Enzyme immunoassay for screening sulfamethazine residues in swine blood

An enzyme immunoassay (EIA) was developed to screen for residues of sulfamethazine (SMT) and its metabolites in swine blood. Swine blood was treated with perchloric acid and centrifuged. The supernatant solution was neutralized with K2HPO4, centrifuged, and applied to a reverse phase C8 cartridge. The analytes were eluted with methanol-water (2 + 3), and the eluate was diluted and assayed. Average recoveries, using 14C-labeled compounds, were 73, 72, 61, and 62% for SMT, N4-glucosylSMT, N4-acetylSMT, and N4-desaminoSMT, respectively. Tubes coated with antibody were incubated with the eluate and an SMT-beta-galactosidase conjugate. Bound enzyme was detected with fluorogenic substrate. When blood was fortified with 0.1 ppm SMT or a molar equivalent of metabolite, the average relative response of the EIA was 100%, control blood; 61%, SMT; 66%, glucosylSMT; 60%, acetylSMT; and 77% desaminoSMT.     

Reverse phase liquid chromatographic assay for calcium pantothenate in multivitamin preparations and raw materials

A reverse phase liquid chromatographic (LC) method has been developed for the assay of calcium pantothenate in commercial multivitamin tablet formulations and raw materials. The assay was validated according to the Pharmaceutical Manufacturers Association Quality Control HPLC Committee guidelines. The chromatographic system includes a C-18 column and a mobile phase consisting of 0.25M sodium phosphate buffer, pH 2.5, and acetonitrile (97 + 3 v/v). The column effluent is monitored by UV detection at 205 nm. The sample preparation involves only extraction in water followed by filtration. The method is stability-indicating with a detection limit of approximately 50 ng/mL of the calcium pantothenate in the samples. The system is linear from at least 0.02 to 0.10 mg/mL. The mean recovery of spiked placebos ranged from 98.7 to 99.8%. The within-day precision of the assay on finished products (N = 6) ranged from 0.3 to 2.0% CV. A system suitability criterion for resolution is based on the separation between calcium pantothenate and 2 closely eluting compounds, saccharin and a saccharin degradation product, 2-sulfamoylbenzoic acid.     

Liquid chromatographic determination of benzoyl peroxide in acne preparations

A rapid, precise, and accurate liquid chromatographic (LC) method is described for the determination of benzoyl peroxide (BP) in acne preparations. BP is extracted from a water dispersion of the preparation with dichloromethane (DCM), and an aliquot is eluted from a C-18 reverse phase LC column with acetonitrile-0.10 M aqueous NaClO4. Selective and sensitive quantitation is accomplished with a reductive mode electrochemical detector. This detector is an order of magnitude more sensitive than a 240 nm UV absorption detector; the lower limit of detection is 2 ng for a 4 microL injection. The recovery of BP is 99.4% and the detector response is linear to at least 2 micrograms per 4 microL injection.     

Nonaqueous reverse phase liquid chromatographic system for separation and quantitation of provitamins A

A reverse phase liquid chromatographic (LC) system has been developed for separating the main naturally occurring carotenoids that have provitamin A activity. The system produces baseline separation of beta-carotene, alpha-carotene, and beta-cryptoxanthin (beta, beta-carotene-3-ol, 472-70-8) from biologically inactive zeinoxanthin (beta, epsilon-carotene-3-ol, 24480-38-4) and from a pigment believed to be alpha-cryptoxanthin (beta, epsilon-carotene-3'-ol). Some cis-isomers are also separated. These separations are obtained on a C-18 column, isocratically, with methanol-chloroform eluant. For quantitation, peak areas from detection at 475 nm are compared with that of an internal standard, 1-(phenylazo)-2-naphthalenol (842-07-9), which elutes prior to the provitamins. Provitamin amounts are calculated from absorbance ratios. Prior to LC, esters are saponified, and interfering pigments are removed from ester-free extracts by adsorption on magnesia.     

Nonaqueous reverse phase liquid chromatographic analysis for cholesterol in milk chocolate

A method using liquid chromatography was developed for the analysis of cholesterol in milk chocolate products. The method involves saponification of the sample with methanolic KOH followed by extraction with ether. Potentially interfering components are eliminated through the use of a silica Sep-Pak cleanup step before injection. The nonaqueous reverse phase LC system consists of a C18 column and an isopropanol-hexane mobile phase with direct detection at 205 nm. Recoveries of 1, 3, and 5 mg cholesterol added to 1 g sample of milk chocolate were 88.6, 102.8, and 110.1%, respectively. Studies conducted with [4-14C]-cholesterol were undertaken to further document the accuracy of the method.     

