Determination of ochratoxin A in wine and beer by immunoaffinity column cleanup and liquid chromatographic analysis with fluorometric detection: collaborative study.

The accuracy, repeatability, and reproducibility characteristics of a liquid chromatographic method for the determination of ochratoxin A (OTA) in white wine, red wine, and beer were established in a collaborative study involving 18 laboratories in 10 countries. Blind duplicates of blank, spiked, and naturally contaminated materials at levels ranging from < or =0.01 to 3.00 ng/mL were analyzed. Wine and beer samples were diluted with a solution containing polyethylene glycol and sodium hydrogen carbonate, and the diluted samples were filtered and cleaned up on an immunoaffinity column. OTA was eluted with methanol and quantified by reversed-phase liquid chromatography with fluorometric detection. Average recoveries from white wine, red wine, and beer ranged from 88.2 to 105.4% (at spiking levels ranging from 0.1 to 2.0 ng/mL), from 84.3 to 93.1% (at spiking levels ranging from 0.2 to 3.0 ng/mL), and from 87.0 to 95.0% (at spiking levels ranging from 0.2 to 1.5 ng/mL), respectively. Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 6.6 to 10.8% for white wine, from 6.5 to 10.8% for red wine, and from 4.7 to 16.5% for beer. Relative standard deviations for between-laboratories reproducibility (RSDR) ranged from 13.1 to 15.9% for white wine, from 11.9 to 13.6% for red wine, and from 15.2 to 26.1% for beer. HORRAT values were < or =0.4 for the 3 matrixes.     

A study of the global distribution of sulfate aerosols with a 2D model including microphysics

Summary The global distribution of sulfate aerosols in the troposphere and lower stratosphere has been calculated using a two-dimensional model. The chemistry includes the main families (NO _x , NO _y , HO _x ) plus the sulfur compounds, while the heterogeneous processes are modelled with a microphysics code which takes into account nucleation, condensation and coagulation. The results are compared with experimental data wherever available. A too low concentration of sulfuric acid is predicted by the model in the troposphere and this is explained by too large a flux of condensation nuclei and partly by the absence of sulfate production from methane sulfuric acid (MSA) in our scheme. Aerosol concentration and size distribution are comparable to observations; however the calculations show a more complex meridional structure than observed, while the size distribution is somewhat shifted toward smaller sizes. This fact is also attributed to the presence of a large number of nuclei and to the absence of any mechanism for evaporation of aerosol particles back to the core. We have also perturbed the anthropogenic fluxes of SO₂, CS₂ and the OCS ground mixing ratios to asses the sensitivity of the aerosol concentration: we have found very little changes in the aerosol distribution because H₂SO₄ chemistry is dominated by heterogeneous processes.     

Simultaneous LC–MS/MS determination of aflatoxin M<Subscript>1</Subscript>, ochratoxin A,deoxynivalenol, de-epoxydeoxynivalenol, α and β-zearalenols and fumonisin B<Subscript>1</Subscript> in urine as a multi-biomarker method to assess exposure to mycotoxins

Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M₁ (AFM₁), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alpha-zearalenol (α-ZOL)/beta-zearalenol (β-ZOL), and fumonisin B₁ (FB₁) can be used as urinary biomarkers. A liquid chromatographic–tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed-phase chromatography using a multi-step linear methanol–water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC–ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03–12 ng mL⁻¹, using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3–20%. Enzymatic digestion with β-glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time.     

Reliable detection of milk allergens in food using a high-resolution, stand-alone mass spectrometer

Reliable methods are needed for detection of allergenic milk proteins in complex food matrixes. The feasibility of an LC/high-resolution MS method for the analysis of milk proteins in a thermally processed model food (incurred cookies) and in white wine spiked, respectively, with milk powder and caseinate is described. Detection of milk proteins was based on the identification of unique peptides in the tryptic digests of cookie/wine extracts using an RP-HPLC separation coupled to an Exactive nonhybrid mass spectrometer using Orbitrap technology. The extremely high mass accuracy and resolution provided by the Orbitrap analyzer allowed a fast preliminary identification of four previously proposed peptide markers of caseins using only accurate values of the m/z of their ions. No interference was observed, despite the complexity of the analyzed matrixes. Moreover, the availability of a high- energy, collisionally activated dissociation cell integrated in the mass spectrometer enabled acquisition of peptide MS/MS-like spectra through post-source fragmentation. Confirmation of peptide marker identity could then be achieved by a comparison between experimental and predicted product ions. The described method shows the great potential of Orbitrap MS as a reliable technique in the field of protein allergen detection once the peptide markers are identified.     

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.     

Identification and characterization of new Fusarium masked mycotoxins,T2 and HT2 glycosyl derivatives,in naturally contaminated wheat and oats by liquid chromatography–high‐resolution mass spectrometry

The presence of glucoside derivatives of T‐2 and HT‐2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high‐resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T‐2 toxin and two monoglucoside derivatives of HT‐2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C‐3 position for T‐2 toxin and at C‐3 or C‐4 position for HT‐2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T‐2 and HT‐2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk. Copyright © 2012 John Wiley & Sons, Ltd.     

The use of conservative coordinates for reconstruction techniques and for the development of a two-dimensional transport model

Summary Conservation properties of Potential Temperature (PT) and Potential Vorticity (PV) in the middle atmosphere allow a Lagrangian approach to the study of tracer transport. A reference frame transformation, from the three-dimensional space to the PT-PV space, is used to analyse experimental measurements allowing reconstruction of data, and to develop a Lagrangian two-dimensional model. Some preliminary results of the application of this technique, that are particulary useful near the polar stratospheric vortex, are shown in this paper.     

Global error estimation for index-1 and -2 DAEs

In this paper, we consider the extension of three classical ODE estimation techniques (Richardson extrapolation, Zadunaisky's technique and solving for the correction) to DAEs. Their convergence analysis is carried out for semi-explicit index-1 DAEs solved by a wide set of Runge-Kutta methods. Experimentation of the estimation techniques with RADAU5 is also presented: their behaviour for index-1 and -2 problems, and for variable step size integration is investigated. This revised version was published online in June 2006 with corrections to the Cover Date.     

High-performance liquid chromatographic separation of cadralazine from its potential metabolites and degradation products. Quantitation of the drug in human plasma and urine

The chromatographic behaviour of cadralazine and its potential metabolites and degradation products with respect to pH, buffer molarity and composition of eluent is described. A selective method with an adequate sensitivity for the determination of the drug in human plasma and urine is also reported. The method includes extraction of biological fluids with chloroform and the analysis of extracts on a reversed-phase column with isocratic elution and detection at 254 nm. The method has been applied to the analysis of plasma and urine of a patient administered a single oral dose of 30 mg of cadralazine.