Preparative enzymatic synthesis of glucuronides of zearalenone and five of its metabolites

The resorcylic acid lactones zearalenone ( 1), alpha-zearalenol ( 2), beta-zearalenol ( 3), alpha-zearalanol (zeranol) ( 4), beta-zearalanol (taleranol) ( 5), and zearalanone ( 6) were converted to their glucuronides on a preparative scale in good yields. Reactions were conducted with bovine uridine 5'-diphosphoglucuronyl transferase (UDPGT) as catalyst and uridine 5'-diphosphoglucuronic acid (UDPGA) as cofactor. The glucuronides were isolated by column chromatography and characterized by NMR spectroscopy and mass spectrometry. Although the principal products were 4- O-glucuronides (i.e., linkage through a phenolic hydroxyl), significant quantities of the 6'- O-glucuronides (i.e., linkage through the aliphatic hydroxyl) of alcohols 2, 4, and 5 were also isolated. In the case of 3, the 2- O-glucuronide was isolated as the minor product. Overall isolated yields of glucuronides, performed on a 20-50 mg scale, were typically ca. 80% based on the resorcylic acid lactone starting material. LC-UV-MS (2) analysis of purified specimens revealed MS (2) fragmentations useful for defining the point of attachment of the glucuronide moiety to the zearalenone nucleus.     

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.     

Metabolic profiles of the mycotoxin zearalenone and of the growth promoter zeranol in urine,liver, and muscle of heifers

A group of five heifers were fed for 84 days with 2 kg of zearalenone-contaminated oats (1370 microg/kg) resulting in an average daily intake of 2740 microg of zearalenone per animal. In a parallel experiment five heifers were implanted with two 25 mg zeranol pellets, one at the beginning of the study and one after 42 days, and fed with 2 kg of "blank" control oats (79 microg/kg, daily intake = 158 microg). A third group of five animals were also fed with 2 kg of "blank" oats and served as control. Urine samples of all animals were collected every 5-6 days during the whole period of the study. Animals of all three groups were killed 84 days after the beginning of the feeding study. Tissue samples (back, femoral region, liver, and residues of implanted pellets) were taken during post-mortem investigations. The content of zearalenone and zeranol and their metabolites in urine and tissue samples was established by an analytical method combining solid-phase extraction and high-performance liquid chromatography-tandem mass spectrometry. Urinary excretion rates of zeralenone and zeranol were calculated from these results.     

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).     

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.     

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.     

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.     

Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo.

The soy isoflavones daidzein and genistein are found in high concentrations in human plasma and urine after soy consumption. However, in vitro and in vivo data regarding the oxidative metabolism of isoflavones in humans are scarce. Therefore, we have studied the oxidative metabolites of these compounds formed in human liver microsomes and excreted in urine of male and female humans ingesting soy products for 2 days. Human liver microsomes transformed the soy isoflavone daidzein to three monohydroxylated and three dihydroxylated metabolites according to GC/MS analysis. On the basis of a previous study with rat liver microsomes and with the help of reference substances, these metabolites were identified as 6,7,4'-trihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone, 7,8,4'-trihydroxyisoflavone, 7,8,3',4'-tetrahydroxyisoflavone, 6,7,8,4'-tetrahydroxyisoflavone, and 6,7,3',4'-tetrahydroxyisoflavone. Significant amounts of the same metabolites except 6,7,8,4'-tetrahydroxyisoflavone were also found in urine of female and male volunteers after soy intake. Genistein was metabolized by human liver microsomes to six hydroxylation products. The main metabolites were the three aromatic monohydroxylated products 5,6,7,4'-tetrahydroxyisoflavone, 5,7,8,4'-tetrahydroxyisoflavone and 5,7,3',4'-tetrahydroxyisoflavone. The aliphatic monohydroxylated metabolite 2,5,7,4'-tetrahydroxyisoflavone and two aromatic dihydroxylated metabolites, 5,7,8,3',4'-pentahydroxyisoflavone and 5,6,7,3',4'-pentahydroxyisoflavone, were formed in trace amounts. The same hydroxylated genistein metabolites except the aliphatic hydroxylated one could also be detected in human urine samples. Methylated forms of the catechol metabolites, which were generated by incubations with catechol-O-methyltransferase in vitro could be detected only in trace amounts in the urine samples. This implies that this reaction does not play a major role in the biotransformation of the hydroxylated daidzein and genistein metabolites in vivo. Most of these oxidative metabolites are described as human in vivo metabolites for the first time. Their biological significance remains to be established.     

