Sample Preparation Protocol Research Articles

Salt-Induced Homogeneous Liquid–Liquid Microextraction of Piroxicam and Meloxicam from Human Urine Prior to Their Determination by HPLC-DAD

A salt-induced homogeneous liquid–liquid microextraction (SI-HLLME) protocol combined with high-performance liquid chromatography–diode array detection is presented for the first time for the determination of piroxicam and meloxicam in human urine. The main parameters affecting the performance of the sample preparation protocol were optimized by means of a two-step experimental design (i.e., 2-level fractional factorial design and Box–Behnken design). Following its optimization, the proposed method was thoroughly validated in terms of the total error concept in order to take into consideration the random and systematic errors. For the target analytes, accuracy profiles were constructed, and they were used as graphical decision-making tools. In all cases, the β-expectation tolerance intervals complied with the acceptance criteria of ±15%, proving that 95% of future results will fall within the defined bias limits. The limits of detection were 0.02 μg mL−1 and 0.03 μg mL−1 for piroxicam and meloxicam, respectively. The relative standard deviations were lower than 4.4% in all cases, and the mean relative biases ranged between −5.7 and 3.4% for both drugs. The proposed scheme is simple and rapid, while it is characterized by high sample throughput. Moreover, SI-HLLME requires reduced sample and reagent consumption, according to the requirements of Green Analytical Chemistry.

Landmark-based retrieval of inflamed skin vessels enabled by 3D correlative intravital light and volume electron microscopy

The nanometer spatial resolution of electron microscopy imaging remains an advantage over light microscopy, but the restricted field of view that can be inspected and the inability to visualize dynamic cellular events are definitely drawbacks of standard transmission electron microscopy (TEM). Several methods have been developed to overcome these limitations, mainly by correlating the light microscopical image to the electron microscope with correlative light and electron microscopy (CLEM) techniques. Since there is more than one method to obtain the region of interest (ROI), the workflow must be adjusted according to the research question and biological material addressed. Here, we describe in detail the development of a three-dimensional CLEM workflow for mouse skin tissue exposed to an inflammation stimulus and imaged by intravital microscopy (IVM) before fixation. Our aim is to relocate a distinct vessel in the electron microscope, addressing a complex biological question: how do cells interact with each other and the surrounding environment at the ultrastructural level? Retracing the area over several preparation steps did not involve any specific automated instruments but was entirely led by anatomical and artificially introduced landmarks, including blood vessel architecture and carbon-coated grids. Successful retrieval of the ROI by electron microscopy depended on particularly high precision during sample manipulation and extensive documentation. Further modification of the TEM sample preparation protocol for mouse skin tissue even rendered the specimen suitable for serial block-face scanning electron microscopy (SBF-SEM).

Quantification and characterization of mineral oil in fish feed by liquid chromatography-gas chromatography-flame ionization detector and liquid chromatography-comprehensive multidimensional gas chromatography-time-of-flight mass spectrometer/flame ionization detector

Mineral oil is an ubiquitous food contaminant potentially toxic. It is generally divided into aromatic hydrocarbons (MOAH) and saturated hydrocarbons (MOSH). These compounds are currently under investigation by the European Union to determine their occurrence and their toxicity before legislating on the matter. Although the discussion mainly focuses on food, animal feed can indirectly contribute to human exposure to such a contaminant.In this study, seven commercial feeds were analyzed. The analyses were carried out in two different Universities (Udine-IT and Liège-BE), performing the same sample preparation protocol: microwave-assisted saponification and extraction followed by epoxidation for the MOAH fraction. The final determination was performed by hyphenated liquid-gas chromatography (LC-GC) and LC coupled to comprehensive multidimensional gas chromatography (LC-GC × GC) with parallel detection, namely flame ionization detector (FID) and time-of-flight mass spectrometer (ToFMS).The results obtained by the two laboratories were generally in good agreement. The results obtained by LC-GC × GC-ToFMS/FID platform provided consistent results, with the advantages of more robust data interpretation that can compensate for problems occurring during purification. Moreover, the coupling of enhanced separation obtained by GC × GC and the MS information allowed for a more in-depth characterization of the contamination.

Approaches to Improve the Quantitation of Oxytocin in Human Serum by Mass Spectrometry.

