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 dioxins and furans in foods and biological tissues: review and update

Determination of trace residues of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) in various matrixes is carried out by a limited number of laboratories in the United States, Canada, and other countries. Current methods for analysis of foods and biological tissues include a combination of preparation, extraction, cleanup, isolation, determination, and identity confirmation procedures. Soxhlet, liquid/liquid, solid-phase, and column extraction procedures are used as well as treatment with acid or base before solvent extraction. Cleanup and isolation steps include sulfuric acid partitioning; adsorption chromatography on Florisil, silica gel, or alumina; gel permeation chromatography; multi-stage column chromatography on sulfuric acid silica and alkali silica; carbon column chromatography; and liquid chromatography fractionation with size exclusion, normal-phase, and reverse-phase columns. Activated carbon and multistage chromatographic columns are widely used in cleanup schemes. Isomer-specific identification and quantitation of PCDD and PCDF congeners at parts-per-trillion levels or lower are carried out by high resolution (capillary) gas chromatography (HRGC) and multiple ion detection mass spectrometry. In addition to chemical methods, bioassay procedures have been recommended (e.g., use of monoclonal antibodies, for immunoassay determination of PCDDs and PCDFs).     

Determination of organochlorine and organophosphorus pesticide residues in eggs using a solid phase extraction cleanup

A multiresidue solid phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of nonpolar organochlorine and polar organophosphorus pesticide residues in eggs is described. The method uses an acetonitrile extraction followed by an SPE cleanup using graphitized carbon black and aminopropyl SPE columns. Organophosphorus pesticides are determined by gas chromatography with flame photometric detection. After further cleanup of the extract using Florisil SPE columns, organochlorine pesticides are determined by gas chromatography with electron capture detection. Studies were performed using eggs containing both fortified and incurred pesticide residues. The average recoveries were 86-108% for 8 fortified organochlorine pesticide residues and 61-149% for 28 fortified organophosphorus pesticide residues.     

Analysis of sulfonamides in animal feeds by liquid chromatography with fluorescence detection

Two analytical methodologies for the simultaneous analysis of eight sulfonamide antibiotics in animal feeds were developed. Analytes were extracted in a simple and rapid procedure by manual shaking with an ethyl acetate/ultrapure water mixture (99:1, v/v) without further sample cleanup. Mean recoveries ranging from 72.7% to 99.4% with relative standard deviations below 9% were achieved from spiked animal feed samples. Determination was carried out by high-performance liquid chromatography using fluorometric detection with precolumn derivatization. The separation of the derivatized compounds was performed using two different chromatographic columns: a conventional C(18) column and a recently available core-shell particle Kinetex C(18) column. Both methods were validated in-house in six different feed matrices, and the two approaches were compared. The experiments showed that the method using the Kinetex column was superior with regard to speed of analysis and precision, both under repeatability and intermediate reproducibility conditions. The limits of detection and quantification were also greatly improved, below 0.10 and 0.34 μg/g, respectively. Finally, this novel approach was successfully applied to the analysis of real feed samples.     

Simple analytical method for organophosphorus pesticide determination in unpolished rice, using removal of fats by zinc acetate

A rapid and simple method is developed for the determination of organophosphorus pesticides in unpolished rice. The new method incorporated acetonitrile-water (1 + 1) extraction, removal of fats by zinc acetate, and further cleanup on an activated charcoal chromatographic column. The higher fatty acids in the extract react rapidly with zinc acetate to form insoluble zinc carboxylates, which precipitate. Additional interferences were cleaned up on an activated charcoal chromatographic column, and organophosphorus pesticides adsorbed on the activated charcoal were eluted with acetone-hexane. Dimethoate is not retained on the activated charcoal and must be extracted with dichloromethane from the first acetonitrile-water eluate. Pesticides are measured by flame photometric gas chromatography. Recoveries from 50 g unpolished rice samples fortified with 5-50 micrograms diazinon, 6-30 micrograms parathion, 8-40 micrograms fenitrothion and IBP, 10-50 micrograms dimethoate and fenthoate, 20-100 micrograms malathion, or 40-200 micrograms EPN ranged from 75.7 to 95.8%.     

Liquid chromatographic determination of zoalene in medicated feeds

A rapid, reliable separation and quantitation of zoalene (3,5-dinitro-o-toluamide) from feeds is accomplished by using reverse phase liquid chromatography (LC) and ultraviolet detection. An extraction technique which is similar to the present AOAC official colorimetric method is used before chromatographic analysis. This extraction is followed by an activated alumina cleanup and LC to separate zoalene from feed matrix. The methodology was applied to a variety of spiked feed matrices, and yielded good recoveries. Liquid chromatographic results were shown to correlate with colorimetric determinations.     

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.     

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.     

