A radial calibration window for analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first-principles based method for studying macromolecules and particles in solution by monitoring the evolution of their radial concentration distribution as a function of time in the presence of a high centrifugal field. In sedimentation velocity experiments, hydrodynamic properties relating to size, shape, density, and solvation of particles can be measured, at a high hydrodynamic resolution, on polydisperse samples. In a recent multilaboratory benchmark study including data from commercial analytical ultracentrifuges in 67 laboratories, the calibration accuracy of the radial dimension was found to be one of the dominant factors limiting the accuracy of AUC. In the present work, we develop an artifact consisting of an accurately calibrated reflective pattern lithographically deposited onto an AUC window. It serves as a reticle when scanned in AUC control experiments for absolute calibration of radial magnification. After analysis of the pitch between landmarks in scans using different optical systems, we estimate that the residual uncertainty in radial magnification after external calibration with the radial scale artifact is ≈0.2 %, of similar magnitude to other important contributions after external calibration such as the uncertainty in temperature and time. The previous multilaboratory study had found many instruments with errors in radial measurements of 1 % to 2 %, and a few instruments with errors in excess of 15 %, meaning that the use of the artifact developed here could reduce errors by 5-to 10-fold or more. Adoption of external radial calibration is thus an important factor for assuring accuracy in studies related to molecular hydrodynamics and particle size measurements by AUC.

Stability of serum, plasma and urine osmolality in different storage conditions: Relevance of temperature and centrifugation.

BACKGROUND: Osmolality reflects the concentration of all dissolved particles in a body fluid, and its measurement is routinely performed in clinical laboratories for the differential diagnosis of disorders related with the hydrolytic balance regulation, the renal function and in small-molecule poisonings. The aim of the study was to assess the stability of serum, plasma and urine osmolality through time and under different common storage conditions, including delayed centrifugation. METHODS: Blood and urine samples were collected, and classified into different groups according to several preanalytical variables: serum or plasma lithium-heparin tubes; spun or unspun; stored at room temperature (RT), at 4°C or frozen at -21°C. Aliquots from each group were assayed over time, for up to 14days. Statistical differences were based on three different international performance criteria. RESULTS: Whole blood stability was higher in the presence of anticoagulant. Serum osmolality was stable for 2days at RT and 8days at 4°C, while plasma was less stable when refrigerated. Urine stability was 5days at RT, 4days at 4°C and >14days when frozen. DISCUSSION: Osmolality may be of great interest for the management of several conditions, such as in case of a delay in the clinical suspicion, or in case of problems in sample collection or processing. The ability to obtain reliable results for samples kept up to 14days also offers the possibility to retrospectively assess baseline values for patients which may require it.

[Biases on community structure during DNA extraction of shrimp intestinal microbiota revealed by high-throughput sequencing].

OBJECTIVES: High-throughput sequencing technology is increasingly applied in intestinal microbiota of aquatic animals including shrimp. However, there is a lack of standard method or kit for DNA isolation from shrimp intestinal microbiota, and little is known about the effectiveness and biases regarding DNA extraction based on high-throughput sequencing. The aim of this study was to study the biases of different DNA extraction kits on community structure of shrimp intestinal microbiota through high-throughput sequencing, and to better understand the structure and composition of bacterial flora associated with healthy Litopenaeus vannamei. METHODS: We extracted the total DNA of intestinal microbiota from L. vannamei with three commercial kits designed for DNA extraction from bacteria, stool and tissue (Omega, USA). DNA quality was evaluated based on the absorbance ratios of 260/280 nm by NanoDrop, while DNA concentration was quantified using PicoGreen. Then Illumina MiSeq high-throughput sequencing was used to examine the intestinal bacterial communities following PCR amplification of 16S rDNA V4 region. RESULTS: The yield and purity of the DNA from the Bacterial Kit (SIB) were superior to those from the Stool Kit (SIS), whereas the DNA from Tissue Kit (SIT) presented too small amount to be amplified efficiently. The average sequence reads obtained from SIB and SIS samples were 52151 ± 5085 and 55296 ± 5147 respectively. After resampling at the same depth of 46800 reads, the operational taxonomic unit (OTU) number and Shannon diversity index of SIS samples were significantly higher than those of SIB samples. By contrast, the reproducibility of OTU among SIB replicates was higher than that among SIS replicates. The dominant phyla of SIS and SIB samples were identical, including Proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes, Actinobacteria, and Cyanobacteria. However, the relative abundances of almost all the dominant groups at various taxonomic levels differed greatly between these two samples. CONCLUSION: Significant biases on community structure of shrimp intestinal microbiota were detected which originated from DNA extraction. And the core microbiota of the healthy L. vannamei in this study was mainly composed of genera Photobacterium, Lactococcus, Aliivibrio, Vibrio, as well as three other unclassified groups.

