Methods for the detection of trichothecenes.

The trichothecenes are a group of fungal metabolites with a tetracyclic, sesquiterpenoid ring system and include a number of compounds which are highly toxic. These compounds are produced by various species of the imperfect fungi including members of the following genera: Calonectria, Fusarium, Myrothecium, Stachybotrys, Trichoderma, and Trichothecium. Both biological and chemical methods for detection of various trichothecenes are reviewed. Some of the bioassay techniques in use for the detection of the various trichothecenes include the rabbit dermal toxicity tests, cytotoxicity, and inhibition of protein synthesis tests; these are highly sensitive but lace specificity. The sensitivity of these tests for the T-2 toxin are 0.005 mug for rabbit skin toxicity, 0.03 mug/ml for inhibition of protein synthesis in rabbit reticulocytes, and less than 1 mug/ml for cytotoxicity to human karyoblast cells. In the present state of development of the chemical assay methods, thin layer and gas-liquid chromatography are specific for various trichothecenes but lack sensitivity. The lower detection limit of the trichothecenes on thin layer plates varies from 0.2 to 2-3 mug/spot while gas-liquid chrommatography has a reported sensitivity of approximately 0.05 mug/injection.     

A gas chromatography/electron ionization-mass spectrometry-selected ion monitoring method for determining the fatty acid pattern in food after formation of fatty acid methyl esters

A method using gas chromatography/electron ionization-mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode was developed for the analysis of fatty acids as methyl esters (FAMEs) in order to determine their percentage contribution to the fatty acid profile in food. In the GC/EI-MS-SIM mode, saturated fatty acids were determined with m/z 87, monoenoic fatty acids were determined with m/z 74, and polyenoic fatty acids were determined via the sum of m/z 79 and m/z 81. The ratios of these fragment ions and the GC retention data provided additional information for tentative structural assignments. The 28 FAME standards tested provided similar results for the novel GC/EI-MS-SIM method and GC/EI-MS in the full scan mode, both of which were slightly worse than GC/flame ionization detection (FID). Analysis of sunflower oil, suet, and cod liver oil verified that both major and minor fatty acids (20-60% and down to 0.001% contribution to the fatty acid pattern) were determined with sufficient quality that justifies application of the GC/EI-MS-SIM method for the analysis of food samples. Furthermore, the method was approximately 20- or approximately 10-fold more sensitive than GC/EI-MS in the full scan mode or GC/FID, respectively. The method is suited for both quantitative purposes and fatty acid identification in samples where only low amounts of lipids are available.     

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.     

Analysis of agricultural residues on tea using d-SPE sample preparation with GC-NCI-MS and UHPLC-MS/MS

This study presents new sample preparation and analytical procedures for the quantification of pesticides on processed tea leaves. The new method includes tea extraction and dispersive solid phase extraction (d-SPE) to prepare gas chromatography (GC) and ultrahigh-performance liquid chromatography (UHPLC)-ready samples, providing a fast and cost-effective solution for time-sensitive industrial analysis to fulfill regulatory requirements. Both GC-negative chemical ionization mass spectrometry (GC-NCI-MS) and UHPLC-tandem mass spectrometry (UHPLC-MS/MS) were employed to produce highly sensitive and reproducible data. Excellent limits of detection (typically below 1 μg/kg for GC and 10 μg/kg for UHPLC), wide linearity ranges, and good recoveries (mostly >70%) were achieved on the selected pesticides. Twenty-seven tea samples purchased from local grocery stores were analyzed using the newly developed methods. Among the pesticides analyzed, endosulfan sulfate and kelthane were the most frequently detected by GC-NCI-MS and imidacloprid and acetamiprid by UHPLC-MS/MS in these teas. The samples were found to be relatively clean, with <1 mg/kg of total pesticide residues. The organic-labeled teas were significantly cleaner than nonorganic ones. The cost per gram of tea did not correlate with pesticide residue levels detected.     

Combination of TLC and HPLC-MS/MS methods. Approach to a rational quality control of Chinese star anise

In this study, a methodological approach for an effective and reliable quality control of Chinese star anise (Illicium verum Hook. F.) is developed and validated. A combined method of TLC and HPLC-MS/MS was used for differentiation of various Illicium species, especially Chinese and Japanese star anise. Species can be distinguished by their TLC flavonoid pattern. A sensitive and selective HPLC/ESI-MS/MS method was developed for the detection and quantification of lower admixtures of I. anisatum and of further toxic Illicium species at a low concentration range using the sesquiterpene lactone anisatin as a marker. The proposed assay includes a solid-phase extraction cleanup procedure with a high recovery (>90%). Chromatographic separation of anisatin was carried out on a C18 column, followed by MS detection using ESI in negative mode. The precursor/product ion transitions m/z 327 --> 127 (quantifier) and m/z 327 --> 297 (qualifier) were monitored. Statistical evaluation of this multireaction-monitoring procedure reveals good linearity and intra- and interday precision. The limits of detection and quantification are 1.2 and 3.9 microg/kg, respectively.     

