Liquid chromatographic determination of amprolium in chicken tissues, using post-column reaction and fluorometric detection

A method is presented for determination of amprolium residues in chicken muscles by a liquid chromatographic post-column reaction system. The drug is extracted from muscles with methanol, and the extract is concentrated to 3-4 mL. This aqueous solution is rinsed with n-hexane and cleaned up by alumina column chromatography. The drug is separated from the interferences on a LiChrosorb RP-8 column, reacted with ferricyanide in alkaline solution, and quantitated by fluorometric detection at 367 nm (excitation) and 470 nm (emission). Recoveries of amprolium added to chicken muscles at levels of 0.1 and 0.2 ppm were 74.9 and 80.9%, respectively. The detection limit was 1 ng for amprolium standard and 0.01 ppm in chicken muscles.     

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.     

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 ampicillin residues in fish tissues by liquid chromatography

A liquid chromatographic (LC) method is described for determination of ampicillin residues in fish tissues. The drug is extracted from tissues with methanol, and the extract is evaporated to dryness. This residue is cleaned up by Florisil cartridge chromatography. LC analysis is carried out on a Nucleosil C18 column, and ampicillin is quantitated by ultraviolet detection at 222 nm. Recoveries of ampicillin added to tissues at levels of 0.2 and 0.1 ppm were 73.2 and 61.5%, respectively. The detection limit was 3 ng for ampicillin standard, and 0.03 ppm in tissues.     

Determination of olaquindox residues in swine tissues by liquid chromatography

A liquid chromatographic (LC) method is described for determination of olaquindox residues in swine tissues. The drug is extracted from tissues with acetonitrile, and the extract is evaporated to dryness. This residue is cleaned up by alumina column chromatography. LC analysis is carried out on a Nucleosil C18 column, and olaquindox is quantitated by ultraviolet detection at 350 nm. The average recoveries of olaquindox added to tissues at levels of 0.2, 0.1, and 0.05 ppm were 74.0, 68.6, and 66.3%, respectively. The detection limit was 2 ng for olaquindox standard and 0.02 ppm in tissues.     

Spectrofluorometric determination of BAY Vp 2674 residues in poultry tissues

A spectrofluorometric (SPF) method is described for determination of residues of BAY Vp 2674 in chicken and turkey tissues. The drug is extracted from tissues with dichloromethane-methanol. The organic extract is concentrated to near dryness and cleaned up by a series of partitionings with n-hexane, then dichloromethane against pH 2 buffer and dichloromethane against pH 12 buffer. The drug is partitioned into dichloromethane from pH 7 buffer and concentrated to dryness. The residue is dissolved in pH 3.5 buffer for SPF analysis at 282 nm (excitation) and 445 nm (emission). Recoveries of BAY Vp 2674 added to chicken and turkey tissues at levels of 0.05, 0.1, and 0.2 ppm range from 86 to 92% with a coefficient of variation of 3.4-10.1%. Detection limit is 0.02 ppm. A liquid chromatographic confirmatory procedure is also described, with ultraviolet and fluorescence detection.     

Liquid chromatographic determination of zoalene and its metabolites in chicken tissues with electrochemical detection

A procedure is described for the quantitation of Zoalene (3,5-dinitro-o-toluamide) and its 2 major monoamino metabolites in chicken tissues. The method includes blender extraction of tissue with chloroformethyl acetate (1 + 1), adsorption of the drug and metabolites on neutral alumina, and subsequent elution of the residues with pH 3.5 formate buffer-methanol (6.5 + 3.5). Recovered residues were separated on a 5 micron C18 column with the alumina eluting solvent as the LC mobile phase. The parent drug and metabolites were detected and quantitated with an electrochemical detector in the reductive mode with a minimum level of reliable measurement of 0.1 ppm. Overall mean recoveries greater than 85% were obtained with Zoalene and its 2 monoamino metabolites in breast, thigh, and liver tissues fortified with 0.25-2.00 ppm. The results on tissues from chickens fed a diet containing 0.0125% Zoalene are presented.     

Liquid chromatographic determination of carbadox residues in animal feed

A liquid chromatographic (LC) method for determining residues of carbadox in the 0.01-10 ppm range in swine feed is described. Carbadox is extracted from ground feed with 25% acidified methanol-CHCl3, removed from emulsion-forming coextractables via an alumina column, separated from highly colored pigments by acid-base liquid-liquid partitioning, and finally isolated from interferences on a second alumina column. Isocratic reverse phase LC at 305 nm is used for quantitation. The average overall recovery at the 0.1, 0.5, and 1.0 ppm spike levels was 83.0% with a standard deviation of 2.04% and a coefficient of variation of 2.46%.     