Gas chromatographic determination of electron capture sensitive volatile industrial chemical residues in foods, using AOAC pesticide multiresidue extraction and cleanup procedures

Electron capture (EC) gas chromatographic (GC) parameters have been developed for determining some of the more volatile industrial chemicals that can be determined by the AOAC multiresidue method for organochlorine and organophosphorus pesticides with modified GC operating conditions. Retention times relative to pentachlorobenzene are reported for 143 industrial chemicals, pesticides, and related compounds on OV-101 GC columns at 130 degrees C. Also reported for most of the compounds are recoveries from fortified samples carried through the AOAC extraction and cleanup procedures for fatty and/or nonfatty foods, Florisil elution characteristics, and GC relative retention times on mixed OV-101 + OV-210 columns at 130 degrees C. Our laboratory has used the modified EC/GC parameters with the AOAC multiresidue extraction/cleanup procedures to determine many volatile halogenated industrial chemical contaminants in foods, chiefly in samples of fresh-water fish. Other modifications of the AOAC method are described to improve the tentative identification and quantitative measurement of these volatile residues.     

Improved method for the determination of hydroxymethylfurfural in baby foods using liquid chromatography-mass spectrometry

An improved analytical method for the rapid, reliable, and sensitive determination of hydroxymethylfurfural (HMF) in baby foods is described. It entailed aqueous extraction from food matrix with simultaneous clarification using Carrez I and II reagents, solid-phase extraction cleanup using Oasis HLB, and analysis by liquid chromatography-mass spectrometry. A narrow-bore column allowed fast chromatographic separation with good resolution of HMF and matrix coextractives. In positive atmospheric pressure chemical ionization conditions, precursor and compound-specific ions were sensitively detected in selected ion monitoring mode. Sample preparation with efficient cleanup followed by fast chromatographic analysis allowed the analysis to be completed in <20 min. Recovery ranged between 91.8 and 94.7% for spiking levels of 0.25, 1.0, and 5.0 mg/kg HMF in cereal-based baby foods. The method was shown to be successful when using liquid chromatography coupled to ultraviolet detection at 285 nm.     

A quantitative method for determination of aflatoxin B in roasted corn.

Roasting aflatoxin-contaminated corn will reduce toxin levels. A quantitative analysis for aflatoxin in roasted corn has been developed by modifying a cleanup technique for green coffee extracts approved as official first action by the AOAC. A chloroform extract is partially purified on a Florisil column, and thin layer chromatographic (TLC) plates are developed with methylene chloride-chloroform-isoamyl alcohol-formic acid (81+15+3+1). Recoveries average 101% and the sensitivity limit is 5 ppb aflatoxin B1. A 2-dimensional TLC procedure can also be used to separate the aflatoxins from background interferences.     

Simultaneous gas chromatographic determination of carbofuran, metalaxyl, and simazine in soils

A method for extraction, cleanup, and simultaneous gas chromatographic detection of carbofuran, metalaxyl, and simazine in soils has been developed. Pesticide residues were extracted from soil with acetone containing 10% 0.2M HCl-KCl buffer (pH 2.0), cleaned up with methylene chloride-carbonate buffer (pH 10.7) solvent partitioning and solid-phase extraction on disposable silica gel columns, and quantitated with gas chromatography using a Supelcowax 10 megabore capillary column and a nitrogen-selective detector. Method limits of detection were 0.02 microgram/g for the 3 pesticides in surface soils (0-30 cm depths) and 0.02, 0.02, and 0.005 microgram/g in soils below 30 cm (subsoils) for carbofuran, metalaxyl, and simazine, respectively. Recoveries for carbofuran, metalaxyl, and simazine were 92.6 +/- 5.5, 93.6 +/- 5.0, and 88.4 +/- 6.7%, respectively, when soil samples were spiked with pesticide concentrations ranging from 0.02 to 2.0 micrograms/g.     

Determination of halogenated contaminants in human adipose tissue

A method has been developed for determination of organochlorine contaminants in human adipose tissue. After fat extraction from the tissue with acetone-hexane (15 + 85, v/v), organochlorines were fractionated from fat by gel permeation chromatography with methylene chloride-cyclohexane (1 + 1, v/v) as solvent. After Florisil column cleanup, the GPC extract was analyzed by capillary column gas chromatography using 2 columns of different polarity. Compound identity was confirmed by gas chromatography-mass spectrometry using selected ion monitoring. Recoveries for fortification levels of 10-500 ng/g were greater than 80% except for trichlorobenzene and hexachlorobutadiene (ca 60%).     

Charcoal column/thin layer chromatographic method for high fructose corn sirup and spectrophotometric method for hydroxymethylfurfural in honey: collaborative studies.

