Standard Sample Preparation Research Articles

Automatization of metabolite extraction for high-throughput metabolomics: case study on transgenic isoprene-emitting birch.

Metabolomics studies are becoming increasingly common for understanding how plant metabolism responds to changes in environmental conditions, genetic manipulations and treatments. Despite the recent advances in metabolomics workflow, the sample preparation process still limits the high-throughput analysis in large-scale studies. Here, we present a highly flexible robotic system that integrates liquid handling, sonication, centrifugation, solvent evaporation and sample transfer processed in 96-well plates to automatize the metabolite extraction from leaf samples. We transferred an established manual extraction protocol performed to a robotic system, and with this, we show the optimization steps required to improve reproducibility and obtain comparable results in terms of extraction efficiency and accuracy. We then tested the robotic system to analyze the metabolomes of wild-type and four transgenic silver birch (Betula pendula Roth) lines under unstressed conditions. Birch trees were engineered to overexpress the poplar (Populus × canescens) isoprene synthase and to emit various amounts of isoprene. By fitting the different isoprene emission capacities of the transgenic trees with their leaf metabolomes, we observed an isoprene-dependent upregulation of some flavonoids and other secondary metabolites as well as carbohydrates, amino acid and lipid metabolites. By contrast, the disaccharide sucrose was found to be strongly negatively correlated to isoprene emission. The presented study illustrates the power of integrating robotics to increase the sample throughput, reduce human errors and labor time, and to ensure a fully controlled, monitored and standardized sample preparation procedure. Due to its modular and flexible structure, the robotic system can be easily adapted to other extraction protocols for the analysis of various tissues or plant species to achieve high-throughput metabolomics in plant research.

Study of microstructural, chemical and microhardness characterization of stainless steels

Today it is almost impossible to find any area of human activity in which metal would not be present and would be closely related to development. Therefore, it is necessary to know its properties and what kind of media will be used in order to select the best type of material to use. This study examines some of the various austenitic stainless steels in terms of composition, microstructure, and hardness. For these analyses, it was necessary to follow a series of standard sample preparation procedures that directly affect the exact result, microstructure, and hardness. The percentage of chromium varied from 17 to 18%, which gives the material good passivity. Molybdenum in grades 304 and 304L is presented as a residual content, and in grades 316 and 316L - as an alloying element. Among all the studied materials, AISI 304 showed the highest hardness. The relationship between the alloy composition, microstructure and hardness, and the specific properties of each steel is discussed.

Assessing a potential to improve data quality for accelerated identification of microorganisms derived from positive blood cultures

Diagnostics of blood-borne infections is still an urgent problem of modern medicine. The main causative agents of septic conditions are gram-positive microorganisms particularly Staphylococcus aureus, enterococci, etc., whereas clinical significance of isolated coagulase-negative staphylococci is ambiguous. Escherichia coli, Klebsiella pneumoniae and other enterobacteria, as well as Acinetobacter baumannii prevail among the gram-negative flora. Modern possibilities of accelerated identification of microorganisms derived from positive blood cultures based on mass-spectrometry consist of two approaches. Firstly, the manufacturers developed consumables for mass spectrometry are proposed, and secondly, there are domestic developments of accelerated sample preparation protocols developed by microbiological laboratories. The approaches used have a number of advantages and disadvantages, but to summarize, the use of the proposed methods in routine practice is quite limited. At the same time, the need to accelerate the issuing a microbiological conclusion related to nosology is great being associated with improved outcomes. In this regard, the aim of the study was to evaluate convergence and accuracy of results for accelerated identification of microorganisms derived from positive blood cultures in blood-borne infections. The study included 87 positive blood cultures, the identification of pathogens from them occurred in four ways: the classical microbiological analysis of blood for sterility, pathogen identification directly from the vial without isolating a pure culture, as well as two sample preparation methods based on ethylenediaminetetraacetic acid (EDTA) and potassium ethylenediaminetetraacetate disubstituted (EDTA-K2) as wash additives. It was found that gram-positive and gram-negative flora were isolated from the blood almost evenly often. When evaluating an influence of biomaterial used for mass spectrometry, it turned out that use of wash additives increases chances of successful identification of bacteria from a blood sample. The influence of tinctorial properties on the results of determining the species assignment of isolates was also evaluated. Identification of gram-positive flora is more accurate, since some pathogens were not identified without washing additives, and when using EDTA-K2 and the corresponding acid, assignment to the genus was obtained in the same samples. A similar pattern was also characteristic of gram-negative flora. At the same time, modern manufacturers of laboratory equipment and reagents allow to standardize sample preparation procedures in the protocols used. The effects of EDTA-K2, which allowing to use it as a washing component, are associated with the binding of calcium and magnesium ions in solution, which reduces the adhesion of bacterial cells to blood cells, thereby contributing to better mass spectrometry of microbial sediments with accelerated identification of microorganisms from positive blood cultures. Thus, use of the described additives can provide high quality, timely and adequate diagnostics of serious and life-threatening conditions such as blood-borne infections.

