Simultaneous analysis of T-2 toxin and HT-2 toxin by an indirect enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of HT-2 toxin in the presence or absence of T-2 toxin is described. In the indirect ELISA, the relative cross-reactivities of antibodies against T-2 toxin (anti-T-2) with T-2 toxin and HT-2 toxin were 1 and 0.1, whereas anti-HT-2 cross-reactivities with T-2 toxin and HT-2 toxin were 0.33 and 1, respectively. Using such relationships, a formula was established that could be used to calculate the individual toxin concentration in a mixed sample after experimentally analyzing for T-2 and HT-2 toxins in the 2 indirect ELISAs. This method was tested by analyzing urine samples spiked with HT-2 toxin alone and samples spiked with both T-2 toxin and HT-2 toxin. A cleanup protocol for treatment of urine samples before ELISA was also established. The overall analytical recovery of HT-2 toxin when it was added at concentrations of 0.1-10 parts per billion (ppb) to the urine samples was ca 89%. When both T-2 and HT-2 toxins were added to the urine samples at equal concentrations of 0.5 to 5.0 ppb, their recoveries were 112 and 109%, respectively.     

Use of small charcoal/alumina cleanup columns in determination of trichothecene mycotoxins in foods and feeds

Small charcoal/alumina cleanup columns have been effectively used to remove interfering materials from grain, feed, and food extracts prior to chromatographic determination of trichothecene mycotoxins. A thin layer chromatographic method has been developed that can simultaneously detect part per billion concentrations of deoxynivalenol, fusarenon X, nivalenol, T-2 toxin, HT-2 toxin, neosolaniol, and diacetoxyscirpenol in food and feed samples. Recoveries of 90-99% can be obtained. The use of charcoal/alumina cleanup columns in conjunction with liquid chromatography and gas chromatography of trichothecenes is also discussed.     

Analysis of the Fusarium mycotoxins fusaproliferin and trichothecenes in grains using gas chromatography-mass spectrometry

A method is described using gas chromatography-mass spectrometry (GC-MS) for the simultaneous detection of the Fusarium mycotoxins fusaproliferin and seven trichothecenes from grains. Sample purification of the raw extract was carried out with commercial solid phase extraction columns, and the recovery of the more polar analytes was increased by rinsing the column with acetonitrile. A significant matrix effect was found for the analysis of fusaproliferin and trichothecenes; thus, the calibrants should be prepared in a blank matrix. The response was linear in the range used. The mean recovery for fusaproliferin was 60.4 or 62.9%, depending on the spiking level. With respect to the trichothecenes, the recovery was generally higher (70.2-125.3%). The method proved to be repeatable for the analysis of fusaproliferin and trichothecenes. The limit of detection for fusaproliferin in the blank matrix mixture was 50 microg/kg, and that for trichothecenes was 5-15 microg/kg. Thirty-eight Finnish grain samples were analyzed for fusaproliferin and trichothecenes with the method developed. Fusaproliferin was not detected in any of the samples. The mean levels of deoxynivalenol, 3-acetyldeoxynivalenol, nivalenol, HT-2 toxin, and T-2 toxin in Finnish grain samples were 272, 17, 150, 40, and <20 microg/kg, respectively.     

Gas chromatographic screening method for T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and related trichothecenes in feeds

A gas chromatographic method for screening trichothecene mycotoxins in feeds is described. Feed is extracted with acetonitrile-water, and the toxins are purified with charcoal-alumina-Celite, Florisil, and silica mini-columns. Deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), T-2 toxin, and their fungal metabolites are hydrolyzed to their corresponding parent alcohols (DON, NIV, scirpentriol, or T-2 tetraol) by alkaline hydrolysis. After derivatization to their pentafluoropropionyl analogs, they are quantitated by capillary gas chromatography with electron capture detection. Identity can be confirmed and sensitivity can be increased by using negative chemical ionization mass spectrometry with no additional sample workup. Recoveries of DAS, DON, and T-2 toxin averaged, respectively, 80, 65, and 85% in corn; 84, 65, and 88% in soybeans; and 70, 57, and 96% in mixed feeds at concentrations ranging from 0.1 to 2.0 ppm. Recoveries of 15-monoacetoxyscirpenol (MAS), HT-2, NIV, and T-2 tetraol were 97, 97, 86, and 56%, respectively, in corn at a concentration of 0.25 ppm: A detection limit of 0.02 ppm in corn, soybeans, and mixed feeds, and 0.05 ppm in silages is estimated.     

