Determination of albendazole and its major metabolites in the muscle tissues of Atlantic salmon,tilapia, and rainbow trout by high performance liquid chromatography with fluorometric detection

A liquid chromatographic procedure for the determination of albendazole ([5-(propylthio)-1H-benzimidazol-2yl]carbamic acid methyl ester) and its major metabolites, albendazole sulfoxide, albendazole sulfone, and albendazole-2- aminosulfone in rainbow trout, tilapia, and salmon muscle with adhering skin tissue is described. The muscle tissue samples are made alkaline with potassium carbonate and extracted with ethyl acetate. The extracts are further subjected to cleanup by utilizing a number of liquid-liquid extraction steps. After solvent evaporation, the residue is reconstituted in mobile phase and chromatographed. The chromatography is carried out on a reversed phase Luna C(18) column, using acetonitrile/methanol/buffer as a mobile phase and a fluorescence detector. The average recoveries from the fortified muscle tissue of the three fish species for albendazole (25-100 ppb), albendazole sulfoxide (15.5-62 ppb), albendazole sulfone (1-10 ppb), and albendazole-2- aminosulfone (10-100 ppb) were 94, 77, 82, and 67%, respectively. The average CV for each compound was < or =10%. The procedure was validated and then applied to the determination of albendazole and its three major metabolites in the muscle tissue of the three fish species obtained after orally dosing with albendazole.     

Determination of gentian violet, its demethylated metabolites, and leucogentian violet in chicken tissue by liquid chromatography with electrochemical detection

A method is presented for determination of residues of gentian violet (GV), its demethylated metabolites (pentamethyl and tetramethyl), and leucogentian violet (LGV) in chicken tissue. The analytes are extracted from tissue with acetonitrile/buffer and partitioned into methylene chloride. Polar lipids are removed on an alumina column followed by partitioning into methylene chloride from a citrate buffer. The compounds of interest are isolated on a disposable carboxylic acid cation exchange column and then eluted with 0.02% HCl in methanol. GV, its metabolites, and LGV are determined by liquid chromatography using isocratic elution with a buffered mobile phase from a cyano column and amperometric electrochemical detection at +1.000 V. Average recoveries of GV and LGV from commercially purchased chicken liver fortified with 20 ppb of each compound were 92% [standard deviation (SD) = 7, coefficient of variation (CV) = 7.6%] and 86% (SD = 7, CV = 8.1%), respectively. Average recoveries of GV, LGV, the pentamethyl metabolite, and 1 of the tetramethyl metabolites from control chicken liver (provided by the Center for Veterinary Medicine) fortified with 20 ppb of each compound were 80% (SD = 7, CV = 8.8%), 76% (SD = 3, CV = 3.9%), 83% (SD = 6, CV = 7.2%), and 76% (SD = 8, CV = 10.5%), respectively. Mean results from 10 analyses of residue-incurred chicken liver were 31 ppb GV (SD = 3, CV = 9.7%), 34 ppb pentamethyl metabolite (SD = 3, CV = 8.8%), and 40 ppb tetramethyl metabolite(s) (SD = 2, CV = 5.0%), for an average value of 105 ppb total residues (SD = 6, CV = 5.7%); no LGV was found. Data are also presented to show applicability of the method to muscle tissue.     

Capillary column gas chromatographic determination of dicamba in water, including mass spectrometric confirmation

A sensitive method is described for determining dicamba at low micrograms/L levels in ground waters by capillary column gas chromatography with electron-capture detection (GC-EC); compound identity is confirmed by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring. Dicamba residue is hydrolyzed in KOH to form the potassium salt. The sample is then extracted with ethyl ether which is discarded. The aqueous phase is acidified to pH less than 1 and extracted twice with ethyl ether. The combined ethyl ether extracts are concentrated, and the residue is methylated using diazomethane to form the corresponding dicamba ester. The derivatized sample is cleaned up on a deactivated silica gel column. The methylated dicamba is separated on an SE-30 capillary column and quantitated by electron-capture or mass spectrometric detection. Average recoveries (X +/- SD) for ground water samples fortified with 0.40 microgram/L of dicamba are 86 +/- 5% by GC-EC and 97 +/- 7% by GC-MS detections. The EDL (estimated detection limit) for this method is 0.1 microgram dicamba/L water (ppb).     

