Fusao Kondo

Factors Influencing the Migration of Bisphenol A from Cans

The objective of this study was to determine whether there is a relationship between bisphenol A (BPA) migration from metal cans and container contents (glucose, sodium chloride, and vegetable oil), heating time, and/or temperature. Cans containing 5 to 20% glucose solution, 1 to 10% sodium chloride solution, and vegetable oils (corn, olive, and soybean oil) were heated at 121 degrees C for 30 min. Water samples were heated at 105 degrees C for 30 min and at 121 degrees C for 15, 30, and 60 min, respectively. In the test involving water samples, it was found that temperatures effect on BPA migration from cans can be more extensive than that of heating time. When cans were heated at 121 degrees C, the presence of 1 to 10% sodium chloride or vegetable oils greatly increased the migration of BPA from the cans. Moreover, the presence of 5 to 20% glucose in cans heated to 121 degrees C resulted in increased BPA migration relative to that for water controls.

Determination of bisphenol A in milk and dairy products by high-performance liquid chromatography with fluorescence detection.

This study was conducted to develop a selective and sensitive method for the determination of bisphenol A (BPA) levels in milk and dairy products. A method based on solvent extraction with acetonitrile and solid-phase extraction (SPE) was developed for the analysis of BPA in milk, yogurt, cream, butter, pudding, condensed milk, and flavored milk, and a method using two SPE cartridges (OASIS HLB and Florisil cartridge) for skim milk was also developed. The developed methods showed good recovery levels (77 to 102%) together with low detection limits (1 microg/liter for milk, yogurt, pudding, condensed milk, flavored milk, and skim milk and 3 microg/liter for cream and butter). These methods are simple, sensitive, and suitable for the analysis of BPA in milk and dairy products. When 40 milk and dairy products were analyzed by the proposed methods, BPA was not identified in noncanned products, but its levels ranged from 21 to 43 microg/kg in canned products, levels that were 60- to 140-fold lower than the migration limits in the European Union and Japan.

Improved Agar Diffusion Method for Detecting Residual Antimicrobial Agents

The improved agar diffusion method for determination of residual antimicrobial agents was investigated, and the sensitivities of various combinations of test organisms and assay media were determined using 7 organisms, 5 media, and 31 antimicrobial agents. Bacillus stearothermophilus and synthetic assay medium (SAM) showed the greatest sensitivity for screening penicillins (penicillin G and ampicillin). The combination of Bacillus subtilis and minimum medium (MM) was the most sensitive for tetracyclines (oxytetracycline and chlortetracycline), B. stearothermophilus and SAM or Micrococcus luteus and Mueller-Hinton agar (MHA) for detecting tylosin and erythromycin, B. subtilis and MHA for aminoglycosides (streptomycin, kanamycin, gentamicin, and dihydrostreptomycin), B. stearothermophilus and SAM for polyethers (salinomycin and lasalocid), and B. subtilis and MM or Clostridium perfringens and GAM for polypeptides (thiopeptin, enramycin, virginiamycin, and bacitracin). However, gram-negative bacterium Escherichia coli ATCC 27166 and MM were better for screening for colistin and polymixin-B. For detecting the synthetic drugs tested, the best combination was B. subtilis and MM for sulfonamides, E. coli 27166 and MM for quinolones (oxolinic acid and nalidixic acid), B. subtilis and MM for furans (furazolidone), and the bioluminescent bacterium Photobacterium phosphoreum and luminescence assay medium for chloramphenicol and oxolinic acid. The results showed that the use of four assay plates, B. stearothermophilus and SAM, B. subtilis and MM, M. luteus and MHA, and E. coli 27166 and MM, was superior to the currently available techniques for screening for residual antimicrobial agents in edible animal tissues.

Occurrence of false-positive results of inhibitor on milk samples using the Delvotest SP assay.

