Enzymatic hydrolysis of T-2 toxin for the quantitative determination of total T-2 and HT-2 toxins in cereals

The selective enzymatic deacetylation of T-2 toxin to give HT-2 toxin has been investigated in aqueous crude extracts of different cereals and exploited to develop an analytical method for the determination of the sum of T-2 and HT-2 toxins. The method has been validated for the analysis of total T-2 and HT-2 toxins in maize, wheat, and oats, showing recoveries from 72 to 97% for maize, from 67 to 84% for wheat, and from 61% to 87% for oats, at spiking levels of 20–400 μg/kg, with relative standard deviation lower than 10%. Liquid chromatography-tandem mass spectrometry was used for quantitative toxin determination. The potential biological role of this enzymatic conversion and its perspectives for application in the development of antibody-based analytical techniques are discussed.      

Production of certified reference material

Summary Carbon has been determined in 31 European reference steels in the range of 11–2561 g/g C, using an absolute calibration method.High-purity calcium carbonate was used as the calibration material and weighing was carried out by means of a balance with extremely small weighing error. Both synthetic calibration samples and test samples were processed alternatingly under identical conditions, and the analyses were completed in the shortest possible time.In 28 of the cases examined, results were lower than the certificate values, by an average of 27 g/g. The interval of uncertainty of the accuracy was 6 g/g (probability 99%).

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Herstellung zertifizierter ReferenzmaterialienGenaue Bestimmung von Kohlenstoff in Stählen im Bereich von 11–2561g/g

Zusammenfassung Die Kohlenstoffgehalte von 31 europäischen zertifizierten Referenzstählen im Arbeitsbereich von 11–2561 g/g Kohlenstoff wurden durch absolute Eichung überprüft.Die hinreichende Reinheit des Eichmittels Calciumcarbonat wurde sichergestellt und ein Wägesystem mit einem hinreichend kleinen Wägefehler benutzt.Das gleiche Verhalten von synthetischen Eichproben und Analysenproben im Analysenprozeß wurde hergestellt und beide Probearten wechselweise unter konstanten Systembedingungen innerhalb kürzester Zeitspanne untersucht. In 28 der untersuchten Fällen wurden im Mittel um 27 ppm tiefere Werte gefunden als in den Zertifikaten angegeben. Das Unsicherheitsintervall der Richtigkeit der gefundenen Werte lag mit einer Wahrscheinlichkeit von 99% im Mittel bei 6 g/g.

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Abbreviations CRM Certified reference material - ppm g/g The author thanks Mr. E. Weichert for his excellent direction of the practical work.     

Gas chromatographic assay with pharmacokinetic applications for monitoring T-2 and HT-2 toxins in plasma

A gas-liquid chromatographic (GLC) method for monitoring T-2 and HT-2 toxins in plasma was developed. The procedure involved extraction of the toxins with ethyl acetate, chromatography on a C18 reversed-phase column and derivatization with heptafluorobutyric anhydride (HFBA). The T-2 and HT-2 HFBA derivatives were chromatographed on OV-17 at various temperatures and measured with an electron-capture detector. Iso-T-2 toxin and iso-HT-2 toxin were used as internal standards. Recoveries averaged 95.1 +/- 8.6% for T-2 toxin and 102.1 +/- 5.2% for HT-2 toxin at levels ranging from 40 to 120 ng/ml. The limits of detection were 30 and 5 ng/ml of T-2 and HT-2 toxin, respectively. The range of the assay covers plasma concentrations at which toxicity becomes manifest. The pharmacokinetic application of this GLC method is illustrated by simultaneous monitoring of T-2 and HT-2 toxins levels in plasma obtained after intravenous administration of T-2 toxin to a dog.     

