Reverse-phase liquid chromatographic determination of lysine in complete feeds and premixes, using manual precolumn derivatization

A sample portion is hydrolyzed with 6N HCl for 23 h and cooled, the pH is adjusted to 7.7 with NaOH, and the solution is diluted with pH 7.7 borate buffer. An aliquot of the sample extract is derivatized with 9-fluorenylmethyl chloroformate (9-FMC). Lysine is separated from other amino acids by isocratic reverse-phase liquid chromatography (LC) using fluorescence detection: 260 nm excitation and 313 nm emission. The mobile phase is acetonitrile-0.1M acetic acid (pH 4.2) buffer (53 + 47). Linearity is satisfactory over a range of 0.4-24 micrograms/mL. Results from 9 feed samples (1.1-2.7% lysine) analyzed by both the LC method and an amino acid analyzer were not significantly different statistically. Recovery of standard lysine, spiked just before derivatization on these same 9 samples (in duplicate), was 100.9% with a coefficient of variation (CV) of 2.4%. A study of within-day and between-day method precision resulted in CVs of 1.1 and 1.8%, respectively. The variation of results was negligible when the method was tested for ruggedness on 7 factors.     

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.     

Sulfamethazine and sulfathiazole determination at residue levels in swine feeds by reverse-phase liquid chromatography with post-column derivatization

Twenty g sample, to which sulfamerazine has been added as internal standard, is extracted with 0.3N HCl + 1.5% diethylamine in 25% methanol. The sample extract is chilled (to aid clarification), centrifuged, and filtered. The sulfonamides are separated from each other and from co-extracted materials on a C-18 reverse-phase column and detected at 450 nm following post-column derivatization with dimethylaminobenzaldehyde. Two isocratic mobile phases have been tested: (1) acetonitrile-2% acetic acid (17 + 83), with an analysis time of 13 min; and (2) acetonitrile-methanol-2% acetic acid (4 + 16 + 80), with an analysis time of 20 min but an improved analysis for some samples. As many as 40 samples have been analyzed at one time unattended with the aid of an autosampler. A total of about 1500 field samples have been assayed using the method. Method sensitivity is 0.1 ppm for either analyte in a hog finishing fed. Linearity for each of the analytes is satisfactory over a range of 0.4-25 ppm in spiked feeds. Coefficients of variation range from 13% at 0.5 ppm to 2% at 13 ppm as tested over a period of time in naturally contaminated samples. The absolute recovery of sulfamerazine varies with sample matrix, but, in the presence of sulfamerazine as internal standard, recovery has been 96.7-99.7% over the range of 0.1-10 ppm. Sulfamerazine and sulfamoxole were tested for their suitability as internal standards. Sulfamerazine is a good internal standard for sulfamethazine; neither is ideal for sulfathiazole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Liquid chromatographic determination of aflatoxins, ochratoxin A, and zearalenone in grains, oilseeds, and animal feeds by post-column derivatization and on-line sample cleanup

A liquid chromatographic method using on-line sample cleanup, reverse flow analytical column loading, gradient elution, and postcolumn derivatization with iodine permits direct, rapid determination of aflatoxins B1, B2, G1, and G2, as well as ochratoxin A and zearalenone. Limits of quantitation are 5 ppb for the aflatoxins and ochratoxin A and 30 ppb for zearalenone. This procedure performs well as a multimycotoxin screen for cereal grains and oilseeds, with more limited success in complete animal feeds.     

On-line trace enrichment for determination of aldicarb species in water, using liquid chromatography with post-column derivatization

Liquid chromatography combining on-line trace enrichment together with a very selective detection technique is used for the determination of aldicarb, aldicarb sulfoxide, and aldicarb sulfone. Sensitivity is increased by loading a 10 mL volume of ground water on a concentrator column installed in the loop position of a 6-port injection valve. Switching the valves allows the concentrated material to be backflushed onto the analytical column by a methanol-water gradient mobile phase. Separation is followed by post-column hydrolysis to yield methylamine, and formation of a fluorophore with o-phthalaldehyde and 2-mercaptoethanol prior to fluorescence detection. The process requires virtually no sample cleanup and provides good precision on recoveries from different matrixes. Minimum detection limit, defined as 5 times baseline noise, is less than 70 ng/L for the 3 compounds.     