Comparison of automated liquid chromatographic and bioassay methods for determining spiramycin concentration in bovine plasma.

The performance of a liquid chromatographic (LC) method for spiramycin measurement in bovine plasma has been compared with that of a microbiological method. Plasma samples were obtained from cattle administered spiramycin intravenously. Comparison tests used were intraclass correlation (r1), correlation (r), and Student's paired t-test. For concentrations lower than 2.5 IU/mL, microbiological values were higher than LC values. This difference in results modified pharmacokinetic interpretation and might be explained by the presence of microbiologically active metabolites.     

Liquid chromatographic determination of three sulfonamides in animal tissue and egg

Sulfonamides are widely used as a feed additive in animal production in Japan. The present paper is a determination of 3 sulfonamides: sulfamethazine (SMZ), sulfamonomethoxine [SMX, 4-amino-N-(3-methoxypyrazinyl)-benzenesulfonamide], and sulfadimethoxine (SDX) in animal tissue and egg by liquid chromatography (LC). Tissues were extracted with acetonitrile and fat was removed by liquid/liquid partition. The sulfonamides were purified by an ODS cartridge column; then each compound was separated by an ODS LC column and detected at 268 nm. Quantification levels were 0.02 ppm for SMZ and SMX, and 0.04 ppm for SDX; detection limits were 0.01 ppm for SMZ and SMX, and 0.02 ppm for SDX. Calibration curves were linear between 2 and 40 ng for SMZ and SMX, and between 4 and 80 ng for SDX. Recoveries from muscle and egg samples spiked with 1-2 micrograms/10 g were 81-98%.     

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 4,4'-dinitrocarbanilide (DNC), the active component of the antifertility agent nicarbazin, in chicken, duck, and goose plasma

4,4'-Dinitrocarbanilide (DNC) was extracted from chicken, duck, and goose plasma and isolated by reversed-phase high-performance liquid chromatography. DNC was detected by ultraviolet absorbance at 347 nm and quantified by comparison to a calibration standard. Recovery data were determined by analyzing DNC-fortified control plasma. The mean recovery of DNC in fortified chicken plasma samples was 99.7 +/- 1.9% for 0.18 and 9.1 ppm DNC, and in fortified duck and goose plasma samples was 99.5 +/- 4.9% and 101.4 +/- 4.5%, respectively, for 0.18, 9.1, and 18 ppm DNC.     

Determination of phenol in honey by liquid chromatography with amperometric detection

A sensitive, selective analytical method has been developed for determination of phenol in honey by liquid chromotography (LC) with amperometric detection (AMD). Phenol is extracted with benzene from the distillate of honey. The benzene extract is washed with 1% sodium bicarbonate solution and then reextracted with 0.1N sodium hydroxide followed by cleanup on a C18 cartridge. Phenol is determined by reverse-phase LC with amperometric detection. An Inertsil ODS column (150 X 4.6 mm, 5 microns) is used in the determination. The mobile phase is a mixture (20 + 80 v/v) of acetonitrile and 0.01M sodium dihydrogen phosphate containing 2mM ethylenediaminetetraacetic acid, disodium salt (EDTA) with the pH adjusted to 5.0. The flow rate is 1 mL/min under ambient conditions. The applied potential of the AMD using a glassy carbon electrode is 0.7 V vs an Ag/AgCl reference electrode. Average recoveries of phenol added to honey were 79.8% at 0.01 ppm spiking level, 90.4% at 0.1 ppm, and 91.0% at 1.0 ppm. Repeatabilities were 3.4, 1.3, and 1.8%, respectively. The detection limit of phenol in honey was 0.002 ppm. For analysis of 112 commercial honey samples, the range and average values of 32 detected samples were 0.05-5.88 ppm and 0.71 ppm, respectively.     

Liquid chromatographic determination of multiresidue fluorescent derivatives of ionophore compounds, monensin, salinomycin, narasin, and lasalocid, in beef liver tissue

A liquid chromatographic (LC) method with fluorometric detection was developed to quantitatively determine residue levels of monensin, salinomycin, narasin, and lasalocid in beef liver tissue. The ionophores are extracted from the tissue, purified by both alumina and Sephadex LH-20 column chromatography, and then derivatized. Lasalocid was directly esterified with 9-anthryldiazomethane (ADAM), but monensin, salinomycin, and narasin were first acetylated with acetic anhydride and then esterified with ADAM. The ADAM derivatives were purified on a silica gel column and separated by LC using an RP C-8 5 micron column. A fluorescence detector set at 365 nm (excitation) and 418 nm (emission) was used to monitor the column effluent. The detection limits were 0.15 ppm, and the calibration curves were linear between 0.5 and 5.0 ppm for all 4 ionophores. Mean recoveries were 57, 70, 75, and 90% for lasalocid (5 ppm), monensin (2.5 ppm), salinomycin (2.5 ppm), and narasin (2.5 ppm), respectively. The ionophores were also separated and semiquantitated by using bioautography and thin layer chromatography with a vanillin spray.     