Determination of zeranol/zearalenone and their metabolites in edible animal tissue by liquid chromatography with electrochemical detection and confirmation by gas chromatography/mass spectrometry

A sensitive method is described for the determination and confirmation of zeranol and zearalenone, as well as their isomers and metabolites, in edible animal tissue. The analytes are extracted from tissue with methanol, hydrolyzed enzymatically, cleaned up by acid-base partitioning, determined by liquid chromatography (LC) with electrochemical (EC) detection, and confirmed by gas chromatography/mass spectrometry (GC/MS). LC analysis is performed by isocratic elution with a buffered mobile phase using a Nova-Pak reverse-phase C18 column with amperometric EC detection at +0.90 V. Capillary GC/MS analysis of the trimethylsilyl derivatives provides mass spectral confirmations.     

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.     

Distribution of resveratrol metabolites in liver, adipose tissue, and skeletal muscle in rats fed different doses of this polyphenol

This study aimed to characterize resveratrol metabolite profiles in liver, skeletal muscle, and adipose tissue in rats treated for 6 weeks with 6, 30, or 60 mg of trans-resveratrol/kg body weight/d. Resveratrol metabolites were quantified by liquid chromatography-tandem mass spectrometry. The greatest number of metabolites was found in liver followed by adipose tissue. A great number of metabolites in muscle was below the limit of detection. The amounts of sulfate conjugates tended to increase when resveratrol dosage was enhanced, while the glucuronide ones increased only between 6 and 30 mg/kg/d. Microbiota metabolites were detected in higher amounts than resveratrol conjugates in liver, while the opposite occurred in adipose tissue and muscle. So, the largest amounts of resveratrol metabolites were found in liver, intermediate amounts in adipose tissue, and the lowest amounts in muscle. Sulfate conjugates, but not glucuronides, showed a dose-response pattern. Microbiota metabolites were predominant in liver.     

Residue depletion of ractopamine and its metabolites in swine tissues, urine, and serum

Ractopamine hydrochloride is a beta-adrenergic leanness-enhancing agent approved for use in swine in the United States. Depletion of ractopamine and its metabolites from animal tissues, urine, and serum is of interest for the detection of illegal use. The objectives of this study were to measure the residues of ractopamine in swine incurred samples after treatment with dietary ractopamine for 28 consecutive days. An efficient and sensitive analytical method was developed for the detection of parent ractopamine and its metabolites in swine tissues, urine, and serum by HPLC-FLD. After extraction, enzymatic digestion, and solid-phase cleanup of the samples, ractopamine residues were determined by liquid chromatography (LC) with fluorescence detector. The limits of detection (LOD) for tissues, urine, and serum were 1 ng g(-1), 0.5 ng mL(-1), and 0.5 ng mL(-1), respectively. Recoveries ranged from 70.5 to 94.5% for samples fortified at 1-50 ng g(-1) or ng mL(-1). Sixty pigs were fed twice daily for 28 consecutive days with feeds containing 18 mg kg(-1) ractopamine HCl. The residue concentrations in urine, liver, and kidney were 650.06 ng mL(-1), 46.09 ng g(-1), and 169.27 ng g(-1), respectively, compared with those in muscle, fat, and serum (4.94 ng g(-1), 3.28 ng g(-1), and 7.48 ng mL(-1), respectively) at the feeding period of 7 days. The residue concentrations at withdrawal period of 0 days in all edible tissues were lower than tolerance values established by the FDA and MRL values listed by the JECFA. These data support the withdrawal time of 0 days established by the FDA for ractopamine used as feed additive in swine.     