Proteome Dynamics During Transition From Exponential to Stationary Phase Under Aerobic and Anaerobic Conditions in Yeast

The yeast Saccharomyces cerevisiae is a widely-used eukaryotic model organism and a promising cell factory for industry. However, despite decades of research, the regulation of its metabolism is not yet fully understood, and its complexity represents a major challenge for engineering and optimizing biosynthetic routes. Recent studies have demonstrated the potential of resource and proteomic allocation data in enhancing models for metabolic processes. However, comprehensive and accurate proteome dynamics data that can be used for such approaches are still very limited. Therefore, we performed a quantitative proteome dynamics study to comprehensively cover the transition from exponential to stationary phase for both aerobically and anaerobically grown yeast cells. The combination of highly controlled reactor experiments, biological replicates, and standardized sample preparation procedures ensured reproducibility and accuracy. In addition, we selected the CEN.PK lineage for our experiments because of its relevance for both fundamental and applied research. Together with the prototrophic standard haploid strain CEN.PK113-7D, we also investigated an engineered strain with genetic minimization of the glycolytic pathway, resulting in the quantitative assessment of 54 proteomes. The anaerobic cultures showed remarkably less proteome-level changes compared with the aerobic cultures, during transition from the exponential to the stationary phase as a consequence of the lack of the diauxic shift in the absence of oxygen. These results support the notion that anaerobically growing cells lack resources to adequately adapt to starvation. This proteome dynamics study constitutes an important step toward better understanding of the impact of glucose exhaustion and oxygen on the complex proteome allocation process in yeast. Finally, the established proteome dynamics data provide a valuable resource for the development of resource allocation models as well as for metabolic engineering efforts.

222Rn calibration procedure for water analyses by liquid scintillation counting

PurposeAmong the several methods to measure 222Rn in water, the liquid scintillation counting (LSC) technique is one of the most sensitive and widely used when analysing non-saline waters by liquid extraction, providing excellent accuracy, precision and low-level detection limits. When using the liquid scintillation counter Tri-Carb 3170 TR/SL (Perkin-Elmer) it is not possible to make an automatic determination of the alpha/beta discrimination parameter, being necessary to evaluate the pulse decay discriminator (PDD) value manually to the 222Rn determination. In this work, we describe the steps to perform the calibration of that type of equipment.MethodsThe method consists in the preparation of standard samples from a 226Ra certified standard solution and the adjustment of the PDD value by hand until the percentage of alpha events reaches a plateau of a near-constant fraction of total events. The process consists in taking several measures between PDD 80 and 200 with 15 min counting time each. Within the plateau, the alpha/beta ratio was used to determine the plateau final range as well as the optimum PDD value. After that, the calibration standards and blanks were measured and the region of interest (ROI) was established by finding the maximum value for the figure of merit (FOM) in the upper and lower limits.ResultsThe optimal PDD value of 148 was established by the alpha/beta ratio, and after that, it was possible to measure 226Ra standards to perform the calibration. An efficiency of (9.758±0.035) × 10–1 was obtained. In routine measurements, a detection limit of about 0.50 Bq L–1 is achieved for a 120 min counting time. To evaluate the uncertainty budget, the uncertainties of the net count rate, the efficiency, the sample amount and the decay correction factor were considered.ConclusionsA method was developed for the calibration of LSC Tri-Carb 3170 TR/SL with the optimization of the PDD using a 226Ra standard solution. The acquisition to obtain the plot of the variation of the events fraction in each window (alpha and beta) in function of PDD allowed to evaluate a plateau where the fraction of events was at a minimum. Afterwards, it was possible to measure the set of 226Ra standards after the equilibrium with 222Rn and determine the efficiency for 222Rn with a detection limit of about 0.50 Bq L–1 in routine analyses.

