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.     

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.     

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).     

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.     

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.     

Rapid confirmatory assay for determining 12 sulfonamide antimicrobials in milk and eggs by matrix solid-phase dispersion and liquid chromatography-mass spectrometry

A rapid confirmatory method for determining 12 sulfonamide (SAs) antibacterials in whole milk and eggs is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-mass spectrometry (MS). The LC-MS instrument was equipped with an electrospray ion source and a single quadrupole. After 4 mL of a milk sample containing the analytes had been deposited on sand (crystobalite), this material was packed into an extraction cell. SAs were extracted by flowing 4 mL of water through the cell heated at 75 degrees C. With some modifications, this procedure was applied also to eggs. After pH adjustment and filtration, 0.5 mL of the final extracts was then injected into the LC column. MS data acquisition was performed in the positive-ion mode and by monitoring at least three ions for each target compound. The in-source collision-induced dissociation process produced confirmatory ions. At the 50 ng/g level, recovery of the analytes in milk and eggs was 77-92% with relative standard deviations ranging between 1 and 11%. Estimated limits of quantification (S/N = 10) were 1-3 ng/g of SAs in milk and 2-6 ng/g in eggs. With both matrices, attempts to reduce the analysis time by using a short chromatographic run time caused severe ion signal suppression for the early-eluted SAs. This effect was traced to competition effects by polar endogenous coextractives, maybe proteinaceous species, which are eluted in the first part of the chromatographic run. This unwelcome effect was almost completely removed by simply adopting more selective chromatographic conditions.     

Analysis of pharmaceutical dosage forms for oxfendazole: II. Simultaneous liquid chromatographic determination of oxfendazole and trichlorfon in equine paste

A reverse phase liquid chromatographic procedure is described for the simultaneous determination of oxfendazole [2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] and trichlorfon [(2,2,2-trichloro-1-hydroxyethyl)phosphonic acid dimethyl ester] in equine paste. The sample is extracted by sonication in methanol. Insoluble excipients are removed by centrifugation and an aliquot plus internal standard are diluted with dilution solvent (water-acetonitrile-phosphoric acid, 80 + 20 + 1). The samples are filtered and injected onto a Partisil-5 ODS-3 column with acetonitrile-0.01 M phosphate buffer pH 6.0 (20 + 80) as mobile phase. Method specificity is confirmed using an absorbance rationing technique. The method yields mean recoveries of 100.9 and 100.0% for trichlorfon and oxfendazole, respectively. Dependence of chromatographic performance characteristics on mobile phase organic content, pH, and buffer concentration is also reported.     

Gas-liquid chromatographic determination of ethopabate in feed premixes

A gas-liquid chromatographic method is presented for determining ethopabate in 0.8 and 8.0% premixes. A sample is extracted with tetrahydrofuran containing an internal standard, by sonication or overnight soaking. The extract is clarified by centrifugation, diluted if necessary, and injected into a gas chromatograph equipped with a flame ionization detector. Average per cent recoveries for spiked blank samples were 100.6 at the 0.8% level and 100.4 at the 8.0% level. Precision, as indicated by replicate analyses of several premixes, ranged from 0.5 to 1.7% relative standard deviation.     

Matrix solid phase dispersion (MSPD) extraction and gas chromatographic screening of nine chlorinated pesticides in beef fat