Liquid chromatographic determination of monensin in chicken tissues with fluorometric detection and confirmation by gas chromatography-mass spectrometry

An accurate, sensitive method is described for the determination of monensin residue in chicken tissues by liquid chromatography (LC), in which monensin is derivatized with a fluorescent labeling reagent, 9-anthryldiazomethane (ADAM), to enable fluorometric detection. Samples are extracted with methanol-water (8 + 2), the extract is partitioned between CHCl3 and water, and the CHCl3 layer is cleaned up by silica gel column chromatography. Free monensin, obtained by treatment with phosphate buffer solution (pH 3) at 0 degrees C, is derivatized with ADAM and passed through a disposable silica cartridge. Monensin-ADAM is identified and quantitated by normal phase LC using fluorometric detection. The detection limit is 1 ppb in chicken tissues. Recoveries were 77.6 +/- 1.8% at 1 ppm, 56.7 +/- 7.1% at 100 ppb, and 46.5 +/- 3.7% at 10 ppb fortification levels in chicken. Gas chromatography-mass spectrometry is capable of confirming monensin methyl ester tris trimethylsilyl ether in samples containing residues greater than 5 ppm.     

High pressure liquid chromatographic determination of aflatoxins in peanut butter using a silica gel-packed flowcell for fluorescence detection

A high pressure liquid chromatographic method has been developed for determining aflatoxins B1, B2, G1, and G2 in peanut butter. The method is based on extraction with acidified aqueous methanol, partition of the aflatoxin into methylene chloride, and purification of the extract on a 2 g silica gel column. The extracted aflatoxins are resolved on a microparticulate (10 micrometer) porous silica gel column in ca 10 min with a water-washed chloroform-cyclohexane-acetonitrile solvent that contains 2% isopropanol. The fluorescence detection system determines aflatoxins B1, B2, G1, and G2 at low levels, i.e., 0.25 ppb B1, 0.5 ppb G1, and 0.2 ppb B2 and G2. Multiple assays of 5 samples of naturally contaminated peanut butters containing total aflatoxins (B1 + B2 + G1 + G2) at levels of 1, 2, 3, 9, and 17 ppb gave intralaboratory coefficients of variation of 7, 4, 4, 11, and 3%, respectively. Samples spiked at levels of 5, 9, and 17 ppb total aflatoxins showed recoveries of 79, 81, and 81%, respectively.     

Determination of oxolinic acid residues in salmon muscle tissue by liquid chromatography with fluorescence detection

The present paper describes a method for determination of oxolinic acid in salmon muscle tissue. Tissue (0.5-2 g) mixed with 2 g anhydrous sodium sulfate is extracted twice with ethyl acetate, centrifuged, and the extract evaporated. The residue is partitioned in a mixture of hexane and 0.01M oxalic acid and the aqueous phase chromatographed using fluorescence detection at 327 nm excitation and 369 nm emission. Calibration and standard curves are linear from 10-200 ppb and 100-2000 ppb at different sensitivity settings. Recoveries ranged from 71-83% in spiked blanks, with a CV of 4-10.3% over a 2-week period. Preliminary results in treated salmon were variable, possibly because some fish refused to eat medicated feed.     

Determination of benzo(a)pyrene in foods

An analytical method was developed for determining benzo(a)pyrene in foods, suitable for routine use. The method consists of 4 cleanup steps: (1) alkali cleavage of sample, (2) preliminary silica gel column chromatography, (3) selective extraction with concentrated sulfuric acid, and (4) further silica gel column chromatography. Recoveries of benzo(a)pyrene added to 50 g (or 10 g) food at levels of 0.4 ppb (or 2 ppb) ranged from 70% for short-necked clam and mackeral to 85% for chicken meat. The sulfuric acid extraction step affords a simple method for isolating benzo(a)pyrene from various kinds of interfering substances which could not be separated by existing methods.     

