APT using pulsed laser assisted evaporation. It is demonstrated that the most important factor in obtaining the most accurate implantation profile was to ensure all phosphorus mass peaks were as free of background noise as possible, with thermal tails from the Si2+ ions obscuring the P2+ ions being the major overlap in the mass spectrum. The false positive contribution to the phosphorus profiles from hydride species appears minimal at the capping layer/substrate interface. The initial capping layer selection of nickel was successful in allowing the analysis of the majority of the phosphorus profile but nickel and phosphorus mass spectra overlaps prevent optimum quantification of phosphorus at the surface.

Automation Highlights from the Literature

Automation Highlights from the Literature

Uncertainty of carboxyhaemoglobin determination in putrefied blood by headspace gas chromatography

The uncertainty of carboxyhaemoglobin (COHb) determination in post-mortem blood samples by headspace gas chromatography with the use of methanizer and flame ionization detector was estimated. Four main components of uncertainty were identified, i.e. calibration, measurement repeatability, standard solutions preparation and sample preparation, and their contributions to the combined uncertainty were determined. The calibration step was the dominating source of uncertainty, counting for more than 39 % of overall variance. The contribution of remaining sources to the combined uncertainty was lower than 31 % (measurement repeatability), 21 % (standards preparation) and 12 % (sample preparation). The calculated absolute expanded uncertainty in samples containing 3.8–82 % mass fraction of COHb ranged between 2.2 % and 2.6 % (coverage factor 2), depending only slightly on the analyte level in the analysed sample.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain.

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Estimating the uniaxial compressive strength of pyroclastic rocks from the slake durability index

Because the preparation of standard samples may not always be possible for weak or soft rocks, the prediction of uniaxial compressive strength (UCS) from indirect methods is widely used for preliminary investigations. In this study, the possibility of predicting UCS from the slake durability index (SDI) was investigated for pyroclastic rocks. For this purpose, pyroclastic rocks were collected from 31 different locations in the Cappadocian Volcanic Province of Turkey. The UCS and SDI tests were carried out on the samples in the laboratory. The UCS values were correlated with the SDI values and a very strong exponential relation was found between the two parameters. Since some data were scattered over the UCS values of 20 MPa, the correlation plot was redrawn for above and below the UCS values of 20 MPa, respectively. Very strong linear correlations were developed for two cases. Our concluding remark is that the UCS of pyroclastic rocks can be estimated from the SDI.

Up-to-date laboratory methods for assessing the safety of perfumery and cosmetic products in the quality assurance system of the Russian Federation

Most of the developed countries as well as Russian Federation have a dynamically developing quality assurance system for testing the quality of laboratory tests and safety of perfumery and cosmetic products entering the market comprising a regulatory and legal framework, physical infrastructure and appropriate methodical basis and staff. At the same time, it is necessary to develop alternative test methods adjusted to perfumery and cosmetic products on a regular basis. In addition, it is necessary to optimize methods for ensuring standard sample preparation conditions in response to new forms of cosmetic products when determining microbiological, physical, chemical and toxicological characteristics, and more accurate observation methods for clinical and laboratory indices to be approved by supervising authorities.