Biofluid infrared spectro-diagnostics: pre-analytical considerations for clinical applications.

Several proof-of-concept studies on the vibrational spectroscopy of biofluids have demonstrated that the methodology has promising potential as a clinical diagnostic tool. However, these studies also show that there is a lack of a standardised protocol in sample handling and preparation prior to spectroscopic analysis. One of the most important sources of analytical errors is the pre-analytical phase. For the technique to be translated into clinics, it is clear that a very strict protocol needs to be established for such biological samples. This study focuses on some of the aspects of the pre-analytical phase in the development of the high-throughput Fourier Transform Infrared (FTIR) spectroscopy of some of the most common biofluids such as serum, plasma and bile. Pre-analytical considerations that can impact either the samples (solvents, anti-coagulants, freeze-thaw cycles…) and/or spectroscopic analysis (sample preparation such as drying, deposit methods, volumes, substrates, operators dependence…) and consequently the quality and the reproducibility of spectral data will be discussed in this report.

Effects of acidification, lipid removal and mathematical normalization on carbon and nitrogen stable isotope compositions in beaked whale (Ziphiidae) bone.

RATIONALE: The analysis of stable isotopes in tissues such as teeth and bones has been used to study long-term trophic ecology and habitat use in marine mammals. However, carbon isotope ratios (δ(13) C values) can be altered by the presence of (12) C-rich lipids and carbonates. Lipid extraction and acidification are common treatments used to remove these compounds. The impact of lipids and carbonates on carbon and nitrogen isotope ratios (δ(15) N values), however, varies among tissues and/or species, requiring taxon-specific protocols to be developed. METHODS: The effects of lipid extraction and acidification and their interaction on carbon and nitrogen isotope values were studied for beaked whale (Ziphiidae) bone samples. δ(13) C and δ(15) N values were determined in quadruplicate samples: control, lipid-extracted, acidified and lipid-extracted followed by acidification. Samples were analyzed by means of elemental analysis isotope ratio mass spectrometry. Furthermore, the efficiency of five mathematical models developed for estimating lipid-normalized δ(13) C values from untreated δ(13) C values was tested. RESULTS: Significant increases in δ(13) C values were observed after lipid extraction. No significant changes in δ(13) C values were found in acidified samples. An interaction between both treatments was demonstrated for δ(13) C but not for δ(15) N values. No change was observed in δ(15) N values for lipid-extracted and/or acidified samples. Although all tested models presented good predictive power to estimate lipid-free δ(13) C values, linear models performed best. CONCLUSIONS: Given the observed changes in δ(13) C values after lipid extraction, we recommend a priori lipid extraction or a posteriori lipid normalization, through simple linear models, for beaked whale bones. Furthermore, acidification seems to be an unnecessary step before stable isotope analysis, at least for bone samples of ziphiids. Copyright © 2015 John Wiley & Sons, Ltd.

Sample normalization methods in quantitative metabolomics.

To reveal metabolomic changes caused by a biological event in quantitative metabolomics, it is critical to use an analytical tool that can perform accurate and precise quantification to examine the true concentration differences of individual metabolites found in different samples. A number of steps are involved in metabolomic analysis including pre-analytical work (e.g., sample collection and storage), analytical work (e.g., sample analysis) and data analysis (e.g., feature extraction and quantification). Each one of them can influence the quantitative results significantly and thus should be performed with great care. Among them, the total sample amount or concentration of metabolites can be significantly different from one sample to another. Thus, it is critical to reduce or eliminate the effect of total sample amount variation on quantification of individual metabolites. In this review, we describe the importance of sample normalization in the analytical workflow with a focus on mass spectrometry (MS)-based platforms, discuss a number of methods recently reported in the literature and comment on their applicability in real world metabolomics applications. Sample normalization has been sometimes ignored in metabolomics, partially due to the lack of a convenient means of performing sample normalization. We show that several methods are now available and sample normalization should be performed in quantitative metabolomics where the analyzed samples have significant variations in total sample amounts.

The application of fuzzy statistics and linear discriminant analysis as criteria for optimizing the preparation of plasma for matrix-assisted laser desorption/ionization mass spectrometry peptide profiling.