Wide-gamut structural colours on oakblue butterflies by naturally tuned photonic nanoarchitectures.

The iridescent structural colours of butterflies, generated by photonic nanoarchitectures, often function as species-specific sexual signals; therefore, they are reproduced precisely from generation to generation. The wing scales of oakblue hairstreak butterflies (genus Arhopala, Theclinae, Lycaenidae, Lepidoptera) contain multi-layer photonic nanoarchitectures, which can generate a wide range of structural colours, from violet to green. By scanning (SEM) and cross-sectional transmission electron microscopy (TEM) investigation, the colour tuning mechanism of the cover scales was explored. We revealed that the characteristic size change of structural elements in similar photonic nanoarchitectures led to different structural colours that were examined by various reflectance spectrophotometry techniques. The measured structural properties of the naturally tuned photonic nanoarchitectures were used to calculate wing reflectances, which were compared with the measurement results. We found that the simulated structural colours were systematically redshifted by 95-126 nm as compared with the measured normal-incidence reflectance results. This is attributed to the swelling of the chitinous multi-layer structures during the standard TEM sample preparation and the tilt of the cover scales, which both affect the apparent layer thicknesses in the TEM cross-sections. We proposed a simulation correction and compared the results with the layer thicknesses measured on cryogenically prepared non-embedded SEM cross-sections.

Optimization of sample preparation conditions in the determination of total arsenic in fish and seafoods by atomic absorption spectrometry with electrothermal atomization

The conditions for sample preparation and determination of total arsenic in fish and seafoods by atomic absorption spectroscopy with electrothermal atomization have been optimized. When using a standard sample preparation procedure (GOST R 53100), a complete decomposition of the organic arsenic compound, arsenobetaine, appeared impossible which leads to underestimated results. A novel sample preparation procedure for the determination of total arsenic, including the addition of concentrated sulfuric acid for complete decomposition of the organic forms of arsenic to inorganic ones is proposed. During the atomization of the sample, an additional stage of drying was introduced with smooth heating from 200 to 250°C, which contributed to a complete removal of sulfuric acid residues from the injected sample volume. The correctness of the proposed methodology was verified using the ICP-MS method and in the analysis of reference samples of fish and seafoods.

Morphofunctional changes in human skin with acneiform dermatoses

The aim of this study was to study the morphofunctional changes in human skin with acneiform dermatoses.Material and methods. We studied the material obtained from 6 volunteers of both sexes aged 26 to 64 years. All subjects had signs of acneiform dermatoses. Pathologically changed areas were studied in comparison with normal skin in the same patient. The subjects were anesthetized and a piece of skin 3×3 mm2 in size was taken with the punch biopsy. After standard histological sample preparation, sections were stained with hematoxylin and eosin. The counting and morphometry of skin cell structures was carried out, the presence of lymphocytes, neutrophils, eosinophils, erythrocytes, vacuolized keratinocytes and their remnants in 6 fields of view in 3 histological slides from each patient was determined from each patient under an Axioskop 2 microscope with a ToupCam TP108500A camera and ToupView software (ToupTek , China). The criterion for the degree of destructive changes in the collagen fibers of the dermis was the determination of their thickness.Results. The morphological structure of the skin in acneiform dermatosis is characterized by a significant increase in the thickness of the spinous (93,7 [82,30; 96,35] microns), granular (28,4 [22,48; 34,44] microns) and corneum (37,8 [25,32; 46,24] microns) layers of the epidermis compared to the norm (66,1 [58,30; 69,42] microns; 6,2 [5,96; 6,63] microns; 10,6 [9,81; 11,26] microns, respectively). Near the sebaceous-follicular complex, inflammatory lymphohistiocytic infiltrates were found in the dermis, represented by lymphocytes (9,5±2,2 units p.f.v.), neutrophils (2,2±0,8 units p.f.v.), eosinophils (17,2±4,0 units p.f.v.), erythrocytes outside the vessels (6,8±1,1 units p.f.v.), vacuolated keratinocytes (22,1±5,7 units p.f.v.) and keratinocyte remnants (15,1±2,4 units p.f.v.) . The number of cells in the infiltrate significantly differed from the norm. On histological preparations of patients with acneiform dermatosis, thinning of the collagen fibers of the dermis (6,0 [4,79; 8,13] microns) was observed compared to unaffected skin (10,4 [10,01; 10,67] microns).Conclusions. Morphological changes in the skin with acneiform dermatosis are manifested by hypertrophic processes of the epidermis, thinning of the collagen fibers of the papillary dermis, the presence of leukocyte infiltrate and a violation of the integrity of the basement membrane of the sebaceous glands. These changes can lead to disruption of the integrity and functioning of the histo-hematic barrier of the skin and an increase in the area of spread of the inflammatory process.

