Liquid chromatographic determination and identification of morantel-related residues as precursors of 3-(3-methyl-2-thienyl) acrylic acid (CP-20,107) in bovine milk

A simple liquid chromatographic (LC) method was developed to determine and identify incurred morantel-related residues in bovine milk by converting them to 3-(3-methyl-2-thienyl) acrylic acid (CP-20, 107). Key techniques in this method involve short-term digestion of milk in HCl to release residues convertible to CP-20, 107, isolation and alkaline hydrolysis of these precursors to CP-20, 107, and recovery of the product for LC analysis. Photochemical conversion of CP-20, 107 to its cis-isomer and separation by LC identifies the residue. A homolog (pyrantel), which is used as an internal standard, is hydrolyzed to 3-(2-thienyl) acrylic acid. These acrylic acid isomers are readily resolved by LC. The method was evaluated over the 1-4 ppb (ng/mL) range for accuracy and precision to assess its utility for withdrawal studies. Bovine milk supplemented with morantel at 1, 2, and 4 ppb and assayed in replicate (n = 7-8) over 4 trials gave mean values and standard deviations of 1.0 +/- 0.11, 2.0 +/- 0.24, and 4.0 +/- 0.44 ppb, respectively. A milk specimen containing physiologically incurred residues of morantel assayed 2.1 +/- 0.19 ppb in replicate (n = 5).     

Determination of depletion and statistical distribution of morantel-related residues in bovine milk following administration of morantel tartrate to dairy cows

Residue depletion studies were conducted in dairy cattle to monitor morantel-related residues in milk following oral administration of morantel tartrate (Rumate. Eleven lactating cows of various ages, periods of lactation, and known milk production were orally dosed with the bolus formulation of morantel tartrate with an actual dose range of 8.4-9.8 mg/kg body weight. Representative samples of milk were collected at 10-14 h intervals post-dose, and subsamples were assayed for the major and minor hydrolysis products of morantel-related residues, MAPA and CP-20,107. Residues assayed as precursors of MAPA peaked at the second milking (24 h post-dose) and were below 25 ppb (range: less than 12-24 ppb). Precursors of CP-20,107, which confirm the identity of morantel, also peaked at 24 h post-dose (range: 2.1-3.3 ppb) and declined rapidly thereafter. A statistical model was used to project the level of residues at the upper limit of 99% of the total target animal (i.e., dairy cattle) population with 95% confidence. The calculated peak levels from this model were 50 and 5.0 ppb for morantel-related residues convertible to MAPA and CP-20,107, respectively.     

Liquid chromatographic determination of sulfamethazine in milk

A simple, relatively rapid liquid chromatographic method has been developed for the determination of sulfamethazine (SMZ) in milk at levels in the low ppb range. The method is based on extracting SMZ from milk with chloroform, evaporating the chloroform, dissolving the residues in hexane, extracting into buffers, and chromatographing the buffer solution. The method has been shown to determine levels as low as 5 ppb reliably. Levels greater than or equal to 7 ppb have been confirmed by gas chromatography/mass spectrometry after derivatization of extracts from fortified, incurred, and shelf milk. Intralaboratory recoveries and percent coefficients of variation are satisfactory. Sulfadimethoxine and sulfaquinoxaline can also be determined by the method. Application of the method to other dairy products is being investigated.     

Multiresidue screen for organophosphorus insecticides using gel permeation chromatography--silica gel cleanup.

A multiresidue screen for quantitative determination of 43 organophosphorus insecticides in 5 g of plant and animal tissues is described. The organophosphorus insecticides are extracted with methanol-dichloromethane (10 + 90, v/v) and cleaned up using automated gel permeation chromatography with hexane-ethyl acetate (60 + 40) eluant and in-line silica gel minicolumns. Concentrated extracts are analyzed by gas chromatography with flame photometric detection. The method recovers 43 organophosphorus insecticides in the range of 72 to 115%. Analysis of fortified bovine liver (n = 5) shows an average 95.9 +/- 4.8% recovery at the 0.05 micrograms/g level and 93 +/- 3.8% at the 0.5 micrograms/g level. Analysis of fortified bovine rumen content (n = 5) shows an average 98 +/- 4.2% recovery at the 0.1 micrograms/g level and 98.7 +/- 2.8% at the 1 micrograms/g level. Method detection limits ranged from 0.01 to 0.05 micrograms/g for the compounds studied using a nominal 5 gram sample.     