The neuropeptide oxytocin (OT) regulates several peripheral and central functions and is a molecule of interest in psychiatric diseases such as autism spectrum disorder, schizophrenia, anxiety and depression. The study of OT in human serum samples is however hampered by inconsistent sample preparation and analysis as well as low endogenous blood concentration (1–10 pM). This results in varying reports on OT’s blood levels and interpretation of OT’s role in different (patho)physiological states. Quantitative mass spectrometry (MS) is a highly promising technology to address this problem but still requires large sample volumes to achieve adequate sensitivity and reliability for the quantitation of compounds at low concentrations. We therefore systematically evaluated sample preparation methods for MS to achieve a reliable sample preparation protocol with good peptide recovery, minimal matrix effects and good overall method efficiency in line with FDA guidelines for bioanalytic method development and validation. Additionally, we investigated a strategy to improve the ionization efficiency of OT by adding charged and/or hydrophobic moieties to OT to improve the lower limit of quantitation. Optimized sample preparation in combination with OT modification with a quaternary pyridinium ion improved the sensitivity of OT by ∼40-fold on a tandem triple quadrupole mass spectrometer (API4000 QTRAP), resulting in a lower limit of quantitation of 5 pM in water (linear range 5 pM – 1 mM) and 2 nM in human serum (linear range 2 nM – 1 mM) compared to 200 pM in water and 86 nM in serum with unmodified OT. This approach and protocol provide a solid foundation towards method development for OT quantitation using MS, which should be of high value for fundamental research as well as clinical monitoring of OT upon drug treatments.

Multicenter Evaluation of Tissue Classification by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Many studies have demonstrated that tissue phenotyping (tissue typing) based on mass spectrometric imaging data is possible; however, comprehensive studies assessing variation and classifier transferability are largely lacking. This study evaluated the generalization of tissue classification based on Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometric imaging (MSI) across measurements performed at different sites. Sections of a tissue microarray (TMA) consisting of different formalin-fixed and paraffin-embedded (FFPE) human tissue samples from different tumor entities (leiomyoma, seminoma, mantle cell lymphoma, melanoma, breast cancer, and squamous cell carcinoma of the lung) were prepared and measured by MALDI-MSI at different sites using a standard protocol (SOP). Technical variation was deliberately introduced on two separate measurements via a different sample preparation protocol and a MALDI Time of Flight mass spectrometer that was not tuned to optimal performance. Using standard data preprocessing, a classification accuracy of 91.4% per pixel was achieved for intrasite classifications. When applying a leave-one-site-out cross-validation strategy, accuracy per pixel over sites was 78.6% for the SOP-compliant data sets and as low as 36.1% for the mistuned instrument data set. Data preprocessing designed to remove technical variation while retaining biological information substantially increased classification accuracy for all data sets with SOP-compliant data sets improved to 94.3%. In particular, classification accuracy of the mistuned instrument data set improved to 81.3% and from 67.0% to 87.8% per pixel for the non-SOP-compliant data set. We demonstrate that MALDI-MSI-based tissue classification is possible across sites when applying histological annotation and an optimized data preprocessing pipeline to improve generalization of classifications over technical variation and increasing overall robustness.

State of Soil Gels in Soddy-Podzolic Soil Samples Prepared for Viscosimetry by Different Methods.

As is well known from the mid-20th century, films of organomineral gels cover and bind soil particles in soils. Soil contact with water has been shown to lead to water absorption by gels and gel swelling. The change of gel properties in soils should manifest itself in a change in the viscosity of soil pastes. A vibrating viscometer was proposed to use to determine the viscosity of soil pastes. The physical meaning laid down in Einstein's formula was used to interpret the results. This made it possible to assess the degree of gel swelling by the amount of water that remained capable of moving independently of soil particles, that is, was not included in soil gels (free water). The effect on the degree of swelling of soil gels was studied for (1) the moisture content in soil samples used to prepare soil pastes, (2) the time after adding water during the preparation of soil samples used subsequently to obtain soil pastes, and (3) the sample preparation of soil samples (initial, autoclaved, or dried to air-dry and absolutely dry states and re-moistened) used to obtain soil pastes. Experiments showed that (1) the degree of swelling of soil gels increased with the increasing moisture content in soil samples, (2) a longer time of interaction of soil samples with water led to a greater degree of swelling of soil gels, and (3) different degrees of swelling of soil gels were observed in pastes prepared from soil samples that had the same moisture content, but differed in sample preparation protocol.