Multiresidue determination of pesticides in malt beverages by capillary gas chromatography with mass spectrometry and selected ion monitoring

A method was developed to determine pesticides in malt beverages using solid phase extraction on a polymeric cartridge and sample cleanup with a MgSO4-topped aminopropyl cartridge, followed by capillary gas chromatography with electron impact mass spectrometry in the selected ion monitoring mode [GC-MS(SIM)]. Three GC injections were required to analyze and identify organophosphate, organohalogen, and organonitrogen pesticides. The pesticides were identified by the retention times of peaks of the target ion and qualifier-to-target ion ratios. GC detection limits for most of the pesticides were 5-10 ng/mL, and linearity was determined from 50 to 5000 ng/mL. Fortification studies were performed at 10 ng/mL for three malt beverages that differ in properties such as alcohol content, solids, and appearance. The recoveries from the three malt beverages were greater than 70% for 85 of the 142 pesticides (including isomers) studied. The data showed that the different malt beverage matrixes had no significant effect on the recoveries. This method was then applied to the screening and analysis of malt beverages for pesticides, resulting in the detection of the insectide carbaryl and the fungicide dimethomorph in real samples. The study indicates that pesticide levels in malt beverages are significantly lower than the tolerance levels set by the United States Environmental Protection Agency for malt beverage starting ingredients. The use of the extraction/cleanup procedure and analysis by GC-MS(SIM) proved effective in screening malt beverages for a wide variety of pesticides.     

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.     

A new LC/MS-method for the quantitation of acrylamide based on a stable isotope dilution assay and derivatization with 2-mercaptobenzoic acid. Comparison with two GC/MS methods

Acrylamide (AA) was found to form a stable thioether in reasonable yields (45-50%) when reacted with 2-mercaptobenzoic acid at 20 degrees C for 3 h. On the basis of this finding and using [(13)C(3)]-acrylamide as the internal standard, a sensitive and selective new stable isotope dilution analysis for AA quantitation in food samples was developed based on single stage LC/MS. Comparison of the quantitative results obtained by applying the new method to potato chips, crispbread or butter cookies with data obtained by two stable isotope dilution analysis, using direct measurement of AA by GC/MS, but differing in the workup procedure, revealed detection limits in the same order of magnitude (6.6 microg/kg). Quantitative data obtained by application of the three methods on the same samples of potato chips or cookies, respectively, were also in very good agreement. Quantitation of AA in crispbreads treated with an amylase/protease mixture did not show increased AA levels, thereby indicating that inclusion of AA in starch/protein gels is not very probable during breadmaking.     

Enzyme‐Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures,Extracts, and Plant Tissues

A rapid technique for trichothecene detection was needed in screening tests of the potential bioherbicide Myrothecium verrucaria (MV), in order to select strains, mutants, or formulations that were void of or that possessed low amounts of these undesirable mycotoxin compounds. Commercially available enzyme‐linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross‐reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L‐2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of Myrothecium verrucaria (MV) in cell cultures, in plant tissues (hemp sesbania and kudzu) treated with purified roridin A, or ethyl acetate fractions of MV cultures. Evaluations of ELISA assays showed linear responses for standards of verrucarin A and roridin A over a concentration range of 0.2 to 20 ppb. Ethyl acetate or aqueous extractions were used to obtain samples from MV cultures and plant tissues for testing. Trichothecenes were detected in conidia and mycelia of MV, and in agar upon which wild‐type MV was grown, indicating secretion into the growth media. Two MV sectors (morphological variants of wild type) also tested positive for trichothecenes. Purified roridin A and concentrated extracts containing trichothecenes from MV spore cultures exhibited phytotoxicity (growth inhibition or necrosis) when applied to excised shoots of hemp sesbania seedlings and intact kudzu leaf tissues. Evidence of some translocation of trichothecenes from the application point in kudzu was found, but translocation to the upper shoot portion of hemp sesbania was not detected at the lowest limit of detection in this assay (0.14 ppb). This assay is also being employed to identify induced mutants and/or other naturally occurring sectors deficient in trichothecene mycotoxin production. Results indicated that ELISA is a sensitive and rapid assay method to quantify trichothecenes produced by this bioherbicidal fungus and in certain plant tissues treated with trichothecenes.     