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.     

Normal-phase liquid chromatographic determination of menadione in animal feeds

A simple method is presented for determination of menadione in vitamin premixes and feedstuffs by normal-phase liquid chromatography (LC). Vitamin K3 is extracted and converted to free menadione, which can be determined directly by LC analysis. Peak area or height is measured at 251 nm, and menadione is quantitated by comparison with the working standard. Menadione can be estimated with a detection limit of 2.5 ppm. Recoveries for premixes ranged from 97.3 to 98.3% and for feedstuffs from 93.7 to 96.8%. The method allows the assay of all commercial K3 compounds in pure or stabilized form and is applicable to a wide variety of feeds and premixes.     

Liquid chromatographic determination of glibenclamide in human plasma

The oral hypoglycemic agent glibenclamide was determined in human plasma by liquid chromatography (LC). Samples, with internal standard added, are extracted with dichloromethane. The organic phase is evaporated, and the residue is reconstituted in mobile phase for injection onto the LC column. Intra- and inter-day variability of the method was assessed at high and low levels of the drug. Although coefficients of variation were similar for both intra- and inter-day studies at both levels, CVs were smaller at the higher concentration level. Recovery of the drug was good at both high and low levels. The minimum level of detection was 5 ng/mL.     

Determination of five macrolide antibiotic residues in eggs using liquid chromatography/electrospray ionization tandem mass spectrometry

A method using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for the determination of trace levels of five macrolide antibiotics (spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin) in eggs is presented. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two or three fragment ion transitions to provide a high degree of sensitivity and specificity for both quantification and confirmation. Matrix-matched standard calibration curves were used to achieve the best accuracy of the method. A fully nested experimental design was used to study the measurement uncertainty arising from intermediate precision and trueness or proportional bias. The overall recoveries, that is, those determined by the nested experiments, of spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin at fortified levels of 60, 100, 200, and 300 microg/kg were 96.8, 98.2, 98.3, 98.8, and 95.4%, respectively. The LC/ESI-MS/MS method detection limits (S/N > or = 3:1) of five macrolides were <1.0 microg/kg.     

Determination of nicarbazin in chicken tissues by liquid chromatography and confirmation of identity by thermospray liquid chromatography/mass spectrometry

A liquid chromatographic method is described for the quantitative measurement of nicarbazin in chicken liver, fat, muscle, and skin tissues. The 4,4'-dinitrocarbanilide (DNC) portion of nicarbazin is extracted from tissues with ethyl acetate. After filtration and evaporation, the extract is purified by liquid-liquid partitioning with acetonitrile-hexane and alumina cartridge chromatography. DNC is separated and measured by reverse-phase liquid chromatography (RP-LC) with an octadecylsilyl (ODS) column and a UV detector set at 340 nm. The overall average recovery of DNC added to tissues was 83.4 +/- 3.1%. The lowest level validated in tissues by this procedure was 0.10 ppm. The limit of detection was estimated to be 0.020 ppm. This method provides a sensitive, selective, rapid, and reproducible alternative to existing purification, separation, and detection techniques, such as differential pulse polarography and colorimetry, for determination of nicarbazin in chicken tissues. Identity of DNC is confirmed by subjecting the purified extracts to thermospray-LC/mass spectrometric analysis using negative-ion detection and selected ion monitoring. Three structural-indicating ions at m/z 302, 272, and 164 are monitored in the thermospray-mass spectrum which are characteristic of the DNC molecule.     

Liquid chromatographic determination of chlortetracycline hydrochloride in ruminant and poultry/swine feeds.

A liquid chromatographic (LC) method is described for the determination of chlortetracycline hydrochloride (CTC) in poultry/swine and ruminant feeds in the 10-100 ppm range and in premix. CTC is extracted from ground feed/premix with acidified acetone, and the extract is filtered through a Millex-HV filter or disposable C18 column. The filtrate is partitioned with methylene chloride when additional cleanup is necessary. A Nova-Pak C18 column is used for LC separation with determination at 370 nm. The average recovery of CTC from premix was 95% with a standard deviation (SD) of 1.70 and a coefficient of variation (CV) of 1.79%. The overall average recovery from feeds was 77% with an SD of 3.18 and a CV of 4.10%.