RESUMO
The interest in LC-MS/MS multi-mycotoxin methods unveiled an urgent need for multi-mycotoxin reference material. A multi-fusariotoxin, including deoxynivalenol (DON); zearalenone (ZEN); T-2 toxin (T-2); HT-2 toxin (HT-2); enniatin A, A1, B, and B1 (ENNs); and beauvericin (BEA), contaminated wheat flour was obtained by inoculation Fusarium spp. strains. The candidate material has successfully passed the homogeneity test and submitted to an international interlaboratory study achieved by 19 laboratories from 11 countries using their routine analytical method. The dispersion of the results for ZEN and BEA did not allow the derivation of reliable consensus values, while the assignment was only possible for DON, HT-2, T-2, and ENN A. No link was found between the methods used by the participants and the results. Significant changes in dry matter contents (≥±1.4 % of the initial dry matter) and significant changes in ergosterol contents (≥±10 %) did not occur. Using the mycotoxin contents in wheat flour stored at -80 °C as reference values, statistically significant decreases were observed only for T-2 contents at +24 °C, in contrast to the storage at -20 and +4 °C. For the other involved toxins, the candidate material was found to be stable at -20, +4, or +24 °C. Based on the T-2 decreases, a shelf life of 6 years was derived from isochronous study when the material is kept at -20 °C. At room temperature (e.g., +24 °C) or higher, this time validity drastically decreases down to 6 months. The development of this metrological tool is an important step towards food and feed quality control using multi-mycotoxin analyses. In vivo animal experiments using multi-mycotoxin-contaminated feeds dealing with the carryover or mitigation could further benefit from the methodology of this work.
Assuntos
Cromatografia Líquida/normas , Análise de Alimentos/métodos , Micotoxinas/análise , Padrões de Referência , Espectrometria de Massas em Tandem/normas , Animais , TemperaturaRESUMO
A direct, fast and sensitive LC-MS/MS method was developed to measure biomarkers for mycotoxin exposure in human urine. In total, 32 biomarkers were quantitatively or semi-quantitatively measured in 32 urine samples of Belgian volunteers using two injections. All urine samples contained deoxynivalenol-15-glucuronide, the major detoxification metabolite of deoxynivalenol, in the ng/mL range. Also deoxynivalenol-3-glucuronide and de-epoxy-deoxynivalenol-glucuronide were present in, respectively, 90 and 25% of the samples, while deoxynivalenol was detected in 60% of the samples, in lower concentrations. Deoxynivalenol glucuronides were the major biomarkers for deoxynivalenol exposure. Ochratoxin A was detected in 70% of the samples in pg/mL. Citrinin and/or dihydrocitrinone were detected in 90% of the samples, also in concentrations of pg/mL. The presence of ochratoxin A and citrinin was confirmed by a second method using sample cleanup by immunoaffinity columns, followed by LC-MS/MS. Our data show that humans are much more exposed to citrinin than realized before and suggest further work on citrinin exposure in relation with ochratoxin A exposure, as both mycotoxins are nephrotoxic.
Assuntos
Cromatografia Líquida/métodos , Micotoxinas/urina , Espectrometria de Massas em Tandem/métodos , Tricotecenos/urina , Adulto , Bélgica , Biomarcadores/urina , Citrinina/análogos & derivados , Citrinina/urina , Feminino , Glucuronídeos/urina , Humanos , Masculino , Ocratoxinas/urinaRESUMO
Fusarium species isolated from Belgian maize were screened for their ability to produce fusarin C, fusaric acid, fumonisins B1 (FB1), FB2 and FB3 in maize grains. First, cultivation of Fusarium species in Myro liquid medium allowed overcoming the shortage of the standard of fusarin C on the market. All Fusarium verticillioides produced much higher contents of mycotoxins in Myro compared to Fusarium graminearum or Fusarium venenatum. The optimization of the LC-MS/MS method resulted in low limits of detection and quantification for fusarin C, fusaric acid, FB1, FB2 and FB3 determination in maize grains. Its application for screening the potential toxin production ability evidenced that the concentrations of the analytes were significantly increased at various levels when F. verticillioides strains were cultivated in maize grains and reached 441 mg kg(-1) for fusaric acid, 74 mg kg(-1) for fusarin C, 1,301 mg kg(-1) for FB1, 367 mg kg(-1) for FB2 and 753 mg kg(-1) for FB3.