Multiresidue analysis of pesticides in soil by gas chromatography with nitrogen-phosphorus detection and gas chromatography mass spectrometry

A rapid multiresidue method for the simultaneous determination of 25 fungicides and insecticides in soil was developed. Soil samples are extracted by sonication with a water-acetonitrile mixture, and the pesticides are partitioned into dichloromethane. Final determination was made by gas chromatography (GC) with nitrogen-phosphorus detection (NPD). Confirmation analysis of pesticides was carried out by GC-MS in the selected ion monitoring (SIM) mode. The identification of compounds was based on retention time and on comparison of the primary and secondary ions. The average recovery by the GC-NPD method obtained for these compounds varied from 68.5% to 112.1% with a relative standard deviation between 1.8% and 6.2%. The GC-NPD method presents good linearity over the range assayed 50-2000 microg/L, and the detection limit for the pesticides studied varied from 0.1 to 10.4 microg/kg. The proposed method was used to determine pesticide levels in soil samples from experimental greenhouse pepper cultivation.     

Purge and trap method for determination of ethylene dibromide in table-ready foods

An improved method has been developed for the determination of ethylene dibromide (EDB, 1,2-dibromoethane) in a variety of table-ready foods. Samples are mixed with water and sparged with nitrogen for 1 h with stirring in a water bath at 100 degrees C. The EDB collected on the adsorbent Tenax TA is eluted with hexane and determined by gas chromatography (GC) with electron capture (EC) and confirmed with Hall electrolytic conductivity (HECD) detection using a second GC column. The highest levels of EDB were also confirmed by full scan GC/mass spectrometry (GC/MS). Twenty-five table-ready foods from the Food and Drug Administration's Total Diet Study that were analyzed by this method exhibited levels up to 70 ppb (pecans). Recoveries from fortified samples ranged from 91 to 104%. Values from this procedure were compared to those obtained by a modified Rains and Holder codistillation method. In all 25 samples this purge and trap procedure showed equivalent or superior recoveries and detected levels of EDB.     

Determination of spinosad and its metabolites in food and environmental matrices. 2. Liquid chomatography-mass spectrometry

A selective and sensitive method utilizing liquid chromatography-mass spectrometry (LC-MS) has been developed for determining residues of the natural insect control agent spinosad in several crop matrices that are difficult to analyze by HPLC with UV detection. The method determines the active ingredients (spinosyns A and D) and three minor metabolites (spinosyns B and K and N-demethylspinosyn D) in alfalfa hay, wheat hay, wheat straw, sorghum fodder, and corn stover. The analytes are extracted from the samples with an acetonitrile/water solution, and the extracts are purified by solid phase extraction with a C(18) disk and a silica cartridge. All five analytes are determined simultaneously in a single injection using positive atmospheric pressure chemical ionization LC-MS with selected ion monitoring. The average recoveries ranged from 69 to 96% with standard deviations ranging from 4 to 15%. The method has a validated limit of quantitation of 0. 01 microgram/g and a limit of detection of 0.003 microgram/g. The LC-MS method can also provide residue confirmation in addition to quantitation.     

Gas chromatography/electron-capture negative ion mass spectrometry for the quantitative determination of 2- and 3-hydroxy fatty acids in bovine milk fat

2- and 3-hydroxy fatty acids (2- and 3-OH-FAs) are bioactive substances reported in sphingolipids and bacteria. Little is known of their occurrence in food. For this reason, a method suitable for the determination of OH-FAs at trace levels in bovine milk fat was developed. OH-FAs (and conventional fatty acids in samples) were converted into methyl esters and the hydroxyl group was derivatized with pentafluorobenzoyl (PFBO) chloride to give PFBO- O-FA methyl esters. These derivatives with strong electron affinity were determined by gas chromatography interfaced to mass spectrometry using electron-capture negative ion in the selected ion monitoring mode (GC/ECNI-MS-SIM). This method proved to be highly sensitive and selective for PFBO-O-FA methyl esters. For the analysis of samples, two internal standards were used. For this purpose, 9,10-dideutero-2-OH-18:0 methyl ester (ISTD-1) from 2-OH-18:1(9 c) methyl ester as well as the ethyl ester of 3-PFBO-O-12:0 (ISTD-2) was synthesized. ISTD-1 served as a recovery standard whereas ISTD-2 was used for GC/MS measurements. The whole-sample cleanup consisted of accelerated solvent extraction of dry bovine milk, addition of ISTD 1, saponification, conversion of fatty acids into methyl esters by use of boron trifluoride, separation of the methyl esters of OH-FAs from nonsubstituted FAs on activated silica, conversion of OH-FAs methyl esters into PFBO-O-FA methyl esters, addition of ISTD-2, and measurement by GC/ECNI-MS-SIM. By this method, ten OH-FAs were quantified in bovine milk fat with high precision in the range from 0.02 +/- 0.00 to 4.49 +/- 0.29 mg/100 g of milk fat.     