A new spectrophotometric method is described for determining hydroxymethylfurfural in honey in which interfering background absorption of honey is corrected for by use of a bisulfite-treated sample as blank. Two procedures for detecting high-fructose corn sirup (HFCS) in honey were also tested. In one, charcoal column pretreatment is used to concentrate trace oligosaccharides, followed by thin layer chromatography to differentiate those of HFCS from those of honey. The other method depends on measurement of the isomaltose/maltose ratio by gas-liquid chromatography. The charcoal/thin layer chromatographic method for HFCS has been adopted official first action. The bisulfite method for hydroxymethylfurfural has been adopted interim first action.     

Residue methodology for AMDRO fire ant insecticide (AC 217,300) in pasture grass and crops

Residue methodology is described for the determination of AC 217,300 residues in pasture grass and crop samples. After extraction and subsequent cleanup on an XAD-2 column, residues of AC 217,300 are determined by liquid chromatography (LC), using a reverse phase paired-ion chromatographic system and detection at 300 nm. The method has a validated limit of sensitivity of 0.05 ppm with corresponding control values for the commodities analyzed of less than 0.01 ppm. Apparent residues over 0.05 ppm can be confirmed by either gas chromatography with an electron capture detector (GC-EC) or gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI). The direct GC-NICI method circumvents the need for sample cleanup on the XAD-2 column, and offers a greatly simplified procedure that is useful for screening samples. Recoveries of AC 217,300 from the commodities analyzed have been satisfactory with all methods of analysis.     

T-2 toxin, a trichothecene mycotoxin: review of toxicity, metabolism, and analytical methods

This review focuses on the toxicity and metabolism of T-2 toxin and analytical methods used for the determination of T-2 toxin. Among the naturally occurring trichothecenes in food and feed, T-2 toxin is a cytotoxic fungal secondary metabolite produced by various species of Fusarium. Following ingestion, T-2 toxin causes acute and chronic toxicity and induces apoptosis in the immune system and fetal tissues. T-2 toxin is usually metabolized and eliminated after ingestion, yielding more than 20 metabolites. Consequently, there is a possibility of human consumption of animal products contaminated with T-2 toxin and its metabolites. Several methods for the determination of T-2 toxin based on traditional chromatographic, immunoassay, or mass spectroscopy techniques are described. This review will contribute to a better understanding of T-2 toxin exposure in animals and humans and T-2 toxin metabolism, toxicity, and analytical methods, which may be useful in risk assessment and control of T-2 toxin exposure.     

High pressure liquid chromatographic determination of sugars in various food products.

A high pressure liquid chromatographic (HPLC) method has been developed which is fast, simple, specific, and reliable over a wide range of sugar concentrations in a variety of food matrices. With few exceptions, sample preparation is simple, requiring only a water-ethanol extraction, followed by a rapid mini-column cleanup before injection into the HPLC system. The majority of samples can be prepared for analysis within 1--1 1/2 hr, and the following sugars are separated in less than 45 min: fructose, glucose, sucrose, maltose, lactose, melibioals, chocolate products, chocolate sirups, cookies, health food products, molasses, preserves, processed fruits, and soy protein products.     

Modified liquid chromatographic method for determination of gentian violet in animal feed

A modified liquid chromatographic method is described for the determination of Gentian Violet (GV) in animal feed. The reliable detection limit is 0.5 ng (reference standards), and 1 ppm GV was reliably determined in feed. The calibration curve was linear between 1 and 40 micrograms/mL. The method, developed in a study by the National Center for Toxicological Research, was modified to use methanol-water (9 + 1) instead of benzene-methanol as the eluting solution in the column cleanup. GV is extracted from feed with methanol-1N HCl (99 + 1), cleaned up on a Sephadex LH-20 column to remove any remaining interferences, separated on a Nova-Pak C18 column fitted with a precolumn filter, and determined at 588 nm. The identity of GV is confirmed by thin-layer chromatography (Rf = 0.47) by comparison with a reference standard. Average recoveries from 3 sets of 5 feed samples containing 2.5, 5.0, and 10.0 ppm GV were 115, 95, and 102%, 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.     

Rapid reverse phase liquid chromatographic determination of aflatoxin M1 in milk

A rapid, economical, and reliable liquid chromatographic (LC) method is described for determination of aflatoxin M1 in milk. The method includes an improved AOAC extraction procedure, cleanup of the extract on a silica cartridge, and LC quantitation. Alternatively, a rapid column cleanup procedure can be used. Milk artificially spiked with aflatoxin M1 at 0.05, 0.1, and 0.5 ppb was analyzed using both new approaches as well as an AOAC method coupled with LC for quantitation of the toxin. Recovery of aflatoxin M1 by the first approach of the new method ranged between 93.4 and 99.1%, and for the alternative procedure between 92.4 and 96.8%. The AOAC method gave lower recovery (85.6-90.7%) of toxin, but the results from this method had a somewhat smaller standard deviation for replicate analyses than did results of the new method.