Mycotoxins in animal feedstuffs: sensitive thin layer chromatographic detection of aflatoxin, ochratoxin A, sterigmatocystin, zearalenone, and T-2 toxin

Improvements have been made to a previously described multi-mycotoxin method that involved a membrane cleanup step. Using 2-dimensional thin layer chromatography and appropriate solvent systems, aflatoxin B1 can be detected in mixed feedstuffs and various ingredients at levels ranging from 0.1 to 0.3 microgram/kg. Corresponding detection limits for ochratoxin A and sterigmatocystin are 5 to 20 microgram/kg and for T-2 toxin and zearalenone 20 to 200 microgram/kg.     

Application of manual and automated systems for purification of ochratoxin a and zearalenone in cereals with immunoaffinity columns.

A manual vacuum manifold and an automated solid phase extraction (ASPEC) system were applied for purification of ochratoxin A and zearalenone in wheat, rye, barley, and oat samples with immunoaffinity columns followed by separation with a high-performance liquid chromatograph and fluorescence detection. The immunoaffinity columns for manual sample purification were purchased from a different manufacturer than were those for the automated system. The limit of detection (LOD) for the method for ochratoxin A with a vacuum manifold and ASPEC was 0.1 microg/kg. For the method for zearalenone, the LODs were 1.5 microg/kg with a vacuum manifold and 3 microg/kg with ASPEC. For the methods for ochratoxin A at spiking levels of 0.6 and 2.5 microg/kg, mean recoveries for different cereals varied from 68 to 106%. For the methods for zearalenone, mean recoveries varied from 78 to 117% at spiking levels of 9 and 25 microg/kg. The relative standard deviations of repeatability with various cereals employing both methods were 2-15 and 2-19% for ochratoxin A and zearalenone, respectively.     

Development of qualitative and semiquantitative immunoassay-based rapid strip tests for the detection of T-2 toxin in wheat and oat

Novel qualitative as well as semiquantitative rapid strip tests for screening of T-2 mycotoxin in agricultural commodities were developed. Colloidal gold particles were coated with monoclonal anti-T-2 antibodies and used as detector reagent, indicating the strip test results by formation of up to two colored lines in a competitive assay format. The test line comprises a protein conjugate of the T-2 mycotoxin and the control line an antispecies-specific antibody to confirm the correct test development. To perform the test, 5 g of sample was extracted in a ratio of 1:5 with methanol/water (70:30) by shaking for 3 min and the extract directly used without further cleanup steps. The T-2 toxin lateral flow device (LFD) presented has a cutoff level around 100 microg/kg for naturally contaminated wheat and oat. The semiquantitative test may be used in the lower micrograms per kilogram range and allows for rapid semiquantitative photometric classification of the level of sample contamination. For both tests, results were obtained within 4 min. The developed LFDs therefore allow for the first time fast and on-site screening for the determination of T-2 toxin in cereals.     

Enzyme-linked immunosorbent assay for T-2 toxin metabolites in urine

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate-bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15-triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2-4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol-4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'-OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.     

Liquid chromatographic determination of aflatoxins in feedstuffs containing citrus pulp

A liquid chromatographic (LC) method was developed for the determination of aflatoxins in feedstuffs containing citrus pulp. The feed-stuff sample is extracted with chloroform, followed by Sep-Pak Florisil cartridge cleanup and Sep-Pak C18 cartridge cleanup. The final eluate (water-acetone, 85 + 15, v/v) is submitted to reverse-phase liquid chromatography with water-methanol-acetonitrile (130 + 70 + 40, v/v/v) as mobile phase and postcolumn derivatization with iodine. Citrus components are removed from the extract efficiently. The limit of detection for aflatoxin B1 is less than 1 microgram/kg. Other aflatoxins can also be detected and measured. Recoveries of aflatoxins B1, B2, G1, and G2 for dairy rations spiked at 13, 5, 10, and 4 micrograms/kg were 87, 86, 81, and 82%, respectively. Corresponding coefficients of variation were 3.1, 3.6, 5.2, and 3.8%, respectively.     