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.     

Determination of Arsenic in Water Samples by Using a Green Hydrophobic-Hydrophilic Switchable Liquid-Solid Dispersive Microextraction Method

A simple and green preconcentration method of hydrophobic to hydrophilic switchable liquid-solid dispersive microextraction (HSL-SDM) has been first time introduced as separation method for arsenic ion in real water samples. Multiwall carbon nanotube (MWCNT) was immobilized with diethylenetriamine (DETA) and then used as solid phase adsorbent for the determination of trace level of arsenic ion by HSL-SDM method prior to analysis by hydride generation atomic absorption spectrometry. Reversibly hydrophobic-hydrophilic switchable of functionalized MWCNT can occur due to the exposing of carbon dioxide (CO₂) as anti-solvent trigger. The reversibly hydrophobic-hydrophilic switchable phenomena of immobilized MWCNT in the liquid-solid dispersive microextraction were checked by using FT-IR and SEM. The optimized analytical condition for arsenic ion analysis such as enrichment factor and limits of detection were obtained 83 and 3.05 ng L^?1, respectively. Accuracy of the developed HSL-SDM method was confirmed by the analysis of certified reference materials. Our developed HSL-SDM method was successfully applicable for measurements of arsenic ions in real water samples.     

Identification of O,O-dialkyl-S-methylphosphorodithioate residues in fish

O,O-Dialkyl-S-methylphosphorodithioates were found in Mississippi River buffalo fish caught near several chemical plants and oil refineries in Hartford and Wood River, IL. These chemicals, which have not been previously recognized as environmental or food contaminants, were identified and quantitated by a procedure similar to the AOAC multiresidue method for organochlorine and organophosphorus pesticides, using gas chromatography (GC) with flame photometric detection (FPD). The key to their identification was a GC/FPD retention time pattern that was virtually the same as that for the diazomethane reaction products of a commercial zinc dialkyl dithiophosphate motor oil additive. GC/mass spectrometry (MS) showed that the compound producing the largest GC/FPD peak contained butoxy groups. The identification of this compound as O,O-di(2-methylpropyl)-S-methyl-phosphorodithioate (Compound C) was confirmed by GC/MS analysis by comparison with the authentic material. The buffalo fish contained 0.15 ppm Compound C and 0.5 ppm total O,O-dialkyl-S-methylphosphorodithioates. Subsequent analyses of fish from other areas showed that these contaminants were not limited to the Hartford-Wood River area. Lower residue levels of Compound C, ranging from 0.01 to 0.05 ppm, were found in fish from the Mississippi River at Sauget, IL, and from the Delaware River and Newark Bay in NJ.     

Analysis of patulin in apple juice by diphasic dialysis extraction with in situ acylation and mass spectrometric determination.

A procedure combining diphasic dialysis extraction with in situ acylation and gas chromatography/mass spectrometry (GC/MS) determination was developed for detection and quantification of the mycotoxin patulin in apple juice. Apple juice samples spiked with 4-N,N-dimethylaminopyridine were dialyzed using methane chloride and acetic anhydride inside dialysis tubing. Patulin was derivatized into its acetate and collected in the tubing after diphasic dialysis and was directly determined using GC/MS with the selective ion monitoring mode without further concentration and cleanup steps. Quantification was carried out by a calibration curve with an internal standard of correlation. The appropriate parameters of both dialysis and derivatization were examined. The linear range of the calibration curve was found to be 10-250 microg/L for patulin, and the limit of quantification was 10 microg/L. Levels of patulin ranging from 0 to 107.2 microg/L with 77-109% recovery were found in 10 apple samples. The technique combining diphasic dialysis extraction and acylation was demonstrated and showed potential for other applications.     

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.     

Determination of daminozide in apples by gas chromatography/chemical ionization mass spectrometry

A method using gas chromatography/chemical ionization mass spectrometry (GC/CIMS) for the determination of daminozide residues in apples has been developed. Daminozide was separated from the sample matrix by water extraction and cation exchange, converted to the methyl ester by treatment with HCl-methanol, and determined by GC/CIMS using succinonitrile as an internal standard. The detection level was 0.05 ppm. Recoveries were 92-104% from apples spiked at the 0.05-0.5 ppm levels. Of the 25 apple samples analyzed, only 2 were positive for daminozide (1.04 and 0.32 ppm).     

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.