Gas-liquid chromatographic determination of thiabendazole and methyl 2-benzimidazole carbamate in fruits and crops.

A method is described for the electron capture gas-liquid chromatographic determination of thiabendazole and methyl 2-benzimidazole carbamate (MBC) after derivatization with pentafluorobenzyl bromide (PFB-Br). The samples are extracted with ethyl acetate, and the residual thiabendazole, benomyl, and MBC are isolated by liquid-liquid extraction into dilute HCl. After neutralization and re-extraction with ethyl acetate, thiabendazole and MBC are reacted with PFB-Br to form the PFB derivatives. Alumina column chromatography was used to clean up extracts, and the derivatives could be detected as low as 5-10 pg. Recoveries were 95-98% from fruits fortified with 0.3-2.0 ppm thiabenzadole; recoveries were 91-97% when 0.05-1.0 ppm benomyl/MBC were added to fruits/crops. The PFB derivatives were identified by gas-liquid chromatography-mass spectrometry.     

Rapid liquid chromatographic determination of patulin in apple juice

A rapid method is described for the quantitative determination of patulin in apple juice. The mycotoxin is extracted from the sample with ethyl acetate and the extract is cleaned up by extraction with a sodium carbonate solution. Patulin is determined by reverse phase liquid chromatography using a muBondapak C18 column and a 254 nm ultraviolet detector. The lower detection limit in patulin standard solution is 0.32 ng and recovery is greater than 75%.     

Sensitive determination of ethopabate residues in chicken tissues by liquid chromatography with fluorometric detection

A liquid chromatographic (LC) method is described for determination of ethopabate residues in chicken tissues. The drug is extracted from tissues with acetonitrile, and the extract is concentrated to 2-3 mL. This aqueous solution is rinsed with ethyl acetate and cleaned up by Florisil column chromatography. LC analysis is carried out on a Zorbax ODS column, and ethopabate is quantitated by using a fluorometric detector set at 306 nm (excitation) and 350 nm (emission). Recoveries of ethopabate added to chicken tissues at levels of 0.01 and 0.05 ppm were 87.8 and 92.7%, respectively. The detection limit was 100 pg for ethopabate standard, and 0.5 ppb in chicken tissues.     

Gas chromatographic determination of picloram in fish

A simple method for determining picloram in fish is described. The sample is homogenized with ethyl acetate, acidified with 1N HCl, and extracted twice more with ethyl acetate. Ethyl acetate fractions are pooled, derivatized with diazomethane, cleaned up by column chromatography, and analyzed by electron capture gas chromatography. Rainbow trout exposed to 14C-picloram were used to evaluate the efficiency of 2 methods of extraction and to provide data on the rate of uptake and the bioconcentration factor. The detection limit for this method is 5 ng/g, using a 4 g sample.     

High pressure liquid chromatographic determination of zearalenone in chicken tissues

A method is reported for the extraction and analysis of zearalenone in chicken fat, heart muscle, and kidney tissue by using high pressure liquid chromatography (HPLC). Zearalenone is extracted with acetonitrile, cleaned up with hexane, and extracted further with ethyl acetate. Zearalenone is determined by HPLC using a reverse phase radial compression separation system, an ultraviolet absorbance detector, and a mobile phase of acetonitrile-water (60 + 40) (v/v). Recoveries of zearalenone added at levels from 50 to 200 ng/g are in the range 82.6-95.1%.     

Gas-liquid chromatographic determination of nifursol in frozen turkey tissues to ten parts per billion.

Nifursol (3,5-dinitrosalicylic acid (5-nitrofurfurylidene) hydrazide) is extracted into ethyl acetate from 10 g tissue in the presence of sodium sulfate. Tissue interferences are removed from the tissue extract by washing with petroleum ether after the extract has been transferred into an aqueous solution by evaporation of ethyl acetate. The drug is hydrolyzed under acid conditions to form 5-nitro-2-furaldehyde (5NF). After partition of 5NF from the aqueous phase into benzene the extract is further cleaned up on a Florisil column. The 5NF is eluted from the Florisil column with benzeneethyl acetate. Electron capture gas-liquid chromatography of a 10 mul injection of the concentrated column eluate is the determinative step. Quantitation is accomplished by comparison of the peak height of the sample to the peak height of the standard which is carried through the method simultaneously. Studies of method performance on turkey muscle, liver, kidney, and skin tissues fortified to contain 10 ppb nifursol show a recovery range of 87.4-95.0% and a coefficent of variation range of 5.7-11.2%.     

High-pressure liquid chromatographic method for the determination of patulin in apple butter.