A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat

A rapid fluorescence polarization (FP) immunoassay has been developed for the simultaneous determination of T-2 and HT-2 toxins in naturally contaminated wheat samples. Syntheses of four fluorescein-labelled T-2 or HT-2 toxin tracers were carried out and their binding response with seven monoclonal antibodies was evaluated. The most sensitive antibody-tracer combination was obtained by using an HT-2-specific antibody and a fluorescein-HT-2 tracer. The developed competitive FP immunoassay in solution showed high cross-reactivity for T-2 toxin (CR% = 100%) while a very low CR% for neosolaniol (0.12%) and no cross-reactivity with other mycotoxins frequently occurring in wheat. A rapid extraction procedure using 90% methanol was applied to wheat samples prior to FP immunoassay. The average recovery from spiked wheat samples (50 to 200 μg kg⁻¹) was 96% with relative standard deviation generally lower than 8%. A limit of detection of 8 μg kg⁻¹ for the combined toxins was determined. Comparative analyses of 45 naturally contaminated and spiked wheat samples by both the FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up showed a good correlation (r = 0.964). These results, combined with the rapidity (10 min) and simplicity of the assay, show that this method is suitable for high throughput screening as well as for quantitative determination of T-2 and HT-2 toxins in wheat.     

加替沙星纯度标准物质研制

研制加替沙星纯度标准物质。采用质量平衡法和氢谱定量核磁法对加替沙星纯度标准物质候选物进行纯度定值,采用卡尔费休法和热重分析法准确测量了影响主成分纯度的水分含量,并考察了水分含量的稳定性。开展了加替沙星纯度定值、均匀性检验、稳定性考察以及不确定度评估。结果表明,加替沙星纯度标准物质的纯度值为93.1%,扩展不确定度为0.6%(k=2),水分含量为6.6%,特性量值均匀性良好,6个月稳定性可靠,被认定为国家二级标准物质,编号为GBW(E) 100476。研制的加替沙星纯度标准物质填补了国内空白,同时也为吸湿性较强的药物纯度标准物质研制过程中水分测定提供了参考与借鉴。     

辛基二茂铁同分异构体标准物质的制备

以辛基二茂铁为原材料,利用常压色谱柱结合环糊精包结分离制得(2-辛基)-二茂铁、(3-辛基)-二茂铁、(4-辛基)-二茂铁3种相对纯度均大于99.5%的辛基二茂铁同分异构体,作为标准物质候选物。利用气相色谱法检测,考察标准物质候选物的均匀性和稳定性,用气相色谱–质谱法、核磁共振法分析候选物中杂质的结构,确定其类型。用内标法和主成分自身对照法对不同的杂质进行定值,并对定值不确定度进行评定。(2-辛基)-二茂铁、(3-辛基)-二茂铁、(4-辛基)-二茂铁的纯度分别为99.5%,99.5%,99.8%,扩展不确定度均为0.2%(k=2)。该标准物质可为相关物质的检测提供量值溯源标准。     

Development of a certified reference material for lead in noodles powder

Accreditation and Quality Assurance - Food Safety and Standards Authority of India has set maximum permissible level of lead in noodle at 2.5 mg/kg. The standardized lead measurement is of high...     

A new certified reference material for benzene measurement in air on a sorbent tube: development and proficiency testing

A certified matrix reference material (CRM) for the measurement of benzene in ambient air has been developed at Laboratoire National de Métrologie et d’Essais. The production of these CRMs was conducted using a gravimetric method fully traceable to the International System of Units. The CRMs were prepared by sampling an accurate mass of a gaseous primary reference material of benzene, using a high-precision laminar flowmeter and a mass flow controller, with a PerkinElmer sampler filled with Carbopack™ X sorbent. The relative standard deviations obtained for the preparation of a batch of 20 tubes loaded with 500 ng of benzene were below 0.2%. Each CRM is considered independent from the others and with its own certified value and an expanded uncertainty estimated to be within 0.5%, lower than the uncertainties of benzene CRMs already available worldwide. The stability of these materials was also established up to 12 months. These CRMs were implemented during proficiency testing, to evaluate the analytical performances of seven French laboratories involved in benzene air monitoring.     