Three hundred twenty-one quarter, 207 whole udder, 310 bulk tank, and 93 tank-lorry milk samples were examined for confirmation of the presence of inhibitor by Delvotest SP assay. Four hundred twenty-six Holstein cows of no drug treatment for at least 30 days from January 1998 to September 1999 were used. Reading time was 2.50, 2.75, and 3.00 h, and results of sampling were recorded by four types according to comparison with the color of the well containing the control milk sample. False-positive outcome was identified by Delvotest SP assay in quarter (13 of 321), whole udder (9 of 207), and bulk tank milk samples (4 of 310), but was not shown on tank-lorry milk samples (0 of 93) at the reading time of 2.50 h. All of the 26 false-positive samples were negative from the examination after heat treatment at 82 degrees C for 5 min. But, two bulk tank milk samples that appeared to have positive results in LacTek and Charm II tests were positive from the test following heat treatment. Somatic cell counts (SCC) were related to the probability of a false-positive result. The more SCC increased, the more the occurrence of a false-positive result increased. In our investigations, 4 of 310 bulk tank milk samples at the reading time of 2.50 h produced false-positive results, and no false-positive results were apparent at a reading time of 2.75 h. Also, the occurrence of false-positive results in quarter and whole udder milk samples decreased when agar was cultured for 2.75 to 3.00 h. There were no false-positive results from tank-lorry milk samples. These results indicate that the Delvotest SP assay may provide a suitable means for the detection of drug residues in not only quarter and whole udder milk of cows but also in bulk tank and tank-lorry milk following reading times of 2.75 to 3.00 h.

Detection, quantitation, and identification of residual aminopenicillins by high-performance liquid chromatography after fluorescamine derivation.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection after precolumn fluorescamine derivation was developed to detect residues of two aminopenicillins, amoxicillin (AMPC) and ampicillin (ABPC), in bovine serum. Proteins in serum samples spiked with each of these penicillins were precipitated with sodium tungstate and sulfuric acid, centrifuged, and removed by passage through a C18 solid-phase extraction cartridge. After precolumn treatment of the extraction products of AMPC and ABPC with fluorescamine solution, HPLC analysis with fluorescence spectrophotometric detection at an excitation wavelength of 390 nm and an emission wavelength of 485 nm was performed to identify these products. Two mobile phases were used for residual analysis by the isocratic HPLC system. An ODP column (polyvinyl alcohol bonded with an octadecyl functional group) that can be used with strongly alkaline mobile phases (pH 2.0 to 13) was selected, and the column temperature was set at 40 degrees C. A mobile phase comprising 100-mM K2HPO4 solution and acetonitrile (72:28, vol/vol), which yielded AMPC and ABPC retention times of 4.1 and 7.9 min, respectively, was suitable for detection of residual ABPC but not for residual AMPC because interference was caused by peaks of other extracted substances. When a mobile phase comprising a different ratio of 100-mM K2HPO4 solution and acetonitrile (78:22, vol/vol) was used, the retention times of AMPC and ABPC were 7.3 and 26.3 min, respectively, and both penicillins could be analyzed using this system. The calculated standard curves of the reaction products with both mobile phases were linear, and the correlation coefficients were greater than 0.999. The lower limit of detection was 10 ng/ml for both penicillins. Analysis of extracts from bovine serum spiked with AMPC and ABPC at a concentration of 1 microg/ml yielded recovery rates of 102.2 +/- 5.5% and 79.0 +/- 5.2%, respectively. This detection method may be useful for routine laboratory testing of AMPC and ABPC.

Simultaneous Determination and Identification of Penicillin Residues by High-Performance Liquid Chromatographic Analysis

A new method was developed for simultaneous determination and identification of seven penicillins (amoxicillin, ampicillin, methicillin, penicillin G, oxacillin, cloxacillin, and dicloxacillin) in bovine serum. Each sample was simply extracted with acetonitrile, and identification of the reaction products of penicillins after treatment with 1,2,4-triazole-HgCl2 solution was then carried out by high-performance liquid chromatography (HPLC) with on ultraviolet spectrophotometric detector at 325 nm. Separation of the penicillin reaction products by HPLC was carried out by using a mobile phase of 0.1 M phosphate buffer and acetonitrile in ratios of 85:15 and 60:40 (vol/vol) in a gradient flow. The retention times of the seven penicillins ranged from 4.3 to 24.6 min, and the detection limits of penicillin concentration ranged from 0.04 to 0.2 μg/ml. The calibration curves for individual penicillins extracted from bovine serum were linear, and the correlation coefficients for each of the drugs were over 0.99. The determination of penicillins extracted from bovine serum spiked with each drug gave a recovery rate from 82.0 ± 5.6% to 105.6 ± 4.6%. This detection method may be useful for routine laboratory testing of residual penicillins.

