DON calibrant: Towards the production of certified B-trichothecene calibrants

Within an EC-funded project calibrants with certified concentrations of Deoxynivalenol (DON), 3-Acetyl-Deoxynivalenol (3-Ac-DON), 15-Acetyl-Deoxynivalenol (15-Ac-DON) and Nivalenol (NIV) in acetonitrile have been produced. So far the project has led to improved isolation and purification of the solid toxins fromFusarium cultures. In addition, conditions for the production, ampouling and transport of the toxin solutions have been optimised. Further investigations should lead to knowledge about storage conditions and internationally accepted molar absorption coefficients for DON, 3-Ac-DON, 15-Ac-DON and NIV in acetonitrile. The intercomparison study which is currently carried out will also help to support knowledge and experience exchange between laboratories in the field ofFusarium mycotoxin analysis.     

Type-B trichothecene calibrants: Comparison of HPLC and GC-results within an intercomparison study

Within the EC-financed project “Feasibility Study for the Production of Certified Calibrants for the Determination of Deoxynivalenol and other B-Trichothecenes”, an intercomparison study was performed with 13 European participants. Main goals of the intercomparison study were to check the feasibility of a small batch of gravimetrically prepared calibrants, to directly compare common and individually prepared calibrants, to test the practicability of toxin mixtures as calibrant solutions and finally to give recommendations for the means of certification. Additionally, it focused on the comparison of gas chromatography (GC) and high performance liquid chromatography (HPLC) for the determination of pure type-B trichothecene solutions, which is described in this publication. The participating laboratories received calibrant solutions as well as toxin solutions of unknown concentration and employed mainly HPLC-UV; GC-ECD (electron capture detection) and GC-MS (mass spectrometry) methods were used less often. The intercomparison study generally suffered from a high rate of outliers (22% of all the data). Throughout the study, 48% of all GC results were classified as outliers and it soon became apparent, that GC results highly infuenced the outcome of the study and that the used GC methods were not robust enough for the certification of type-B trichothecene calibrants. The high discrepancy between HPLC and GC results in the intercomparison study presumably lies in the crucial step of derivatisation. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005. Financial support: European Commission, Project No. GRD1-2002-70018     

Discrepancy between radioimmunoassay and high performance liquid chromatography tandem-mass spectrometry for the analysis of androstenedione

The discrepancy of results for the quantification of androstenedione in human serum between a radioimmunoassay (RIA) method and high performance liquid chromatography tandem-mass spectrometry (LC–MS/MS) was investigated. RIA overestimated concentrations compared to LC–MS/MS on 59 clinical samples (RIA = 1.79 × LC–MS/MS + 0.94). RIA kit and LC–MS/MS calibrants were also determined by both methods. The RIA performed with improved accuracy on the calibrants (RIA = 1.35 × LC–MS/MS − 0.28). Lipid, protein, electrolyte content, and pH of the two sets of calibrants were further investigated. The RIA calibrants contained little lipid material, while the LC–MS/MS calibrant material contained the same levels expected in normal serum/plasma. The pH and sex hormone binding globulin (SHBG) values were different between the RIA calibrants and the LC–MS/MS calibrant material (SHBG, 31 ± 2 and 38 ± 2 nmol/l; pH, 8.27 ± 0.18 and 8.66 ± 0.03, respectively). No correlation was observed between androstenedione RIA and LC–MS/MS discrepancy and lipid or protein. LC–MS/MS sample preparation was tested for the removal of protein-bound material and recovery determined (99–108%). The corresponding RIA results overestimated androstenedione by 52–174% compared to LC–MS/MS. The results here demonstrate that LC–MS/MS is the more accurate method.     

Sucrose utilization during potato microtuber growth in bioreactors

 Potato microtubers are used as pathogen-tested in vitro stocks for certified seed potato production. Microtubers grown in a rotating bioreactor grew at a faster rate when the medium was replaced frequently. Although the total microtuber number was not affected, the number of microtubers over 1 g quadrupled when 75% of the medium was replaced every 2 weeks when compared with no medium refreshment. Significantly slower microtuber growth rates resulted when a lower sugar concentration (40 g 1⁻¹ instead of 80 g 1⁻¹) was used or when a mixture of glucose and fructose replaced sucrose. Although high sucrose levels are necessary for optimal microtuber production, the sucrose supplied was rapidly hydrolyzed into glucose and fructose, making the long-term maintenance of desirable sucrose levels difficult. These results indicate that successful strategies to reduce sucrose hydrolysis without inhibiting microtuber growth will improve the efficiency of sucrose utilization in potato microtuber bioreactors. Received: 1 December 1998 / Revision received: 6 May 1999 · Accepted: 19 May 1999     