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 narasin in feed premixes

A liquid chromatographic (LC) method has been developed to determine narasin in feed premixes. Narasin is extracted from the premix with a methanol-water solvent, and the extracted solution is assayed by using LC. Recovery of narasin from a 12.5 g/lb premix is quantitative (100%), with a relative standard deviation of 1.44%. The results correlated well (coefficient 0.92) with a turbimetric bioassay method.     

Fluorometric determination of benzylideneacetone in fragrance products by liquid chromatography with post-column derivatization

A method is described for the liquid chromatographic (LC)-fluorometric determination of benzylideneacetone in fragrance products. Benzylideneacetone is first separated from other fragrance ingredients by LC and then reacted post-column with a methanolic solution of isonicotinic acid hydrazide and aluminum nitrate. The reactants are maintained at 65 degrees C for about 1.5 min to quantitatively form the fluorescent isonicotinoyl hydrazone derivative of benzylideneacetone. The aluminum ion forms a complex with the hydrazone to enhance the fluorescence of the derivative. The amount of benzylideneacetone is determined by measuring the intensity of the fluorescence emitted by the hydrazone derivative and comparing that value with those obtained for derivatized standards. Recovery studies were conducted by spiking commercial fragrances with benzylideneacetone at concentrations of 0.01, 0.05, and 0.1% (w/v). Recoveries ranged from 98 to 104% with a mean recovery of 100.2% and a standard deviation of 2.4%.     

Ion-pair reverse-phase liquid chromatographic determination of amprolium in complete feeds and premixes

Amprolium is extracted with methanol-water (2 + 1) containing 5mM dioctylsulfosuccinate (DOSS) and 10mM CaCl2. The analyte is separated from coextracted materials by isocratic ion-pair reverse-phase liquid chromatography, following removal of late-eluting materials on an acid alumina cleanup column, and is detected at 270 nm. The mobile phase contains 4mM DOSS with 0.3% diethylamine and 1% acetic acid in 40% acetonitrile. Linearity is satisfactory over the range of 2.5-50 micrograms/mL. Mean recovery, as determined by standard addition to commercial samples, is 100.1%. Accuracy was further tested in studies comparing the LC method to the official AOAC colorimetric method, using commercial samples, and was found to be satisfactory. Studies show that common poultry feed additives, grass meals, and some pelletization aids do not interfere with the analysis; however, when bentonite is present, recovery is decreased. The precision of the method, measured over several experiments on commercial samples, is satisfactory as indicated by coefficients of variation ranging from less than 1 to 4.5. A ruggedness test resulted in an overall CV of 3.2%, indicating the probable success of the method in a collaborative study.     

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%).     

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.     

High-speed liquid chromatographic determination of phenothiazine in commercial pesticide formulations.

Phenothiazine (thiodiphenylamine) in commercial pesticide formulations is analyzed by high-speed liquid chromatography and ultraviolet absorption detection. Phenothiazine is extracted from the formulation with methanol and injected into the liquid chromatograph. Methanol-water (1+1) is the mobile phase. The amount of phenothiazine is calculated from peak height ratios. The liquid chromatographic procedure is much faster than the infrared or colorimetric method and yields values in close agreement with both of these methods.     

High-speed liquid chromatographic determination of biphenyl residues in citrus products.

A simple, direct, and rapid method is given for the analysis of citrus oils for the fungistat biphenyl by high-speed liquid chromatography. The method is extended to orange juice and various "dry flavors" by an extraction procedure. Analytical limits are less than 1 ppm without need for any cleanup or concentration steps.     

