High pressure liquid chromatography of zearalenone and zearalenols in rat urine and liver

A high pressure liquid chromatographic technique with internal standardization has been developed for determining zearalenone and metabolites in rat urine and liver. Following extraction with methylene chloride and solvent partition, samples are cleaned up by applying the extract to a Sephadex LH-20 column and eluting with a mixture of benzene-methanol (85 + 15). Compounds were resolved on 2 Part-isil-10 columns (25 cm x 4.6 mm id) in series with a mobile phase of isooctane-chloroform-methanol (35 + 25 + 3), and detected at 280 nm. The internal standard was 6'alpha-acetoxyzearalane. Limits of detection were about 2.0 ng for zearalenone and 5.0 ng for zearalenols (6'-hydroxyzearalane). Zearalenone and zearalenols were excreted mainly in free form with relatively little glucuronide conjugation. Metabolism of zearalenone to free zearalenol was minor compared with formation of bound forms.     

Quantitative liquid chromatographic method using fluorescence detection for determining zearalenone and its metabolites in blood plasma and urine

The liquid chromatographic (LC) method described, suitable for use with both blood plasma and urine, is applicable for determination of zearalenone and alpha-zearalenol at levels as low as 0.5 ng/mL plasma and 5 ng/mL urine. The sample is incubated overnight with beta-glucuronidase to analyze for both conjugated and unconjugated forms of zearalenone. The next day, the sample is acidified with H3PO4, extracted with chloroform, and evaporated to dryness. The residue is dissolved in toluene and loaded onto a silica gel cartridge which is washed with toluene and eluted with toluene-acetone (88 + 12). The eluate is evaporated, and the residue is dissolved in chloroform, extracted with 0.18M NaOH, neutralized with H3PO4, and re-extracted with chloroform. The chloroform extract is evaporated, dissolved in mobile phase for LC, and injected onto a normal phase column under the following chromatographic conditions: mobile phase of water-saturated dichloromethane containing 2% 1-propanol, and fluorescence detector, excitation wave-length 236 nm, and 418 nm cut-off emission filter. Recoveries of zearalenone and its metabolites from blood plasma and urine are 80-89% in the range 2.0-10 ng standard/mL plasma, and 81-90% in the range 10-30 ng standard/mL urine. This method was used to analyze blood and urine samples from a pig fed zearalenone-contaminated feed (5 mg/kg), corresponding to 80 micrograms/kg body weight. Zearalenone was rapidly metabolized to alpha-zearalenol, which appeared in the blood only 30 min after feeding. Almost all zearalenone and alpha-zearalenol was found conjugated with glucuronic acid in both blood plasma and urine.     

Rapid, sensitive liquid chromatographic method for determination of zearalenone and alpha- and beta-zearalenol in wheat

A rapid, sensitive liquid chromatographic (LC) method is described for quantitative determination of zearalenone and alpha- and beta-zearalenol in wheat. The procedure incorporates an internal standard, zearalenone oxime, to facilitate quantitation and automated analysis. A sample, buffered with pH 7.8 phosphate, is extracted with water-ethanol-chloroform (2 + 50 + 75) and cleaned up. The final residue is dissolved in LC mobile phase and injected onto a reverse phase RP-18 column under the following conditions: water-methanol-acetonitrile (5 + 3 + 2) mobile phase; fluorescence (excitation wavelength 236 nm, 418 nm cut-off emission filter) and UV (254 nm, range 0.0025 AU) detectors. The limit of detectability (twice background) is 0.5 ng for zearalenone and alpha-zearalenol standards on the fluorescence detector and 4 ng for beta-zearalenol on the UV detector, which is equivalent to 20 micrograms zearalenone and 20 micrograms alpha-zearalenol/kg, and 160 micrograms beta-zearalenol/kg feed. Standard curves are linear over the range 0-35 ng zearalenone and alpha-zearalenol on the fluorescence detector and 0-50 ng beta-zearalenol on the UV detector. Recoveries of all compounds are 87.5-101% in the range 0.1-3.0 mg/kg (ppm).     