Residue depletion study and withdrawal period for flunixin-N-methyl glucamine in bovine milk following intravenous administration

The objective of this study was to establish a withdrawal period for flunixin in milk by quantifying 5-hydroxyflunixin, the marker residue, in bovine milk as a function of time, following intravenous treatment of lactating dairy cows with flunixin-N-methyl glucamine (Banamine or Finadyne). Lactating dairy cows were dosed on three consecutive days at 2.2 mg of flunixin free acid/kg of body weight/day. Milk was collected twice daily and assayed using a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) procedure. The method was validated at concentrations in the range 0.5-250 ppb. The concentrations for 5-hydroxyflunixin measured 12 h after the last administration of drug ranged from 1.56 to 40.6 ppb for all cows. Milk concentrations for 5-hydroxyflunixin were used to establish withdrawal periods of 36 h using guidelines established by the U.S. Food and Drug Administration/Center for Veterinary Medicine and 24 h using guidelines established by the European Medicinal Evaluation Agency/Committee on Veterinary Medicinal Products.     

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).     

Direct competitive enzyme-linked immunosorbent assay for sulfamethazine residues in milk

A direct competitive enzyme-linked immunosorbent assay (ELISA) is described for the detection and estimation of sulfamethazine residues in milk. Samples are cleaned up rapidly by acidifying and centrifuging the milk, adjusting the supernatant liquid to pH 7.0, and centrifuging again. The supernate is then assayed using set points to estimate sulfamethazine levels in the sample in the range of 1 ppb to 1 ppm. Multiple samples of milk can be screened in 1.5-2 h by this ELISA method.     

Simple and rapid liquid chromatography-tandem mass spectrometry confirmatory assay for determining amoxicillin and ampicillin in bovine tissues and milk

A simple specific and rapid confirmatory method for determining the two amphoteric penicillins, that is, amoxicillin and ampicillin, in bovine muscle, liver, kidney, and milk is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry. With this instrumentation, the selected reaction monitoring acquisition mode with two fragmentation reactions for each analyte was adopted. After acidification and filtration of the aqueous extracts, 25 microL of the tissue final extracts and 50 microL of the milk final extract were injected into the LC apparatus. Absolute recovery of the two analytes in any biological matrix at the 50 ppb level in tissues and the 4 ppb level in milk was 74-95% with relative standard deviations (RSDs) of no larger than 9%. When penicillin V was used as surrogate internal standard, relative recovery of the targeted compounds present in bovine tissues and milk at, respectively, 25 and 2 ppb levels ranged between 100 and 106% with RSDs of no larger than 11%. When fractionation of analytes by using a short chromatographic run was attempted, remarkable signal weakening for the two analytes was experienced. This effect was traced to polar endogenous coextractives eluted in the first part of the chromatographic run that interfered with the gas-phase ion formation of the two penicillins. Slowing the chromatographic run eliminated this unwelcome effect. Limits of quantification of the two analytes in bovine milk were estimated to be <1 ppb, whereas amoxicillin and ampicillin could be quantified in bovine tissues down to 3.1 and 0.8 ppb levels, respectively.     

Gas chromatographic determination of systemic fungicide tricyclazole in soil and water

A method is described for determination of tricyclazole residues in soil and water. Tricyclazole is extracted from soil by refluxing with ethyl acetate-acetone (80 + 20 v/v) and from water by partitioning into dichloromethane. The soil extract is purified by coagulation. The compound is detected and measured by gas chromatography using a flame photometer operated in the sulfur mode. Detection limits are 8 ppb for soil and 0.8 ppb for water. Recoveries for control samples fortified with tricyclazole at 0.1-5.0 ppm averaged 97.1% for soil and 108.1% for water.     

