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.     

Multiresidue method for determination of eight neutral beta-lactam penicillins in milk by fluorescence-liquid chromatography

A method of determining total penicillins begins with an enzymatic hydrolysis of the beta-lactam ring to form their respective penicilloate product. Acetonitrile precipitates much of the casein and protein, which are then separated from the liquid by centrifugation. The lipids are removed from the aqueous fraction with methylene chloride. Mercuric chloride is added, which reacts with the penicilloate to liberate the side chain that has a terminal aldehyde. These penilloaldehyde products are extracted with methylene chloride and are subsequently reacted with dansyl hydrazine. The resulting fluorolabeled side chains are separated by liquid chromatography on a C18 column with acetonitrile-water as mobile phase. The fluorescence is measured by the mercury line at 254 nm excitation wavelength and a 500 nm filter on the emission side. The overall average recoveries from milk spiked at 25, 50, and 100 ppb are benzyl penicillin 79.4%; phenoxymethyl penicillin 59.7%; phenethicillin 75.9%; nafcillin 87.7%; methacillin 47.5%; oxacillin 57.6%; cloxicillin 37.3%; and dicloxicillin 26.4%.     

Identification and semiquantitation of monensin sodium in animal feeds by thin layer bioautography

A bioautographic technique for the determination of monensin sodium contamination in animal feeds is described. The feeds are extracted in aqueous methanol and the initial monensin extracts are isolated by filtration through an alumina column. These eluates are partitioned between 5% NaCl and methylene chloride, and are further purified through a Sephadex LH-20 column. A 10 mL eluate containing the monensin is collected from the Sephadex column and evaporated, and the residue is dissolved in methylene chloride. Aliquots are spotted on a thin layer plate and monensin is detected by a thin layer bioautographic technique, using Bacillus subtilis as the test organism. The reliable limit of sensitivity is 100 ppb, but 10 ppb can be detected. This technique can be used to semiquantitate monensin by comparing the zones of inhibition of unknown test samples against monensin standards.     

Determination of alpha-amanitin in serum and liver by multistage linear ion trap mass spectrometry

This paper describes a rapid LC-MS/MS/MS method for the analysis of alpha-amanitin in serum and liver. Serum was initially prepared by precipitation of proteins with acetonitrile and subsequent removal of acetonitrile with methylene chloride. Liver was prepared by homogenization with aqueous acetonitrile and subsequent removal of acetonitrile using methylene chloride. For both matrices, the aqueous phase was then extracted using mixed-mode C18/cation exchange SPE cartridges and analyzed on a linear ion trap LC-MS system. Standards were prepared in extracts of control matrix. Seven replicate fortifications of serum at 0.001 mug/g (1 ng/g) of alpha-amanitin gave a mean recovery of 95% with 8.8% CV (relative standard deviation) and a calculated method detection limit of 0.26 ng/g. Seven replicates of control liver fortified at 1 ng/g gave a mean recovery of 98% with 17% CV and a calculated method detection limit of 0.50 ng/g. This is the first report of a positive mass spectrometric identification and quantitation of alpha-amanitin in serum and liver from suspect human and animal intoxications.     

Determination of oleandrin in tissues and biological fluids by liquid chromatography-electrospray tandem mass spectrometry

A rapid LC-MS/MS method, using a triple-quadrupole/linear ion trap mass spectrometer, was developed for the quantitative determination of oleandrin in serum, urine, and tissue samples. Oleandrin, the major cardiac glycoside of oleander (Nerium oleander L.), was extracted from serum and urine samples with methylene chloride and from tissues with acetonitrile. The tissue extracts were cleaned up using Florisil solid-phase extraction columns. Six replicate fortifications of serum and urine at 0.001 microg/g (1 ppb) oleandrin gave average recoveries of 97% with 5% CV (relative standard deviation) and 107% with 7% CV, respectively. Six replicate fortifications of liver at 0.005 microg/g (5 ppb) oleandrin gave average recoveries of 98% with 6% CV. This is the first report of a positive mass spectrometric identification and quantitation of oleandrin in tissue samples from oleander intoxication cases. The sensitivity and specificity of the LC-MS/MS analysis enables it to be the method of choice for toxicological investigations of oleander poisoning.     