Analysis of T-2 and HT-2 toxins in cereal grains by immunoaffinity clean-up and liquid chromatography with fluorescence detection

A sensitive, precise and accurate method has been developed for the simultaneous determination of T-2 and HT-2 toxins in cereal grains at ppb levels using high-performance liquid chromatography (HPLC) with fluorescence detection and 1-antroylnitrile (1-AN) as labeling reagent after immunoaffinity clean-up. Cereal samples were extracted with methanol/water (90:10, v/v), and the extracts were cleaned-up through commercially available immunoaffinity columns containing monoclonal anti-T-2 antibodies (T-2 test HPLC, Vicam). T-2 and HT-2 toxins were quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. The monoclonal antibody showed 100% cross-reactivity with both T-2 and HT-2 toxin, and the immunoaffinity column clean-up was effective up to 1.4 microg of both toxins. The method was successfully applied to the analysis of T-2 and HT-2 toxins in wheat, maize and barley. Recoveries from spiked samples with toxin levels from 25 to 500 microg/kg ranged from 70% to 100%, with relative standard deviation generally lower than 8%. The limit of detection of the method was 5 microg/kg for T-2 toxin and 3 microg/kg for HT-2 toxin, based on a signal-to-noise ratio 3:1. HT-2 toxin was detected in ten naturally contaminated wheat samples out of 14 samples analyzed, with toxin levels ranging from 10 to 71 microg/kg; three of them contained also T-2 toxin up to 12 microg/kg.     

Arsenic species in certified reference material MURST-ISS-A2 (Antarctic krill)

Arsenic compounds were quantified in the certified reference material MURST-ISS-A2 (Antarctic krill) using HPLC/ICPMS. The data should prove useful for assessing the accuracy of arsenic speciation procedures.     

NIES certified reference material for microcystins,hepatotoxic cyclic peptide toxins from cyanobacterial blooms in eutrophic water bodies

A certified reference material (CRM) for microcystins has been prepared by the National Institute for Environmental Studies (NIES). Microcystins are hepatotoxic cyclic peptides produced by cyanobacteria in eutrophic water bodies. At least seven microcystin variants were found by HPLC analysis of the NIES CRM, of which [Dha⁷]microcystin-RR and -LR were the major microcystins present. Because of the lack of available standards we determined the total microcystin concentration in the CRM by the MMPB method, and elucidated the structures of the main individual microcystin variants following their isolation. Analyses of NMR and MS spectra indicated that the remaining minor variants in the CRM were [D-Asp³, Dha⁷]microcystin-RR and -LR, and [Dha⁷]microcystin-YR, -ThTyrR, and -HilR. The CRM is valuable not only as a standard material for the quantitation of total microcystins but also for the identification of individual [Dha⁷]microcystin variants.     

Development of bovine serum albumin certified reference material

We present the development process for National Institute of Metrology (NIM) bovine serum albumin (BSA) certified reference material (CRM). Each CRM unit contains about 200 mg of purified BSA. The moisture, ignition residue, molecular weight, and high-performance liquid chromatography (HPLC) purity were analyzed and mass spectrometry based protein identification was carried out to ensure the material was BSA. Both amino acid based isotope dilution mass spectrometry (IDMS) and a purity deduction method were selected for value assignment. The certified value was the average of the IDMS and the purity deduction result. HPLC purity analysis was used to examine the homogeneity and stability of solid BSA CRM. Fifteen units were selected for between-bottle homogeneity examination and seven subsamples from the same bottle were selected for within-bottle homogeneity examination. Statistics showed the CRM passed both the between-bottle and the within-bottle homogeneity examination. The CRM stability under storage conditions (-20 °C) was tested for 18 months and no trend was observed. Uncertainties from the balance, amino acid purity, hydrolysis, method reproducibility, homogeneity, and stability were taken into account in uncertainty evaluation. The final certified value of NIM BSA CRM is (0.963±0.038) g/g.     