Simple and rapid method for determination of oxolinic acid in tiger shrimp (Penaeus monodon) by high-performance liquid chromatography

A simple high-performance liquid chromatographic method for determining oxolinic acid in tiger shrimp ( Penaeus monodon ) tissue has been developed. The sample was simply extracted with ethyl acetate without further clean-up. The extract was analyzed using reversed-phase chromatography with UV detection at 330 nm. An extraction efficiency of 97.4 ± 10.3% was obtained. The within-run precision ranged from 1.46 to 4.21% and the between-run precision ranged from 6.01 to 9.77%. Detection of oxolinic acid in spiked shrimp was linear from 0.007 to 4 μg/g with a correlation coefficient of 0.998. The minimal detection limit of this procedure was 0.0035 μg/g.

Simple Continuous and Simultaneous Determination of Multiple Sulfonamide Residues

A new continuous separation method was developed for the determination of nine different sulfonamides (sulfaguanidine, sulfamethazine, sulfapyridine, sulfadiazine, sulfathiazole, sulfamethizole, sulfamethoxazole, sulfadimethoxine, and sulfaquinoxaline). Bioassay on minimum medium seeded with Bacillus subtilis ATCC 6633 was carried out for detection. An extract taken from an agar block of the clear inhibition zone on minimum medium produced by a mixture of sulfonamides was then subjected to high-performance liquid chromatography. For identification, high-performance liquid chromatography analyses were performed using two different columns and analytical conditions. Using a μ-Bondapak C18 column, the sulfonamides were separated at room temperature using a mobile phase of methanol: 0.1 M KH2PO4 (30:70, vol/vol) at a flow rate of 1.0 ml/min. A variable wavelength detector set at 265 nm and recorder set at 4 mm/min were used for the detection. The entire mixture was resolved as eight peaks from 4.68 to 50.78 min. When an Asahipak GS-320 column was employed, nine peaks were separated with retention times ranging from 12.62 to 54.43 min using a mobile phase of acetonitrile: 1% acetic acid (25:75, vol/vol) at a flow rate of 2.0 ml/min. Correlation coefficients of standard curves for individual sulfonamides were linear (>0.99) with recoveries ranging from 25.2 ± 8.6% to 64.1 ± 8.6% for a concentration range of 1.0-25 μg/ml.

Improved Method for the Determination of Residual Aminoglycoside Antibiotics in Animal Tissues by Electrophoresis and Bioautography

The use of agar gel electrophoresis and bioautography for the identification of small amounts of seven aminoglycoside antibiotics (streptomycin, dihydrostreptomycin, kanamycin, bekanamycin, gentamicin, fradiomycin and paromomycin) was studied. The procedure used for the electrophoretic separation of these components is described. The antibiotics were detected with high sensitivity using Bacillus subtilis ATCC 6633 as a test organism, Tris buffer at pH 8.0, and Bacto-Agar as the supporting medium for each of the components. The limits of detection obtained for the various antibiotics ranged between 0.078 and 0.313 μg/ml and the standard deviation ranged from 0.05 and 0.34. Recoveries from extracts of bovine kidney tissue containing each drug ranged from 59.0% to 90.2%. The sensitivity of this method can be adjusted through minor modifications, thus allowing it to be used for routine residual analysis of aminoglycosides in biological samples.

Morphology of goat milk-derived mammary epithelial cells cultured in collagen gel.

Mammary epithelial cells derived from goat milk were cultured without contamination of fibroblasts using the collagen gel. Cells grown on the fixed collagen gel showed similar morphology to those grown in plate cultures, with elongation and pleomorphism and producing a monolayer. After detaching the gels from the dishes cells became rounded. No cellular growth was seen when embedded in the collagen gel. Cells grown on both fixed and floating gel cultures were positive for antikeratin and antiactin immune serum. By electron microscopy bells grown on the fixed gel culture had poor organelles, whereas those on floating gel cultures had moderately developed Golgi apparatuses and laminar structures. No secretory granules were seen in any type of cultures with collagen gel. The floating collagen gel culture might be useful for in vitro studies on differentiation of mammary epithelial cells.