An alternative bioinformatics approach based on fuzzy theory statistics and linear discriminant analysis is proposed for the interpretation of MALDI MS spectra in peptide profiling. When applied, the methodology enables the establishment of a reproducible plasma preparation protocol appropriate for the evaluation of small data sets. The samples were collected from pregnant women affected by gestational diabetes mellitus (GDM), n=18 and control group, n=13. The following pre-treatment sets were tested: pipette tips with C18 stationary phase (ZipTip, Millipore and Omix, Agilent) and magnetic bead-based weak cation exchange chromatography kit (MB WCX, Bruker Daltonics). The spectra were recorded using a MALDI TOF mass spectrometer (UltrafleXtreme, Bruker Daltonics) for a mass range of m/z from 1000 to 10,000. The significant features were selected using the wrapper selection method, and two classification systems were tested: discriminant analysis (DA) and fuzzy inference system (FIS). ClinProTools software was employed to compare the usefulness of the proposed methodology. The study showed that the optimum results for MS spectra were obtained after the use of the ZipTip as pre-treatment method in plasma preparation. Chemometric analysis allowed the differentiation of the GDM group from the control with a high degree of accuracy: 0.7333 (DA) and 0.8065 (FIS).

Sampling and sample processing in pesticide residue analysis.

Proper sampling and sample processing in pesticide residue analysis of food and soil have always been essential to obtain accurate results, but the subject is becoming a greater concern as approximately 100 mg test portions are being analyzed with automated high-throughput analytical methods by agrochemical industry and contract laboratories. As global food trade and the importance of monitoring increase, the food industry and regulatory laboratories are also considering miniaturized high-throughput methods. In conjunction with a summary of the symposium "Residues in Food and Feed - Going from Macro to Micro: The Future of Sample Processing in Residue Analytical Methods" held at the 13th IUPAC International Congress of Pesticide Chemistry, this is an opportune time to review sampling theory and sample processing for pesticide residue analysis. If collected samples and test portions do not adequately represent the actual lot from which they came and provide meaningful results, then all costs, time, and efforts involved in implementing programs using sophisticated analytical instruments and techniques are wasted and can actually yield misleading results. This paper is designed to briefly review the often-neglected but crucial topic of sample collection and processing and put the issue into perspective for the future of pesticide residue analysis. It also emphasizes that analysts should demonstrate the validity of their sample processing approaches for the analytes/matrices of interest and encourages further studies on sampling and sample mass reduction to produce a test portion.

Interlaboratory assessment of cryomilling sample preparation for residue analysis.

The effectiveness of the comminution approach used for bulk field samples limits the size of the subsample that must be extracted and analyzed to ensure an adequately representative and reproducible measurement. In many cases this subsample size restricts the residue method to the use of larger vessel formats, limiting downstream throughput. The introduction of a secondary fine-milling step to this process using a subsample size already known to be representative can further improve sample homogeneity and allow direct method scaling to small high-throughput formats. Dramatic increases in method throughput can then be achieved through the simultaneous processing of numerous samples in parallel. This approach was evaluated across a diverse grouping of crop matrices using two substantially different pesticide types. Both fortified and field-collected samples demonstrated a high degree of precision and reproducibility across laboratories. Additional benefits of this approach include significant reductions in cost and solvent waste generation, as well as improvements in assay quality and transferability.

Estimation of sampling uncertainty of pesticide residues based on supervised residue trial data.

Typical sampling uncertainties were calculated as the average of relative standard deviations (CV) of residues measured in individual crops tested in supervised residue trials and from their pooled variance for crop groups. The relative confidence intervals of the sampling uncertainty for different crops were estimated from the random duplicate composite samples generated with computer modeling from residues in 182 independent primary sample sets, each consisting of 100-320 residue data. The relative 95% confidence intervals were found to be independent from the CV of primary residue data populations; therefore, the calculated values are generally applicable. In view of the potentially serious consequences of underestimated sampling uncertainties, their upper confidence limits are recommended for practical use to verify the compliance of products and for planning statistically based sampling programs. Sampling uncertainties are reported for 24 crop groups and 106 individual crops.

Development of the performance reference compound approach for the calibration of "polar organic chemical integrative sampler" (POCIS).

