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.     

Analysis of zearalenone and alpha-zearalenol in urine of ruminants using gas chromatography-tandem mass spectrometry

The present paper describes a sensitive procedure for quantitative analysis of the Fusarium mycotoxins zearalenone and alpha-zearalenol in urine of ruminants. Extraction is done with an octadecyl (C18) column and cleanup with a silica column providing a preparation that is analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS). The trimethylsilyl ether derivatives of zearalenone and alpha-zearalenol yield molecular ions with m/z 462 and 536, respectively. These ions are selected in the first mass analyzer and then fragmented in a collision cell to give characteristic daughter ions (m/z 151, 333, 318, and 446). The method is known as multiple reaction monitoring (MRM). Elimination of chemical background noise by selecting proper fragment ions produces chromatograms in which identification and quantitation in a biological matrix is possible. The method was tested with sheep urine from an experimental feeding trial and was used to confirm natural mycotoxicosis of cows affected with zearalenone. Zearalenone (1 ppb) and alpha-zearalenol (14 ppb) were found in 2 different cow urine samples. The detection limit for both zearalenone and zearalenol is 1 ppb (1 ng/mL) in urine and is linear between 1 and 20 ppb for the former and 1 and 10 ppb for the latter.     

Quantitative determination and structure elucidation of type A- and B-trichothecenes by HPLC/ion trap multiple mass spectrometry.

A method is presented for the quantification and structure confirmation of trichothecenes in wheat by high-performance liquid chromatography combined with multiple mass spectrometry (MS(n)()). Nine type A- and B-trichothecenes were determined (nivalenol, deoxynivalenol, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin). Extraction was carried out with acetonitrile/water. The extract was purified on a MycoSep column. Quantification was based on an internal standard and atmospheric pressure chemical ionization in the positive ion mode. Recoveries from spiked wheat were in the range of 80-106% at levels of 500 ppb. The limits of quantification for the whole method were between 10 and 100 ppb. Ion adduct formation with ammonium and acetate ions and MS(n) experiments provided information about substitution and fragmentation behavior of the mycotoxins. A scheme has been established for the partial structure elucidation of type A- and B-trichothecenes in fungal cultures.     

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.     

Determination of T-2 and HT-2 toxins in cereals including oats after immunoaffinity cleanup by liquid chromatography and fluorescence detection

A reliable method for the determination of T-2 toxin and HT-2 toxin in different cereals, including oats, as well as in cereal products was developed. After extraction with methanol/water (90/10, v/v) and dilution with a 4% NaCl solution, the toxins were purified with immunoaffinity columns, derivatized with 1-anthroylnitrile, separated by HPLC, and determined using fluorescence detection. Due to the unspecific derivatization reagents, validation parameters were matrix dependent: in the range 10-200 microg/kg, recovery rates of 74-120% with relative standard deviations between 0.5 and 20.3% were obtained. On average, the limit of quantitation was shown to be 8 microg/kg for each toxin. For naturally contaminated samples, comparable results were obtained when analysis was performed according to this method without derivatization as well as according to a method based on a SPE cleanup utilizing tandem mass spectrometric detection in both cases. Using aqueous acetonitrile as extractant resulted in incorrectly high toxin concentrations due to water absorption of dry samples and toxin accumulation in the organic phase in the subsequent phase separation of the extractant. Furthermore, when comparing the commercially available immunoaffinity columns for T-2 and HT-2 toxins, significant differences regarding capacity and cleanup performance were observed.     

Development of liquid chromatography-electrospray mass spectrometry for the determination of patulin in apple juice: investigation of its contamination levels in Japan

Patulin is a mycotoxin produced by mainly Penicillium species, for example, P. expansum, and Aspergillus species. There are several reports of patulin contamination in apple juice. Last year, the Ministry of Health, Labour and Welfare of Japan set the maximum allowable level of patulin in apple juice at 50 ppb and decided that the measurement of patulin levels in apple juice products should be conducted. To this end, a simple, accurate, and selective analytical method for the detection of patulin at levels lower than 5 ppb, the detection limit, is desired. This paper reports the development of an analytical method that employs solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS). When MS measurements were conducted with the selected ion monitoring (SIM) mode, the pseudomolecular ions at m/z 153 and 156 were used to monitor patulin and (13)C(3)-labeled patulin, respectively. The detection limit (S/N = 3) and the quantification limit (S/N = 10) of patulin at injection levels into LC-MS were 12.5 and 25 pg, respectively. However, when the actual sample was applied for the analysis based on the developed method including the sample preparation, the detection limit (S/N = 3) and quantification limit (S/N = 10) were 2.5 and 5 pg in sample, respectively. The calibration curve obtained for concentrations ranging from 5 to 500 ppb showed good linearity with a coefficient of determination (r (2)) of 0.999. In addition, the recovery was >95% when an internal standard was used. The method was applied to the analysis of 76 apple juice samples from Japan, and as a result, patulin levels ranging from <1.0 to 45 ppb (detection frequency = 15/76) were detected. In this study, it was found that patulin was a greater contaminant in concentration/reduction than in "not from concentrate" apple juice.     