Assuntos
Grão Comestível/microbiologia , Fumonisinas/metabolismo , Ácido Fusárico/metabolismo , Fusarium/metabolismo , Polienos/metabolismo , Zea mays/microbiologia , Cromatografia Líquida , Meios de Cultura/química , Fusarium/crescimento & desenvolvimento , Fusarium/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
An LC-MS/MS method was developed and validated for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin, HT-2-toxin and metabolites, including 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol-3-glucoside, α-zearalenol, ß-zearalenol, zearalenone-4-glucoside, α-zearalenol-4-glucoside, ß-zearalenol-4-glucoside and zearalenone-4-sulfate in maize, wheat, oats, cornflakes and bread. Extraction was performed with acetonitrile/water/acetic acid (79/20/1, v/v/v) followed by a hexane defatting step. After filtration, the extract was evaporated and the residue was redissolved in mobile phase for injection. The mobile phase, which consisted of a mixture of methanol and water with 10 mM ammonium acetate, was adjusted to pH 3 with glacial acetic acid. A sample clean-up procedure was not included because of the low recoveries of free and masked mycotoxins and their differences in polarity. The method allowed the simultaneous determination of 13 Fusarium mycotoxins in a one-step chromatographic run using a Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer. The method was validated for several parameters such as linearity, apparent recovery, limit of detection, limit of quantification, precision, expanded measurement uncertainty and specificity. The limits of detection varied from 5 to 13 ng g⻹; those for the limit of quantification from 10 to 26 ng g⻹. The results of the performance characteristics of the developed LC-MS/MS method were in good agreement with the criteria mentioned in Commission Regulation (EC) No. 401/2006. Thirty samples of a variety of food and feed matrices were sampled and analysed between July 2010 and January 2011.
Assuntos
Pão/análise , Grão Comestível/química , Contaminação de Alimentos , Inspeção de Alimentos/métodos , Toxina T-2/análise , Tricotecenos/análise , Zearalenona/análise , Ração Animal/análise , Cromatografia Líquida de Alta Pressão , União Europeia , Inspeção de Alimentos/normas , Fumonisinas/análise , Fumonisinas/química , Fumonisinas/metabolismo , Fusarium/metabolismo , Limite de Detecção , Ocratoxinas/análise , Ocratoxinas/química , Ocratoxinas/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray , Toxina T-2/análogos & derivados , Toxina T-2/química , Toxina T-2/metabolismo , Espectrometria de Massas em Tandem , Tricotecenos/química , Tricotecenos/metabolismo , Zearalenona/química , Zearalenona/metabolismoRESUMO
Seven commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns (IACs) were tested for cross-reactivity to conjugated forms (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, DON-3-glucoside, DON-3-glucuronide, ZEN-glucosides, ZEN-glucuronide) and metabolites (de-epoxydeoxynivalenol, α-zearalenol, ß-zearalenol) and nivalenol (NIV), using a semi-quantitative multi-mycotoxin ultra-performance liquid chromatography-tandem mass spectrometry method. The DON IACs showed cross-reactivity for nearly all DON derivatives tested. The ZEN IACs showed limited cross-reactivity to some of the ZEN derivatives. The IACs were evaluated for their potential use as sample clean-up for mycotoxins in serum.