Rapid liquid chromatographic determination of dimetridazole and ipronidazole in swine feed

A simple and rapid method is described for the determination of dimetridazole (DMZ) and ipronidazole (IPR) in swine feeds at various levels (0.11-110 ppm). The drugs are released from feed by prewetting with a buffer, followed by extraction with either methanol or methylene chloride, depending on the drug level; if necessary, an acid-base cleanup is used before the liquid chromatographic analysis. The analytes are separated on a C18 column and monitored at 320 nm for detection and quantitation. Recoveries of DMZ from several feed formulations averaged 108% at the 92.8 ppm level with a standard deviation (SD) of 4.00% and a coefficient of variation (CV) of 3.70%, 101% at the 11.2 ppm level with an SD of 11.9% and a CV of 11.8%, and 100% at the 0.112 ppm level with an SD of 9.27% and a CV of 9.25%. Recoveries of IPR averaged 77.1% at the 12.9 ppm level with an SD of 1.75% and a CV of 2.27%; IPR recoveries averaged 35.2% at the 0.129 ppm level with an SD of 3.39% and a CV of 9.63%.     

Determination of acidic herbicides and related compounds in water and soil by capillary gas chromatography using a nitrogen-phosphorus detector

Methods are described for the determination of acidic herbicides and related compounds in water and soil. Eight acidic herbicides and related compounds were extracted from water using either dichloromethane or an XAD-2 resin column. The acidic moieties were derivatized with 2-cyanoethyldimethyl(diethyl)aminosilane. The derivatized compounds were separated using capillary gas chromatography and quantitated using a nitrogen-phosphorus detector. Extraction from water using dichloromethane or an XAD-2 resin column resulted in recoveries greater than 90% at 0.1 ppb with an average coefficient of variation (CV) of 6%. In soils extracted with aqueous acetonitrile-acetic acid and partitioned into dichloromethane, recoveries at 500 ppb were greater than 75% with an average CV of 3.3%. The methods are rapid and there are few interferences.     

Validation study of gas chromatographic determination of pentachlorophenol in animal liver

A validation study was conducted of a gas chromatographic procedure for the determination of pentachlorophenol (PCP) in chicken, pork, and beef liver. Five analysts representing 5 laboratories analyzed randomly numbered blind duplicates at 3 fortified tissue concentrations and one incurred tissue on 2 consecutive days. The PCP concentrations ranged from approximately 40 to 400 parts per billion (ppb). All data were reported to 3 significant figures in ppb. The coefficients of variation for repeatability were between 2.8 and 8.5%, except for the beef liver, at a mean value of 80 ppb PCP, where the CV was 11.3%. The CVs for reproducibility were in the range of 9.7-16.5% with little significant difference by species. The CV asymptotically approached 10% as the PCP level increased.     

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.     

Determination of flunixin in edible bovine tissues using liquid chromatography coupled with tandem mass spectrometry

An accurate, reliable, and reproducible assay was developed and validated to determine flunixin in bovine liver, kidney, muscle, and fat. The overall recovery and percent coefficient of variation (%CV) of twenty-eight determinations in each tissue for flunixin free acid were 85.9% (5.9% CV) for liver, 94.6% (9.9% CV) for kidney, 87.4% (4.7% CV) for muscle, and 87.6% (4.4% CV) for fat. The theoretical limit of detection was 0.1 microg/kg (ppb, ng/g) for liver and kidney, and 0.2 ppb for muscle and fat. The theoretical limit of quantitation was 0.3, 0.2, 0.6, and 0.4 ppb for liver, kidney, muscle, and fat, respectively. The validated lower limit of quantitation was 1 ppb for edible tissues with the upper limit of 400 ppb for liver and kidney, 100 ppb for fat, and 40 ppb for muscle. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. Briefly, the method involves an initial acid hydrolysis, followed by pH adjustment ( approximately 9.5) and partitioning with ethyl acetate. A portion of the ethyl acetate extract was purified by solid-phase extraction using a strong cation exchange cartridge. The eluate was then evaporated to dryness, reconstituted, and analyzed using LC/MS/MS. The validated method is sensitive and specific for flunixin in edible bovine tissue.     