CUT&RUN for Chromatin Profiling in Caenorhabditis elegans.

Cleavage under targets and release using nuclease (CUT&RUN) is a recently developed chromatin profiling technique that uses a targeted micrococcal nuclease cleavage strategy to obtain high-resolution binding profiles of protein factors or to map histones with specific post-translational modifications. Due to its high sensitivity, CUT&RUN allows quality binding profiles to be obtained with only a fraction of the starting material and sequencing depth typically required for other chromatin profiling techniques such as chromatin immunoprecipitation. Although CUT&RUN has been widely adopted in multiple model systems, it has rarely been utilized in Caenorhabditis elegans, a model system of great importance to genomic research. Cell dissociation techniques, which are required for this approach, can be challenging in C. elegans due to the toughness of the worm's cuticle and the sensitivity of the cells themselves. Here, we describe a robust CUT&RUN protocol for use in C. elegans to determine the genome-wide localization of protein factors and specific histone marks. With a simple protocol utilizing live, uncrosslinked tissue as the starting material, performing CUT&RUN in worms has the potential to produce physiologically relevant data at a higher resolution than chromatin immunoprecipitation. This protocol involves a simple dissociation step to uniformly permeabilize worms while avoiding sample loss or cell damage, resulting in high-quality CUT&RUN profiles with as few as 100 worms and detectable signal with as few as 10 worms. This represents a significant advancement over chromatin immunoprecipitation, which typically uses thousands or hundreds of thousands of worms for a single experiment. The protocols presented here provide a detailed description of worm growth, sample preparation, CUT&RUN workflow, library preparation for high-throughput sequencing, and a basic overview of data analysis, making CUT&RUN simple and accessible for any worm lab. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth and synchronization of C. elegans Basic Protocol 2: Worm dissociation, sample preparation, and optimization Basic Protocol 3: CUT&RUN chromatin profiling Alternate Protocol: Improving CUT&RUN signal using a secondary antibody Basic Protocol 4: CUT&RUN library preparation for Illumina high-throughput sequencing Basic Protocol 5: Basic data analysis using Linux.

Calibration of K and alpha (α) for bulk commodities

In order to minimise the sampling error and sampling bias associated with the sampling of metal bearing ores it is essential that the heterogeneity characteristics of the ores be fully appreciated. Heterogeneity tests were carried out on the significantly different manganiferous ores produced at Wessels and Mamatwan mines near Hotazel, South Africa, for the purpose of establishing an optimal sampling protocol for the ores. The method referred to as the Segregation Free Analysis (SFA) was used for the determination of the parameters K and Alpha by construction of calibration curves. The method involves crushing a sufficient amount of ore so that after passing it through a set of fifteen nested screens there is sufficient material to then be split into 32 samples of mass 2-5 kg, using a riffle splitter, and analysing each of the samples. Thus, for fifteen nested screens there are fifteen series each consisting of 32 samples, making a total of 480 samples for analysis. Of the eighteen elements that were analysed in each sample only %Mn3O4, %FeO, %K2O, %P, and %SO2 were calibrated, the first two being the main paying elements and the last three being deleterious elements for the smelting processes in which the ores are used. Calibration curves indicate that for the coarse fraction, above 1 cm, manganese ores have alpha values close to 3, whereas those less than 1 cm in diameter have alpha values closer to 1. Reasons for this behaviour are uncertain but it could be related to the behaviour of the crystal structure in the very pure ores as the ores are progressively crushed and screened to finer size fractions. Separate nomograms were therefore prepared for the coarse and fine fractions. Net conclusions indicate that both Wessels and Mamatwan ores are relatively easy to sample and that simple two or three stage processing will suffice when preparing the final 2 g aliquot at 75 microns. Apart from minor modifications in the sample preparation protocols, there is no evidence to suggest that the Wessels and Mamatwan ores require different sample preparation protocols, or that they should be assayed differently. The calibration curves for manganese ore are compared with the calibration curves for gold bearing ores which generally have alpha values close to 1. The difference in alpha between the gold ores and bulk commodities is considered to be related to the primary distribution of the metals in nature, lognormal for gold and normal for manganese.