ТИНКТОРИАЛЬНЫЕ И МОРФОЛОГИЧЕСКИЕ ХАРАКТЕРИСТИКИ МИКРОБНЫХ ОБЪЕКТОВ ДЛЯ ФОРМИРОВАНИЯ ЭЛЕКТРОННОГО АТЛАСА В СИСТЕМЕ АВТОМАТИЗИРОВАННОГО АНАЛИЗА

The authors present the results of automated analysis of gram positive and gram negative cocci and bacilli (Staphylococcus aureus, Neisseria spp., Corynebacterium spp., Escherichia coli) for a database that facilitates detection of changes in normal microflora and diagnosis of disbiotic conditions of various human biotopes. Following the standard sample preparations and Gram staining, microbial smears were investigated using an automated and computerized analyzer developed by the Mintz Radio Engineering Institute in Moscow. Morphological and tinctorial analysis established significant differences in cell area, perimeter and limit. Significant differences in tinctorial properties of microbial cells make possible image research using the RGB and HSV color spaces. Automated analysis enables identification of microbial cells simultaneously by digital images of morphological and color/brightness parameters. The proposed techniques can be helpful in constructing an electronic microflora atlas of various human biotopes.

Development and Validation of an LC-MS/MS Method for the Quantitative Determination of Contezolid in Human Plasma and Cerebrospinal Fluid

To develop and verify a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining contezolid in plasma and cerebrospinal fluid (CSF). Protein precipitation was performed on samples using linezolid as the internal standard. We used an Agilent EclipsePlus C18 column operating at 0.4 mL/min in conjunction with acetonitrile and water mobile phases for the LC-MS/MS analysis. Using the precursor-product ion pairs 409.15→269.14 (contezolid) and 338.14→195.1 (linezolid), multiple reaction monitoring was used to quantify the compounds. Plasma linearity range was 50.0 to 5000 ng/mL, and CSF was 20.0 to 1000 ng/mL (r2 = 0.999). The inter-batch and intra-batch precisions were ≤2.57% and ≤5.79%, respectively. Plasma recovered 92.94%, and CSF recovered 97.83%. Plasma, CSF, hemolytic plasma, and hyperlipidemic plasma all showed a coefficient of variation ≤ 7.44%. The stability and dilution integrity of this method were also acceptable. The study also demonstrated that artificial CSF can be used as a matrix for the preparation of standard curve samples. A simple and accurate method was developed and validated for the determination of contezolid concentrations in human plasma and CSF, which may be useful for monitoring the therapeutic effect of central nervous system medications.

A skew in poo: Biases in primate fecal isotope analysis and recommendations for standardized sample preparation.