Absorption, disposition, metabolism, and excretion of [3-(14)C]caffeic acid in rats

Male Sprague-Dawley rats ingested 140 × 10(6) dpm of [3-(14)C]trans-caffeic acid, and over the ensuing 72 h period, body tissues, plasma, urine, and feces were collected and the overall levels of radioactivity determined. Where sufficient radioactivity had accumulated, samples were analyzed by HPLC with online radioactivity and tandem mass spectrometric detection. Nine labeled compounds were identified, the substrate and its cis isomer, 3'-O- and 4'-O-sulfates and glucuronides of caffeic acid, 4'-O-sulfates and glucuronides of ferulic acid, and isoferulic acid-4'-O-sulfate. Four unidentified metabolites were also detected. After passing down the gastrointestinal tract, the majority of the radiolabeled metabolites were excreted in urine with minimal accumulation in plasma. Only relatively small amounts of an unidentified (14)C-labeled metabolite were expelled in feces. There was little or no accumulation of radioactivity in body tissues, including the brain. The overall recovery of radioactivity 72 h after ingestion of [3-(14)C]caffeic acid was ～80% of intake.     

Nature of the residue of [(14)C]Cloransulam-methyl in lactating goats

Two lactating goats were given a daily oral dose of either [UL-aniline-(14)C; AN] or [triazolopyrimidine-7,9-(14)C; TP]cloransulam-methyl for 5 consecutive days. Each animal received a dietary equivalent of approximately 10 mg/kg of test material, approximately 2225 times the realistic maximum dietary exposure for a dairy animal. Milk, urine, and feces samples were collected in the morning and afternoon for each animal. Each goat was sacrificed within 23 h of receiving the last dose, and the liver, kidneys, samples of blood, fat, muscle, and gastrointestinal tract contents, and urine from the bladder were collected. All of these samples were analyzed for (14)C content. Cloransulam-methyl (CM) was rapidly excreted by the animals, with 99.9% of the recovered radioactivity appearing in the urine and feces. Radiochemical analysis showed very low residues, with the highest being in the kidneys at 0.122 and 0. 128 mg equiv of CM/kg (AN and TP labeled compounds, respectively). Radioactive residues were extracted and fractionated from kidney, liver, and milk. Analysis showed approximately 0.066 mg/kg CM in the kidney but <0.003 mg/kg in the liver. Only one metabolite, cloransulam, was identified (in liver, 9.5% of total radioactive residue; 0.005 mg/kg). All other metabolites were present at lower levels. Sulfonanilide bridge cleavage was not a significant degradation route for cloransulam-methyl in ruminants. These data indicated a very low bioaccumulation potential for cloransulam-methyl and its metabolites in ruminants. For a ruminant exposed to anticipated levels of cloransulam-methyl in its diet, parent and metabolites, in total, would not be expected to exceed 50 ng/kg in the kidney and liver.     

High performance liquid chromatographic method using fluorescence detention for quantitative analysis of zearalenone and alpha-zearalenol in blood plasma

A sensitive, high performance liquid chromatographic method is described for quantitative determination of zearalenone and alpha-zearalenol in blood plasma. Blood plasma is extracted with 2-propanol in ether, the extract is evaporated to dryness, and the residue is dissolved in 0.18N NaOH. The aqueous phase is washed with chloroform, dichloromethane, and benzene, neutralized with 0.10M H3PO4, and extracted with benzene. The extract is evaporated, dissolved in methanol, and injected onto a reverse phase column containing LiChrosorb RP-8 under the following conditions: methanol-acetonitrile-water mobile phase, fluorescence detector, excitation wavelength 236 nm, and 418 nm cut-off emission filter. The limit of detectability (twice background) is 0.5 ng standard which is equivalent to 0.6 ng standard/mL blood plasma. Linear standard curves are observed over the range of 0-35 ng of injected zearalenone and alpha-zearalenol. The recoveries from blood plasma are 76-101% in the range of 1.5-6.0 ng standard/mL blood.     