A GC/MSD method for the analysis of metolachlor in cabbage,broccoli, and tomato

Abstract

Metolachlor (2‐chloro‐N‐(2‐ethyl‐6‐methylphenyl)‐N‐(2‐meth‐oxy‐1‐methylethyl)acetamide) is being considered for control of eastern black nightshade (Solarium ptycanthum Dun.) in cabbage (Brassica oleracea L. var. capitata), broccoli (B. oleracea L. var. Botrytis) and tomato (Lycopersicon esculentum Mill.). We have developed a rapid, sensitive method to determine metolachlor residues in these commodities to ensure its absence in the edible tissue. The crop material was extracted with methanol, the extract partitioned with hexane, then passed through a silica cartridge and analysed for metolachlor by GC/MSD in selected ion mode at m/z 161. Recovery of metolachlor from fortified plant material was greater than 84%. The method detection limit was calculated to be 0.6 ppb for tomato, 1.1 ppb for cabbage, and 4.2 ppb for broccoli. Metolachlor residues were below the detection limit in all commodities harvested from treatments which received 2 or 3 kg metolachlor/ha.     

High pressure liquid chromatographic determination of the rodenticide brodifacoum in rat tissue

An HPLC method was developed to determine residues of individual isomers of brodifacoum (3-[3-4'-bromo[1,1'-biphenyl]-4-yl)-1, 2, 3, 4-tetrahydro-1-naphthalenyl]-4-hydroxy-2H-1-benzopyran-2-one) in rat tissue. The compound was extracted twice with 10% methanol in chloroform, filtered, and cleaned up by using automated gel permeation chromatography. A final cleanup on a silica gel SEP-PAK was added to protect the analytical column from irreversible adsorption and to reduce analysis time. The analysis was done on a microPorasil column; a UV detector was used for quantitation. Recoveries of brodifacoum added to rat tissue in concentrations of 0.12-5.0 ppm were greater than 90%.     

Rapid immunochemical screening method for aflatoxin B1 in human and animal urine

A method has been developed to determine the presence of aflatoxin B1 in the urine of animals (including humans) by utilizing commercial immunochemical kits that can be used in the field. Urine is treated with diatomaceous earth and filtered to clarify the sample; 2-3 ppb aflatoxin B1, corresponding to about 300 ppb in the ingested feed/food, can be detected in the filtered urine without further purification. To improve sensitivity, the urine filtrate is passed through a C18 solid phase column to extract the aflatoxin. The column is washed with acetonitrile-water (15 + 85) and water, aflatoxin B1 is eluted with methanol-water (7 + 3), and water is added to the eluate, which is then tested for aflatoxin with the test kit. The limit of detection is 0.2 ppb, reflecting consumption of 40 ppb or more aflatoxin in the feed/food. When the initial sample volume is adequate, purification through the C18 column step is usually sufficient. For limited sample volumes, the eluate from the C18 column is mixed with water, added to an immunosorbent affinity column, and washed with water to remove excess sample matrix and impurities. Aflatoxin B1 is eluted with acetonitrile. The extract is evaporated under nitrogen and the residue is redissolved in methanol-water (25 + 75). At this purification stage, the limit of detection is reduced to 0.05 ppb.     

Liquid chromatographic determination of ochratoxin A in coffee beans and coffee products

A liquid chromatographic method for the determination of ochratoxin A in coffee beans (green and roast), instant coffee, and coffee drink is described. The sample is subjected to extraction with methanol-1% aqueous sodium bicarbonate (1 + 1) and C18 cartridge cleanup. The extract is chromatographed on a Nucleosil 5C18 column with a mobile solvent of acetonitrile-water-0.2M phosphate buffer pH 7.5 (50 + 47 + 3) containing 3 mM cetyltrimethylammonium bromide as an ion-pair reagent. Ochratoxin A is detected with a fluorometer (excitation 365 nm, emission 450 nm). The sensitivity was increased 20-fold by using ion-pair resolution. The detection limits corresponded to 2 micrograms/kg for coffee beans, 5 micrograms/kg for instant coffee, and 0.2 microgram/kg for coffee drink. The recoveries from coffee products were generally better than 80.7% and the relative standard deviations were 3.43-5.93%. The peak coinciding with ochratoxin A can be confirmed by treatment using alcohol (methanol, ethanol, or n-propanol) and H2SO4.     