Ion-pair reverse phase liquid chromatographic determination of sodium acifluorfen in feed

Ion-pair reverse phase liquid chromatography (LC) and UV detection at 280 nm have been used to determine sodium acifluorfen (sodium-5-[2-chloro-4-(trifluoromethyl)-phenoxy]-2-nitrobenzoate), an experimental diphenyl ether herbicide, in dog feed. Sodium-5-(2,4-dichlorophenoxy)-2-nitrobenzoate is used as the internal standard. The feed is homogenized in 0.01N HCl, followed by ethyl acetate extraction, and centrifugation. The organic layer is removed and evaporated and the residue is reconstituted in methanol and filtered before LC analysis (mobile phase methanol-water (58 + 42), 0.005M in tetrabutylammonium phosphate and 0.045M in (NH4)2HPO4, at pH 7.4). The ion-pair technique offers a high degree of control over the retention characteristics of the herbicide and internal standard. The use of the internal standard permits precise and accurate quantitation and substantially reduces analysis time compared with the external standard method.     

Determination of carbofuran and its metabolites in rice paddy water by using solid phase extraction and liquid chromatography

A procedure for the determination of carbofuran and its metabolites (carbamate and phenolic) in rice paddy water is described. Water samples are concentrated on a C-18 solid phase extraction (SPE) column and eluted with methanol-water. The eluate is analyzed by reverse-phase liquid chromatography (LC) and measured by a wavelength programmable ultraviolet (UV) detector. The limit of detection for the method is 0.4 micrograms/L. Recovery studies were carried out at levels ranging from 1 to 15 micrograms/L in both rice paddy water and distilled water; recoveries ranged from 85.9 to 112.9%.     

Classification of octadecyl-bonded liquid chromatography columns

The differences in performance characteristics exhibited by columns available under the same octadecyl (C-18) label cause difficulties for the authors and users of official methods. This paper presents a simple scheme for classifying C-18 columns on the basis of the 2 predominant reverse phase retention mechanisms-hydrophobic and silanophilic interactions, which are dependent on the hydrocarbon coverage and the unreacted silanol sites, respectively. Hydrophobic interactions are represented by the ratio of anthracene k:benzene k in an acetonitrile-water (65 + 35) mobile phase. The unreacted silanols are represented by the N,N-diethyltoluamide k:anthracene k ratio with acetonitrile as the mobile phase. Column efficiency, which is largely dependent on particle size and packing technique, is included as a third classification criterion. Twelve brands of C-18 packings were subjected to this test scheme and qualitatively classified into 3 major groups on the basis of 3 criteria:hydrophobicity index, free silanol index, and efficiency. This scheme is intended as an aid in the selection of similar or equivalent columns and in defining the type of column suitable for an analytical method.     

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%.     

Determination of folacin in foods and other biological materials

The objective of most methods for determination of folates in foods and other biological materials is to estimate the total folacin content of the sample. Because folacin comprises a diverse group of related compounds exhibiting similar biological activity, the analytical method must be capable of measuring all of the folates. Methods have been developed for separation of folates in their monoglutamyl form by using anion-exchange, paired-ion reverse phase, or conventional reverse phase liquid chromatography (LC). The application of these separations to determination of folates in foods and other biological materials has been limited largely by the need for development of adequate preparative methods and sufficiently sensitive and specific detection procedures. Although LC with ultraviolet absorption detection has been successful in certain limited applications, the development of fluorometric detection methods has permitted LC determination of folates in a wide range of materials. Tetrahydrofolic acid and its substituted derivatives are detected by monitoring their native fluorescence in an acid mobile phase, while folic acid and certain other folates are measured by using an oxidative post-column fluorogenic derivatization system. Methods also have been developed for determination of the polyglutamyl chain length distribution of folates in biological materials. In total, these procedures permit a direct determination and characterization of folacin compounds.     

Determination of fluazifop-butyl and fluazifop acid in soybeans and soybean oil using liquid chromatography with oxidative amperometric detection

A new method is described for the determination of the herbicide fluazifop-butyl, and its metabolite fluazifop acid, in soybeans and soybean oil as fluazifop acid. Liquid chromatography with amperometric detection (LC/AD) is used to determine fluazifop acid produced from the metabolism or base hydrolysis of fluazifop-butyl in soybeans and soybean oil. These foods were spiked with fluazifopbutyl at 0.05, 0.10, and 0.50 ppm and hydrolyzed with 0.2N NaOH in methanol. The hydrolysate (adjusted to pH less than or equal to 1) is extracted with dichloromethane and the extract is washed with 1.0% NaHCO3. The NaHCO3 is acidified to pH less than or equal to 1 and extracted with dichloromethane; the partitioning is repeated 2 more times. The dichloromethane is removed, mobile phase solvent is added, and aliquots are injected onto a PRP-1 liquid chromatographic column; fluazifop acid is separated from coextracted compounds and detected at an applied potential of + 1.25 V, using an amperometric electrochemical detector in the oxidation mode. Recoveries ranged from 69 +/- 6.5 to 101 +/- 18% and from 72 +/- 7.5 to 88 +/- 11% for soybeans and soybean oil, respectively. Accuracy of these recoveries was confirmed by use of 14C-radiolabeled fluazifop-butyl and by liquid scintillation spectrometry of the 14C-fluazifop acid released.     