Calibration of mass spectrometric peptide mass fingerprint data without specific external or internal calibrants

Background  

Peptide Mass Fingerprinting (PMF) is a widely used mass spectrometry (MS) method of analysis of proteins and peptides. It relies on the comparison between experimentally determined and theoretical mass spectra. The PMF process requires calibration, usually performed with external or internal calibrants of known molecular masses.     

Reference materials and reference measurement procedures: an overview from a national metrology institute

An outline of the processes involved in both certified clinical reference material production and clinical reference measurement procedure development at the National Institute of Standards and Technology (NIST), the national metrology institute of the United States, is presented. The role that NIST and other national metrology institutes play in the metrological traceability of certified reference material is discussed. Highlighted are the challenges associated with the development of reference measurement systems for complex clinical analytes, such as proteins, and examples of existing efforts in this area are given. Examples of recent international collaborations in developing certified reference materials for analytes such as cardiac troponin I, brain natriuretic peptide, and serum creatinine demonstrate the close cooperation that national metrology institutes must have with the clinical community to establish complete reference measurement systems.     

Development of a 3D‐printed single‐use separation chamber for use in mRNA‐based vaccine production with magnetic microparticles

Laboratory protocols using magnetic beads have gained importance in the purification of mRNA for vaccines. Here, the produced mRNA hybridizes specifically to oligo(dT)‐functionalized magnetic beads after cell lysis. The mRNA‐loaded magnetic beads can be selectively separated using a magnet. Subsequently, impurities are removed by washing steps and the mRNA is eluted. Magnetic separation is utilized in each step, using different buffers such as the lysis/binding buffer. To reduce the time required for purification of larger amounts of mRNA vaccine for clinical trials, high‐gradient magnetic separation (HGMS) is suitable. Thereby, magnetic beads are selectively retained in a flow‐through separation chamber. To meet the requirements of biopharmaceutical production, a disposable HGMS separation chamber with a certified material (United States Pharmacopeia Class VI) was developed which can be manufactured using 3D printing. Due to the special design, the filter matrix itself is not in contact with the product. The separation chamber was tested with suspensions of oligo(dT)‐functionalized Dynabeads MyOne loaded with synthetic mRNA. At a concentration of c_B = 1.6–2.1 g·L^–1 in lysis/binding buffer, these 1 μm magnetic particles are retained to more than 99.39% at volumetric flows of up to 150 mL·min^–1 with the developed SU‐HGMS separation chamber. When using the separation chamber with volumetric flow rates below 50 mL·min^–1, the retained particle mass is even more than 99.99%.     

The Biological Stain Commission's Quality Control Laboratory operations and improved traceability of certified stains

Abstract

The Biological Stain Commission (BSC) is a quality control laboratory that certifies biological dyes for staining cells and tissues. Originally, a single lot of a certified dye was sold to histologists. Today, companies frequently change their lot numbers as part of regulatory efforts. When a certified dye undergoes a lot number change, the BSC must re-certify this dye to verify that it is identical to the one certified earlier. The BSC has improved how these lot changes are monitored using a redesigned BSC certification label. Certification labels always have been issued by the BSC and are attached to every bottle of “BSC certified dye” that is sold. The new BSC certification label has added security features and currently bears both the BSC certification number and the manufacturer batch lot number. The result is improved security and traceability of certified dyes.     

Characteristics of continuous cell line 293 used for preparation of antineoplastic agent Cancerolysin]

Certification of continuous cell 293 culture used for cultivation of antineoplastic preparation Cancerolysin was carried out. The seeding and working banks of cells 293 were established and deposited for storage at the Vector Centre. The cells were certified in accordance with the WHO requirements. The cell 293 culture was shown to have high proliferative activity; morphology typical of the line; its karyotype and enzymogram are typical of human cells; the culture is not contaminated with bacteria, fungi, mycoplasms and viruses including oncogenic ones; it has high virus-producing activity; it preserves stability of all the biological properties in long-term cultivation. The seeding and working cell banks were recommended for the use in production of drugs for the treatment of oncologic patients.     