Assuntos
Especificidade de Anticorpos , Cromatografia de Afinidade , Imunoensaio , Tricotecenos/imunologia , Zearalenona/imunologia , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Soro , Espectrometria de Massas em Tandem , Tricotecenos/análise , Tricotecenos/química , Zearalenona/análise , Zearalenona/químicaRESUMO
The glucosinolate profile of black radish (Raphanus sativus L. niger) based dietary supplements has been investigated by HPLC-PDA, LC-ESI-MS/MS and LC-APCI-MS/MS systems. Optimization of the MS/MS parameters and LC conditions was performed using sinigrin reference standard and rapeseed certified reference material (BC190) respectively. An LC-ESI-MS/MS system was used to detect (screen) and identify the naturally occurring intact glucosinolates (GLs). The intact GLs identified were then desulfated and quantified on an HPLC-PDA system as desulfo-glucosinolates (DS-GLs). Prior to quantification, the DS-GLs were identified using an APCI-MS/MS. The HPLC-PDA method performance criteria were evaluated using glucotropaeolin potassium salt. The validated method was applied for the analysis of six dietary supplements. In total, six glucosinolates were identified and quantified in the dietary supplements; glucoraphasatin (0.2-0.48 mg/g), glucosisaustricin (0.37-0.91 mg/g), glucoraphenin (0.84-1.27 mg/g), glucoputrajivin (0.14-0.28 mg/g), glucosisymbrin (0.70-0.99 mg/g) and gluconasturtiin (0.06-0.12 mg/g). Glucoraphenin was the most abundant glucosinolate in all samples.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Suplementos Nutricionais/análise , Glucosinolatos/análise , Raphanus/química , Espectrometria de Massas em Tandem/métodos , Glucosinolatos/química , Concentração de Íons de Hidrogênio , Análise de Regressão , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10-400 µg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 µg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 µg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements.
Assuntos
Antioxidantes/farmacologia , Células CACO-2/efeitos dos fármacos , Citocromo P-450 CYP1A1/biossíntese , Enterócitos/efeitos dos fármacos , Ginkgo biloba/química , Extratos Vegetais/farmacologia , Células CACO-2/enzimologia , Células CACO-2/patologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Enterócitos/enzimologia , Enterócitos/patologia , Indução Enzimática , HumanosRESUMO
A capture enzyme-linked immunosorbent assay (cELISA) was developed using intimin-specific monoclonal antibodies to detect specific antibody in rabbits that have been in contact with enteropathogenic Escherichia coli (EPEC). Sera from 121 EPEC-negative, minimum-disease-level (MDL) rabbits were used for negative controls, and sera from 25 MDL rabbits, experimentally infected with EPEC of bio-/serotype 3-/O15, for positive controls. These were used to determine a cut-off value for a positive cELISA result. The value selected gave the test a sensitivity of 80.0% and a specificity of 98.4% on an individual level. At this value, a flock level sensitivity and specificity of 79.2 and 85.2%, respectively were calculated for a flock with a prevalence of seven per cent, if 40 animals were tested, and a minimum of two reactors were obtained. The test characteristics improve with increasing prevalence. To evaluate the diagnostic potential of the cELISA, sera from 40 to 50 slaughter rabbits per flock from 25 rabbit flocks with bacteriologically determined EPEC status were tested. The results demonstrated that this test can be a useful tool to determine the EPEC status of a rabbitry, provided that it is used at regular intervals.
Assuntos
Adesinas Bacterianas/imunologia , Proteínas de Transporte/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli , Escherichia coli/isolamento & purificação , Coelhos/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Proteínas da Membrana Bacteriana Externa , Eletroforese em Gel Bidimensional/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/imunologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Espectrometria de Massas/veterinária , Camundongos , Prevalência , Sensibilidade e EspecificidadeRESUMO
Four anthocyanins from Ajuga reptans flowers and its cell cultures were isolated, and a fifth was also characterized by HPLC-mass spectrometry. By means of chemical and spectroscopic analyses, their structures were identified as delphinidin 3-(p-coumaroyl-feruloyl)sophoroside-5-malonylglucoside, delphinidin 3-(diferuloyl)sophoroside-5-malonylglucoside, and cyanidin 3-(di-p-coumaroyl)sophoroside-5-glucoside, respectively. The other two were tentatively identified as delphinidin 3-(diferuloyl)sophoroside-5-glucoside and cyanidin 3-(feruloyl-p-coumaroyl)sophoroside-5-malonylglucoside. In neutral aqueous solution, the crude extract from A. reptans flower cell cultures and the major anthocyanin cyanidin 3-(di-p-coumaroyl)sophoroside-5-malonylglucoside were more stable than cyanidin 3-glucoside, and also prevented more efficiently peroxidation than did the latter. A. reptans flower cell culture anthocyanins may have a potential as natural colorants for food utilities or other purposes.