Rapid gas chromatographic method for determining ethyl carbamate in alcoholic beverages with thermal energy analyzer detection

A rapid column elution method has been developed for the determination of ethyl carbamate (EC) in alcoholic beverages. The beverage is mixed with Celite and packed in a column containing deactivated alumina capped with a layer of sodium sulfate. EC is then eluted with methylene chloride. The method, using a gas chromatograph-thermal energy analyzer with a nitrogen converter for detection and quantitation of EC, has been applied to a variety of alcoholic beverages. Recoveries +/- standard deviations of EC in wine and whisky fortified at the 20 and 133 micrograms/kg (ppb) levels averaged 87.3 +/- 5.3 and 88.7 +/- 3.6%, respectively. The method has a limit of detection of 1.5 ppb. Gas chromatography/mass spectrometry/mass spectrometry was used to confirm the identity and quantitation of EC in selected beverage extracts.     

Gas chromatographic determination of ethylene dibromide in flour products

A method based on gas chromatography with electron capture detection was developed for the determination of ethylene dibromide (EDB) extracted from flour products. The procedure relies on the organic extraction of flour/water mixtures and uses an internal standard, 1-bromo-3-chloropropane. Recoveries of EDB at 10 and 100 ppb were 80.1 +/- 2.8% (SD) and 84.4 +/- 4.3%, respectively; recovery of the internal standard at the working concentration 500 ppb was 98.3 +/- 6.7%. Calibration curves were linear over the range 5-400 ppb, with a mean overall coefficient of variation of less than 5%. The reliability of the procedure was assessed by using gas chromatography combined with mass spectrometry. Results are shown for determination of EDB in locally milled flour products.     

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.     

Gas chromatographic method for ethylene dibromide in grains and grain-based products: collaborative study

Nine laboratories analyzed samples of whole grain, intermediate, and ready-to-eat products for ethylene dibromide (EDB) residues. Supplied samples of wheat, rice, and flour contained both fortified and incurred EDB; corn bread mix, baby cereal, and bread contained only fortified EDB. The whole grains and intermediates were analyzed by the same basic procedural steps as in the official method for multifumigants: They were extracted by soaking in acetone-water (5 + 1). The baby cereal and bread were analyzed by a modification of the Rains and Holder hexane co-distillation procedure. EDB was determined by electron capture gas chromatography operated with an SP-1000 column. All products contained 3 different levels of EDB and were analyzed as blind duplicates. Overall mean recoveries ranged from 85.2% for 69.6 ppb to 105.0% for 4.35 ppb, both in baby cereal. Interlaboratory relative standard deviations ranged from 5.7% for 869 ppb in wheat to 20.2% for 69.6 ppb in baby cereal, both fortified. Mean levels of incurred EDB in wheat, rice, and flour were 926.7, 982.0, and 49.9 ppb, respectively; corresponding relative standard deviations were 9.9, 7.7, and 13.1%. The method was adopted official first action.     

Analysis of fenbendazole residues in bovine milk by ELISA

Fenbendazole residues in bovine milk were analyzed by ELISAs using two monoclonal antibodies. One monoclonal antibody (MAb 587) bound the major benzimidazole anthelmintic drugs, including fenbendazole, oxfendazole, and fenbendazole sulfone. The other (MAb 591) was more specific for fenbendazole, with 13% cross-reactivity with the sulfone and no significant binding to the sulfoxide metabolite. The limit of detection of the ELISA method in the milk matrix was 7 ppb for MAb 587 and 3 ppb for MAb 591. Fenbendazole was administered in feed, drench, and paste form to three groups of dairy cattle. Milk was collected immediately before dosing and then every 12 h for 5 days. The ELISA indicated that residue levels varied widely among individual cows in each group. Fenbendazole levels peaked at approximately 12-24 h and declined rapidly thereafter. Metabolites were detected at much higher levels than the parent compound, peaked at approximately 24-36 h, and declined gradually. Residue levels were undetectable by 72 h. The ELISA data correlated well with the total residues determined by chromatographic analysis, but the use of the two separate ELISAs did not afford an advantage over ELISA with the single, broadly reactive MAb 587. The ELISA method could be used to flag high-residue samples in on-site monitoring of fenbendazole in milk and is a potential tool for studying drug pharmacokinetics.     