Identification of urinary metabolites of the red clover isoflavones formononetin and biochanin A in human subjects

Dietary supplements manufactured from red clover are widely marketed to provide the beneficial health effects of isoflavones without changing the original diet. In this study the metabolism of formononetin and biochanin A, the principal isoflavones of red clover, was studied in human subjects. Seven women ingested four red clover dietary supplements, and the metabolites of the isoflavones were identified in their urine samples. The structures of trimethylsilyl derivatives of the metabolites were established by GC-MS. New reduced metabolites of formononetin (dihydroformononetin and angolensin) and biochanin A (dihydrobiochanin A and 6'-hydroxyangolensin) were identified in urine samples using authentic reference compounds. Possible metabolic pathways are presented for the red clover isoflavones formononetin and biochanin A.     

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.     

兽药“8405”有效成份洗必太Chlorhexidinum及其代谢物残留的动态变化

采用G-T-洗必太研究了兽药新剂型“8405”的有效成份洗必太,及其代谢物在大白鼠主要脏器,组织,尿和血液中残留的变化。测定结果表明,连续注药三日后第2小时,在所观察的器官、组织和尿液中,发现有不同数量的洗必太存在。第240小时,在肝、肾、肌肉、脂肪体和尿液中,洗必太趋于消失。但是,直至第1080小时(第45天),在所观察的脏器、组织和尿液中,仍有洗必太的代谢物残留。     

Urinary and plasma levels of resveratrol and quercetin in humans, mice, and rats after ingestion of pure compounds and grape juice

The present study investigates the bioavailability of resveratrol and quercetin in humans, mice, and rats after oral ingestion of grape juice preparations or pure aglycones. Oral administration of resveratrol and quercetin to humans yielded detectable levels of resveratrol, quercetin, and their derivatives in the plasma and urine. Urinary levels of resveratrol, quercetin, and their metabolites were observed in human subjects receiving 600 and 1200 mL of grape juice, whereas quercetin metabolites were identified in urine samples even after receiving 200 mL of grape juice. The cumulative amounts of resveratrol and quercetin excreted in the urine of mice receiving concentrated grape juice for 4 days were 2.3 and 0.7% of the ingested doses, respectively. After i.g. administration of resveratrol to rats (2 mg/kg), up to 1.2 microM resveratrol was observed in the plasma. The study demonstrates that the glycoside forms of resveratrol and quercetin in grape juice are absorbed to a lesser extent than the aglycones.     

Occurrence of zearalenone-4-beta-D-glucopyranoside in wheat

An LC-MS method was developed for the analysis of zearalenone-4-beta-D-glucopyranoside and zearalenone in wheat (Triticum aestivum). The limit of determination for zearalenone-4-beta-D-glucopyranoside and zearalenone was 10 microg/kg. The recovery rates were calculated to be 69% and 89% at a concentration of 100 microg/kg for zearalenone-4-beta-D-glucopyranoside and zearalenone, respectively. Twenty-four Bavarian wheat samples from a 1999 harvest were analyzed. Zearalenone was present in 22 of 24 field samples, the levels ranged from 11 to 860 microg/kg. Zearalenone-4-beta-D-glucopyranoside was found in 10 of the zearalenone positive samples (42%) at levels ranging from 17 to 104 microg/kg. The amounts of zearalenone-4-beta-D-glucopyranoside were correlated to those of zearalenone (r2 = 0.86, b = 0.10). After gastrointestinal hydrolyzation, zearalenone-4-beta-glucopyranoside might be implicated in the development of a zearalenone-syndrome. Therefore, more attention should be focused on conjugated mycotoxins in food and feed.