Protein profiling of hemolymph in Haemaphysalis flava ticks

BackgroundTick hemolymph bathes internal organs, acts as an exchange medium for nutrients and cellular metabolites, and offers protection against pathogens. Hemolymph is abundant in proteins. However, there has been limited integrated protein analysis in tick hemolymph thus far. Moreover, there are difficulties in differentiating tick-derived proteins from the host source. The aim of this study was to profile the tick/host protein components in the hemolymph of Haemaphysalis flava.MethodsHemolymph from adult engorged H. flava females was collected by leg amputation from the Erinaceus europaeus host. Hemolymph proteins were extracted by a filter-aided sample preparation protocol, digested by trypsin, and assayed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). MS raw data were searched against the UniProt Erinaceidae database and H. flava protein database for host- and tick-derived protein identification. Protein abundance was further quantified by intensity-based absolute quantification (iBAQ).ResultsProteins extracted from hemolymph unevenly varied in size with intense bands between 100 and 130 kDa. In total, 312 proteins were identified in the present study. Therein 40 proteins were identified to be host-derived proteins, of which 18 were high-confidence proteins. Top 10 abundant host-derived proteins included hemoglobin subunit-α and subunit-β, albumin, serotransferrin-like, ubiquitin-like, haptoglobin, α-1-antitrypsin-like protein, histone H2B, apolipoprotein A-I, and C3-β. In contrast, 169 were high-confidence tick-derived proteins. These proteins were classified into six categories based on reported functions in ticks, i.e., enzymes, enzyme inhibitors, transporters, immune-related proteins, muscle proteins, and heat shock proteins. The abundance of Vg, microplusin and α-2-macroglobulin was the highest among tick-derived proteins as indicated by iBAQ.ConclusionsNumerous tick- and host-derived proteins were identified in hemolymph. The protein profile of H. flava hemolymph revealed a sophisticated protein system in the physiological processes of anticoagulation, digestion of blood meal, and innate immunity. More investigations are needed to characterize tick-derived proteins in hemolymph.Graphical

Quantifying up to 90 polyphenols simultaneously in human bio-fluids by LC-MS/MS

Establishing a method for human biomonitoring (HBM) of polyphenols enables the assessment of internal concentrations of these food bio-actives and the correlation with potential health effects such as antioxidant or anti-inflammatory properties. Thus, a targeted LC-MS/MS method for quantifying up to 90 analytes, representing the main polyphenol classes including flavanones, isoflavones, stilbenes, and phenolic acids, was developed for human urine, serum, and plasma. The method was established for low sample volumes and with a cost and time efficient sample preparation protocol for high-throughput, which is critical for its application in large cohort and exposome-wide association studies. On average, the sample preparation yielded extraction efficiencies of 98% for urine, 98% for serum, and 87% for plasma. Limits of detection were between 0.11 ng mL−1 and 300 ng mL−1 for urine, 0.12 ng mL−1 and 190 ng mL−1 for serum, and 0.12 ng mL−1 and 340 ng mL−1 for plasma, excluding one analyte. In-house validation revealed that 66, 49, and 64 analytes for urine, serum, and plasma, respectively, fulfilled all stringent requirements, that are usually utilized for tailored single analyte methods, at all evaluated concentration levels. After validation, this method was applied in a proof-of-principle study that detected 39 polyphenols in urine. Changes in the concentrations of the analytes after the ingestion of a high polyphenol smoothie was examined over 24 h. The study further confirmed that the majority of polyphenols detected were phenolic acids, and phase II conjugated metabolites were more abundant than their respective non-conjugated forms.