Simultaneous determination of oxytetracycline, tetracycline, and chlortetracycline in milk by liquid chromatography

A simple, rapid procedure is described for the simultaneous quantification of 3 tetracycline drugs in bovine milk. Samples are prepared by dilution with an EDTA/phosphate buffer solution and filtration through a molecular weight cutoff filter. Analytes are concentrated on-column using a reverse-phase gradient elution system of oxalic acid, acetonitrile, and methanol. The limits of quantitation are approximately 15-50 ng/mL and the limits of detection are 10-20 ng/mL, depending on the compound. For oxytetracycline, over the range 50-1200 ng/mL, the average recovery and intralaboratory coefficient of variation were 97% and 4.1%, respectively. Over the same range, these parameters were, respectively, 97% and 5.0% for tetracycline, and 89% and 6.4% for chlortetracycline. The applicability of this procedure is demonstrated by separation and detection of incurred tetracycline residues in milk from treated animals.     

High pressure liquid chromatographic determination of furazolidone in turkey tissue.

A high pressure liquid chromatographic method for determining furazolidone in turkey tissue has been developed. Tissues are ground with methanol and centrifuged. For lower levels of furazolidone, 2--40 ppb, the supernate is evaporated to dryness and redissolved before it is injected onto the liquid chromatographic column. Using a reverse phase column and an ultraviolet absorption detector set at 365 nm, the assay is linear over the concentration range 2--400 ppb with a coefficient of variation of less than 4%. Average recovery from fortified tissues was 96% with a coefficient of variation of 6% at the 50--400 ppb level, and 105% with a coefficient of variation of 11% at the 2--40 ppb level.     

Confirmation of phorate, terbufos, and their sulfoxides and sulfones in water by capillary gas chromatography/chemical ionization mass spectrometry

A gas chromatographic/mass spectrometric method capable of confirming phorate, terbufos, their sulfoxides, and sulfones in water is reported. Parents and their metabolites are separated in less than 5 min using a short capillary GC column and high carrier gas linear velocities. Positive ion chemical ionization mass spectrometry generates (M + H) ions indicative of the different molecular weights of the analytes and at least one confirmatory fragment ion for each analyte. Residues have been qualitatively confirmed at the 1 ppb level in fortified water samples from a variety of sources. Apparent residues in control water were less than 0.1 ppb.     

Liquid chromatographic determination of multiple sulfonamide residues in bovine milk

A liquid chromatographic method has been developed for simultaneous determination of residues of 10 sulfonamide drugs at 10 ppb and above in raw bovine milk. The method is based on a chloroform-acetone extraction, evaporation of organic phase, dissolution of residues in an aqueous potassium phosphate solution, and extraction of fatty residue into hexane. The aqueous layer is collected, filtered, injected onto an LC system, and detected by ultraviolet absorption at 265 nm. To elute all 10 sulfonamides isocratically, 2 chromatographic conditions are required. Seven sulfonamides can be quantitated with 12% methanol in the mobile phase; 4 sulfonamides can be quantitated with 30% methanol. Sulfamethazine, the most widely used sulfonamide, is detected on both systems. Recoveries are 44-87% for individual sulfonamides, with only 2 below 60%. Coefficients of variation are 3-13% at 10 ppb.     

A screening method for determining nitrofuran drug residues in animal tissues.

A method was developed for measuring low levels of total nitrofurans in animal tissues and milk. The antimicrobial nitrofurans (5 or more products) used in agriculture are extracted from tissue with aqueous acid in the presence of ethyl acetate. After centrifugation and evaporation, the organic residue is washed with hexane and the nitrofurans are hydrolyzed to 5-nitrofuraldehyde in aqueous acid at 70 degrees C. The hydrolysis product is extracted with benzene and measured by gas-liquid chromatography with electron capture detection. Recoveries of nitrofurazone and furazolidone from fortified poultry and swine tissues at the levels of 0.5 and 0.1 ppm are 75 and 65%, respectively. This procedure can be used to detect the total nitrofuran content of as little as 10 ppb muscle tissues and milk, 100 ppb liver, and 50 ppb fat with no interference from related veterinary nitrodrugs.     

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.