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 ochratoxin A in animal feed and cereal grains by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method is described for determination of ochratoxin A in animal feeds and cereal grains. Samples are initially extracted with chloroform-ethanol (8 + 2) and 5% acetic acid in water. Extracts are purified using a silica gel cartridge followed by a cyano cartridge. The samples are evaporated, diluted to a known volume, and analyzed using a 10 cm column of 3 micron C18 and a fluorescence detector. The method was applied to a variety of animal feeds and cereal grains at levels of 1.0-0.005 ppm added ochratoxin A. The overall recovery was 90.6% +/- 3.6.     

High-speed liquid chromatographic determination of monensin, narasin, and salinomycin in feeds, using post-column derivatization

A high-speed liquid chromatographic (LC) method using post-column derivatization is described for the determination of monensin, narasin, and salinomycin in a variety of animal feeds. The ionophores are extracted with hexane-ethyl acetate (90 + 10). A portion of the sample is evaporated, diluted to a known volume, and analyzed using a 6 cm 3 microns C18 column and an absorbance detector after post-column reaction with vanillin. The method has been applied to poultry and swine feeds with levels of 3-100 ppm added antibiotic. A comparison was also carried out with medicated poultry feed and beef feed lot supplement samples previously analyzed by 2 separate bioassay methods for monensin and salinomycin, respectively. Recoveries for the LC method ranged from 92.1 to 103% with an average recovery of 98.1% and a coefficient of variation of 3.65%.     

Liquid chromatographic determination of tetracycline residues in animal feeds

A liquid chromatographic method for the multiresidue determination of tetracyclines (TCs) in feeds is described. The levels of quantitation were 10 ppm each for tetracycline-HCl (TC), oxytetracycline (OTC), and chlortetracycline-HCl (CTC); the detection limit was 40 ppb for each. The calibration curves were linear between 2.5 and 100 ppm. The procedure involved double extraction with pH 2.0 and pH 4.5 McIlvain buffers, cleanup on a Sephadex LH-20 column, separation on a Nova-Pak C18 column, and detection at 370 nm. Recoveries of 10 micrograms/g of each TC in multiresidue feed samples ranged from 55.8 to 75.5% for OTC, 71.6 to 100% for TC, and 22.4 to 60.6% for CTC. The identities of the TCs were confirmed by thin layer chromatography.     

Improved turbidimetric assay of tylosin in premix and animal feeds not containing urea

A turbidimetric method is described for the determination of tylosin in premix and animal feeds not containing urea. This method includes several modifications of existing tylosin turbidimetric and AOAC plate assays to remove interferences from the feed, to concentrate low levels of tylosin, and to reduce the variability of the assay results. An acidic alumina column cleanup step has been incorporated into the method to remove interferences from feed ingredients. A disposable C18 column was used to concentrate the tylosin from low-level feeds, and the use of a larger analytical sample size has decreased the variability of the assay results. Average recoveries of tylosin added to chicken and swine rations were 98 and 101%, respectively.     

Liquid chromatographic determination of oxfendazole in swine feeds

A relatively simple analytical method is presented for determination of oxfendazole (2-(methoxycarbonylamino)-5-phenylsulfinyl-benzimidazole) at levels as low as 0.012% in swine feeds, using cation exchange liquid chromatography (LC). The sample was extracted with a solvent mixture of methanol-glacial acetic acid (90 + 10) at 45 degrees C, using a gyrorotory shaker. Plant pigments and other feed excipients were removed using zinc acetate treatment and pH-controlled extraction. Oxfendazole was further separated from the remaining interferences and quantitatively determined by LC on a Partisil SCX column with acetonitrile-0.01M phosphate buffer as mobile phase. The method is stability-specific, linear, precise, and accurate at 80-120% labeled strength (relative standard deviation 0.9-1.7 with mean recovery of 98-99%). Supporting data at a level of 0.0135% oxfendazole in swine feed indicated that this method is capable of complete recovery of oxfendazole from medicated swine feeds.     

Reverse phase liquid chromatographic determination of bisacodyl in dosage forms

A method is described for the determination of bisacodyl in enteric-coated tablets and suppositories by liquid chromatography (LC). The method will also determine the hydrolysis degradation products monoacetylbisacodyl and desacetylbisacodyl. The sample is dissolved in 2-propanol, and the extract is diluted with the mobile phase and injected into a liquid chromatograph fitted with a mu Bondapak C18 column and an ultraviolet detector set at 254 nm. The column is eluted with methanol-acetonitrile-0.01M citric acid (25 + 25 + 50). The pooled mean recovery value for bisacodyl from commercial enteric-coated tablets and suppositories was 99.7% with a pooled coefficient of variation (CV) of 0.72%. For content uniformity assays, the CVs were 0.7 and 1.0% for groups of 10 individual commercial suppositories and tablets, respectively. Differences between assay values by the LC and USP XX methods were 0.2% of declared for enteric-coated tablets (n = 5) and 1.0% of declared for suppositories (n = 2). The LC method can determine as little as 0.015 microgram of the monoacetyl or desacetyl degradation product.     

