A simple and rapid method for the simultaneous determination of five penicillins (ampicillin, penicillin G, penicillin V, oxacillin and cloxacillin) in muscle, liver and kidney tissues using high-performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) was developed. Mass spectral acquisition was done in the negative ion mode by applying selected reaction monitoring (SRM). The five penicillins were extracted with water, and the extracted solution was cleaned up on a C18 cartridge. Phenethicillin was added as an internal standard, and the extract was diluted with water for injection into the LC-ESI-MS/MS. The recoveries of the five penicillins were in the range of 77.3∼99.8% from muscle, liver and kidney fortified at 10∼250 ng/g. The detection limits for ampicillin were 6 ng/g in muscle and kidney and 15 ng/g in liver. For penicillin G and penicillin V, the detection limits were 2 ng/g in muscle and kidney and 5 ng/g in liver. For oxacillin and cloxacillin, the detection limits were 4 ng/g in muscle and kidney and 10 ng/g in liver. Twenty-three muscle, fourteen liver and twenty-two kidney samples from the markets were analyzed by this method. No penicillins were detected in any sample.
We studied the effect of cleaning and cooking on the residues of flutolanil, fenobucarb, silafluofen and buprofezin in rice. The rice had been sprayed in a paddy field in Wakayama city, with 3 kinds of pesticide application protocols: spraying once at the usual concentration of pesticides, repeated spraying (3 times) with the usual concentration of pesticides and spraying once with 3 times the usual concentration of pesticides. The residue levels of pesticide decreased during the rice cleaning process. Silafluofen, which has a higher log Pow value, remained in the hull of the rice. Fenobucarb, which has a lower log Pow value, penetrated inside the rice. The residue concentration of pesticide in polished rice was higher than that in pre-washed rice processed ready for cooking. During the cooking procedure, the reduction of pesticides in polished rice was higher than that in brown rice.
We describe a method of mechanical agitation to determine rates of dialkyl phthalate migration from polyvinyl chloride (PVC) products into saliva simulant. The method consists of rotary shaking of a sample with 30 mL of saliva simulant (pH 7.0) at 35°C in a 50 mL glass tube at 300 rpm for 15 min, then measuring the amount of dialkyl phthalate in the saliva simulant by HPLC with a UV detector. The migration rates of diisononyl phthalate (DINP), di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) from PVC plates containing about 45% (w/w) plasticizer (molded in our laboratory) were identical. However, the migration rates from molded plates containing 13% (w/w) DBP were almost double those of DINP and DEHP at the same ratios. In addition, the amounts of DINP that migrated in vitro after rotary shaking for 15 min were equivalent to those in vivo determined in saliva from volunteers who chewed plates for 60 min. The migration rates of dialkyl phthalates from 11 commercially available toys ranged from 15.6 to 85.2 μg/cm2/h [relative standard deviation (RSD), 3 to 12%].
An analytical method using GC/MS for the detection of 4 kinds of dietary emulsifiers, glycerin, sucrose, sorbitan and propylene glycol monoesters of fatty acids (GE, SuE, SE, PGE), in beverages was developed. The emulsifiers were extracted from beverages with tetrahydrofuran-ethyl acetate (6 : 4) by homogenizing. The extract was cleaned up on a silica gel column and subsequently a C8 cartridge column, followed by acetylation. The derivatives were then detected by GC/MS. Our newly established method enabled to characterize 4 kinds of emulsifiers and also to identify their fatty acids without hydrolysis or de-esterification. When this method was applied to various beverages on the market, many GE and SuE with different fatty acids were detected. These results suggested that several dietary emulsifiers are used as food additives at the same time in beverages on the market.
A simple and rapid method using HPLC was developed for the determination of tricresyl phosphate (TCP) in polyvinyl chloride (PVC) articles. A test sample was extracted with acetonitrile at 37°C overnight. The extract solution diluted with an equivalent amount of water was applied to a Sep-Pak C18 cartridge, and TCP was eluted with acetonitrile-water (2 : 1) mixture. The eluate was analyzed by HPLC with an Inertsil Ph-3 column, using 65% acetonitrile/water as the mobile phase, with UV detection (264 nm). The calibration curve was rectilinear from 0.5 to 100 μg/mL. The recoveries of TCP added to various kinds of PVC articles at the level of 1,000 μg/g were 84.7∼98.6%. The determination limit of TCP was 50 μg/g in samples. This method was applied to products including 3.1, 6.6 and 8.8% TCP and the recoveries of TCP were 87.3∼91.4%. This method is very simple, and it seems suitable for a regulatory test.