Assay of the set of all Sudan azodye (I, II, III, IV, and Para-Red) contaminating agents by liquid chromatography-tandem mass spectrometry and isotope dilution methodology

A high-throughput mass spectrometric method is presented for the simultaneous detection of Sudan I, II, III, IV, and Para-Red azodyes in foodstuff. The method is based on the use of deuterium-labeled internal standards and atmospheric pressure chemical ionization (APCI) in a triple-quadrupole instrument. The gas-phase breakdown pattern of each labeled and unlabeled analogue displays the naphthoic moiety as a common fragment. The search for the parents of this common species (parent ion scans) allows, by flow injection and in a single run, the evaluation of the presence of each polluting species spiked in typical foodstuffs. A detailed assay of each azodye was performed by LC-APCI and isotope dilution method, through the multiple reaction monitoring approach, using deuterium-labeled internal standards. Sudan dyes can be quantified above the threshold of 10 ppb except for Sudan Para-Red, for which the limit of quantification was 20 ppb, likely due to the different ionization efficiency.     

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.     

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 alachlor, metolachlor, atrazine, and simazine in water and soil by isotope dilution gas chromatography/mass spectrometry

A multiresidue method was developed for the simultaneous determination of low parts per billion (ppb) concentrations of the herbicides alachlor, metolachlor, atrazine, and simazine in water and soil using isotope dilution gas chromatography/mass spectrometry (GC/MS). Known amounts of 15N,13C-alachlor and 2H5-atrazine were added to each sample as internal standards. The samples were then prepared by a solid phase extraction with no further cleanup. A high resolution GC/low resolution MS system with data acquisition in selected ion monitoring mode was used to quantitate herbicides in the extract. The limit of detection was 0.05 ppb for water and 0.5 ppb for soil. Accuracy greater than 80% and precision better than 4% was demonstrated with spiked samples.     

Extraction, gas-liquid chromatographic detection, and quantitation of hexachlorophene residues from plant tissues high in lipid content

A method has been developed for the extraction, cleanup, derivatization, detection, and quantitation of hexachlorophene (HCP) residues from 2 types of plant storage tissue high in lipid content. Wet soybean or peanut tissue was homogenized and extracted with ethyl ether and chromatographed on silica gel to remove the neutral lipids. The cleaned up sample was methylated with diazomethane and the dimethoxyhexachlorophene was eluted from a second silical gel column and chromatographed on a 6' glass column packed with 3% OV-1 or 3% SE-30 on Gas-Chrom Q. The instrument detection limit for the 63Ni electron capture detector was less than 0.1 ng for dimethoxyhexachlorophene and about 1 ppb HCP residue in plant issue. Recovery of 10-420 ppb HCP added to tissue averaged 90.9 +/- 5.7%. Interfering substances were removed, column life was increased, peak sharpness was increased, and tailing of the parent compound was decreased by using appropriate column chromatography.     

Fast and sensitive screening method for detection of trichothecenes in maize by using protein synthesis inhibition in cultured fibroblasts

A fast and sensitive bioassay with hamster (BHK-21 C13) fibroblasts for the detection of toxic trichothecenes in maize is described. Cells are exposed to pure toxins or crude maize extracts for 30 min. The mixture is then incubated with [1-14C]-leucine for an additional 60 min and the radioactivity incorporated into the protein of the washed cells is determined. The sensitivity of the assay was in the range 1-10 ng/mL (or 50 ppb in maize) for T-2, HT-2, and diacetoxyscirpenol. At least 1000-fold higher concentrations of non-trichothecene mycotoxins and plant toxins were necessary to cause an inhibition of protein synthesis in the cells. Of 24 maize samples tested, 14 gave a positive response in this assay and the presence of trichothecenes could be confirmed chemically in 11 samples. Therefore, the described bioassay is proposed as a useful screening method for cytotoxic trichothecenes in maize.     

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.     

Immunochromatography of fusarochromanone mycotoxins

The present paper describes an enzyme-linked immunoassay (ELISA) used in combination with thin-layer chromatography (TLC) and liquid chromatography (LC) for determination of fusarochromanone (TDP) mycotoxins in barley, wheat, and a Fusarium culture grown in rice and corn. The mycotoxins were first extracted from the sample with 100% methanol and subjected to TLC or LC without additional cleanup treatment. Individual fractions eluted from TLC or LC were acetylated, then analyzed by ELISA. Determinations of TDP toxins at levels as low as 0.1 and 0.5 ng were achieved by ELISA in combination with LC and TLC, respectively. The detection limit for TDP-1 in barley and wheat was about 20 ppb by ELISA alone as compared with a detection limit of 5 ppb by a combination of ELISA with either TLC or LC. Overall analytical recovery (% of added) of TDP-1 added to barley and wheat at 5, 10, and 20 ppb of TDP-1 was 106.9 +/- 15.3 and 113.2 +/- 11.6 by LC-ELISA and 108.8 +/- 9.1 and 110.4 +/- 4.9 by TLC-ELISA, respectively. Analysis of extracts obtained from Fusarium equiseti R6137 grown in corn and rice by the combination of TLC and ELISA revealed that diacetyl-TDP was also produced by this fungus in addition to TDP-1 and TDP-2. Comparable results were obtained when fungal extracts were subjected to ELISA, LC, and immunochromatography (i.e., combination of ELISA with either TLC or LC).     