Development of a candidate certified reference material of cypermethrin in green tea

This paper presents the preparation of a candidate certified reference material (CRM) of cypermethrin in green tea, GLHK-11-01a according to the requirements of ISO Guide 34 and 35. Certification of the material was performed using a newly developed isotope dilution mass spectrometry (IDMS) approach, with gas chromatography high resolution mass spectrometry (GC–HRMS) and gas chromatography–tandem mass spectrometry (GC–MS/MS). Statistical analysis (one-way ANOVA) showed excellent agreement of the analytical data sets generated from the two mass spectrometric detections. The characterization methods have also been satisfactorily applied in an Asia-Pacific Metrology Program (APMP) interlaboratory comparison study. Both the GC–HRIDMS and GC–IDMS/MS methods proved to be sufficiently reliable and accurate for certification purpose. The certified value of cypermethrin in dry mass fraction was 148 μg kg⁻¹ and the associated expanded uncertainty was 14 μg kg⁻¹. The uncertainty budget was evaluated from sample in homogeneity, long-term and short-term stability and variability in the characterization procedure. GLHK-11-01a is primarily developed to support the local and wider testing community on need basis in quality assurance work and in seeking accreditation.     

Improvement of detection sensitivity of T-2 and HT-2 toxins using different fluorescent labeling reagents by high-performance liquid chromatography

T-2 and HT-2 toxins are Fusarium mycotoxins that can occur in cereals and cereal-based products. Three fluorescent labeling reagents, i.e. 1-naphthoyl chloride (1-NC), 2-naphthoyl chloride (2-NC) and pyrene-1-carbonyl cyanide (PCC), were used for the determination of T-2 and HT-2 toxins by high-performance liquid chromatography (HPLC) with fluorescence detection (FD). Pre-column derivatization of T-2 and HT-2 toxins was carried out under mild conditions (50 °C, 10 min) in toluene with 4-dimethylaminopyridine (DMAP) as catalyst. All fluorescent derivatives were identified and characterized by HPLC-tandem mass spectrometry (HPLC-MS/MS). Optimal stoichiometric ratios (toxin:derivatizing reagent:catalyst), linear range and repeatability of the reaction, stability and sensitivity of the derivatives were determined. A wide linear range (10–1000 ng of either derivatized T-2 or HT-2 toxin), good stability (up to 2 weeks at −20 °C or 5 days at room temperature) of the fluorescent derivatives and good repeatability of the reaction (RSD ≤ 8%) were observed. Detection limits (based on a signal-to-noise ratio of 3:1) were 10.0, 6.3 and 2.0 ng for derivatized T-2 toxin and 6.3, 2.3 and 2.8 ng for derivatized HT-2 toxin with 1-NC, 2-NC and PCC, respectively. In terms of sensitivity and repeatability, PCC and 2-NC reagents showed better performance than 1-anthroylnitrile (1-AN), a previously reported labeling reagent for T-2- and HT-2 toxins. Preliminary studies also showed the applicability of PCC and 2-NC as fluorescent labeling reagents for the simultaneous determination of T-2 and HT-2 toxins in cereal grains by HPLC/FD following immunoaffinity column clean-up.     

Development of a certified reference material for the determination of perfluorooctanoic acid

A certified reference material (CRM) for the determination of perfluorooctanoic acid (PFOA) has been issued as NMIJ CRM 4056-a by the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). Purity (kg kg^?1) based on a titration method was determined by subtracting the mass fractions of impurities measured using liquid chromatography/mass spectrometry (LC/MS) from those of acids expressed as PFOA measured by a neutralization potentiometric titration. To validate an obtained result, purity based on a mass balance method was determined by subtracting the mass fractions of impurities, measured using LC/MS, Karl-Fischer titration (KFT), and vacuum evaporation, from 1 kg kg^?1. Results from both titration and mass balance methods were in agreement within the accepted limits of uncertainty. The certified purity of NMIJ CRM 4056-a was determined to be 0.959 kg kg^?1, calculated as the mean of the results obtained with the two methods. The standard uncertainty of the certified purity was evaluated from purity evaluations as well as from sample homogeneity and stability obtained from LC/MS and KFT analyses. Consequently, the expanded uncertainty was estimated to be 0.005 kg kg^?1 with a coverage factor of k = 2.     