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer’s disease biomarkers

BackgroundCerebrospinal fluid (CSF) is an important biofluid for biomarkers of neurodegenerative diseases such as Alzheimer’s disease (AD). By employing tandem mass tag (TMT) proteomics, thousands of proteins can be quantified simultaneously in large cohorts, making it a powerful tool for biomarker discovery. However, TMT proteomics in CSF is associated with analytical challenges regarding sample preparation and data processing. In this study we address those challenges ranging from data normalization over sample preparation to sample analysis.MethodUsing liquid chromatography coupled to mass-spectrometry (LC–MS), we analyzed TMT multiplex samples consisting of either identical or individual CSF samples, evaluated quantification accuracy and tested the performance of different data normalization approaches. We examined MS2 and MS3 acquisition strategies regarding accuracy of quantification and performed a comparative evaluation of filter-assisted sample preparation (FASP) and an in-solution protocol. Finally, four normalization approaches (median, quantile, Total Peptide Amount, TAMPOR) were applied to the previously published European Medical Information Framework Alzheimer’s Disease Multimodal Biomarker Discovery (EMIF-AD MBD) dataset.ResultsThe correlation of measured TMT reporter ratios with spiked-in standard peptide amounts was significantly lower for TMT multiplexes composed of individual CSF samples compared with those composed of aliquots of a single CSF pool, demonstrating that the heterogeneous CSF sample composition influences TMT quantitation. Comparison of TMT reporter normalization methods showed that the correlation could be improved by applying median- and quantile-based normalization. The slope was improved by acquiring data in MS3 mode, albeit at the expense of a 29% decrease in the number of identified proteins. FASP and in-solution sample preparation of CSF samples showed a 73% overlap in identified proteins. Finally, using optimized data normalization, we present a list of 64 biomarker candidates (clinical AD vs. controls, p < 0.01) identified in the EMIF-AD cohort.ConclusionWe have evaluated several analytical aspects of TMT proteomics in CSF. The results of our study provide practical guidelines to improve the accuracy of quantification and aid in the design of sample preparation and analytical protocol. The AD biomarker list extracted from the EMIF-AD cohort can provide a valuable basis for future biomarker studies and help elucidate pathogenic mechanisms in AD.

Detection and assessment of alpha-synuclein in Parkinson disease

PurposeDifferent studies have reported varying alpha-synuclein values in the cerebrospinal fluid (CSF), serum, and plasma, making determination of the alpha-synuclein cutoff value for Parkinson's disease difficult and rendering identifying the cause of variation essential. MethodWe searched PubMed from inception to June 2021 and identified 76 eligible studies. Included studies reported data on total, phosphorylated, and oligomeric alpha-synuclein in the CSF, serum, or plasma from individuals with Parkinson's disease and healthy controls. The mean or median alpha-synuclein values from the included studies were summarized and categorized through laboratory assays to visualize potential trends. ResultsThe enzyme-linked immunosorbent assay (ELISA) is the most common assay used to determine alpha-synuclein concentrations. Less common assays include Luminex, single molecule arrays, electrochemiluminescence, and immunomagnetic reduction (IMR). IMR is a single-antibody and wash-free immunoassay designed for determining the extremely low concentration of bio-molecules. For patients with Parkinson's disease, the median or mean testing values ranged from 60.9 to 55,000 pg/mL in the CSF, 0.446 to 1,777,100 pg/mL in plasma, and 0.0292 to 38,200,000 pg/mL in serum. The antibody selection was diverse between studies. The tendency of distribution was more centralized among studies that used the same kit. Studies adopting specific antibodies or in-house assays contribute to the extreme values. Only a few studies on phosphorylated and oligomeric alpha-synuclein were included. ConclusionThe type of assay and antibody selection in the laboratory played major roles in the alpha-synuclein variation. Studies that used the same assay and kit yielded relatively unanimous results. Furthermore, IMR may be a promising assay for plasma and serum alpha-synuclein quantification. A consensus on sample preparation and testing protocol unification is warranted in the future.

Sample preparation optimization of insects and zebrafish for whole-body mass spectrometry imaging.

Appropriate sample preparation is one of the most critical steps in mass spectrometry imaging (MSI), which is closely associated with reproducible and reliable images. Despite that model insects and organisms have been widely used in various research fields, including toxicology, drug discovery, disease models, and neurobiology, a systematic investigation on sample preparation optimization for MSI analysis has been relatively rare. Unlike mammalian tissues with satisfactory homogeneity, freezing sectioning of the whole body of insects is still challenging because some insect tissues are hard on the outside and soft on the inside, especially for some small and fragile insects. Herein, we systematically investigated the sample preparation conditions of various insects and model organisms, including honeybees (Apis cerana), oriental fruit flies (Bactrocera dorsalis), zebrafish (Danio rerio), fall armyworms (Spodoptera frugiperda), and diamondback moths (Plutella xylostella), for MSI. Three cutting temperatures, four embedding agents, and seven thicknesses were comprehensively investigated to achieve optimal sample preparation protocols for MSI analysis. The results presented herein indicated that the optimal cutting temperature and embedding agent were -20°C and gelatin, respectively, providing better tissue integrity and less mass spectral interference. However, the optimal thickness for different organisms can vary with each individual. Using this optimized protocol, we exploited the potential of MSI for visualizing the tissue-specific distribution of endogenous lipids in four insects and zebrafish. Taken together, this work provides guidelines for the optimized sample preparation of insects and model organisms, facilitating the expansion of the potential of MSI in the life sciences and environmental sciences.