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 N-methylcarbamate pesticides in liver by liquid chromatography

A liquid chromatographic (LC) method using a 2-step purification technique for the simultaneous determination of 10 carbamates in bovine, swine, and duck livers has been developed. Carbamates are extracted from liver samples with methylene chloride. After evaporation, the residues from the extract are dissolved in methylene chloride-cyclohexane (1 + 1) and cleaned up by gel permeation chromatography. The eluate containing carbamate residues is evaporated to dryness, reconstituted in methylene chloride, further purified by passing it through an aminopropyl Bond Elut extraction cartridge, and analyzed by liquid chromatography using post-column derivatization with orthophthalaldehyde and fluorescence detection. Excitation and emission are set at 340 and 418 nm, respectively. Liver samples for beef, pork, and duck were fortified with 5, 10, and 20 ppb of mixed carbamate standards. The average of 10 recoveries of 10 carbamates at all 3 levels of fortification was greater than 80% with coefficients of variation less than 17%.     

Liquid chromatographic determination of vitamins D2 and D3 in fortified milk and infant formulas

A liquid chromatographic (LC) method was developed for determining vitamins D2 and D3 in fortified milk and infant formulas. The lipid-soluble components were extracted from the aqueous phase by homogenizing in isopropanol-methylene chloride with magnesium sulfate added to remove water. The vitamins were fractionated from the lipid material by using gel permeation chromatography (GPC) followed by further cleanup of the combined GPC fractions on a muBondapak/NH2 column. Four muStyragel (100 A) columns connected in series were used for GPC fractionation of sample extracts in methylene chloride. Injection and collection were repeated 3 times to collect enough vitamin D for quantitation. The muBondapak/NH2 column, using a mobile phase of methylene chloride-isooctane-isopropanol (600 + 400 + 1), resolved vitamin D from other UV-absorbing compounds and soy sterols in infant formula and from cholesterol in milk. Vitamins D2 and D3 coeluted as one peak, with the resolution and vitamin level sufficient for visual monitoring (280 nm/0.02 absorbance unit full scale) in a collection time of 22-26 min. A Zorbax ODS (6 micron) column and a methylene chloride-acetonitrile-methanol (300 + 700 + 2) mobile phase were used for LC quantitation; vitamins D2 and D3 were baseline resolved in about 11 min. The infant formula samples included ready-to-use and concentrated liquids prepared in nonfat milk base or soy base fortified with vitamins D2 or D3 at 400 IU/qt or L (10 micrograms). The mean percent recovery of added vitamin D3 (400-500 IU/qt) from infant formula (n = 7) was 89.6 +/- 6.7 (coefficient of variation (CV) 7.5%).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Capillary column gas chromatographic determination of ethyl carbamate in alcoholic beverages with confirmation by gas chromatography/mass spectrometry

A method is described for determining ethyl carbamate at low microgram/kg levels in several types of alcoholic beverages by capillary column gas chromatography with Hall electrolytic conductivity detection and confirmation by mass spectrometry. Samples are diluted to obtain a uniform concentration of ethanol (ca 10%) then saturated with NaCl and extracted with methylene chloride. Extracts are evaporated to a small volume and injected in ethyl acetate solution for chromatographic analysis. The method was evaluated by 5 laboratories, 4 employing the Hall detector and one using mass spectrometric detection. Overall between-laboratory mean percent recoveries were: wine, 85.3 +/- 21.0% coefficient of variation (CV) (spiking level 20-45 micrograms/kg); sherry, 83.8 +/- 16.1% CV (spiking level, 81-142 micrograms/kg); whiskey, 79.5 +/- 13.9% CV (spiking level 127-190 micrograms/kg); and brandy, 85.0 +/- 12.5% CV (spiking level 297-446 micrograms/kg). Mass spectrometric results agreed well with the Hall results for all commodities. Detection limits were about 5 micrograms/kg for the Hall detector and about 0.5 microgram/kg for mass spectrometric detection.     

Beta-cyclodextrin post-column fluorescence enhancement of aflatoxins for reverse-phase liquid chromatographic determination in corn

beta-Cyclodextrin enhances the fluorescence of aflatoxins B1 and G1 in aqueous systems. This effect was utilized in developing a unique reverse-phase liquid chromatographic (LC) method for determination of aflatoxins B1, B2, G1, and G2 (B1 detection limit 1 ppb), without preparing derivatives of B1 and G1. The aflatoxins are dissolved in methanol or the mobile phase for injection onto the LC system. Using a mobile phase of methanol-beta-cyclodextrin (1 + 1), the aflatoxins are resolved on a C18 column. Fluorescence of the aflatoxins is enhanced by post-column introduction of an aqueous concentrated beta-cyclodextrin solution. All 4 aflatoxins elute within 10 min in the order G2, G1, B2, B1. Fluorescence responses for B1 and G1 standards were linear over the concentration range 0.5-10 ng, yielding correlation coefficients (r) of 0.9989 and 1.000, respectively. The average peak response ratio for G1:B1 for the mobile phase-enhancement solution described was 0.765 with a coefficient of variation (CV) of 0.98%. CVs were 6.2, 9.0, and 7.5% for multiple assays of aflatoxin B1 in 3 samples of naturally contaminated corn. For samples of corn spiked to a total B1 content of 8.3 ng/g, average B1 recovery was 90% (CV 11.7%).     