Liquid chromatographic determination of flucytosine in capsules: collaborative study

A liquid chromatographic method for the determination of flucytosine in capsules was collaboratively studied by 7 laboratories. The method uses a C18 reverse phase column, water-methanol-acetic acid mobile phase containing 1-octanesulfonic acid sodium salt, p-aminobenzoic acid as internal standard, and photometric detection at 285 nm. The mean recovery value (+/- SD) of flucytosine from a synthetic formulation representing capsules was 99.2 +/- 1.72% (CV = 1.73%). Composited samples of 250 and 500 mg commercial capsules gave assay values of (mean +/- SD) 103.17 +/- 2.21 and 99.29 +/- 1.29% of declared, respectively. CV values were 2.15 and 1.30%. Reproducibility and repeatability CVs were 2.19 and 1.50%, respectively, for the 250 mg capsules, and 1.34 and 0.63%, respectively, for the 500 mg capsules. The method has been adopted official first action.     

Matrix solid phase dispersion (MSPD) isolation and liquid chromatographic determination of furazolidone in pork muscle tissue

A method for the isolation and liquid chromatographic (LC) determination of furazolidone in pork muscle tissue is presented. Blank or furazolidone-fortified pork muscle tissue samples (0.5 g) were blended with octadecylsilyl (C18, 18% load, endcapped, 2 g) derivatized silica. A column made from C18/pork matrix was first washed with hexane (8 mL), followed by elution of furazolidone with ethyl acetate. The ethyl acetate extract was then passed through an activated alumina column. The eluate contained furazolidone that was free from interfering compounds when analyzed by LC with UV detection (photodiode array, 365 nm). Detector response with increasing concentrations of furazolidone isolated from fortified samples was linear (r = 0.998 +/- 0.002) with an average percentage recovery of 89.5 +/- 8.1% for the concentration range (7.8-250 ng/g) examined and resulted in a minimum detectable limit of 390 pg on column, and a detector response of more than 5 times baseline noise. The inter-assay variability was 9.9 +/- 5.4% with an intra-assay variability of 1.5%.     

Application of a tandem mass spectrometer and core-shell particle column for the determination of 151 pesticides in grains

A comparison of ultrahigh performance liquid chromatography (UHPLC) with a 2.6 μm core-shell particle column (Kinetex C(18)) and conventional liquid chromatography (LC) with a 3 μm porous particle column (Atlantis dC(18)), coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS), for the determination of 151 pesticides in grains is presented in this study. Pesticides were extracted from grain samples using a procedure known as QuEChERS (quick, easy, cheap, effective, rugged, and safe). Quantification, with an analytical range from 5 to 500 μg/kg, was achieved using matrix-matched standard calibration curves with isotopically labeled standards or a chemical analogue as internal standards. The method performance parameters that included overall recovery, intermediate precision, and measurement uncertainty were evaluated using a designed experiment, that is, the nested design. The UHPLC (Kinetex C(18)) was superior to conventional LC (Atlantis dC(18)) as it yielded a shorter analytical run time, increased method sensitivity, and improved method performance. For UHPLC/ESI-MS/MS (Kinetex C(18)), 90% of the pesticides studied had recoveries between 81 and 110%, 88% of the pesticides had intermediate precision ≤20%, and 84% of the pesticides showed measurement uncertainty ≤40%. As compared to UHPLC/ESI-MS/MS (Kinetex dC(18)), the LC/ESI-MS/MS (Atlantis dC(18)) showed a relatively lower sensitivity, less repeatability, and larger measurement uncertainty. UHPLC/ESI-MS/MS with 2.6 μm core-shell particle column and scheduled MRM proved to be a good choice for quantification or determination of pesticides in grains.     

Liquid chromatographic determination of taurine in vitamin premix formulations

A liquid chromatographic (LC) method is described for the determination of taurine in vitamin and vitamin-mineral premix formulations. The method involves extraction of taurine with 0.1 M bicarbonate buffer, followed by precolumn derivatization with dansyl chloride and LC using fluorescence detection. 6-Aminocaproic acid is used as an internal standard. A reverse phase analytical column and a mobile phase of 0.1 M acetate buffer solution (pH 7.2)-acetonitrile (75 + 25) are used. Vitamins, minerals, and other excipients in the premix formulations do not interfere in the determination. The method is simple, precise, and accurate.     

Liquid chromatographic determination of morantel tartrate in cattle feed

A liquid chromatographic (LC) method is proposed for measuring 0.485-0.970% morantel tartrate in cattle feeds. The drug is leached from feed, diluted, separated from interfering substances on a silica column, and measured in the effluent stream by 313 nm spectrophotometric detection. Two potential degradation products, i.e., cis-isomer of morantel tartrate and N-(3-methylaminopropyl)-trans-3-(3-methyl-2-thienyl)acrylamide, and a related anthelmintic, i.e., pyrantel tartrate, do not interfere. Average recovery of drug from liquid spiked samples and laboratory blends was 98-100% with a maximum coefficient of variation (CV) of 2.3%. Results for pelleted and crumbled commercial scale feeds ranged from 94 to 102% of label claim, with a maximum CV of 1.5%.     