Assuntos
Glucosídeos/isolamento & purificação , Isoflavonas/isolamento & purificação , Lamiaceae/química , Acilação , Antocianinas , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Sequência de Carboidratos , Células Cultivadas , Glucosídeos/química , Glucosídeos/farmacologia , Isoflavonas/química , Isoflavonas/farmacologia , Lamiaceae/citologia , Dados de Sequência Molecular , Análise EspectralRESUMO
Four anthocyanins were isolated from Ajuga reptans flowers and one from the cell cultures. By FAB mass spectrometry measurements, the structures of these pigments were determined as delphinidin and cyanidin glucosides acylated with two cinnamic acids, while three of them were also malonylated. A delphinidin-based pigment in the crude extract from cell cultures was identical to the major flower pigment as shown by HPLC co-chromatography. Moreover, by application of 1H and 13C NMR consisting of DQF-COSY, NOESY, ROESY, 2D-HOHAHA, HSQC and HMBC methods, the structures of two new anthocyanins were identified as delphinidin and cyanidin 3-O-(2-O-(6-O-(E)-p-coumaryl-beta-D-glucopyranosyl)-(6-O-(E)-p- coumaryl)-beta-D-glucopyranosyl)-5-O-(6-O-malonyl-beta-D-glucopyranoside ). The deacylated anthocyanins were confirmed as delphinidin and cyanidin 3-sophoroside-5-glucosides.
Assuntos
Antocianinas/química , Plantas/química , Acilação , Antocianinas/isolamento & purificação , Configuração de Carboidratos , Sequência de Carboidratos , Células Cultivadas , Dados de Sequência Molecular , Estrutura MolecularRESUMO
Lactose utilisation by cucumber cell suspension cultures starts only after a long lag phase and is accompanied by an increase of an extracellular lactosespecific ß-galactosidase activity. Supplementing the lactose medium with sucrose shortens the lag phase.Milk whey permeate seems to contain a factor(s) which inhibits lactose utilisation. After supplementing the medium with sucrose or its hydrolysis products, growth and substrate utilisation is as efficient as in Murashige and Skoog medium. Galactose also induces growth, but growth and substrate utilisation are slower. In whey medium, supplemented with sucrose, the extracellular ß-galactosidase activity again accompanies growth induction.
RESUMO
The evolution of the total amount of DNA in epicotyls and of the amount of DNA per cell nucleus in epicotyl cortex cells during germination was followed in two closely related pea varieties, Pisum sativum cv. Finale and Pisum sativum cv. Rondo. Under etiolating conditions, growth of the cv. Rondo occurs only by cell elongation which is preceded by endomitotic DNA synthesis, while in the cv. Finale growth is the result of cell elongation accompanied by endomitotic DNA synthesis and cell division. The maximum C-level attained in both cultivars under etiolating conditions is 8 C (C=haploid amount of DNA in a gamete cell). Both the maximum C-level reached and the percentage of cells reaching this C-level seem to be under strict genetic control. In both cultivars, light inhibits the endomitotic DNA replication.Neither gibberellic acid (GA3), nor AMO 1618 alter the maximum C-level or the percentage distribution of the C-classes. Both growth regulators are effective, although in an opposite way, only in tissues where cell division occurs or where endomitotic DNA synthesis is blocked, as in light-grown pea epicotyls.
RESUMO
Large doses of gamma-irradiation, given to air-dried pea seeds, inhibit the endomitotic DNA synthesis in pea epicotyls during germination in darkness. The cortex cells of the etiolated epicotyls reach only the 4 C DNA level, whereas cortex cells of unirradiated seeds reach the 8 C DNA level. Epicotyl elongation and cell elongation are also reduced.Application of gibberellic acid restores the endomitotic DNA synthesis and the cell elongation in epicotyls of irradiated seeds. The cortex cells reach again the 8 C DNA level in darkness.The results suggest that gamma-irradiation blocks endomitotic DNA synthesis and cell elongation by lowering the concentration of endogenous gibberellins.