A multiresidue technique is presented for the extraction and quantitative gas chromatographic screening of 9 insecticides (lindane, heptachlor, aldrin, heptachlor epoxide, p,p'-DDE, dieldrin, endrin, p,p'-TDE, and p,p'-DDT) as residues in beef fat. Beef fat was fortified by adding the 9 insecticides, plus dibutyl chlorendate as internal standard, to 0.5 g portions of beef fat and blending with 2 g C18 (octadecylsilyl)-derivatized silica. The C18/fat matrix blend was fashioned into a column by adding the blend to a 10 mL syringe barrel containing 2 g activated Florisil. The insecticides were then eluted from the column with 8 mL acetonitrile, and a 2 microL portion of the acetonitrile eluate was then directly analyzed by gas chromatography with electron capture detection. Unfortified blank controls were treated similarly. The acetonitrile eluate contained all of the pesticide analytes (31.25-500 ng/g) and was free of interfering co-extractants. Correlation coefficients for the 9 extracted pesticide standard curves (linear regression analysis, n = 5) ranged from 0.9969 (+/- 0.0021) to 0.9999 (+/- 0.0001). Average relative percentage recoveries (85 +/- 3.4% to 102 +/- 5.0%, n = 25 for each insecticide), inter-assay variability (6.0 +/- 1.0% to 14.0 +/- 6.7%, n = 25 for each insecticide), and intra-assay variability (2.5-5.1% n = 5 for each insecticide) indicated that the methodology is acceptable for the extraction, determination, and screening of these residues in beef fat.     

Multiresidue matrix solid phase dispersion (MSPD) extraction and gas chromatographic screening of nine chlorinated pesticides in catfish (Ictalurus punctatus) muscle tissue

A multiresidue technique for extraction and gas chromatographic screening of 9 insecticide (lindane, heptachlor, aldrin, heptachlor epoxide, p,p'-DDE, dieldrin, endrin, p,p'-TDE, and p,p'-DDT) residues in catfish (Ictalurus punctatus) muscle tissue is presented. The 9 insecticides, plus dibutyl chlorendate internal standard, were fortified into catfish muscle tissue (0.5 g) and blended with 2 g C18 (octadecylsilyl derivatized silica reverse-phase material). The C18/muscle tissue matrix blend was fashioned into a column by adding the blend to a 10 mL syringe barrel containing 2 g activated Florisil. The insecticides were then eluted from the column with acetonitrile (8 mL), and a portion (2 microL) of the acetonitrile eluate was then directly analyzed by gas chromatography with electron capture detection. Unfortified blank controls were treated similarly. The resultant extracts contained pesticide analytes (31.25-500 ng/g) free of interfering compounds when analyzed. Correlation coefficients for the 9 extracted pesticide standard curves (linear regression analysis, n = 5) ranged from 0.9967 (+/- 0.0018) to 0.9999 (+/- 0.0001). Average percentage recoveries (82 +/- 4.8% to 97 +/- 3.6%, n = 25 for each insecticide), interassay (5.0 +/- 2.7% to 16.9 +/- 6.5%, n = 25 for each insecticide) and intraassay (1.8 to 4.7%, n = 5 for each insecticide) variabilities were indicative of an acceptable methodology for the analysis and screening of these residues in catfish muscle tissue.     

Chlorinated compounds and phenols in tissue, fat, and blood from rats fed industrial waste site soil extract: methods and analysis

A method was developed to analyze rat tissue, fat, and blood for some of the chlorinated compounds found in an extract of soil from an industrial waste site. Extraction with hexane and then with ethyl ether-hexane (1 + 1) was followed by concentration over steam, and gas chromatographic analysis with an electron capture detector. Volatile compounds were analyzed in a glass column coated with 6% SP-2100 plus 4% OV-11 on Chromosorb W. Semivolatile compounds, chlorinated compounds, and pesticides were analyzed in a 70 m glass capillary column coated with 5% OV-101. Phenols were analyzed in a glass column packed with 1% SP-1240 DA on Supelcoport. However, the most efficient means of separation was to use the same glass column for volatile compounds, a DB-5 fused silica capillary column for semivolatile compounds, pesticides, and phenols, and the same 1% SP-1240 DA glass column for separation of beta-BHC and pentachlorophenol. Recoveries ranged from 86.3 +/- 9.1% (mean +/- standard deviation) to 105 +/- 10.4%. Sensitivities for semivolatile chlorinated compounds, pesticides, and phenols were about 4 ng/g for fat, 1 ng/g for tissue, and 0.2 ng/mL for blood. Sensitivities for volatile compounds were about 4-fold higher (16, 4, and 0.8, respectively). Sensitivities for dichlorobenzenes and dichlorotoluenes were 8 ng/g for fat, 2 ng/g for tissue, and 0.4 ng/mL for blood.     