Determination of subnanomolar concentrations of vanadium in environmental water samples using flow injection with luminol chemiluminescence detection

A flow injection chemiluminescence method is described for the determination of subnanomolar concentrations of vanadium in environmental water samples. The procedure is based on the oxidation of luminol in the presence of dissolved oxygen catalyzed by vanadium(IV). Vanadium(V) reduction and preconcentration of vanadium(IV) was carried out using in‐line silver reductor and 8‐hydroxyquinoline chelating columns at pH 3.15, respectively. The calibration graph for vanadium(IV) was linear in the concentration range of 0.025–10 µg/L with relative standard deviation in the range of 0.4–5.58%. The detection limit (3s blank) was 3.8 × 10^?3 µg/L without preconcentration; when the vanadium(IV) was preconcentrated with an 8‐HQ column for 1 min (2.0 mL of sample loaded), the detection limit of 5.1 × 10^?4 µg/L was achieved. One analytical cycle can be completed in 2.0 min. The analysis of certified reference materials (CASS‐4, NASS‐5 and SLRS‐4) by the proposed method showed good agreement with the certified values. The method was successfully applied to the determination of total dissolved vanadium in environmental water samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Environmental-economic benefits and trade-offs on sustainably certified coffee farms

Coffee with diverse shade trees is recognized as conserving greater biodiversity than more intensive production methods. Sustainable certification has been proposed as an incentive to conserve shade grown coffee. With 40% of global coffee production certified as sustainable, evidence is needed to demonstrate whether certification supports the environmental benefits of shade coffee. Environmental and economic data were taken from 278 coffee farms in Nicaragua divided between non-certified and five different sustainable certifications. Farms were propensity-score matched by altitude, area of coffee and farmer education to ensure comparability between non-certified and certified farms. Farms under all certifications had better environmental characteristics than non-certified for some indicators, but none were better for all indicators. Certified farms generally received better prices than non-certified farms. Farms with different certifications had different investment strategies; C.A.F.E. Practice farms had high investment and high return strategies, while Utz and Organic farms had low investment, low productivity strategies. Tree diversity was inversely related to productivity, price and net revenue in general, but not for certified farms that received higher prices. Certification differentiates farms with better environmental characteristics and management, provides some economic benefits to most farmers, and may contribute to mitigating environment/economic trade-offs.     

Room Temperature Ionic Liquid-Based Dispersive Liquid Phase Microextraction for the Separation/Preconcentration of Trace Cd2+ as 1-(2-pyridylazo)-2-naphthol (PAN) Complex from Environmental and Biological Samples and Determined by FAAS

The current work develops a new green methodology for the separation/preconcentration of cadmium ions (Cd²⁺) using room temperature ionic liquid-dispersive liquid phase microextraction (RTIL–DLME) prior to analysis by flame atomic absorption spectrometry with microsample introduction system. Room temperature ionic liquids (RTIL) are considered “Green Solvents” for their thermally stable and non-volatile properties, here 1-butyl-3-methylimidazolium hexafluorophosphate [C₄mim][PF₆] was used as an extractant. The preconcentration of Cd²⁺ in different waters and acid digested scalp hair samples were complexed with 1-(2-pyridylazo)-2-naphthol and extracted into the fine drops of RTILs. Some significant factors influencing the extraction efficiency of Cd²⁺ and its subsequent determination, including pH, amount of ligand, volume of RTIL, dispersant solvent, sample volume, temperature, and incubation time were investigated in detail. The limit of detection and the enhancement factor under the optimal conditions were 0.05 μg/L and 50, respectively. The relative standard deviation of 100 μg/L Cd²⁺ was 4.3 %. The validity of the proposed method was checked by determining Cd²⁺ in certified reference material (TM-25.3 fortified water). The sufficient recovery (>98 %) of Cd²⁺ with the certified value. The mean concentrations of Cd in lake water 13.2, waste water 15.7 and hair sample 16.8 μg/L, respectively and the developed method was applied satisfactorily to the preconcentration and determination of Cd²⁺ in real samples.     

