Norihisa Kawamura

Improvement of chemical analysis of antibiotics : XV. Isocratic high-performance liquid chromatographic methods for the analysis and preparative separation of the components of bacitracin☆

Isocratic high-performance liquid chromatographic (HPLC) systems were established for analytical and preparative separation of the components of the antibiotic preparation bacitracin (BC). The best analytical results were obtained using a C18 modified silica gel column (Capcell Pak C18) with a solvent system of 0.04 M disodium hydrogenphosphate buffer and methanol (4:6), pH 9-10. The calibration graphs showed good linear relationships between 50 and 1000 ng for BC-A and between 65 and 1000 ng for BC-F. With respect to the preparative HPLC, a Capcell Pak C18 column with methanol-0.05 M aqueous sodium sulphate solution (6:4) as a mobile phase gave satisfactory results. The isolation of BC-A and -F was readily achieved without decomposition of the components by using the present preparative HPLC followed by desalting on a prepacked C18 cartridge.

Identification of unlawful food dyes by thin-layer chromatography-fast atom bombardment mass spectrometry

A thin-layer chromatographic-fast atom bombardment mass spectrometric (TLC-FAB-MS) method incorporating an analyte condensation technique was established for the identification of the 27 food dyes consisting the twelve dyes permitted for use in foods and the fifteen unlawful dyes in Japan. The use of magic bullet [1,4-dithiothreitol-1,4-dithioerythritol (3:1)] as a matrix allowed the measurement of the FAB mass spectra of the food dyes except for Food Blue No. 2 (Indigo Carmine). The separation was performed on a C18-modified silica gel TLC plate using the following two solvent systems: methanol-acetonitrile-5% aqueous sodium sulphate solution (3:3:10) and methyl ethyl ketone-methanol-5% aqueous sodium sulphate solution (1:1:1). The condensation technique for concentration of a diffuse sample spot on the TLC plate improved the detection limit 4-20-fold with good reproducibility. The method was successfully applied to the identification of unlawful dyes in imported foods.

Simple method for the analysis of food dyes on reversed-phase thin-layer plates

A technique for the determination of food dyes using reversed-phase thin-layer chromatography on octadecyl-modified silica is described. A solvent system containing 5.0% aqueous sodium sulphate solution enables good separation of the food dyes. Their separation is dependent on the pH of the solvent system, good separation among all dyes being obtained between pH 6.0 and 7.0. The determination of thirteen dyes can be achieved by a combination of methanol-acetonitrile-5.0% aqueous sodium sulphate solution (3:3:10) (solvent system A) and methanol-methyl ethyl ketone-5.0% aqueous sodium sulphate solution (1:1:1) (solvent system B). Solvent system A was used for the determination of Tartrazine, Amaranth, Indigo Carmine, New Coccine, Sunset Yellow FCF, Allura Red AC, Fast Green FCF and Brilliant Blue FCF and for the screening of Acid Red (R-106), Eosin (R-103), Erythrosin (R-3), Rose Bengale (R-105) and Phloxine (R-104). When the spots of R-3, R-103, R-104, R-105 and R-106 appear on the plate, their determination can be achieved by using solvent system B. Each calibration graph was linear between 0.2 and 1.0 micrograms.

Improvement of chemical analysis of antibiotics: XVI. Simple and rapid determination of residual pyridonecar☐ylic acid antibacterials in fish using a prepacked amino cartridge

A simple and rapid method for the determination of residual pyridonecarboxylic acid antibacterials (PCAs) (oxolinic acid, nalidixic acid and piromidic acid) in fish was developed using a combination of high-performance liquid chromatography (HPLC) and clean-up with an amino-type prepacked cartridge. PCAs were extracted with n-hexane-ethyl acetate (1:3) and the extract was applied to a Baker 10 amino cartridge. PCAs were eluted from the cartridge with acetonitrile-methanol-0.01 M aqueous oxalic acid solution (pH 3.0) (3:1:6) and were determined by HPLC. The separations were performed on Nucleosil 3C18 (3 microns, 75 x 4.6 mm I.D.) using a mobile phase containing oxalic acid. The recoveries of PCAs from various fishes fortified at the level of 1.0 ppm were 77.1-95.5%, and the detection limits were 0.05 ppm. The analytical time per sample was less than 30 min.