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.     

Matrix solid phase dispersion isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue

A method for the isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue is presented. Blank control and sulfadimethoxine-fortified fish muscle tissue samples (0.5 g) were blended with octadecyisilyl (C18, 40 micrograms, 18% load, endcapped) derivatized silica packing material. A column made from the C18/fish tissue blend was first washed with hexane (8 mL), following which the sulfadimethoxine was eluted with dichloromethane (8 mL). The eluant contained sulfadimethoxine analyte that was free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array, 270 nm). Standard curves for sulfadimethoxine isolated from fortified samples were linear (0.999 +/- 0.001) with an average relative percentage recovery of 101.1 +/- 4.2% for the concentration range (50, 100, 200, 400, 800, and 1600 ng/g) examined using sulfamethoxazole as the internal standard. The interassay variability was 10.7 +/- 8.2% with an intra-assay variability of 2.2%.     

Immunosorbents coupled on-line with liquid chromatography for the determination of fluoroquinolones in chicken liver.

Four fluoroquinolones were analyzed in fortified chicken liver using an automated, on-line immunoaffinity extraction method. The fluoroquinolones were extracted from the liver matrix using an immunoaffinity capture column containing anti-sarafloxacin antibodies covalently cross-linked to protein G. After interfering liver matrix components had been washed away, the captured fluoroquinolones were automatically eluted directly onto a reversed phase column. Liquid chromatographic analyses were performed by isocratic elution using 2% acetic acid/acetonitrile (85:15) as the mobile phase and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 280 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Overall recoveries from fortified liver samples (20, 50, and 100 ng/g) ranged between 85.7 and 93.5% with standard deviations of <5%. The limit of quantification for each fluoroquinolone was 1 ng/mL. The limits of detection, based on a signal-to-noise ratio of 5:1, were 0.47, 0.32, 0.87, and 0.53 ng/mL for ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin, respectively.     

High pressure liquid chromatographic determination of ochratoxin A and zearalenone in cereals.

A high pressure liquid chromatographic (HPLC) method has been developed for determining ochratoxin A and zearalenone in cereals. The sample is extracted with phosphoric acid and chloroform. The extract is cleaned by washing on a silica gel column with cyclohexane-ethylene dichloride-ethyl ether. After eluting zearalenone with chloroform, ochratoxin A is eluted with chloroform-formic acid. Zearalenone is extracted into alkaline solution, washed with chloroform, the pH is adjusted, and the zearalenone is extracted back into chloroform. Ochratoxin A is purified by chromatography on aqueous sodium biarbonate-Celite. The mycotoxins are determined by using a liquid chromatograph with 2 columns in series packed with Spherisorb ODS 10 micrometer and 5 micrometers, respectively. Ochratoxin A is detected with a speftrophotofluorometer, coupled in series with an ultra-violet detector for estimation of zearalenone. Detection limits are 1-5 micrograms/kg for ochratoxin A and 2 micrograms/kg for zearalenone.     