Migration from can-coatings into retorted canned food simulants (canned oil and water, 121°C, 30 min) was investigated through HPLC with a fluorescence detector and evaporative light scattering detector, and by measurements of residue on evaporation and consumption of potassium permanganate. HPLC analysis revealed that migration into the canned oil was hundreds of times more than that into n-heptane (25°C, 60 min, the official test conditions according to the Japanese Food Sanitation Law), whereas it was similar to the migration into isooctane-butyl acetate mixtures (60°C, 60 min), and that migration into the canned water was several times more than that into water (95°C, 30 min, the official test conditions). Residue on evaporation for the n-heptane extract was several-fold lower than 30 ppm (the official limit), whereas that for the isooctane-butyl acetate mixtures exceeded 30 ppm. Consumption of potassium permanganate for the canned water was 30 times higher than that for the water extract (95°C, 30 min). The official test conditions for can-coatings, in particular the use of n-heptane as an oil simulant, were suggested to lead to substantial underestimation of migration into canned food.
An improved method for determination of bisphenol A (BPA), phenol (PH), p-tert-butylphenol (PTBP) and diphenylcarbonate (DPC) in polycarbonate products was developed without using the hazardous solvent dichloromethane. Polycarbonate samples were ground to powder or cut into small pieces, and 0.5 g was soaked in 5 mL of acetonitrile for 24 hours at 40°C. The test solution was then filtered and subjected to HPLC analysis. The proposed method was evaluated by comparison of the results with those of the standard method for 14 polycarbonate products. Extraction ratios (average values obtained by the present method/average values obtained by the standard method) of BPA, PH, PTBP and DPC were 0.89∼1.19, 0.89∼1.14, 0.94∼1.30 and 1.08∼1.11, respectively. While 120 mL/sample of organic solvent is required in the standard method, only 5 mL/sample of acetonitrile was used in our new method.
‘Magic mushrooms’ (MMs) are psychoactive fungi containing the hallucinogenic compounds, psilocin (1) and psilocybin (2). Since June 6, 2002, these fungi have been regulated by the Narcotics and Psychotropics Control Law in Japan. Because there are many kinds of MMs and they are sold even as dry powders in local markets, it is very difficult to identify the original species of the MMs by morphological observation. Therefore, we investigated the internal transcribed spacer (ITS) region in the ribosomal RNA gene of MMs obtained in Japanese markets to classify them by a genetic approach. Based on the size and nucleotide sequence of the ITS region amplified by PCR, tested MMs were classified into 6 groups. Furthermore, a comparison of the DNA sequences of the MMs with those of authentic samples or with those found in the databases (GenBank, EMBL and DDBJ) made it possible to identify the species of tested MMs. Analysis by LC revealed that psilocin (1) was contained at the highest level in Panaeolus cyanescens among the MMs, but was absent in the Amanita species.
The effect of exogenous polyamines (cadaverine, putrescine, norspermidine, spermidine, and spermine) on the growth, toxicity, and toxin profile of the dinoflagellate Alexandrium minutum T1 was examined. It was found that cadaverine at concentrations of 0.1-2.0 μmol/L enhanced the growth of A. minutum T1. Putrescine and norspermidine at a low level (0.1 μmol/L) also promoted the algal growth. Spermidine depressed the algal growth. However, the cell toxicity levels of A. minutum T1 cultured with or without cadaverine, putrescine, norspermidine, and spermidine were almost the same. The toxic components of A. minutum T1 were GTXs 1-4 only, and GTXs 1 and 4 were predominant (74.6±7.1%) in all cultures. On the other hand, spermine did not effect the growth of A. minutum T1, though it decreased the cell toxicity and the ratio of GTX 2+GTX 3 (15.0±6.6%).