Quantitative amino acid analysis of feedstuff hydrolysates by reverse phase liquid chromatography and conventional ion-exchange chromatography

Corn, soybean meal, and isolated soybean protein samples were acid-hydrolyzed and analyzed for amino acid content by reverse phase liquid chromatography (LC) and by conventional ion-exchange chromatography (IEC) using an amino acid analyzer. The former method employed pre-column derivatization with orthophthalaldehyde (OPTA)/ethanethiol and fluorescence detection. In the LC procedure, glycine and threonine were not resolved, and proline and cyst(e)ine were not detected. In general, amino acid values obtained by LC and IEC compared closely within and across feedstuffs, and both agreed well with published amino acid composition data. The notable exceptions were aspartic acid, glutamic acid, and alanine. Results of this study suggest that reverse phase LC with pre-column OPTA derivatization can be applied to accurately measure primary amino acids in individual feedstuffs.     

Rapid, sensitive liquid chromatographic method for determination of zearalenone and alpha- and beta-zearalenol in wheat

A rapid, sensitive liquid chromatographic (LC) method is described for quantitative determination of zearalenone and alpha- and beta-zearalenol in wheat. The procedure incorporates an internal standard, zearalenone oxime, to facilitate quantitation and automated analysis. A sample, buffered with pH 7.8 phosphate, is extracted with water-ethanol-chloroform (2 + 50 + 75) and cleaned up. The final residue is dissolved in LC mobile phase and injected onto a reverse phase RP-18 column under the following conditions: water-methanol-acetonitrile (5 + 3 + 2) mobile phase; fluorescence (excitation wavelength 236 nm, 418 nm cut-off emission filter) and UV (254 nm, range 0.0025 AU) detectors. The limit of detectability (twice background) is 0.5 ng for zearalenone and alpha-zearalenol standards on the fluorescence detector and 4 ng for beta-zearalenol on the UV detector, which is equivalent to 20 micrograms zearalenone and 20 micrograms alpha-zearalenol/kg, and 160 micrograms beta-zearalenol/kg feed. Standard curves are linear over the range 0-35 ng zearalenone and alpha-zearalenol on the fluorescence detector and 0-50 ng beta-zearalenol on the UV detector. Recoveries of all compounds are 87.5-101% in the range 0.1-3.0 mg/kg (ppm).     

Rapid liquid chromatographic determination of aflatoxins M1 and M2 in artificially contaminated fluid milks: collaborative study

An international collaborative study involving 14 collaborators from 5 different countries was conducted to test a rapid liquid chromatographic (LC) method for detecting aflatoxins M1 and M2 in fluid milk. Each collaborator prepared artificially contaminated milk samples (0.078-1.31 ng M1/mL and 0.030-0.13 ng M2/mL) by adding solutions containing various concentrations of aflatoxins M1 and M2 to fresh milk. Recoveries ranged from 85.2 to 102.5% (av. 93.7%) for aflatoxin M1 and from 99.5 to 126.7% (av. 109.8%) for aflatoxin M2. Coefficients of variation averaged 21.4% (M1) and 35.9% (M2). An analysis of variance was calculated from combined data to determine variance components. The within-laboratory variations (So) (repeatability) were 27.9% (M1) and 23.9% (M2), and the among-laboratory variations (Sx) (reproducibility) were 44.5% (M1) and 64.7% (M2). No visual differences were determined between normal or reverse phase LC for contaminated samples; however, there were an insufficient number of collaborators using normal phase to give meaningful separate statistical data. For 26 observations of uncontaminated milk, 3 false M1 positives were reported for normal phase LC determinations and 2 false M1 positives were reported for reverse phase LC determinations. Three normal phase and 11 reverse phase false M2 positives were reported for 104 observations in uncontaminated milk. The reverse phase LC method for determination of aflatoxins M1 and M2 in fluid milk has been adopted official first action.     

