A simple method employing aminoglycoside-inactivating enzymes from Escherichia coil for identification of aminoglycosides (AGs) such as dihydrostreptomycin (DHSM), kanamycin (KM) and fradiomycin (FRM) in food was developed. The crude enzyme (S105) fraction was prepared by ultrasonic treatment of cells of drug-resistant E. coil, followed by centrifugation at 105, 000×g. DHSM was inactivated by the S105 fraction of SM-resistant E. coli in the presence of both adenosine triphosphate (ATP) and Mg2+, KM and FRM were also inactivated by the S105 fraction of KM-resistant E. coli in the presence of both ATP and Mg2+ Discrimination of KM from FRM was done by the inactivation of the former by commercial kanamycin-6′-acetyltransferase (AAC(6′)-1) in the presence of ATP, Mg2+ and acetyl CoA. KM was inactivated by the action of AAC(6′)-1, while FRM was not. Treatment of serum and kidney samples of rats, which had been intramuscularly injected with DHSM and KM and FRM, with these S105 fractions and AAC(6′)-1 resulted in the loss of the respective antimicrobial activities. When this method was applied to 4 bovine kidney samples in which AGs had been detected by the ordinary method, the AGs were identified as DHSM in two samples and KM in two other samples. This method is useful for identification of DHSM, KM and FRM in AGs-contaminated samples.
An analytical method for triacetin in foods by gas chromatography (GC) was developed. The extraction method from foods is as follows. Samples including water and emulsifier were completely dried and solidified by the use of anhydrous sodium sulfate, and oily dry samples were ground to fine powder. These samples were homogenized in 100ml of ethyl ether and centrifuged two times. The centrifuged solution containing triacetin was employed for GC analysis. The analysis on a GC column of 25% PEG-20M was interfered with ethyl caprate and o-ethylphenol, which are flavour constituents in foods, but this interference was successfully removed by use of a silica gel column. Analytical recovery of triacetin from various foods by this method was 82.1-91.8%. The detection limit was 10ppm.
An electrophoretic procedure using thin layer plates of silica-gel 60F254 (Merck) and cellulose gel (Asahi Kasei Co., Ltd., Japan) has been developed for the separation and detection of 7 tetracyclines (TC, CTC, OTC, MTC, DMCTC, DOXY and MINO). Seventeen electrolyte solutions with different pH values were examined. The results obtained were as follows. 1. With the silica-gel plate (10×20cm), fairly good separations of tetracyclines were obtained in 0.2M sodium phosphate (dibasic)-0.1M citric acid (20:980), pH=2.2, for 40min at 1, 000V and in 1.0M sodium carbonate-1.0M sodium bicarbonate-water (50:50:900), pH=9.8, for 60min at 500V 2. With the cellulose plate (10×20cm), good results were obtained when the electrophoresis was done in pyridine-acetic acid-water (1:11:89), pH=3.6, for 20min at 2, 000V and in 0.05M succinic acid-0.05M sodium tetraborate (778:222), pH=4.2, for 40min at 2, 000V. 3. Five tetracyclines (TC, CTC, OTC, DMCTC, MINO) except for MTC and DOXY were separated from each other under the above 4 electrophoretic conditions. 4. All tetracyclines on TLC plates were well detected by UV absorption measurement at 365nm.
A method of isocratic high performance liquid chromatography (HPLC) using an ion-pair partition system was established for simultaneous determination of 11 food coal-tar dyes (FD: including Amaranth, R-2; Erythrosine, R-3; New Coccine, R-102; Phloxine, R-104; Rose Bengal, R-105; Acid Red, R-106; Tartrazine, Y-4; Sunset Yellow FCF, Y-5; Fast Green FCF, G-3; Brilliant Blue FCF, B-1 and Indigo Carmine, B-2). Conditions for HPLC analysis were as follows: column, Develosil ODS-5 (5μm, 4.6mm i. d. ×250mm); mobile phase, acetonitrile-0.05M sodium dihydrogen phosphate (3:2) containing 0.002M cetyltrimethylammonium chloride and 0.003M tetra-n-hexylammonium bromide, pH adjusted to 3.0 with phosphoric acid; column temperature, 50°C; flow rate, 1ml/min; detection wavelength, 254nm. Soft drinks were pretreated with polyamide column chromatography prior to HPLC analysis for FD. Recoveries of FD from soft drinks were over 89.0%. The detection limits were 0.05μg/g for R-106, B-2, Y-4 and Y-5, 0.10μg/g for B-1, R-2, R-3, R-102 and R-104, and 0.20μg/g for G-3 and R-105. This method is considered to be applicable for routine analysis of food coal-tar dyes.
Titanium trichloride titration is employed as the official method of determination of azo colors and Indigo Carmine. The method was modified to use automatic potentiometric titration. The injection droplet volume and the time intervals between an injection and the next injection were decided as 30μl and 3sec, respectively. The temperature during titration was set at 70°C. When determination of Tartrazine by the presented method was carried out repeatedly, the standard deviation was 0.15 and the coefficient of variation was 0.17%. In the determination of other azo colors and an indigo color, the coefficients of variation were 0.16-0.61%. The method presented here is simple, rapid and accurate, and should be useful for the determination of food coal-tar dyes.
Two methods for the gas chromatographic determination of organochlorine pesticides in egg were evaluated. The first method consists of silicagel dry column chromatography for extraction and removal of fat, followed by clean-up with a Florisil cartridge. The second method consists of extraction with acetonitrile, hexane/acetonitrile partitioning and clean-up with a Florisil cartridge. The recoveries of pesticides from egg spiked at the level of 0.01ppm were 85-104% and 26-76% on the silicagel dry column and by the hexane/acetonitrile partitioning method, respectively. The concentrations of HCH, DDT and DDE in the egg sample analyzed by the hexane/acetonitrile partitioning method were 60-71% of those found by the silicagel dry column method. The silicagel dry column method gave not only superior recovery of pesticides but also simple and rapid sample preparation; however, a large amount of residue remained after evaporation of the sample solution. Eggs imported from China were analyzed and the results were compared with the data on domestic eggs.
An analytical procedure for glycyrrhizic acid (GA) in foods by liquid-liquid extraction is presented. GA in a sample was extracted with 1% ammonium hydroxide solution. After the sample extract was adjusted pH to 2.0 with 3N hydrochloric acid, GA was extracted with 1-butanol-ethyl acetate (3:7) solvent mixture. GA in the organic layer was re-extracted with 1% ammonium hydroxide solution and the solvent was removed under reduced pressure. The residue was dissolved in 60% (v/v) methanol and GA was determined by high performance liquid chromatography. Recoveries of GA from a variety of food samples spiked at 20 and 100μg/g ranged from 86.9 to 98.7%, and averaged 93.4% with a coefficient of variation of 3.1%. The detection limit of GA was 1μg/g. In a survey of commercial foods, GA was detected at levels from 2 to 224μg/g in 16 out of 56 samples.