Detection of twelve mycotoxins in mixed animal feedstuffs, using a novel membrane cleanup procedure.

A multimycotoxin thin layer chromatographic screening method is described which is applicable to most animal feedstuffs. Interference from nonspecific lipid, pigment, and other components of simple and mixed feeds is reduced to a minimum by using a membrane cleanup step. Aflatoxins B1, B2, G1, and G2, citrinin, diacetoxyscirpenol, ochratoxin A, patulin, penitrem A, sterigmatocystin, T-2 toxin, and zearalenone may be reliably detected. The sensitivity of the method is generally low for mixed feeds but even so aflatoxin B1 can be detected at a level of 3 ppb and ochratoxin A at 80 ppb. While the basic method is less sensitive for sterigmatocystin (330 ppb), patulin (600 ppb), zearalenone (1000 ppb), and the trichothecenes (1000-4000 ppb), it may be adapted so as to reduce the above detection limits when the presence of these toxins is suspected. Lower levels may be detected in extracts of simple feeds.     

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.     

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.     

A gas chromatography-flame ionization detection method for detection of fusaproliferin in corn

A sensitive and accurate detection method is of great importance in monitoring fusaproliferin levels in foods and animal feeds and evaluating its potential hazard to human and animal health. Several methods have been developed to detect fusaproliferin in cereals and cereal-related products, including thin-layer chromatography, high-performance liquid chromatography, enzyme-linked immunosorbent assay, liquid chromatography-mass spectrometry (MS), gas chromatography (GC), and GC-MS. However, these detection methods either suffer from low sensitivity, need expensive instruments, or are susceptible to interfering substances in the sample matrix. The GC-flame ionization detector method developed herein is sensitive, reliable, and easy to use for detecting fusaproliferin in corn and corn-based samples. Its detection limits were 0.04 ng for standard trimethylsilyl-fusaproliferin and about 5 ppb for fusaproliferin in corn samples. The limits of quantitation of this method were 0.15 ng fusaproliferin/injection and 20 ppb of fusaproliferin in corn samples. The recovery rates of fusaproliferin from corn samples spiked with 200, 1000, and 5000 ppb standard fusaproliferin were 109, 85.7, and 98.9% on average. The repeatability of the method was acceptable when evaluated by the Horwitz equation. Of the tested corn samples, three out of five sweet corn and the three yellow corn samples were found to have low levels of fusaproliferin (9.4-45.3 ppb). A moldy corn sample had a fusaproliferin content of 297 ppb.     

Analysis of fenbendazole residues in bovine milk by ELISA

Fenbendazole residues in bovine milk were analyzed by ELISAs using two monoclonal antibodies. One monoclonal antibody (MAb 587) bound the major benzimidazole anthelmintic drugs, including fenbendazole, oxfendazole, and fenbendazole sulfone. The other (MAb 591) was more specific for fenbendazole, with 13% cross-reactivity with the sulfone and no significant binding to the sulfoxide metabolite. The limit of detection of the ELISA method in the milk matrix was 7 ppb for MAb 587 and 3 ppb for MAb 591. Fenbendazole was administered in feed, drench, and paste form to three groups of dairy cattle. Milk was collected immediately before dosing and then every 12 h for 5 days. The ELISA indicated that residue levels varied widely among individual cows in each group. Fenbendazole levels peaked at approximately 12-24 h and declined rapidly thereafter. Metabolites were detected at much higher levels than the parent compound, peaked at approximately 24-36 h, and declined gradually. Residue levels were undetectable by 72 h. The ELISA data correlated well with the total residues determined by chromatographic analysis, but the use of the two separate ELISAs did not afford an advantage over ELISA with the single, broadly reactive MAb 587. The ELISA method could be used to flag high-residue samples in on-site monitoring of fenbendazole in milk and is a potential tool for studying drug pharmacokinetics.     

Determination of aflatoxin M1 in milk and milk products contaminated at low levels

A new method is described for the determination of aflatoxin M1 in milk and dairy products by thin layer chromatography. The main characteristic is the extraction system using an alkaline solution. Lipids are removed by centrifuging at low temperatures, and the aflatoxins are then extracted with CHCl3. The method has 2 options: Technique II (detection limit 0.02 ppb) requires cleanup on a chromatographic column; this is not necessary in Technique I (detection limit 0.1 ppb). The recovery rate in both techniques is over 92.8% in milk and yoghurt. This method may also be used for other aflatoxins. Because of the advantages of the method, Technique II is recommended for aflatoxin M1 control in milk, where a low detection limit is necessary. Technique I is proposed for experimental aflatoxin production studies in dairy products, which require analysis of a large number of samples but which do not require a very low detection limit.