Sample preparation protocol for wood and phloem formation analyses

This technical note and its corresponding video show the procedure for optimal sample preparation to perform wood and phloem formation analyses.

The use of surrogate standards as a QA/QC tool for routine analysis of microplastics in sewage sludge

The efficient retention of microplastic particles (MP) during wastewater treatment results in their accumulation in the sewage sludge. Thus, sewage sludge represents a key matrix for understanding MP flows between engineered and natural systems. Building on previous reports, we present a sample preparation protocol optimized for digested sewage sludge. The key steps include acid digestion supported by Fenton reagents, enzymatic digestion, and density separation using sodium polytungstate (density of 1.9 gcm−3). We use colored polyethylene (PE) spheres as surrogate standards to assess sample specific recoveries in terms of number and size, based on visible light (vis) microscopy and focal plane array - micro-Fourier transform - infrared (FPA-μ-FT-IR) imaging.The FT-IR spectra of common MP were identical before and after the digestion procedures and morphological changes were observed for polylactide fibers only. Average recovery rates for PE spheres, polypropylene fibers and polyethylene terephthalate fragments extracted from spiked digested sewage sludge and determined using (automated) vis microscopy ranged from 80% to 100%. Similar recovery rates of around 80% were also obtained for PE spheres based on FPA-μ-FT-IR measurements. The median diameters of red and blue PE spheres in dry state and recovered from spiked deionized water and from extracts of spiked digested sewage sludge determined using vis microscopy ranged between 46 μm and 67 μm. These diameters were similar to 54 μm and 61 μm obtained from the FPA-μ-FT-IR measurements of the corresponding deionized water samples and digested sludge extracts and in line with data from the producer (53 μm–63 μm). Using our digestion protocol in combination with surrogate standards, we measured MP number concentrations of around 10,000 #/g in dried, digested sewage sludge, in agreement with recent results from other studies.

A comprehensive review of wheat phytochemicals: From farm to fork and beyond.

The health benefits of whole wheat consumption can be partially attributed to wheat's phytochemicals, including phenolic acids, flavonoids, alkylresorcinols, carotenoids, phytosterols, tocopherols, and tocotrienols. It is of increasing interest to produce whole wheat products that are rich in bioactive phytochemicals. This review provides the fundamentals of the chemistry, extraction, and occurrence of wheat phytochemicals and includes critical discussion of several long-lasting issues: (1) the commonly used nomenclature on distribution of wheat phenolic acids, namely, soluble-free, soluble-conjugated, and insoluble-bound phenolic acids; (2) different extraction protocols for wheat phytochemicals; and (3) the chemistry and application of in vitro antioxidant assays. This review further discusses recent advances on the effects of genotypes, environments, field management, and processing techniques including ultrafine grinding, germination, fermentation, enzymatic treatments, thermal treatments, and food processing. These results need to be interpreted with care due to varied sample preparation protocols and limitations of in vitro assays. The bioaccessibility, bioavailability, metabolism, and potential health benefits of wheat phytochemicals are also reviewed. This comprehensive and critical review will benefit scientific researchers in the field of bioactive compounds of cereal grains and also those in the cereal food industry to produce high-quality functional foods.

Preparation and Characterization of Metastable trans-Dinitrogen Tetroxide.

Under atmospheric conditions, NO2 is in equilibrium with its dimers, N2O4, which can exist in the form of constitutional isomers and stereoisomers whose relative stabilities and reactivities are still being debated. Experimental limitations facing the spectroscopic characterization of the isomers of N2O4 prevent us from determining their relative contributions to reaction mechanisms possibly causing discrepancies in the reported reaction orders and rates. Using reflection-absorption infrared spectroscopy, molecular beam deposition, and matrix isolation techniques, it is shown that the relative abundances of NO2 and its dimers can be controlled by heating or cooling the deposited gas. The comparison of spectra acquired from samples prepared using molecular beam deposition with those obtained using tube dosing deposition demonstrates how the N2O4 isomer distributions are sensitive to details of the experimental conditions and sample preparation protocols. These observations not only provide a better understanding of a possible source for the disagreements found in the literature, but also a methodology to control and quantify the chemical speciation in NO2 vapors in terms of the relative abundances of NO2 and of the various isomers of N2O4.