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.     

Liquid chromatographic determination of alpha-zearalenol and zearalenone in corn: collaborative study

The liquid chromatographic determination of alpha-zearalenol and zearalenone in corn was collaboratively studied. Each of 13 collaborators received 7 corn samples; 2 were blanks and 5 were spiked to contain 50, 100, and 200 ng alpha-zearalenol/g and 50, 100, 500, 1000, and 4000 ng zearalenone/g. Four sets (including blanks) of blind duplicates were included in the study. Five naturally contaminated corn samples (one in duplicate) were also provided. All collaborators detected both mycotoxins at 50 ng/g. Average recoveries reported by all collaborators ranged from 81.9% at 200 ng/g to 100.3% at 50 ng/g for alpha-zearalenol and from 77.8% at 1000 ng/g to 123% at 50 ng/g for zearalenone. Three collaborators reported false positives for both alpha-zearalenol and zearalenone. The within-laboratory CV values based on blind duplicates were 22.6% for alpha-zearalenol and 31.4% for zearalenone. The CV values based on laboratory-sample interaction were 25.6 and 33.8% for alpha-zearalenol and zearalenone, respectively. The CV values for naturally contaminated samples (including duplicates) were 47.0% for alpha-zearalenol and 37.7% for zearalenone. The method has been adopted official first action.     

Comparison evaluation of liquid chromatographic and bioassay methods of analysis for determination of paralytic shellfish poisons in shellfish tissues

A liquid chromatographic (LC) method was compared with the AOAC mouse bioassay method (18.086-18.092) for determination of paralytic shellfish toxins in shellfish tissues. Shellfish samples were collected from Massachusetts coastal waters as part of a state surveillance program, and extracts of shellfish meat were analyzed for toxins by using both analytical methods. Overall correlation of the LC and bioassay methods is good (r = 0.943), but for samples with toxicities less than 100 micrograms saxitoxin/100 g shellfish meat, the correlation is significantly less (r = 0.531). Limits of detection are 10 micrograms saxitoxin/100 g shellfish meat and 40 micrograms saxitoxin/100 g shellfish meat for the LC and bioassay methods, respectively. Analytical capacity of the LC method is limited to 12 samples/person-day compared with 30 samples/person-day for the bioassay. Sampling capacity of the LC method could be increased by using a fluorescence detector with a wider response range, which would eliminate the need for dilution of concentrated samples.     

Liquid chromatographic determination of chlortetracycline hydrochloride in ruminant and poultry/swine feeds.

A liquid chromatographic (LC) method is described for the determination of chlortetracycline hydrochloride (CTC) in poultry/swine and ruminant feeds in the 10-100 ppm range and in premix. CTC is extracted from ground feed/premix with acidified acetone, and the extract is filtered through a Millex-HV filter or disposable C18 column. The filtrate is partitioned with methylene chloride when additional cleanup is necessary. A Nova-Pak C18 column is used for LC separation with determination at 370 nm. The average recovery of CTC from premix was 95% with a standard deviation (SD) of 1.70 and a coefficient of variation (CV) of 1.79%. The overall average recovery from feeds was 77% with an SD of 3.18 and a CV of 4.10%.     

Quantification of zearalenone in various solid agroenvironmental samples using D6-zearalenone as the internal standard