Gas production in lactose-containing medium (e.g., BGLB, LB, or EC medium) is the most important characteristic in the E. coli/Coliform group (C.F.G.) test on food and water. Generally a Durham tube is used as the fermentation tube, and the collected gas is analyzed. However, difficulties can arise, such as insufficient gas volume or muddy precipitates. Since air clings well to fibers in water, the Tsunoda tube (T-tube), a stalk-like tube stuffed with synthetic fibers which can capture the gas more precisely and easily, was designed. With this T-tube, air elimination during the preparation of the medium and detection of gas, even in small quantities, are both simplified. Our results show that the T-tube can replace the Durham tube as a device for detecting gas-production by the E. coli/Coliform group, with improved accuracy and sensitivity.
Water extracts of 32 herbs that are constituents of curry and curry powder were screened for superoxide anion radical (O2·-) scavenging activity. Among the screened samples, only clove, allspice, and basil were shown to decrease DMPO-O2·- adduct yields by more than 50% at 0.25 mg/mL as measured by an ESR spin trapping technique based on the HPX-XOD reaction. To study the mechanism of the O2·- scavenging activity, Km values were obtained from a Lineweaver-Burk plot for XOD in the presence of different concentrations of HPX, and the IC50 values at different DMPO concentrations were compared. Clove and basil directly eliminated O2·- like superoxide dismutase (SOD), whereas allspice reduced the amount of O2·- by inhibition of formation of O2·-.
A simplified simultaneous analytical method of imazalil (IZ) and its major metabolite, α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol (IZM), in citrus fruits was developed, and commodities samples were investigated. A homogenate of citrus fruits was extracted with ethyl acetate under basic conditions. The crude extract was partitioned between 0.025 mol/L of sulfuric acid and ethyl acetate. The analytes were extracted from the aqueous fraction under basic conditions with ethyl acetate. The extract solution was purified with an ENVI-CarbTM cartridge, and then analyzed by GC-FTD and GC/MS. Recoveries of IZ and IZM added to grapefruit at the level of 0.05 μg/g were 90.0 and 108.7%, and those in the case of lemon were 100.4 and 93.0%, respectively. The detection limits were 0.01 μg/g in samples. By this method, IZ and IZM were analyzed in 46 citrus fruits on the market and were detected simultaneously in some samples.
An analytical method was developed for determination of residual avoparcin in chicken muscle by measuring α- and β-avoparcin, major components of the pharmaceutical preparation avoparcin, using HPLC with UV and amperometric detectors. The analytical HPLC was run on a Cosmosil 5C18-AR column (4.6 mm×25 cm) with a gradient formed from A: 2.5% acetic acid, 0.01 mol/L sodium heptane sulfonic acid-acetonitrile (88.5 : 11.5) (pH 4.0) and B: 2.5% acetic acid-acetonitrile (10 : 90), using UV and amperometric detection (AMD) with glassy-carbon electrode (+900 mV). Avoparcin was extracted from chicken muscle by homogenization with methanol-0.2 mol/L sulfuric acid (6 : 4) followed by centrifugation after pH adjustment to 4 with 1 mol/L sodium hydroxide. The supernatant was evaporated to dryness, and the residue was dissolved in water. The aqueous layer was adjusted to pH 4 by adding 1 mol/L sodium hydroxide. Then it was purified on a Sep-Pak tC18 plus ENV cartridge. The cartridge was washed with water, and retained substances were eluted with 50% methanol. The eluate was evaporated to dryness under reduced pressure. The residue was dissolved in water and determined by HPLC. Recoveries of avoparcin spiked in chicken muscle were 73.1∼88.1% at levels of 2∼10 μg/g. The detection limits were 0.5 μg/g (UV) and 0.2 μg/g (AMD).
The natural food color gardenia red is manufactured through the hydrolysis of the methyl ester of iridoid glucoside. Gardenia blue is also made from iridoid glucoside. Therefore, there is a possibility that the commercial products contain methanol. To determine methanol in gardenia red and gardenia blue, a headspace GC method with standard addition was developed. In the case of gardenia blue with the methyl ester, methanol may be formed during the analytical procedures for methanol. Thus, conditions in which methanol would not be produced in the headspace-GC method were investigated. Vials containing 1 g of the color preparation, 1 mL of water, and a standard solution were sealed. Equilibrium temperature was an important factor among the conditions for analyzing methanol in gardenia blue. Although at room temperature and 50°C, the contents of methanol were equal, the content increased 1.2 times at 80°C. Methanol contents determined at 50°C were 8 and 9 μg/g in two gardenia red products and 25∼34 μg/g in three gardenia blue products, which were below the residual limit of 50 μg/g set for many other natural food additives.