Qualitative Screening of Volatile Thiols in Wine by Selective Silver Ion Solid-Phase Extraction with Heart-Cutting Multidimensional Gas Chromatography Mass Spectrometry/Olfactometry.

The chemical analysis of odorous volatile thiols is intrinsically challenging. Substantial progress has been made in quantitative analysis of targeted thiols at ultra-trace concentrations (ng/L), but lesser analytical attention has been given to the qualitative screening of unknown thiols. This work presents a solid-phase extraction (SPE) method using a silver ion (Ag+)-based sorbent to isolate volatile thiols from red wine. This proposed Ag+ SPE method was effective (recovery: 87-101% for four non-furan thiols and 35-49% for two furan thiols), simple, safe, and greatly reduced artifacts, testifying to its suitability as the sample preparation protocol for a qualitative screening experiment. Separation and detection were conducted using heart-cutting multidimensional gas chromatography coupled to mass spectrometry/olfactometry (H/C MDGC-MS/O). Key parameters including H/C width, main host oven temperature, and cryogenic trapping temperature were investigated for optimal instrument performance. The developed Ag+ SPE H/C MDGC-MS/O strategy was readily applicable for qualitative screening of odorous volatile thiols in wine, as demonstrated by two case studies.

Nafion Swelling in Salt Solutions in a Finite Sized Cell: Curious Phenomena Dependent on Sample Preparation Protocol.

When a membrane of Nafion swells in water, polymer fibers “unwind” into the adjoining liquid. They extend to a maximum of about ~300 μm. We explore features of Nafion nanostructure in several electrolyte solutions that occur when the swelling is constrained to a cell of size less than a distance of 300 μm. The constraint forces the polymer fibers to abut against the cell windows. The strongly amphiphilic character of the polymer leads to a shear stress field and the expulsion of water from the complex swollen fiber mixture. An air cavity is formed. It is known that Nafion membrane swelling is highly sensitive to small changes in ion concentration and exposure to shaking. Here we probe such changes further by studying the dynamics of the collapse of the induced cavity. Deionized water and aqueous salt solutions were investigated with Fourier IR spectrometry. The characteristic times of collapse differ for water and for the salt solutions. The dynamics of the cavity collapse differs for solutions prepared by via different dilution protocols. These results are surprising. They may have implications for the standardization of pharmaceutical preparation processes.

Requirements for accurate quantification of nitrate and nitrite in molasses: Insights from an interlaboratory comparison

The quantification of nitrate and nitrite in molasses is an emerging requirement for the sugar industry. The establishment of a validated analytical methodology is necessary to comply with the EU-guidelines on maximum permissible levels currently under discussion. In the present study, validation of an anion exchange chromatographic method to determine nitrate and nitrite in molasses was performed determining key validation parameters via several stages of an interlaboratory comparison for the first time. Since a stable, homogeneous sample material was necessary for establishing quantitative accuracy, a nitrite-spiked molasses material was prepared and evaluated independently across two laboratories in a parallel study. This preliminary study revealed that quantification of nitrate and nitrite content in molasses could be achieved with excellent precision under repeatability conditions of measurement (1.21% RSD for nitrate; 1.26% RSD for nitrite) in independent laboratories but requires preservation of samples at −20 °C for long-term (>several days) storage prior the analysis. A subsequent stage involving five different participating laboratories was then undertaken to evaluate anion exchange chromatography with conductivity detection for this application within a round robin study. The first phase focused on the analysis of nitrite-spiked molasses to optimize the individual analytical practices of laboratories and to elaborate a harmonized sample preparation protocol. In the second phase the participating laboratories analyzed seven molasses samples with different pH-value, dry substance, factory origin, with nitrate levels ranging from 2931 mg/kg – 6436 mg/kg and nitrite levels ranging from 25.3 mg/kg – 109 mg/kg. The determination of precision of reproducibility conditions of measurement revealed average relative interlaboratory standard deviations of 2.5% for nitrate and of 13% for nitrite. The present study clearly demonstrates the necessity of harmonization and standardization regarding measurement and data evaluation procedures applied for controlling nitrate and nitrite in molasses. • The challenging determination of nitrite with IC in molasses is addressed. • Storage of molasses at −20 °C is necessary to ensure sample stability. • Interlaboratory comparison with five laboratories is performed. • Harmonization and standardization of methods are identified to be necessary.