Rapid determination of fumigant and industrial chemical residues in food

A gas chromatographic (GC) method is described for the determination of 22 fumigant and industrial chemical residues in a variety of foods. The fumigants and industrial chemicals determined are methyl bromide, methylene chloride, carbon disulfide, chloroform, 1,1-dichloroethane, ethylene dichloride, methyl chloroform, carbon tetrachloride, methylene bromide, propylene dichloride, 2,3-dichloropropene, trichloroethylene, 1,3-dichloropropylene, 1,1,2-trichloroethane, chloropicrin, ethylene dibromide, tetrachloroethylene, propylene dibromide, 1,1,2,2-tetrachloroethane, p-dichlorobenzene, o-dichlorobenzene, and 1,2-dibromo-3-chloropropane. Except for the latter three, the fumigants are determined at 90 degrees C on 3.6 m 20% loaded OV-101 columns with electron-capture and Hall-electroconductivity detectors. The other 3 compounds (o-dichlorobenzene, p-dichlorobenzene, and 1,2-dibromo-3-chloropropane), which elute beyond 30 min on the above columns, are determined at 90 degrees C on 1.8 m 5% loaded OV-101 columns with the same detectors. The ng/g-level fortifications have an overall mean analyte recovery of 70% and a coefficient of variation of 40%. The variety of foods examined includes both fatty and nonfatty food types (e.g., off-the-shelf cooked and uncooked grain-based items, dairy products, fresh and canned fruits and vegetables, and meats). Samples are extracted and cleaned up according to fat content and food type. Samples containing less than 71% fat are extracted by using an aqueous: nonaqueous shakeout (20% acetone solution under isooctane). Most extracts (isooctanes) are analyzed directly. Extracts from samples containing from 21 to 70% fat (e.g., ground beef, pecans, and corn chips) are cleaned up further on micro-Florisil columns to remove excess fat. A few other samples containing more than 71% fat or oil (e.g., butter, salad dressing, and vegetable oil) are diluted directly in isooctane and, depending on the degree of dilution, can be cleaned up further on micro-Florisil columns. Also, clear beverages (e.g., soda and tea) are extracted directly with isooctane. These extraction and cleanup techniques were tested on 231 different table-ready foods. Three-hundred incurred residues of 10 different fumigants were found in 138 items examined; 93 items had no detectable residues. The main advantage of the method is rapid semiquantitative determination of multiple fumigants from all food types.     

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.     

Multiresidue assay for benzimidazole anthelmintics by liquid chromatography and confirmation by gas chromatography/selected-ion monitoring electron impact mass spectrometry

A multiresidue method utilizing all-disposable labware has been developed for 8 benzimidazole anthelmintics from ovine, bovine, and swine muscle and liver tissues. After an initial extraction with ethyl acetate and subsequent evaporation, a 3-component extraction using hexane, ethanol, and 0.2N HCl was used for final cleanup. Clean extracts were produced for separation and determination by reverse-phase liquid chromatography at 298 nm, using methanol and aqueous buffer as mobile phase. A synthesized internal standard, 2-(n-butylmercapto)benzimidazole, was used for quantitation of all drugs. Results are included along with statistical information verifying the performance of the method. Spiked control tissues and incurred drug tissues were used for an intralaboratory study with a concentration range of 50-1470 ppb. A series of standard curves at 0, 50, 100, and 200 ppb were analyzed. Overall recovery at the 100 ppb level averaged 92% (CV 8%) in liver tissues, across all 3 species and 88% (CV 5%) in muscle tissues across all 3 species. Results were confirmed by gas chromatography/mass spectrometry with acid hydrolysis of the remaining extract in 2N HCl followed by re-extraction of the amine and derivatization to the tert-butyldimethylsilyl derivative. The anthelmintics were identified by gas chromatography/selected ion monitoring electron-impact mass spectrometry. Ion ratio measurements were taken and compared to standard material. CVs averaged 10% or less for all drugs tested.     

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.