Comparative study of three different methods for the determination of aflatoxins in tahini

Aflatoxins spiked at three different levels (6.5, 13.0, and 19.5 microg/kg) in tahini, a sesame butter, were analyzed by using three different methods: high-performance liquid chromatography (HPLC), fluorometry, and enzyme-linked immunosorbent assay (ELISA). An immunoaffinity column was used for cleanup and purification of extracts prior to detection by HPLC and fluorometry. All methods were statistically evaluated for accuracy, precision, and simple correlations. Additionally, 14 tahini samples randomly obtained from Turkish retail markets were analyzed using an immunoaffinity column cleanup procedure coupled with the HPLC detection method. The fluorometric determination method involving an immunoaffinity column cleanup step was found to be highly correlated with the HPLC method (r = 0.978). Both methods were found to be effective due to their high recoveries and low variance for the prediction of total aflatoxin contamination in tahini samples. The ELISA method, due to its high variation in replicates, was found to be applicable only as a screening method. The survey study demonstrated the need for control of aflatoxin contamination of foodstuffs involving sesame seeds as an ingredient.     

Determination of 12 type A and B trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass spectrometry

A new sensitive method for the simultaneous determination of 12 trichothecenes (deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, fusarenon X, T-2 toxin, HT-2 toxin, neosolaniol, monoacetoxyscirpenol, diacetoxyscirpenol, T-2 triol, and T-2 tetraol) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The development of the method and investigations on the matrix influence on the MS signal are described in particular. The matrix effect was thereby minimized by using an internal standard, a special mobile phase, and specific fragmentation parameters. The sample was extracted with acetonitrile/water (84:16, v/v), and the extract was cleaned up with a MycoSep 227 column. Quantification was based on the internal standard de-epoxy-deoxynivalenol. Calibration curves were linear between 16 and 1600 ng/g, and the limits of detection ranged from 0.18 to 5.0 ng/g. The developed method was applied for the determination of trichothecenes in 120 naturally contaminated wheat and oat samples.     

Aflatoxins and ochratoxin A in red paprika for retail sale in Spain: occurrence and evaluation of a simultaneous analytical method

Aflatoxins are the only mycotoxins with legal limits for spices in the European Union. A further limit for ochratoxin A is expected to be adopted soon. Thus, rapid simultaneous methods for quantifying the five mycotoxins are sought. Liquid extraction, immunoaffinity column cleanup, and HPLC-FD with a wavelength program were optimized for the analysis of the five mycotoxins in paprika, a complex fatty matrix. The limits of detection ranged from 0.3 to 0.6 microg/kg. Repeatability (RSDr) ranged from 7.9 to 13.4%, and recoveries were between 61.4 and 77.8%, in both cases at the lower spike level. Aflatoxins, when found, were far below the two legal limits of 5 microg/kg for aflatoxin B 1 and 10 microg/kg for total aflatoxins. Ochratoxin A was more frequently found, with a mean of 11.8 microg/kg, and in a more varied range (SD = 18.9 microg/kg). When an automation of the precolumn derivatization step was attempted, the procedure proved to be unfeasible, but experience derived from this trial and from the general employment of this reaction enables some comments on the possibilities and limitations of this procedure and on research for an alternative one to be made.     

Enzyme-linked immunosorbent assay for the detection of yessotoxin and its analogues

Polyclonal antibodies were produced for the development of competitive enzyme-linked immunoassays for use in quantifying yessotoxins in shellfish, algal cells, and culture supernatants. Immunizing and plate coating antigens were prepared by derivatization of yessotoxin either by ozonolysis or bromination and conjugation to proteins. Two assays that were the most sensitive for yessotoxin were optimized and characterized. Cross-reactivity studies indicated that the antibodies raised have broad specificity and that binding to analogues was strongly affected by changes to the A-ring and, to a lesser extent, the K-ring regions of the toxin molecule. ELISA provides a sensitive and rapid analytical method that is suitable for screening large numbers of samples and detects all the yessotoxin analogues that the European Commission currently requires shellfish to be tested for. The assay limit of quantitation for yessotoxin in whole shellfish flesh is 75 microg/kg; therefore, assay sensitivity is sufficient to measure toxin levels well below the maximum permitted level set by the European Commission. The antibodies produced can be used in additional applications such as the immunolocalization of yessotoxins in shellfish and preparation of immunoaffinity columns.     