The Use of Fungal Biomass Agaricus bisporus Immobilized on Amberlite XAD-4 Resin for the Solid-Phase Preconcentration of Thorium

Solid-phase extraction method was developed for the preconcentration of thorium (Th). Fungal biomass Agaricus bisporus was immobilized to Amberlite XAD-4 as solid-phase sorbent. The critical parameters such as pH of the sample solution, flow rate of the sample, volume of the sample, and the effect of major ions that affect the preconcentration of thorium in this system were evaluated. The optimum pH for the sorption of Th is 6.0, and quantitative elution occurs with 1.0 mol L^?1HCl. The loading capacity was determined as 0.079 mmol g^?1. The optimized method was validated through analysis of the certified reference material of tea leaves (NCS ZC73014) and successfully applied to the determination of Th in a real ore sample with satisfactory results.     

Localization of phaseolin genes in the polytene chromosomes ofPhaseolus coccineus (Leguminosae)

The location of the genes for the major storage protein, phaseolin, was determined in the polytene chromosomes of the embryo-suspensor ofPhaseolus coccineus, cv. Hammond's Dwarf Scarlett, byin situ hybridization with a³⁵S-labelled cloned probe. The genes were adjoined to chromosome pair no. 3. The distribution of the label indicates a puffed state of the sites during embryogenesis of the legume studied. The location of the phaseolin genes was certified by hybridization of a ribosomal DNA probe to the polytene chromosomes, because their position is well known.     

On-line preconcentration/determination of zinc from water,biological and food samples using synthesized chelating resin and flame atomic absorption spectrometry

An on-line flow injection pre-concentration-flame atomic absorption spectrometry method was developed to determine trace zinc in water (tap, dam, and well water), biological (hair and nail), and liver samples. As a solid phase extractant, a synthesized new chelating resin, poly(2-thiozylmethacrylamide-co-divinylbenzene-co-2-acrylamido-2-methyl-1-propane sulfonic acid) was used. The resin was characterized by Fourier transform infrared spectroscopy, elemental analysis, and surface area by nitrogen sorption. A pre-concentration factor of 40-fold for a sample volume of 12.6 mL was obtained by using the time-based technique. The detection limit for the pre-concentration method was found to be 2.2 μg L^?1. The precision (as RSD,%) for 10 replicate determinations at the 0.04 μg mL^?1 Zn concentration was 1.2%. The calibration graph using the pre-concentration system for zinc was linear with a correlation coefficient of 0.998 in the concentration range from 0.005 to 0.05 μg mL^?1. The applicability and accuracy of the developed method were estimated by the analysis spiked water, biological, liver samples (83–105%), and also certified reference material TMDA-70 (fortified lake water) and SPS-WW1 Batch 111-Wastewater. The results were in agreement with the certified values.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.     

Atmospheric Production of Glycolaldehyde Under Hazy Prebiotic Conditions

The early Earth’s atmosphere, with extremely low levels of molecular oxygen and an appreciable abiotic flux of methane, could have been a source of organic compounds necessary for prebiotic chemistry. Here, we investigate the formation of a key RNA precursor, glycolaldehyde (2-hydroxyacetaldehyde, or GA) using a 1-dimensional photochemical model. Maximum atmospheric production of GA occurs when the CH₄:CO₂ ratio is close to 0.02. The total atmospheric production rate of GA remains small, only 1×10⁷ mol yr^???1. Somewhat greater amounts of GA production, up to 2 × 10⁸ mol yr^???1, could have been provided by the formose reaction or by direct delivery from space. Even with these additional production mechanisms, open ocean GA concentrations would have remained at or below ~1 μM, much smaller than the 1–2 M concentrations required for prebiotic synthesis routes like those proposed by Powner et al. (Nature 459:239–242, 2009). Additional production or concentration mechanisms for GA, or alternative formation mechanisms for RNA, are needed, if this was indeed how life originated on the early Earth.     

Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T

We have designed a new low-pressure Diamond Anvil Cell (DAC), calibrated two novel pressure calibrants and validated the use of semi-quantitative Raman and X-ray spectroscopies to monitor the fate of microbes, their metabolism or their cellular components under controlled pressures and temperatures in the 0.1-1.4 GPa and 20-300 degrees C P,T range. The low-pressure DAC has a 250- to 600-microm-thick observation diamond window to allow for lower detection limits and improved microscopic imaging. This new design allows the determination of cellular growth parameters from automated image analysis, which can be correlated with the spectroscopic data obtained on metabolism, ensuring high quality data collection on microbial activity under pressure. The novel pressure sensors offer the ease of use of the well-known ruby scale, while being more sensitive and reacting to pressure variations instantaneously.