Patulin is extracted from apple butter samples with ethyl acetate and the extract is cleaned up on a silica gel column, using benzene-ethyl acetate (75+25) as the eluant. High-pressure liquid chromatography, using a 25 cm ZorbaxSil column, isooctane-ethyl ether-acetic acid (750+250+0.5) as the mobile solvent, and a 254 nm ultraviolet detector, is used for the determinative step. Under these conditions, patulin is eluted before 5-hydroxymethylfurfural, a component of apple butter which interferes with other liquid chromatographic and thin layer chromatographic methods. Recoveries of patulin added at levels of 34.6, 138.4, and 276.8 mug/kg ranged from 89.0 to 112.1%.     

Gas-liquid chromatographic determination of captan and two metabolites in milk and meat

A gas-liquid chromatographic (GLC) method has been developed for the determination of captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) and 2 metabolites, tetra-hydrophthalimide (THPI) and tetrahydrophthalamic acid (THPMA), in milk and meat. The sample is extracted with ethyl acetate and is cleaned up by acetonitrile partition and silica gel chromatography where captan, THPI, and THPMA are separated. Captan is directly determined by GLC. THPI and THPMA are separately derivatized in an acetone solution of pentafluorobenzyl bromide. The resultant derivatives are purified separately on an Al2O3 column and quantitated by GLC, using an electron capture detector. Recoveries from milk samples fortified at 0.02-10 ppm ranged from 71 to 102%; recoveries from meat samples fortified at 0.04-10 ppm ranged from 75 to 99%.     

Collaborative study of a spectrofluorometric method for lasalocid sodium in feeds.

A collaborative study has been completed on a spectrofluorometric method for determining lasalocid sodium in finished poultry feeds. After a brief treatment of feed samples with pH 4.7 buffer, the drug is extracted with ethyl acetate. The ethyl acetate extract is further cleaned up by treatment with HCI and NaOH and quantitatively measured by spectrofluorometry. Nonspecific fluorescence is corrected for via the lasalocid-boric acid complex. The method is rapid, sensitive, and free of interference. A number of other feed additives, including monensin and ethoxyquin, do not interfere. Nine collaborators participated in the study and agreement between laboratories was satisfactory. The average recoveries of lasalocid sodium from the mash feeds added at levels of 0.0064, 0.0080, and 0.0096% were 100, 98, and 99%, respectively, and the corresponding coefficients of variation were 12.8, 8.2, and 8.0% (all results included). The method has been adopted as official first action.     

Liquid chromatographic determination of ergot alkaloids in wheat

A method is described for the determination of individual ergot alkaloids in wheat. The sample is extracted with ethyl acetate-4% ammonium hydroxide (100 + 10), and the extract is cleaned up by liquid-liquid partition. The ergot alkaloids are resolved by liquid chromatography (LC), using a porous cross-linked polystyrene-divinylbenzene resin column and a mobile phase consisting of acetonitrile-0.05 M dibasic ammonium phosphate (55 + 45) buffered at pH 10.0. The ergot alkaloids ergonovine, ergonovinine, ergotamine, ergotaminine, alpha-ergocryptine, alpha-ergocryptinine, ergocristine, and ergocristinine are separated by LC and detected with a fluorescence detector. Recovery of ergot alkaloids added to wheat at levels of 16-760 ng/g averaged 85.6% with a coefficient of variation of 11.1%.     

High pressure liquid chromatographic determination of nitrofurazone in milk.

A rapid high pressure liquid chromatographic (HPLC) screening method for the quantitative determination of nitrofurazone in milk has been developed. The drug is extracted with ethyl acetate from a 2.0 ml milk serum sample, the organic layer is evaporated to dryness, and the residue is dissolved in the mobile phase and injected into the liquid chromatogarph. A reverse phase muBondapak C18 column is used with monitoring at 365 nm. The detection limit is 5 ppb and recoveries are 57--67%. Mass spectroscopic confirmation of the HPLC nitrofuran peak is described.     

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.     

Liquid chromatographic method for determining the macrolide antibiotic sedecamycin and its major metabolites in swine plasma and tissues

A liquid chromatographic (LC) method was developed to determine sedecamycin, a 17-membered macrolide antibiotic used for treating swine dysentery, and its major metabolites (lankacidin C, lankacidinol A, and lankacidinol) in swine plasma and tissues. Plasma is directly extracted with ethyl acetate and analyzed by liquid chromatography without purification. Tissues are homogenized in a phosphate buffer containing sodium chloride, and then extracted with ethyl acetate. The extracts are subjected to silica gel-Florisil, double-layered column chromatography to remove endogenous interfering substances. The LC determination uses silica gel and ODS-silica as a stationary phase. The detection limits for sedecamycin and its metabolites were less than or equal to 0.05 ppm, and average recoveries and coefficients of variation (0.2-1 ppm range) were greater than 75% and less than 10%, respectively.     

Determination of levamisole in animal tissues using liquid chromatography with ultraviolet detection.