Determination of aflatoxins in high-pigment content samples by matrix solid-phase dispersion and high-performance liquid chromatography

A fast, efficient, and cost-effective method was developed for the analysis of aflatoxins in farm commodities with high-pigment content, such as chili powder, green bean, and black sesame. The proposed method involved matrix solid-phase dispersion (MSPD) and high-performance liquid chromatography (HPLC)-fluorescence detection (FLD) with postcolumn electrochemical derivatization in a Kobra cell. The MSPD procedure combined the extraction with neutral alumina and pigment cleanup with graphitic carbon black (GCB) in a single step. The recoveries of aflatoxins ranged from 88% to 95% with the relative standard deviations (RSD) less than 6% (n = 6). The limits of detection (LODs) were 0.25 ng/g aflatoxin B1, G1, and 0.10 ng/g aflatoxin B2, G2, respectively. The analytical results obtained by MSPD were compared to those of the immunoaffinity column (IAC) cleanup method. No significant differences were found between the two methods by t-test at the 95% confidence level.     

Gas-liquid chromatographic/mass spectrometric screening method for T-2 toxin in milk

A method for the analysis of T-2 toxin in milk is presented. Ethyl acetate extracts of milk samples which had been spiked with T-2 toxin were purified by thin layer chromatography and derivatized with N,O-bis(trimethylsilyl)acetamide to produce the T-2 toxin trimethylsilyl ether (T-2 toxin-TMS). N,O-bis(trimethylsilyl-d9)acetamide was used to make T-2 toxin d9-trimethylsilyl ether (T-2 toxin-d9 TMS) which was added to the derivatized milk extract as an internal standard. Samples were analyzed by combined gas-liquid chromatography/mass spectrometry using either electron impact ionization or chemical ionization mass spectrometry. In electron impact ionization analyses, simultaneous monitoring of the T-2 toxin-TMS fragment ion at m/z 436 and the T-2 toxin-d9TMS fragment ion at m/z 445 gave a T-2 toxin-TMS detectability estimated at 6 microgram/kg. In chemical ionization analyses, the T-2 toxin-TMS fragment ion at m/z 377 and the T-2 toxin-d9TMS fragment ion at m/z 386 were simultaneously monitored to give a T-2 toxin-TMS detectability estimated at 3 microgram/kg. Average recovery was 85% at 200 microgram/kg and 65% at 20 microgram/kg.     

Detection and quantitation of T-2 mycotoxin in rat organs by radioimmunoassay

A standard radioimmunoassay was compared with radiochromatography for the ability to detect unlabeled T-2 mycotoxin in organs from exposed animals. When 10% of HT-2, the only known metabolite that cross-reacts with T-2, was included and expressed as T-2 equivalents in the radiochromatographic detection, correlation between toxin detection in liver, spleen, and kidney by the 2 techniques was r = 0.98. An unknown metabolite was detected in heart extract by radiochromatography. Inclusion of this material in the T-2 equivalents detected by radiochromatography indicated a near-perfect correlation (r = 0.95; p greater than 0.05; slope = 0.82; y = intercept = 72) among all 4 tissues.     