Feces are a treasure trove in the study of animal behavior and ecology. Stable carbon and nitrogen isotope analysis allows to assessthe dietary niches of elusive primate species and primate breastfeeding behavior. However, some fecal isotope data may unwillingly be biased towardthe isotope ratios of undigested plant matter, requiring more consistent sample preparation protocols. We assess the impact of this potential data skew in 114 fecal samples of wild bonobos (Pan paniscus) by measuring the isotope differences (Δ13 C, Δ15 N) between bulk fecal samples containing larger particles (>1 mm) and filtered samples containing only small particles (<1 mm). We assess the influence of fecal carbon and nitrogen content (ΔC:N) and sample donor age (subadult, adult) on the resulting Δ13 C, Δ15 N values (n = 228). Additionally, we measure the isotope ratios in three systematically sieved fecal samples of chimpanzees (Pan troglodytes verus), with particle sizes ranging from 20 μm to 8 mm (n = 30). We found differences in fecal carbon and nitrogen content, with the smaller fecal fraction containing more nitrogen on average. While the Δ13 C values were small and not affected by age or ΔC:N, the Δ15 N values were significantly influenced by fecal ΔC:N, possibly resulting from the differing proportions of undigested plant macroparticles. Significant relationships between carbon stable isotope ratios (δ13 C) values and %C in large fecal fractions of both age groups corroborated this assessment. Δ15 N values were significantly larger in adults than subadults, which should be of concern in isotope studies comparing adult females with infants to assess breastfeeding. We found a random variation of up to 3.0‰ in δ13 C and 2.0‰ in nitrogen stable isotope ratios within the chimpanzee fecal samples separated by particle sizes. We show that particle size influences isotope ratios and propose a simple, cost-effective filtration method for primate feces to exclude larger undigested food particles from the analysis, which can easily be adopted by labs worldwide.

Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution

Biological systems are the sum of their dynamic three-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these methods require specialized equipment and personnel to complete them. Here, we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution, focusing on Arabidopsis clathrin-coated vesicles (CCVs). While CCVs are essential trafficking organelles, their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments. First, we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this method in any electron microscopy lab. Secondly, we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis method to screen the “pseudo 3D” morphologies of CCVs imaged with 2D modalities. Collectively, our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.

Estimation of mode I static fracture toughness of carbonate rock using small rock fragments

Fracture toughness is the key to analysis of failure progress, which can be defined under different stress conditions as fracture toughness modes. The mode I fracture toughness (KIC) of rock is an important parameter in petroleum and reservoir engineering; however, preparation of standard samples for determination of this parameter is costly and time consuming and is not feasible under all conditions. At times, core samples are not available for such destructive tests. In such situations, the use of small rock fragments for measurement or estimation of the mechanical properties of intact rock can be a practical method. The present study evaluated the single compressive strength test (SCS) as an indirect method for estimating KIC using small rock fragments. Spherical carbonate rock samples of 2–10 mm in diameter from the Asmari Formation were produced from rock blocks and were tested. The behavior of each was studied and the fracture mechanisms were determined for calculation of the amount of stress (σSCS) at the failure point. Semicircular bending tests were used to determine the KIC for each sample and regression models were established between σSCS and KIC where R2 ≥ 0.95. Of the 16 samples studied, 10 were used to develop a regression model and six were used for validation. It was found that the regression models were able to estimate KIC with good accuracy. To evaluate the best model more accurately, the error of estimation of each model was calculated and it was found that the linear and exponential correlations had the lowest errors of estimation. Based on the range of estimated values, linear correlation was selected as the best correlation method. A correlation was made between all σSCS and KIC values for the carbonate rock samples using the linear regression model where R2 = 0.91 and a final empirical relation has been proposed.

Mesowestern Blot: Simultaneous Analysis of Hundreds of Submicroliter Lysates.