Liquid chromatographic-electrochemical detection screening procedure for six nitro-containing drugs in chicken tissues at low ppb level

A screening procedure is described for the detection of furazolidone, nitrofurazone, aklomide, zoalene, nitromide, and sulfanitran residues in a single extract of chicken liver, breast, or thigh muscle at the low ppb level. The method includes extraction of tissue with chloroformethyl acetate-dimethyl sulfoxide (50 + 50 + 0.8), adsorption on neutral alumina, and subsequent elution of the residues with pH 6.0 phosphate buffer-methanol (1 + 1). Eluants are separated on a 25 cm, 5 microns C18 column with pH 6.0 phosphate buffer-methanol (57.5 + 42.5) as mobile phase. The drugs are detected with an electrochemical detector in the reductive mode at -0.8 V. Mean recoveries from all tissues ranged from 76.5% for nitrofurazone to 97.1% for zoalene.     

Determination of gibberellic acid residues on fruits by synchronous scanning derivative spectrofluorometry

A synchronous derivative spectrofluorometric method is described for the determination of the plant growth regulator, gibberellic acid (GA3). The method is based on the formation of a fluorogen in concentrated sulfuric acid. The reaction is carried out at 85% sulfuric acid and in aqueous medium. The common fluorometric method with a linear dynamic range of 137-400 ppb, and a detection limit of 48 ppb is described. The synchronous first and second derivative method has linear dynamic ranges between 7.6-40 ppb and 12-40 ppb, with detection limits of 3.5 and 6.7 ppb, respectively. The influence of reaction variables and of other plant growth regulators present, and the application to residues on oranges, lemons, and grapes, are also described.     

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.     

Determination of ethylene dibromide in foods and grains by high resolution capillary gas chromatography with electron capture detection

Ethylene dibromide (EDB) levels in food samples were determined by gas chromatography with a high-resolution capillary column and electron capture detector. The capillary column used was 3 mm id X 25 m cross-linked 5% phenylmethyl silicone. Column temperature was set at 40 degrees C by a coolant containing carbon dioxide gas. Optimum temperatures of the injection port and detector were 200 and 350 degrees C, respectively. The detection limit was 0.5 ppb and linear from 1 to 20 pg on the dynamic range. EDB residues in food samples were extracted with n-hexane by steam distillation. A few impurity peaks appeared near EDB on the chromatogram; however, the EDB peak was resolved. Recoveries of EDB from wheat and brown rice ranged from 66.1 to 99.6%. EDB was detected in 3 samples of imported wheat at a range of 0.74-1.70 ppb, and was not detected at all in 37 samples. The EDB remaining in EDB-fortified cookies after baking was examined. The amounts of EDB were reduced to 30 to 50% of the original amounts by kneading the dough, and to below 1.5% by baking.     

Modified method for electron capture gas-liquid chromatographic determination of diethylstilbestrol residues in urine of fattened bulls

A gas-liquid chromatographic (GLC) method with electron capture (EC) detection was developed for determining diethylstilbestrol residues in the urine of fattened bulls. Diethylstilbestrol (DES) is extracted into benzene, and then into 1N sodium hydroxide. The pH of the phenolic fraction (alkaline phase) is adjusted to 10.2 and DES is extracted again into benzene. Sample extracts are cleaned up on silica gel. Trifluoroacetic anhydride (TFAA) is used as acylation reagent, and the derivatized sample is chromatographed on a 3% OV-17 column and measured with a 63Ni EC detector. The method is suitable for determining residues at levels as low as 2 ppb.     