High-speed liquid chromatographic determination of o-phenylphenol residues in citrus products.

A method is presented for the quantitative analysis of o-phenylphenol residues in citrus oils, encapsulated flavors, and dried meal. The method utilizes high-speed liquid chromatography for the determination after specific sample preparations for each material. These preparations include hexane extraction of acidified basic extracts or steam distillation and extraction. The limit of the analysis is less than 1 ppm with an analysis time of less than 45 min.     

Liquid chromatographic determination of sulfamethazine in feeds

A reverse-phase liquid chromatographic method for the assay of sulfamethazine (SMZ) in feeds is described. Feed samples are extracted with 50% methanol solution, centrifuged, filtered, and diluted when necessary, and chromatographed on a C-18 column. Samples are eluted with a mobile phase of 20% methanol and 80% of a solution containing acetic acid and tetramethylammonium chloride. The average recovery from spiked samples was 97.2% with a coefficient of variation of 1.2%. Linearity was very good (correlation coefficient 0.9997). Within-day and between-day coefficients of variation averaged 1.3 and 2.6%, respectively. The results for samples assayed by this method compared closely with the results from the same extracts assayed by the AOAC colorimetric method.     

High-performance liquid chromatographic method for determination of biogenic amines in feedstuffs, complete feeds, and animal tissues

Numerous methods to analyze biogenic amines in biological materials have been described. A versatile and rapid methodology to analyze these compounds in feedstuffs, complete feeds, and animal tissues, however, has not been reported. The current method was developed to address this need. Biogenic amines in feedstuffs, complete animal feeds, and animal tissues were extracted with 10% trichloroacetic acid, reacted with O-phthaladehyde using high-performance liquid chromatographic employing a cation exchange column. Detection limits were 50 pmol/mL for tyramine, histamine, putrescine, and spermine; 40 pmol/mL for cadaverine; and 25 pmol/mL for spermidine. Extraction efficiency of biogenic amines in feedstuffs, duodenum, liver, ileum + jejunum, and whole shrimp and shrimp hepatopancreas ranged between 99-105, 93-135, 80-85, 65-102, 88-98, and 88-97%, respectively. It can be concluded that the current method can be applied to individual feedstuffs, complete feeds, and animal tissues for the rapid and accurate determination of concentration of biogenic amines.     

Liquid chromatographic determination of zearalenone and zearalenol in animal feeds and grains, using fluorescence detection

A liquid chromatographic (LC) method was developed for the determination of zearalenone and zearalenol in grains and mixed animal feeds. Samples are extracted with chloroform and purified by a base-acid liquid-liquid partition. Zearalenone and zearalenol are separated by reverse phase LC and determined by fluorescence detection, excitation wavelength 236 nm with a 418 nm cutoff filter. The method was applied to the determination of zearalenone and zearalenol in 395 survey samples of corn, oats, barley, sorghum, silage, and finished feeds. The limit of detection is 10 ng/g for both toxins. The range of naturally occurring toxins found was 10-4,000 ng/g. Average recoveries were 84% for zearlenone and 69% for zearalenol. Coefficients of variation were 24.6% for zearalenone and 30.8% for zearalenol for crop year 1980, and 28.3% for zearalenone and 22.0% for zearalenol for crop year 1981.     

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.     

Liquid chromatographic determination of zoalene in medicated feeds

A rapid, reliable separation and quantitation of zoalene (3,5-dinitro-o-toluamide) from feeds is accomplished by using reverse phase liquid chromatography (LC) and ultraviolet detection. An extraction technique which is similar to the present AOAC official colorimetric method is used before chromatographic analysis. This extraction is followed by an activated alumina cleanup and LC to separate zoalene from feed matrix. The methodology was applied to a variety of spiked feed matrices, and yielded good recoveries. Liquid chromatographic results were shown to correlate with colorimetric determinations.