Trace elements in bovine muscle: an ongoing project for a new certified reference material

In the framework of the activities of the Community Reference Laboratory (CRL) for residues at the Istituto Superiore di Sanità (ISS) of Rome, a number of proficiency tests were performed in order to assess and improve, wherever necessary, the quality of regulatory residue analysis within the EU. In this context, a pilot study was undertaken with the purpose of ascertaining the feasibility of a project for the certification of a new reference material for trace elements in bovine muscle. On behalf of the ISS, approximately 70 kg of bovine muscle, collected at a slaughterhouse in Geel (Belgium), were freeze-dried, homogenized, stabilized and bottled under argon atmosphere by the Institute for Reference Materials and Measurements, Joint Research Centre, European Commission (IRMM-JRC-EC). Preliminary analyses were carried out with Quadrupole (Q) and High Resolution (HR) Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to evaluate the content of As, Cd, Cu and Pb, whereas the Flow Injection Mercury System (FIMS) was used to quantify Hg. Digestion of the matrix was achieved by acid-assisted microwave irradiation. The mean values of experimentally obtained data were the following (in nanograms per gram): As, 24.2±1.2; Cd, 5.48±0.29; Cu, 4765±95; Hg, 3.37±0.73; and Pb, 184±7. Subsequently, 16 National Reference Laboratories (NRLs) for residues in the Member States plus the Norwegian one and eight Italian public laboratories were accepted to participate in the project and received one bottle of the freeze-dried bovine muscle. The laboratories were requested to perform three different analytical runs (in three different days), each consisting of three individual measurements. Preliminary results showed good agreement and fair evidence for homogeneity of the entire mass, thus setting the stage for the future certification of this candidate CRM.     

Interactions between cyclodextrins and fluorescent T-2 and HT-2 toxin derivatives: a physico-chemical study

T-2 and HT-2 toxins are mycotoxins produced by several Fusarium species that are commonly found in various cereal grains, including oats, barley, wheat and maize. Intake estimates indicate that the presence of these mycotoxins in the diet can be of concern for public health. In this work, the inclusion processes occurring between fluorescent anthracene-derivatives of T-2 and HT-2 toxins and different cyclodextrin (CD) molecules were investigated in aqueous solutions by means of UV–Vis absorption, fluorescence emission and dynamic light scattering. Binding constant values and chemico-physical parameters were calculated. It was found that β-CDs give stronger inclusion reactions with both T-2 and HT-2 derivatives, as stated by important emission intensity increments. Such interactions were found to be fundamentally enthalpy-driven. Among β-CDs, the effect of the methylation at hydroxyl groups was tested: as a result, the di-methyl form of β-CD was found to induce the best fluorescence intensity enhancements.     

Determination of deoxynivalenol, T-2 and HT-2 toxins in a bread model food by liquid chromatography-high resolution-Orbitrap-mass spectrometry equipped with a high-energy collision dissociation cell

A sensitive and accurate method employing a single stage high resolution mass spectrometer equipped with a high-energy collision-dissociation cell (HCD) for the simultaneous determination of deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in a processed bread model food has been developed. Two sample pre-treatment routes for the extraction of these mycotoxins were investigated, based on Mycosep^® column clean up or QuEChERS-like procedure, respectively. The former approach suffered less from matrix effects and allowed to achieve in bread samples LODs of 7, 12 and 17 ng/g for T-2, HT-2 and DON, respectively, with 0.5 ppm mass accuracy. Two acquisition modes, full scan MS and all ion fragmentation, exploiting the fragmentation features offered by an HCD chamber and integrated within the Orbitrap analyser, were compared for quantitative purposes. The method was applied to investigate the degradation of these mycotoxins during bread processing using a bread model food. Most T-2 hydrolyzed to HT-2 during dough preparation, and about 20–30% of HT-2 and DON was degraded during bread baking.