Western blotting is a widely used technique for molecular-weight-resolved analysis of proteins and their posttranslational modifications, but high-throughput implementations of the standard slab gel arrangement are scarce. The previously developed Microwestern requires a piezoelectric pipetting instrument, which is not available for many labs. Here, we report the Mesowestern blot, which uses a 3D-printable gel casting mold to enable high-throughput Western blotting without piezoelectric pipetting and is compatible with the standard sample preparation and small (∼1 μL) sample sizes. The main tradeoffs are reduced molecular weight resolution and higher sample-to-sample CV, making it suitable for qualitative screening applications. The casted polyacrylamide gel contains 336, ∼0.5 μL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution profiles. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder and cell lysate (RIPA buffer) demonstrate that the protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semidry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is at least 32-fold, and at least ∼500 attomols of β-actin can be detected (∼29 ng of total protein from mammalian cell lysates: ∼100–300 cells). Because the gel mold is 3D-printable, users with access to additive manufacturing cores have significant design freedom for custom layouts. We expect that the technique could be easily adopted by any typical cell and molecular biology laboratory already performing Western blots.

Deciphering the Virus Signal Within the Marine Dissolved Organic Matter Pool.

Viruses are ubiquitously distributed in the marine environment, influencing microbial population dynamics and biogeochemical cycles on a large scale. Due to their small size, they fall into the oceanographic size-class definition of dissolved organic matter (DOM; <0.7 μm). The purpose of our study was to investigate if there is a detectable imprint of virus particles in natural DOM following standard sample preparation and molecular analysis routines using ultrahigh-resolution mass spectrometry (FT-ICR-MS). Therefore, we tested if a molecular signature deriving from virus particles can be detected in the DOM fingerprint of a bacterial culture upon prophage induction and of seawater containing the natural microbial community. Interestingly, the virus-mediated lysate of the infected bacterial culture differed from the cell material of a physically disrupted control culture in its molecular composition. Overall, a small subset of DOM compounds correlated significantly with virus abundances in the bacterial culture setup, accounting for <1% of the detected molecular formulae and <2% of the total signal intensity of the DOM dataset. These were phosphorus- and nitrogen-containing compounds and they were partially also detected in DOM samples from other studies that included high virus abundances. While some of these formulae matched with typical biomolecules that are constituents of viruses, others matched with bacterial cell wall components. Thus, the identified DOM molecular formulae were probably not solely derived from virus particles but were partially also derived from processes such as the virus-mediated bacterial cell lysis. Our results indicate that a virus-derived DOM signature is part of the natural DOM and barely detectable within the analytical window of ultrahigh-resolution mass spectrometry when a high natural background is present.

Evaluation of the Vitek Ms system for the identification of filamentous fungi.

The use of MALDI-TOF for fungal identification remains a challenge. In this study, using a commercial protein extraction kit and updated database, VITEK MS system was able to identify up to 89.0% of a diverse collection of 200 filamentous fungi representing 43 fungal species.

Extraction of anabolic steroids with aqueous-organic mixtures from hexanic solutions

The extraction of anabolic steroids (1,4-androstadiene-3,17-dione; 17α-methyltestosterone; 19-norethiocholanolone; 4-hydroxytestosterone; 4-androstene-3,17-dione; 5α-androstane-3β,17β-diol; dehydroepiandrosterone; calusterone; clostebol; methandienone; methyldienolone; nandrolone; testosterone; epitestosterone; tibolone; 19-norandrostenedione; 1-methylene-5α-androstan-3α-ol-17-one) by aqueous-organic mixtures from hexane solutions was studied at the temperature (20 ± 1) °C. Based on the experimental data obtained, the partition ratios of anabolic steroids were calculated, which were used to optimise the standard sample preparation procedure in the process of determining the compounds under study in biologically active dietary supplements and specialised sports nutrition. It was found that the most effective and selective extractants from hydrocarbon solutions are water-acetonitrile mixtures containing from 10 to 20 % by volume of water. An extraction technique has been developed for sample preparation of biologically active dietary supplements for subsequent gas chromatographic determination of anabolic steroids in them using a triple quadrupole mass spectrometric detector. The proposed method is characterised by a standard deviation of 10–15 % and a detection limit about 10 μg/kg of biologically active dietary supplements, which makes it possible to reliably determine impurities of prohibited anabolic steroids in them.