Because of its pronounced estrogenicity, zearalenone may be of concern not only in the aqueous but also in the terrestrial environment. Therefore, we developed several analytical methods to quantify zearalenone in different solid matrices of agroenvironmental relevance (i.e., plant organs, soil, manure, and sewage sludge). The use of D(6)-zearalenone as the internal standard (IS) was essential to render the analytical method largely matrix-independent because it compensated for target analyte losses during extract treatment and ion suppression during ionization. Soil and sewage sludge samples were extracted with Soxhlet, whereas plant material and manure samples were extracted by liquid solvent extraction at room temperature. Absolute recoveries for zearalenone were 70-104% for plant materials, 105% for soil, 76% for manure, and 30% for sewage sludge. Relative recoveries ranged from 86 to 113% for all matrices, indicating that the IS was capable to largely compensate for losses during analysis. Ion suppression, between 8 and 74%, was in all cases compensated by the IS but influenced the method quantification levels. These were 3.2-26.2 ng/g(dryweightdw) for plant materials, 0.7 ng/g(dw) for soil, 12.3 ng/g(dw) for manure, and 6.8 ng/g(dw) for sewage sludge. Plant material concentrations varied from 86 ng/g(dw) to more than 16.7 microg/g(dw), depending on the organ and crop. Soil concentrations were between not detectable and 7.5 ng/g(dw), depending on the sampling depth. Zearalenone could be quantified in all manure samples in concentrations between 8 and 333 ng/g(dw). Except for two of the 85 investigated sewage sludge samples, zearalenone concentrations were below quantification limit.     

Liquid chromatographic determination of three sulfonamides in animal tissue and egg

Sulfonamides are widely used as a feed additive in animal production in Japan. The present paper is a determination of 3 sulfonamides: sulfamethazine (SMZ), sulfamonomethoxine [SMX, 4-amino-N-(3-methoxypyrazinyl)-benzenesulfonamide], and sulfadimethoxine (SDX) in animal tissue and egg by liquid chromatography (LC). Tissues were extracted with acetonitrile and fat was removed by liquid/liquid partition. The sulfonamides were purified by an ODS cartridge column; then each compound was separated by an ODS LC column and detected at 268 nm. Quantification levels were 0.02 ppm for SMZ and SMX, and 0.04 ppm for SDX; detection limits were 0.01 ppm for SMZ and SMX, and 0.02 ppm for SDX. Calibration curves were linear between 2 and 40 ng for SMZ and SMX, and between 4 and 80 ng for SDX. Recoveries from muscle and egg samples spiked with 1-2 micrograms/10 g were 81-98%.     

Ultrahigh-performance liquid chromatographic-tandem mass spectrometric multimycotoxin method for quantitating 26 mycotoxins in maize silage

A multianalyte method was developed to identify and quantitate 26 mycotoxins simultaneously in maize silage by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The extraction and cleanup procedure consists of two extraction steps followed by purification on a Waters Oasis HLB column. The method developed was validated with the requirements of Commission Decision 2002/657/EC taken into account. The limit of detection and quantitation ranges were 5-348 and 11-695 ng/g, respectively. Apparent recovery varied between 61 and 116%, whereas repeatability and reproducibility were within the ranges of 3-45 and 5-49%, respectively. The method developed was successfully applied for maize silage samples taken at the cutting surface and 1 m behind that surface. Mainly Fusarium toxins (beauvericin, deoxynivalenol, enniatins, fumonisins, fusaric acid, and zearalenone) were detected, but postharvest toxins such as mycophenolic acid and roquefortine C were identified as well.     

Liquid chromatographic determination of zearalenone and alpha- and beta-zearalenols in milk

Previous research has demonstrated transmission of zearalenone and alpha- and beta-zearalenols into the milk of cows and other animals. Since human intake of zearalenone and its metabolites via milk is an unknown factor in risk assessment of zearalenone and because appropriate methodology for their determination in milk is not available, a rapid and sensitive analytical method has been developed. Essentially, the method includes extraction with basic acetonitrile, acidification, partition into methylene chloride on a hydrophilic matrix, cleanup on an aminopropyl solid phase extraction column, and reverse-phase liquid chromatography with fluorescence detection. Recoveries from milk averaged 84% for zearalenone, 93% for alpha-zearalenol, and 90% for beta-zearalenol at spiking levels of 0.5 to 20 ng/mL. As little as 0.2 ng/mL of zearalenone and alpha-zearalenol and 2 ng/mL of beta-zearalenol can be detected in milk. These 3 compounds are stable in refrigerated milk for at least 2 weeks and in milk brought to boiling. Enzymes (beta-glucuronidase and aryl sulfatase) may be added to milk prior to extraction to hydrolyze any conjugates.     