Simultaneous determination of ethidimuron, methabenzthiazuron, and their two major degradation products in soil

An analytical method has been developed for the quantification of two herbicides (ethidimuron and methabenzthiazuron) and their two main soil derivatives. This method involves fluidized-bed extraction (FBE) prior to cleanup and analysis by reverse-phase liquid chromatography with UV detection at 282 nm. FBE conditions were established to provide efficient extraction without degradation of the four analytes. (14)C-labeled compounds were used for the optimization of extraction and purification steps and for the determination of related efficiencies. Extraction was optimal using a fexIKA extractor operating at 110 degrees C for three cycles (total time = 95 min) with 75 g of soil and 150 mL of a 60:40 v/v acetone/water mixture. Extracts were further purified on a 500 mg silica SPE cartridge. Separation was performed on a C18 Purosphere column (250 mm x 4 mm i.d.), at 0.8 mL min(-1) and 30 degrees C with an elution gradient made up of phosphoric acid aqueous solution (pH 2.2) and acetonitrile. Calibration curves were found to be linear in the 0.5-50 mg L(-1) concentration range. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. Recovery values, determined from spiked samples, were close to 100%. Limits of detection ranged between 2 and 3 microg kg(-1) of dry soil and limits of quantification between 8 and 10 microg kg(-1) of dry soil. An attempt to improve these performances by using fluorescence detection following postcolumn derivatization by orthophthalaldehyde-mercaptoethanol reagent was unsuccessful.     

Identification and apoptotic potential of T-2 toxin metabolites in human cells

The mycotoxin T-2 toxin, produced by various Fusarium species, is a widespread contaminant of grain and grain products. Knowledge about its toxicity and metabolism in the human body is crucial for any risk assessment as T-2 toxin can be detected in processed and unprocessed food samples. Cell culture studies using cells of human origin represent a potent model system to study the metabolic fate of T-2 toxin as well as the cytotoxicity in vitro. In this study the metabolism of T-2 toxin was analyzed in a cell line derived from human colon carcinoma cells (HT-29) and primary human renal proximal tubule epithelial cells (RPTEC) using high-performance liquid chromatography coupled with Fourier transformation mass spectrometry (HPLC-FTMS). Both cell types metabolized T-2 toxin to a variety of compounds. Furthermore, cell cycle analysis in RPTEC proved the apoptotic effect of T-2 toxin and its metabolites HT-2 toxin and neosolaniol in micromolar concentrations.     

Substances interfering with the gas-liquid chromatographic determination of T-2 mycotoxin.

Two substances interfering with the gas-liquid chromatographic (GLC) detection of T-2 mycotoxin were identified as 1-glyceryl-monooleate and 1-glycerylmonolinoleate. These monoglycerides are natural products formed by species of Fusarium growing on cereal grains and are also additives contained in liquid vegetable and animal fats added to the feed mixture. The monoglycerides can be removed from the analytical sample by resolution by thin layer chromatography prior to separation by GLC. Trimethylsilyl ether derivatives of the monoglycerides and T-2 toxin have almost identical retention times on 3% OV-1 columns, whereas the trifluoroacetyl and pentafluoropropionyl derivatives give baseline separation on the same column. The monoglycerides can be misidentified as the T-2 toxin in analyses involbing GLC.     

Determination of pesticides and PCBs in virgin olive oil by multicolumn solid-phase extraction cleanup followed by GC-NPD/ECD and confirmation by ion-trap GC-MS

A multicolumn solid-phase extraction cleanup for the determination of organophosphorus (OP) and organochlorine (OC) pesticides plus PCB congeners in virgin olive oil is presented. The method involves dissolution of the olive oil in hexane, followed by a cleanup system using a diatomaceous earth column (Extrelut-QE) with reversed (C(18)) and normal (alumina) phase SPE columns. Determination of OPs was by GC-NPD, while the OCs and PCBs were analyzed using GC-ECD. Recovery assays for OPs varied from 81.7% to 105.3%, for OCs ranged between 74.3% and 99.4%, while for PCBs were from 60.1% to 119.2%. Quantitation limits ranged from 10 to 25 microg/kg olive oil for OPs, and from 1 to 6 microg/kg olive oil for OCs and PCBs. In the case of positive samples, the confirmation of pesticide identity was performed by ion-trap GC-MS/MS. The applicability of the method was assayed with 19 virgin olive oil samples collected from different olive mills of Aragón (Spain). Only one OP pesticide (acephate) was detected in one sample at a concentration of 10 microg/kg. Organochlorine pesticides were found in 5-47% of samples at very low levels ranging from 1.5 to 5.2 microg/kg. PCBs were found in 20-90% of samples, showing concentrations between 2.3 and 17.3 microg/kg.