Sensitive UHPLC-MS/MS Technique for Monitoring Levothyroxine (T4) in Human Serum Against Endogenous Thyroxin Level

Background: Levothyroxine is a synthetic thyroid hormone that is chemically identical to Thyroxine (T4), which is secreted by the follicular cells of the thyroid gland. Levothyroxine is used to treat deficiency of thyroid hormone and prevent the recurrence of thyroid cancer. Levothyroxine is present endogenously in the human body. Method: It requires a treated matrix for the preparation of calibration curve standard and quality control samples. The method was developed using LC-MS/MS and validated in human charcoal stripped serum. Charcoal stripped matrix was used for the preparation of Calibration curve standards and Quality control samples. The method involves Solid-Phase Extraction technique. Levothyroxine D3 is used as an internal standard (ISTD). Result: Chromatographic separation was achieved using reversed phase analytical column Gemini NX-C18 110Å, 3μm (50x3.6) mm. Mobile phase consisted of acetonitrile and water in a ratio of 70:30 with 150μL of formic acid in 1000 mL of the mobile phase. Mobile phase achieved shorter run-time of 0.9 minute due to the use of Ultra-high performance liquid chromatography (UHPLC). Positive electro-spray ionization technique detected MRM ion pair transitions 777.60→731.65 for Levothyroxine and 780.70→734.6 for Levothyroxine- D3 (ISTD) were used. AB SCIEX Triple Quad™ API-4000 LC-MS/MS system and the bioanalytical method with 10ng/mL as the limit of quantification have been applied successfully to pharmacokinetics studies. Conclusion: Chromatographic separation was achieved using reversed phase analytical column Gemini NX-C18 110Å, 3μm (50x3.6) mm. Mobile phase consisted of acetonitrile and water in a ratio of 70:30 with 150μL of formic acid in 1000 mL of the mobile phase. Mobile phase achieved shorter run-time of 0.9 minutes due to the use of Ultra-high performance liquid chromatography (UHPLC). AB SCIEX Triple Quad™ API-4000 LC-MS/MS system and the bioanalytical method with 10ng/mL as the limit of quantification have been applied successfully to pharmacokinetics studies.

Iron Speciation in Animal Tissues Using AC Magnetic Susceptibility Measurements: Quantification of Magnetic Nanoparticles, Ferritin, and Other Iron-Containing Species.

The simultaneous detection and quantification of several iron-containing species in biological matrices is a challenging issue. Especially in the frame of studies using magnetic nanoparticles for biomedical applications, no gold-standard technique has been described yet and combinations of different techniques are generally used. In this work, AC magnetic susceptibility measurements are used to analyze different organs from an animal model that received a single intratumor administration of magnetic nanoparticles. The protocol used for the quantification of iron associated with the magnetic nanoparticles is carefully described, including the description of the preparation of several calibration standard samples of nanoparticle suspensions with different degrees of dipolar interactions. The details for the quantitative analysis of other endogenous iron-containing species such as ferritin or hemoglobin are also described. Among the advantages of this technique are that tissue sample preparation is minimal and that large amounts of tissue can be characterized each time (up to hundreds of milligrams). In addition, the very high specificity of the magnetic measurements allows for tracking of the nanoparticle transformations. Furthermore, the high sensitivity of the instrumentation results in very low limits of detection for some of the iron-containing species. Therefore, the presented technique is an extremely valuable tool to track iron oxide magnetic nanoparticles in samples of biological origin.

Impact of Pre-Analytical and Analytical Variables Associated with Sample Preparation on Flow Cytometric Stainings Obtained with EuroFlow Panels.

Simple SummaryObjective interpretation of flow cytometry may be hampered by a lack of standardized sample preparation procedures. The EuroFlow consortium conducted a series of experiments to determine the potential impact of different pre-analytical and analytical factors on the variability of results in terms of relative cell populations distribution and marker expression levels. The experiments were performed on healthy donors and patients with different hematological malignancies (e.g., acute leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome) to mimic real-world clinical settings. Overall, the results showed that sample storage conditions, anticoagulant use, and sample processing protocol might need to be tailored for sample and cell type(s), as well as to the specific markers evaluated. However, defining of well-balanced boundaries for storage time to 24 h, staining-acquisition delay to 3 h, and choosing a washing buffer of pH within the range of 7.2 to 7.8 would be a valid recommendation for most applications and circumstances described herein.Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.