Matrix solid phase dispersion isolation and liquid chromatographic determination of five benzimidazole anthelmintics in fortified beef liver

A multiresidue method for isolation and liquid chromatographic determination of 5 benzimidazole anthelmintics (thiabendazole, oxfendazole, mebendazole, albendazole, and fenbendazole) in beef liver tissue is presented. Blank or benzimidazole-fortified liver samples (0.5 g) were blended with octadecylsilyl derivatized silica packing material (C18, 18% load, endcapped, 2 g). A column made from the C18/liver matrix was first washed with hexane (8 mL), following which the benzimidazoles were eluted with acetonitrile. The acetonitrile extract was then passed through an activated alumina column. The eluate contained benzimidazole analytes that were free from interfering compounds as determined by UV detection (photodiode array, 290 nm). Correlation coefficients of standard curves for individual benzimidazoles isolated from fortified samples, using internal standardization, were linear (0.996 +/- 0.002 to 0.999 +/- 0.001) with average relative percentage recoveries from 62.0 +/- 6.7 to 86.8 +/- 8.6% for the concentration range (100-3200 ng/g) examined. The interassay variability was 7.0 +/- 4.1 to 12.9 +/- 10.2% with an intra-assay variability from 2.2 to 4.0%.     

Liquid chromatographic determination of the estrogens daidzein, formononetin, coumestrol, and equol in bovine blood plasma and urine

A liquid chromatographic (LC) method is described for the determination of the plant estrogens diadzein, formononetin, and coumestrol and the estrogenically active metabolite equol in bovine blood plasma and urine. The blood and urine samples are incubated overnight with and without beta-glucuronidase/sulfatase for analysis of both free and conjugated forms of estrogens. Samples are applied to Extrelut columns, extracted with ethyl acetate, and evaporated to dryness. Residues from urine samples are dissolved in methanol, diluted with water, acidified with HCl, and purified by injection through a Sep-Pak C18 cartridge. This eluate is used for LC analysis. Residues from blood samples are dissolved in benzene-petroleum ether (1 + 1), extracted with ammonium hydroxide, acidified with glacial acetic acid, and extracted with ethyl acetate. The ethyl acetate extract is evaporated, dissolved in 80% methanol, injected onto a LC reverse-phase column, and separated in a linear gradient system between 40 and 80% methanol in phosphate buffer. Quantitation is performed by means of UV and fluorescence responses. The method was sensitive enough to determine 0.4 ng/mL of daidzein and formononetin and 0.1 and 13 ng/mL of coumestrol and equol, respectively, in blood, and 130, 80, and 7 ng/mL of daidzein, formononetin, and coumestrol, respectively, and 4 micrograms/mL of equol in urine. The applicability of the method was checked by the determination of total and free plant estrogens in blood samples from a dairy cow fed a normal diet.     

Normal phase liquid chromatographic determination of nanogram quantities of ivermectin in cattle blood or plasma

A method has been developed for determining ivermectin in 5 mL samples of cattle blood by a 2-step process: cleanup solvent extraction followed by direct injection onto a normal phase liquid chromatography (LC) system with UV detection. Recovery was 77-80% +/- 5.5% standard deviation. Endogenous interference that may be present caused the lower limit of detection to be set at 4-5 ng/mL. The method was used to show that in blood the distribution of ivermectin favors plasma in a fixed proportion over cellular material, and further to provide a time-course profile of ivermectin in the whole blood of injected cattle. In whole blood, ivermectin concentration peaked between 3 and 5 days and dissipated slowly with a half-life of 3 days.     

Gas chromatographic analysis of milk for deoxynivalenol and its metabolite DOM-1

A gas chromatographic method is described for the determination of deoxynivalenol (DON) and its metabolite DOM-1 in milk. Milk samples were extracted with ethyl acetate on a commercially available disposable extraction column, followed by hexane-acetonitrile partitioning. Final purification was accomplished on a reverse phase C-18 cartridge. The trimethylsilyl ether (TMS) derivatives of DON were prepared, chromatographed on an OV-17 column, and quantitated with an electron capture detector. Chromatography of the TMS derivatives of milk extracts was compared to that of the corresponding heptafluorobutyryl derivatives. The limit of detection using TMS derivatives was 1 ng/mL for both toxins with recoveries averaging 82% +/- 9% at 2.5 and 10 ng/mL milk for DON and 85% +/- 6% at 10 ng/mL for DOM-1.     

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.