An efficient and sensitive liquid chromatographic method is described for the determination of the anthelminthic drug levamisole, in muscle, liver, kidney and fat of sheep, pigs and poultry, using thiabendazole as internal standard. Samples were extracted by homogenizing with chloroform, and were applied to Supelco Si solid-phase extraction columns and eluted with methanol. Chromatographic analysis was performed on a LiChrospher 60 RP-Select B column using methanol/ammonium acetate buffer 0.05 M (55/65, v/v) as mobile phase and reading at 220 nm. The quantification limit for the assay was 4 ng/g. Mean recoveries were about 84% for liver, 85% for kidney, 89% for muscle and 84% for fat. The assay has been used for statutory testing purposes.     

Liquid chromatographic determination of rotenone in fish, crayfish, mussels, and sediments

An analytical procedure is described for determining residues of rotenone in fish muscle, fish offal, crayfish, freshwater mussels, and bottom sediments. Tissue samples were extracted with ethyl ether and extracts were cleaned up by gel permeation chromatography and silica gel chromatography. Sediment samples were extracted with methanol, acidified, partitioned into hexane, and cleaned up on a silica gel column. Rotenone residues were quantitated by liquid chromatography, using ultraviolet (295 nm) detection. Recoveries from sediment samples fortified with rotenone at 0.3 microgram/g were 80.8%, whereas recoveries from tissue samples fortified with 0.1 microgram/g ranged from 87.7 to 96.8%. Samples fortified with 0.3 microgram/g and stored at -10 degrees C for 6 months before analysis had recoveries ranging from 83.2 to 90.5%. Limits of detection were 0.025 microgram/g for sediments and 0.005 microgram/g for tissue samples.     

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.     

Enzyme-linked immunosorbent assay of benomyl and thiabendazole in some foods

Enzyme-linked immunosorbent assays were developed for benomyl as its decomposition product, methyl 2-benzimidazole carbamate, and thiabendazole in foods. Immunogens consisting of human serum albumin coupled to 2-succinamidobenzimidazole or 2-(2'-succinamido-4'-thiazolyl)benzimidazole were used in rabbits to raise antisera that were specific for the respective fungicides. Lower limits of quantitation of 0.35 ppm for benomyl and 0.03 ppm for thiabendazole were established without cleanup of the ethyl acetate extract. Recoveries of benomyl from 3 crops spiked at 0.5 to 10 ppm averaged 89% (range 73-109%) and of thiabendazole from 5 crops spiked at 0.1 to 2.0 ppm were 93% (range 81-105%).     

Capillary gas chromatographic-mass spectrometric determination of fluoroacetate residues in animal tissues

A method for the quantitative determination of fluoroacetate (FAC) residues in animal tissues is described. The procedure involves tungstic acid extraction, partitioning into ethyl acetate, evaporation of ethyl acetate, derivatization with pentafluorobenzyl bromide (PFB), and analysis of the resulting derivative (PFB-FAC) by capillary gas chromatography-mass spectrometry (CGC-MS) with specific ion monitoring (SIM). The tungstic acid system extracted 96.8 +/- 4.2% of the endogenous 14C-1080 residues in rat tissues. Recovery of FAC during the extraction, purification, and derivatization procedures is established by use of a 14C-FAC spike. 1,2-Dibromobenzene is used as an internal standard for the CGC-MS analysis. PFB-FAC is identified on the basis of comparative retention times and the relative intensities of m/z 257.9 and 181.0. PFB-FAC is quantitated by comparing the response at m/z 257.9 to a PFB-FAC standard curve. Routine sensitivity of the method allows determination of 10 ppb fluoroacetate in tissue.     

Liquid chromatographic determination of the processing aid 4-hexylresorcinol in shrimp.

A rapid, sensitive, liquid chromatographic (LC) method has been developed for determination of residuals of the processing aid, 4-hexylresorcinol, on shrimp meat. An aqueous homogenate of shrimp meat is extracted with ethyl acetate followed by precolumn preparation on a silica Sep-Pak cartridge. LC determination is preformed with a Nova-Pak C18 column, with UV detection at 214 nm. Sensitivity was 0.006 micrograms, and recovery from shrimp meat samples of known 4-hexylresorcinol addition was 94%. Shrimp treated with 4-hexylresorcinol under the recommended dip protocol had mean residuals of 1.18 ppm, with a standard deviation of 0.13 ppm.     

Preparation of deoxynivalenol (vomitoxin) from field-inoculated corn

A process was developed for production of gram quantities of deoxynivalenol (DON) from corn that had been inoculated in the field with Fusarium graminearum and was estimated to contain 400-500 mg DON/kg. Steps in the purification procedure included extraction with methanol-water (1 + 1), partition from an aqueous solution into ethyl acetate by using a hydrophilic matrix, defatting, Florisil column chromatography, methylene chloride-water partition, semipreparative liquid chromatography (LC), and crystallization from ethyl acetate. The average yield of crystalline DON produced by this method (or with minor variations) was 281 mg/kg.