The purpose of the Water Framework Directive is to ensure the quality of the natural water across Europe. In this context, passive samplers have shown interesting capacities for the monitoring of contaminants in aqueous ecosystems. They allow the measurement of time-weighted average concentrations, overcoming many drawbacks of the spot-sampling techniques known to be expensive and time consuming. However, application of passive samplers such as polar organic chemical integrative samplers (POCIS) for the monitoring of hydrophilic contaminants requires calibration to define compound sampling rates; key parameters to deduce the pollutant water concentrations from the amounts of pollutants accumulated by the device. Unfortunately, sampling rates are influenced by a range of environmental factors; in that respect, a question remains: is it not evident to know to what extent the sampling rates obtained in laboratory experiments can be used in field conditions? The problem can be solved for hydrophobic samplers by using performance reference compounds (PRCs), and an ongoing challenge for POCIS is focused on the improvement of the quantitative aspect of this family of samplers. In this study, potential PRCs have been selected during a specific experiment and their performance was tested in the laboratory under two hydrodynamic conditions. Results revealed a good proportionality between elimination rates of PRCs and sampling rates of chemicals. Afterwards, the application of the approach under environmental conditions was assessed by deploying POCIS in the Arcachon Bay (France) where POCIS-PRC-derived water concentrations appear to be close to the simultaneous grab-sampling results.

Optimization of a novel setup for an on-line study of elemental mercury adsorption by cold-vapor atomic absorption spectrometry.

BACKGROUND: The objective of this work was developing a simple and stable time-based on-line setup for assessing the potential of mercury (Hg) vapor adsorption of the commercial sorbents used in air sampling and control operation followed by cold vapor atomic absorption spectrometry (CVAAS). METHODS: A special designed separation chamber was used where reduction of the injected Hg (II) solution took place. Purge gas passes through this chamber resulting to a prompt release of mercury vapor, purging into the adsorbent that regulated at the desired adsorption temperature. After sorbent saturation, in order to study the adsorption parameters of sorbents (activated carbon and bone char) such as breakthrough time (BTT), and adsorptive capacity, mercury gas stream was passed through the sorbents, directly transport to the CVAAS. RESULTS: Preliminary experiments concerning the reductant solution showed that SnCl2 offers higher stability than NaBH4. Around the loading range 0.125-2.5 ml min⁻¹ of 100 µg l⁻¹ Hg(II) solution, a linear calibration curve with the equation peak area=0.134; loading flow=-0.017 and a correlation coefficient r=0.996 was obtained, and the detection limit was improved up to c(L)=1 µg l⁻¹. The relative standard deviation of five measurements of lowest flow loading of Hg (II) was RSD=2.8%. The significant differences were observed in the breakthrough time and mercury adsorptive capacity between activated carbon and bone char (P=0.010). CONCLUSION: This novel setup is suitable for an on-line study of elemental mercury adsorption, determination of breakthrough time and adsorption capacity, and because of its stable performance during all experiments; it can be applied to the time based studies.

The use of a xylosylated plant glycoprotein as an internal standard accounting for N-linked glycan cleavage and sample preparation variability.

RATIONALE: Traditionally, free oligosaccharide internal standards are used to account for variability in glycan relative quantification experiments by mass spectrometry. However, a more suitable internal standard would be a glycoprotein, which could also control for enzymatic cleavage efficiency, allowing for more accurate quantitative experiments. METHODS: Hydrophobic, hydrazide N-linked glycan reagents (both native and stable-isotope labeled) are used to derivatize and differentially label N-linked glycan samples for relative quantification, and the samples are analyzed by a reversed-phase liquid chromatography chip system coupled online to a Q-Exactive mass spectrometer. The inclusion of two internal standards, maltoheptaose (previously used) and horseradish peroxidase (HRP) (novel), is studied to demonstrate the effectiveness of using a glycoprotein as an internal standard in glycan relative quantification experiments. RESULTS: HRP is a glycoprotein containing a xylosylated N-linked glycan, which is unique from mammalian N-linked glycans. Thus, the internal standard xylosylated glycan could be detected without interference to the sample. Additionally, it was shown that differences in cleavage efficiency can be detected by monitoring the HRP glycan. In a sample where cleavage efficiency variation is minimal, the HRP glycan performs as well as maltoheptaose. CONCLUSIONS: Because the HRP glycan performs as well as maltoheptaose but is also capable of correcting and accounting for cleavage variability, it is a more versatile internal standard and will be used in all subsequent biological studies. Because of the possible lot-to-lot variation of an enzyme, differences in biological matrix, and variable enzyme activity over time, it is a necessity to account for glycan cleavage variability in glycan relative quantification experiments.

Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.

Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-α-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard.

Platelet-stored angiogenesis factors: clinical monitoring is prone to artifacts.

BACKGROUND: The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing. METHODS: Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated. RESULTS: Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF-4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P=0.006; P< 0.001). CONCLUSION: The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.