Improvement of chemical analysis of antibiotics. XVII : Application of an amino cartridge to the determination of residual sulphonamide antibacterials in meat, fish and egg

A simple, rapid and reliable method for the determination of residual sulphonamide antibacterials (SAs) (sulphathiazole, sulphisozole, sulphamethoxazole, sulphadiazine, sulphamerazine, sulphadimidine, sulphamonomethoxine, sulphadimethoxine, sulphamethoxypyridazine and sulphaquinoxaline) in meat, fish and egg was developed using a combination of high-performance liquid chromatography (HPLC) and clean-up with an amino-type prepacked cartridge. SAs were extracted with ethyl acetate and applied to a Baker 10 amino cartridge. After elution from the cartridge, SAs were determined by HPLC. The recoveries at the level of 0.5 ppm were 73.7-99.1% and the detection limits were 0.05 ppm. The analysis time per sample was about 45 min.

Improvement of chemical analysis of antibiotics : XIII. Systematic simultaneous analysis of residual tetracyclines in animal tissues using thin-layer and high-performance liquid chromatography

A simultaneous analytical system for residual tetracycline antibiotics (TCs) in animal tissues including liver has been developed. This system consists of three methods using Baker 10 C18 clean-up followed by high-performance liquid chromatography (method A), thin-layer chromatography (TLC)-UV densitometry (method B) and TLC with spray reagents (method C). Methods A and B were established for the precise determination of TCs and method C for universal screening. The recoveries from beef liver fortified at the level of 1.0 ppm and their coefficients of variation were 60.5-83.3% and 2.3-3.9% (method A) and 58.1-92.8% and 1.0-3.7% (method B), respectively. The detection limits in animal tissues were 0.01 ppm (method A) and 0.1 ppm (methods B and C). The time required for the analysis of four samples was 2.5-3 h with each method.

Improvement of chemical analysis of antibiotics. XII. Simultaneous analysis of seven tetracyclines in honey.

An analytical system for the simultaneous determination of residual oxytetracycline, tetracycline, chlortetracycline, doxycycline, methacycline, demethylchlortetracycline and minocycline in honey has been established by a combination of simple thin-layer chromatographic (TLC) and precise high-performance liquid chromatographic (HPLC) methods. In this system, screening by TLC can detect tetracyclines (TCs) at a level of 0.1 ppm in honey without the need for special equipment, and the quantitative method by HPLC can determine TCs with good recovery (83.7-99.6%) and coefficient variation (0.9-4.3%).

Improvement of chemical analysis of antibiotics. IX. A simple method for residual tetracyclines analysis in honey using a tandem cartridge clean-up system.

A simple, rapid and precise analytical method for the residual tetracyclines in honey has been established using a tandem cartridge clean-up system (prepacked reversed-phase and ion-exchange cartridges) followed by high-performance liquid chromatography. The recoveries of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC) from honey spiked at a level of 1.0 ppm are 87.1, 85.3, 98.0 and 99.0%, respectively, with coefficients of variation of 1.1-3.9%. The detection limits in honey are 0.02 ppm for OTC and TC, and 0.05 ppm for CTC and DC, respectively. The time required for the analysis of four samples is only 1 h.

Relationship between chlordane and its metabolites in blood of pest control operators and spraying conditions

SummaryChlordane has been widely used to protect soil and house foundations against termite infestation. Pest control operators (PCOs) are occupationally exposed to chlordane. The relationship between chlordane and its metabolites in blood of PCOs and spraying conditions were investigated. Chlordane and its metabolites were detected in the blood of some chlordane-exposed PCOs, but not in that of the controls. Trans-nonachlor and the metabolites oxychlordane and heptachlor epoxide were detected in the blood of PCOs. Total concentration of chlordane and its metabolites in blood (trans-nonachlor + oxychlordane + heptachlor epoxide) was less than 5.6 ppb (mean: 0.89 ppb). The concentration of chlordane and its metabolites in blood of chlordane-exposed PCOs was significantly correlated with the number of spraying days and the amount of chlordane sprayed, particularly with a large correlation coefficient (r = 0.81, P < 0.001) with the spraying days in the three months prior to the medical examination. The concentration of chlordane and its metabolites in blood is considered to be a useful indicator of biological monitoring for chlordane exposed workers (PCOs).

Simultaneous determination of nine food additives using high-performance liquid chromatography

A technique for the simultaneous determination of sorbic acid, benzoic acid, dehydroacetic acid, p-hydroxybenzoic acid esters (ethyl, isopropyl, n-propyl, isobutyl and n-butyl p-hydroxybenzoate) and saccharin sodium using ion-pair reversed-phase high-performance liquid chromatography is described. The nine food additives were separated on a Nucleosil 3C18 (3 microns) column (75 x 4.6 mm I.D.) using methanol-acetonitrile-0.05 M aqueous acetonic acid solution (pH 4.5) (1.5:1:3.1) containing 2.5 mM cetyltrimethylammonium chloride as the mobile phase at a flow-rate of 1.0 ml/min, and detected at 233 nm. The food additives were separated within 18 min and their calibration graphs were linear between 2 and 200 ng.