Production and isolation of aflatoxin M1 for toxicological studies

One hundred mg aflatoxin M1 was produced and purified for toxicological studies. Aspergillus flavus NRRL 3251 was cultured on rice to produce aflatoxins B1, B2, M1, and M2, B1 and B2 were separated from M1 and M2 by a normal phase low pressure liquid chromatography (LC) column. M1 was then separated from M2 by a reverse phase low pressure LC column. Recoveries of aflatoxins from the LC columns were about 90%. The purified M1 was confirmed by ultraviolet-visible spectrometry, mass spectrometry, nuclear magnetic resonance spectrometry, optical rotation, and its mutagenicity to Salmonella typhimurium TA98.     

Liquid chromatographic determination of aflatoxicol in porcine liver

A liquid chromatographic (LC) method for determination of aflatoxicol in porcine liver was developed. Liver sample is homogenized with water, diluted with saturated Na2SO4 solution, and extracted with acetone. After filtration, less polar interferences are removed by partition with isooctane. Aflatoxicol in the aqueous fraction is partitioned into CHCl3. The extract is dried over anhydrous Na2SO4 and evaporated nearly to dryness at 35 degrees C under a gentle flow of dry filtered air or nitrogen. Residue is dissolved in CHCl3-hexane and applied to a hexane-activated silica cartridge. The cartridge is washed with hexane-CHCl3, then aflatoxicol is eluted with CHCl3-acetone. Purified extract is evaporated to dryness, dissolved in methanol, and analyzed by C18 reverse phase liquid chromatography using a water-CH3CN-acetic acid mobile phase and fluorescence detection. Recovery of aflatoxicol from spiked liver samples at levels ranging from 0.25 to 4.0 ng aflatoxicol/g wet tissue averaged 92% with a limit of detection of about 0.1 ng aflatoxicol/g liver.     

Analysis of metsulfuron-methyl in soil by liquid chromatography/tandem mass spectrometry. Application to a field dissipation study.

An analytical method is described for the extraction of metsulfuron-methyl from soil at sub-parts per billion levels (LOQ = 0.2 microgram kg(-1)). The herbicide was quantitatively determined and identified by ESI LC/MS/MS. The method has been applied to a field dissipation study in which metsulfuron-methyl was applied to spring barley at three dosage rates: 4, 8, and 16 g of active ingredient ha(-)(1). The results of 2 years are presented. The dissipation rate of metsulfuron-methyl in topsoil was very rapid, with a calculated half-life of 6.5 days. Laboratory mineralization studies with native soils in contrast to autoclaved soils indicated that microbial degradation of (14)C-labeled metsulfuron-methyl and (14)C-labeled 2-amino-4-methoxy-6-methyl-1,3,5-triazine in soil microcosms is an important factor for the complete degradation of metsulfuron-methyl in the field. However, the mineralization rate of the sulfonamide was much higher.     

Synthesis, identification, and acute toxicity of some N-alkyl derivatives of 3,4-methylenedioxyamphetamine

A series of N-alkyl derivatives of 3,4-methylenedioxyamphetamine (MDA) was prepared in an effort to characterize these potential drugs of abuse. These secondary amines were synthesized via reductive amination of the corresponding ketone with alkylamines. The ultraviolet absorption spectra for these compounds produced almost equally intense absorbance at 234 and 285 nm. The compounds were separated by liquid chromatography using reverse phase (C18) procedures with a ternary mobile phase mixture. Toxicity studies showed all of the compounds to have LD50 values similar to N-methyl MDA (MDMA).     

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.     

A study of ten toxins associated with paralytic shellfish poison using prechromatographic oxidation and liquid chromatography with fluorescence detection.

Ten paralytic shellfish toxins [saxitoxin, neosaxitoxin, B-1, B-2, gonyautoxin 1, 2, and 3 (i.e., GTX-1, GTX-2, and GTX-3), C-1, C-2, and C-3] were oxidized at room temperature under mildly basic conditions with hydrogen peroxide or periodic acid. The products were then analyzed by liquid chromatography (LC). The N-1-hydroxylated toxins (neosaxitoxin, B-2, GTX-1, and C-3) formed fluorescent products after periodate oxidation at ca pH 8.7, but did not form fluorescent derivatives with peroxide oxidation. The non-N-1-hydroxylated toxins (saxitoxin, B-1, GTX-2, GTX-3, C-1, and C-2) formed highly fluorescent derivatives with both peroxide and periodate oxidations. Individual toxins produced mainly single fluorescent peaks by reverse-phase LC. However, all GTX toxins eluted with the same retention time. Also, C-1 and C-2 eluted together, as did neosaxitoxin and B-2. The non-N-1-hydroxylated toxins could be detected in quantities as low as 20-50 pg/injection, while the N-1-hydroxy analogues could be detected at levels as low as 100-500 pg/injection. UV absorption and fluorescence emission spectra were similar for the oxidation products of all toxins examined (max. 333 +/- 2 nm absorption, 389 +/- 4 nm fluorescence emission).