Simultaneous liquid chromatographic screening of five coccidiostats in chicken liver

A reverse-phase liquid chromatographic (LC) method is described for simultaneously determining 5 coccidiostats--aklomide, dinsed, ethopabate, nitromide, and zoalene in chicken liver. The method entails blender extraction of 10 g liver with ethyl acetate, column chromatography through Sephadex LH-20 and neutral alumina, and LC analysis on a C18 column with UV detection at 260 nm. The drugs were eluted from Sephadex with methanol-benzene (10 + 90), from alumina with methanol-dichloromethane (10 + 90), and from C18 with acetonitrile-water (linear gradient: 25% acetonitrile for 10 min, increasing to 55% over 15 min; flow rate 1 mL/min). Liquid chromatography was completed in 40 min and calculations were based on peak height measurements. Average recoveries of the coccidiostats from fortified liver ranged from 72 to 97%, except for dinsed, which showed a relatively constant average recovery of 57%. The detection limit for the standards was 2.5 ng on column. Levels as low as 50 ng/g were detected in fortified liver samples.     

Determination of xanthomegnin in grains and animal feeds by liquid chromatography with electrochemical detection

A sensitive, highly selective liquid chromatographic (LC) method is described which uses electrochemical (EC) reduction of the analyte in the determinative step. The method is capable of determining xanthomegnin in mixed animal feeds and grains at levels ranging from 15 to 1200 ng/g. The method can detect as little as 0.5 ng xanthomegnin injected on the LC column. Xanthomegnin is extracted with chloroform and 0.1M phosphoric acid. An aliquot of the crude extract is purified by silica gel column chromatography using a Sep-Pak silica gel cartridge. A novel feature of the method is that xanthomegnin is "backed off" the column by reversing the flow of the eluant through the column. LC is then used to separate xanthomegnin from other interfering substances. Xanthomegnin is detected by EC reduction at -0.16 V. Recoveries of xanthomegnin added to samples at levels ranging from 15 to 1200 ng/g averaged 79% with a coefficient of variation of 7.9%. Results also demonstrate that this LC system can separate the related metabolites viomellein and rubrosulphin from each other and from xanthomegnin and that the same EC detection system can be used to detect these metabolites.     

Sephadex LH-20 cleanup, high pressure liquid chromatographic assay, and fluorescence detection of zearalenone in animal feeds

A high pressure liquid chromatographic (HPLC) method is described to determine zearalenone in animal feeds at levels as low as 0.01 ppm. Samples are extracted with chloroform-ethanol and initially purified using a SEP-PAK silica cartridge, followed by column chromatography using Sephadex LH-20. Separation by normal phase HPLC is followed by fluorescence detection. Recoveries at levels of 1.0-0.01 ppm averaged greater than 90%. Confirmation included HPLC analysis of the sample and a zearalenone standard, using 3 different excitation wavelengths, and comparison of fluorescence responses obtained. The method was successfully applied to the analysis of 1 corn and 3 cornmeal samples. Zearalenone was detected in all 4 samples at levels of 0.379-19.2 ppm.     

Comparison of an ELISA-based screening test with liquid chromatography for the determination of aflatoxins in corn.

An enzyme-linked immunosorbent assay (ELISA) screening test (CITE PROBE) was compared to liquid chromatography (LC) for the determination of aflatoxins in naturally contaminated corn samples. The CITE PROBE, with a positive/negative cutoff of 5 ng/g aflatoxin B1, was correct (based on LC results) on 47 of 51 samples. Two of the incorrect responses by the CITE PROBE were false positives on samples containing 4.4 ng/g and 4.1 ng/g aflatoxins by LC. Another incorrect response was a false negative on a sample containing 5.5 ng/g aflatoxins by LC. The fourth incorrect response was a false positive on a sample containing 1.9 ng/g aflatoxins by LC. On the basis of these results, the CITE PROBE was determined to be a reliable screening method for the detection of greater than or equal to 5 ng/g aflatoxins in corn.     