Analysis of pharmaceutical dosage forms for oxfendazole: I. Reverse phase liquid chromatographic determination of oxfendazole in swine premix

A reverse phase liquid chromatographic (LC) procedure is described for quantitating oxfendazole (2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] in swine premix. Sample preparation consists of extracting oxfendazole with an acetone-methanol mixture. An aliquot of the extract is then centrifuged to separate undissolved premix excipients. Internal standard is added to the supernate and the sample is further diluted with water-acetonitrile-phosphoric acid (80 + 20 + 1). Oxfendazole is quantitatively determined using a Partisil-5-ODS-3 column with acetonitrile-0.01 M phosphate buffer (pH 6.0) as the mobile phase. The method is stability specific and yields a mean recovery of 101.1 +/- 0.4% for the 1.35% premix formulation. The dependence of chromatographic performance characteristics on mobile phase organic content, pH, and buffer concentration is also reported.     

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.     

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.     

Determination of virginiamycin in feeds.

Virginiamycin was extracted from the feed by ethanol-pH 2.5 phosphate buffer (1 + 1). The pH during extraction was adjusted (when necessary) to between 4 and 5. Sample dilutions and the standard dose response line were prepared to contain ethanol pH 6 phosphate buffer (2 + 8), and the test organism was Sarcina lutea. Three feeds (a poultry ration, a swine finishing ration, and a swine starter ration) showed virginiamycin recovery of 88.8--108.9% when standard solutions were added at concentrations of 4.54--90.8 g/ton. The coefficient of variation (4--20%) was larger for low potency feeds (10 g/ton) compared to the higher feeds (100 g/ton). Similarly, excellent recovery was obtained when the swine starter feed was fortified by a commercial premix. Amprolium, roxarsone, and monensin can be present at 20 times the concentration of virginiamycin with little or no interference in the antibiotic determination. Lasalocid at 10 times the concentration of virginiamycin caused a slightly positive bias (recovery, 107.4%).     

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%).     

Simultaneous liquid chromatographic screening of five coccidiostats in chicken liver

A reverse-phase liquid chromatographic (LC) method is described for simultaneously determining 5 coccidiostats--aklomide, dinsed, ethopabate, nitromide, and zoalene in chicken liver. The method entails blender extraction of 10 g liver with ethyl acetate, column chromatography through Sephadex LH-20 and neutral alumina, and LC analysis on a C18 column with UV detection at 260 nm. The drugs were eluted from Sephadex with methanol-benzene (10 + 90), from alumina with methanol-dichloromethane (10 + 90), and from C18 with acetonitrile-water (linear gradient: 25% acetonitrile for 10 min, increasing to 55% over 15 min; flow rate 1 mL/min). Liquid chromatography was completed in 40 min and calculations were based on peak height measurements. Average recoveries of the coccidiostats from fortified liver ranged from 72 to 97%, except for dinsed, which showed a relatively constant average recovery of 57%. The detection limit for the standards was 2.5 ng on column. Levels as low as 50 ng/g were detected in fortified liver samples.     

Matrix solid-phase dispersion (MSPD) isolation and liquid chromatographic determination of oxytetracycline, tetracycline, and chlortetracycline in milk

A multiresidue method for the isolation and liquid chromatographic determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) antibiotics in milk is presented. Blank and tetracycline (OTC, TC, and CTC) fortified milk samples (0.5 mL) were blended with octadecylsilyl (C18, 40 microns, 18% load, endcapped, 2 g) derivatized silica packing material containing 0.05 g each of oxalic acid and disodium ethylenediaminetetraacetic. A column made from the C18/milk matrix was first washed with hexane (8 mL), following which the tetracyclines were eluted with ethyl acetate-acetonitrile (1 + 3; v/v). The eluate contained tetracycline analytes that were free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array, 365 nm). Correlation coefficients of standards curves for individual tetracycline isolated from fortified samples were linear (from 0.982 +/- 0.009 to 0.996 +/- 0.004) with average percentage recoveries from 63.5 to 93.3 for the concentration range (100, 200, 400, 800, 1600, and 3200 ng/mL) examined. The inter-assay variability ranged from 8.5 +/- 2.4% to 20.7 +/- 13.0% with an intra-assay variability of 1.0-9.3%.     

Turbidimetric assay for virginiamycin in feeds and premixes

A turbidimetric assay method applicable to virginiamycin at level ranging from 5 g/ton in feeds to 50% in a premix is described. Incubation period is 4 h. Test organism is Streptococcus faecium ATCC 8043. On 6 levels of feed-grade material, the overall mean recovery was 6.75% Standard recovery studies resulted in RSD values ranging from 2.01 to 3.88% and a mean standard recovery of 100%.