Reverse phase liquid chromatographic determination and confirmation of aflatoxin M1 in cheese

A systematic method is proposed for determination and confirmation of aflatoxin M1 in cheese by liquid chromatography (LC). A sample of cheese is extracted with chloroform, cleaned up on 2 silica gel columns followed by a Sep-Pak C18 cartridge, and chromatographed on a 5 microns octadecyl silica column with fluorometric detection. The sample extract or standard is treated with n-hexane-trifluoroacetic acid (TFA) (4 + 1) for 30 min at 40 degrees C. Analysis by LC with TFA-treatment of the extract provides quantitative data. Multiple assays of 5 samples of Gouda cheese spiked with aflatoxin M1 at levels of 0.5, 0.1, and 0.05 ng/g showed average recoveries of 93.2, 91.6, and 92.4%, with coefficients of variation of 2.63, 3.97, and 4.52%, respectively. Assay of 5 naturally contaminated cheeses resulted in 0.051-0.448 ng/g of aflatoxin M1. Limit of quantitation is about 0.01 ng/g. The identity of aflatoxin M1 is confirmed by treating aflatoxin M1 or the M2a derivative with TFA-methanol (or ethanol) (3 + 1). The TFA-methanol reaction products of M2a could be detected quantitatively.     

Development of multiclass methods for drug residues in eggs: hydrophilic solid-phase extraction cleanup and liquid chromatography/tandem mass spectrometry analysis of tetracycline, fluoroquinolone, sulfonamide, and beta-lactam residues

A method was developed for detection of a variety of polar drug residues in eggs via liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). A total of twenty-nine target analytes from four drug classes-sulfonamides, tetracyclines, fluoroquinolones, and beta-lactams-were extracted from eggs using a hydrophilic-lipophilic balance polymer solid-phase extraction (SPE) cartridge. The extraction technique was developed for use at a target concentration of 100 ng/mL (ppb), and it was applied to eggs containing incurred residues from dosed laying hens. The ESI source was tuned using a single, generic set of tuning parameters, and analytes were separated with a phenyl-bonded silica cartridge column using an LC gradient. In a related study, residues of beta-lactam drugs were not found by LC/MS/MS in eggs from hens dosed orally with beta-lactam drugs. LC/MS/MS performance was evaluated on two generations of ion trap mass spectrometers, and key operational parameters were identified for each instrument. The ion trap acquisition methods could be set up for screening (a single product ion) or confirmation (multiple product ions). The lower limit of detection for screening purposes was 10-50 ppb (sulfonamides), 10-20 ppb (fluoroquinolones), and 10-50 ppb (tetracyclines), depending on the drug, instrument, and acquisition method. Development of this method demonstrates the feasibility of generic SPE, LC, and MS conditions for multiclass LC/MS residue screening.     

Improved gas chromatographic method for quantitation of deoxynivalenol in wheat, corn, and feed

A gas chromatographic (GC) method is described to determine deoxynivalenol in wheat and corn at levels as low as 20 ppb. Ground samples are extracted with water, adsorbed onto a Clin Elut column, extracted with ethyl acetate, and passed through a silica gel Sep-Pak cartridge. The final extract is then derivatized with N-heptafluorobutyrylimidazole and quantitated by GC using an electron capture detector. Recoveries are greater than 85% for spiked samples at levels of 50-1000 ppb. Results for wheat, corn, and mixed feed samples are given as well as the results of an interlaboratory study on a naturally contaminated wheat sample.     

Gas chromatographic determination of mecarbam and its metabolites mecarboxon, diethoate, and diethoxon in cottonseeds

A gas chromatographic method is described for determining residues of mecarbam and 3 of its metabolites, mecarboxon, diethoate, and diethoxon, in cottonseeds. For mecarbam analysis, following Soxhlet extraction with chloroform (after blending), the oily extract is partitioned with propylene carbonate and cleaned up on a silica gel column. Metabolites are extracted by the same method, followed by cleanup of mecarboxon on a silica gel column or diethoxon on an alumina column; cleanup of diethoate can be performed on either column. All 4 compounds are determined using a flame photometric detector equipped with a phosphorus filter. Average recoveries for cottonseed samples fortified with 0.03-1.0 ppm mecarbam ranged from 80 to 88%. Average recoveries were 81-88% for mecarboxon and 90-92% for diethoate (alumina column) and diethoxon from samples fortified with 0.05-1.0 ppm. Average recovery of diethoate from samples cleaned up on the silica gel column were 84-88% in the range of 0.05-0.2 ppm. Values obtained for mecarbam residues in field-treated samples are also presented.