Simplified cleanup and liquid chromatographic determination of oxamyl residues in potato tubers

A simple and efficient method is presented for the extraction, cleanup, and liquid chromatographic (LC) determination of oxamyl residues in potato tubers. Samples are extracted with methanol, partitioned into dichloromethane, and cleaned up using Sep-Pak Florisil cartridges. LC determination is performed using a Zorbax PSM 60 size exclusion column with an acetonitrile-water (1 + 9) mobile phase and UV detection at 254 nm. Recovery of oxamyl from spiked control tubers averaged 94.1 and 85.9% at fortification levels of 0.4 and 0.08 micrograms oxamyl/g tuber, respectively. The minimum detectable concentration of oxamyl by this method is 0.01 micrograms/g.     

Mycotoxins in grain dust: method for analysis of aflatoxins, ochratoxin A, zearalenone, vomitoxin, and secalonic acid D

A multimycotoxin method is presented to quantitate aflatoxins, ochratoxin A, zearalenone, secalonic acid D, and vomitoxin in grain dust. Dust spiked with these mycotoxins was extracted sequentially with methylene chloride followed by acetonitrile-water (86 + 14). Vomitoxin was recovered in the latter extract and all other mycotoxins were recovered in the methylene chloride. Aflatoxins and ochratoxin were quantitated by fluorescence measurement on silica thin layer chromatographic plates. The other mycotoxins were quantitated after cleanup by reverse phase liquid chromatography and ultraviolet detection. Recoveries from dust spiked in the parts per billion (ng/g) range were approximately 80% (SD = 15-29%) for all mycotoxins. Minimum detectable amounts ranged from less than 0.5 ng/g for aflatoxins to 20 ng/g for zearalenone.     

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.     

Fusarium mycotoxins: overlooked aquatic micropollutants?

Deoxynivalenol and zearalenone are among the most prevalent toxins produced by Fusarium spp. They have been investigated in food and feed products for decades but rarely in the environment. We therefore established solid-phase extraction and liquid chromatography-mass spectrometry (LC-MS) methods to quantify these mycotoxins at trace concentrations in aqueous natural samples. In a model emission study, we inoculated a winter wheat field with Fusarium graminearum and subsequently monitored deoxynivalenol and zearalenone in its drainage water. Before during and after harvest in June and July 2007, these toxins were emitted in concentrations from 23 ng/L to 4.9 microg/L for deoxynivalenol and from not detected to 35 ng/L for zearalenone. Simultaneously, in July and August 2007, deoxynivalenol was also detected in a number of Swiss rivers in concentrations up to 22 ng/L and zearalenone was present in several river samples below the method quantification limit. Other mycotoxins might be emitted from Fusarium-infected fields as well, because some of them are produced in similar amounts as deoxynivalenol and zearalenone and exhibit similar or even higher water solubility than deoxynivalenol. The ecotoxicological consequences of the presence of mycotoxins in surface waters remain to be elucidated.     

Thin layer chromatographic determination of aflatoxins, ochratoxins, sterigmatocystin, zearalenone, citrinin, T-2 toxin, diacetoxyscirpenol, penicillic acid, patulin, and penitrem A.

A general method is described for determining 16 mycotoxins in mixed feeds and other food products used in the manufacture of these feedstuffs. The mycotoxins are extracted and cleaned up by extracting with solvents of different pH. Thin layer chromatography is used to separate the toxins; toxins are then quantitated by the limit detection method. The minimum detectable concentration of mycotoxins in various products is: aflatoxin B1 or G1, 4--5 micrograms/kg; ochratoxin A or ethyl ester A 140--145 micrograms/kg; citrinin 600--750 micrograms/kg; zearalenone, 410--500 micrograms/kg; sterigmatocystin, 140--145 micrograms/kg; diacetoxyscirpenol, 2400--2600 micrograms/kg; T-2 toxin, 800--950 micrograms/kg; patulin, 750--800 micrograms/kg; penitrem A 14,000--14,500 micrograms/kg; penicillic acid 3400--3650 micrograms/kg.