Effects of processing and cooking on the reduction of dinotefuran concentration in Japanese rice samples.

Dinotefuran is an insecticide belonging to the neonicotinoid class, which is frequently used to control pests in paddy rice owing to its permeability and effectiveness against sucking insects. Since 2002, this insecticide has been commercially available in Japan, and has become controversial due to its high detection frequency in brown rice for primary consumption. In this study, the effects of processing and cooking on the reduction of dinotefuran residues in commercially available brown rice were investigated. Boiled rice is difficult to homogenise and extract with acetonitrile. Using pre-freezing and cryogenic milling with powdered dry ice, dinotefuran in boiled rice was extracted well. A measurement method comprising sample preparation (acetonitrile extraction, gel permeation chromatography, and SPE) and detection with anLC-MS/MS system was used. In 10 out of 25 commercial brown rice samples, dinotefuran was detected at a concentration of 0.04 µg/g (mean), which was more than the limit of quantitation of 0.01 µg/g. The dinotefuran levels were significantly less than the MRL of 2 µg/g in Japan. Even after polishing, washing, and boiling, dinotefuran was detected in 10 brown rice samples, with mean residue levels of 74.7%, 60.8%, and 39.6%, respectively, of the original concentration in brown rice. Based on these data, the processing factor of dinotefuran in boiled rice has been estimated to be approximately 0.4. Dinotefuran residues were reduced in the boiled rice, but less so than other pesticides. Although the maximum daily intake of dinotefuran in boiled rice was 0.0065 mg/person/day, its percent ratio to the ADI of dinotefuran in Japan was less than 0.05%. These results suggest that the daily intake of dinotefuran from rice might not be a critical problem at present, in spite of its relatively high detection frequency in boiled rice.

[Validation of ELISA Kits for Pesticide Residue Analysis in Vegetables and Fruits].

Five kinds of commercially available ELISA kits (acetamiprid, azoxystrobin, chlorothalonil, fenitrothion and imidacloprid) were validated for determination of pesticide residues in vegetables and fruits. The reaction characteristics were also examined to evaluate their influence on the determinations. The trueness value was 91-162%, the repeatability was 2.1-16.2%, and the reproducibility was 4.0-20.3%. The desired values were achieved for 18 among 30 combinations (60%) of the ELISA kits and the agricultural products examined. A standard curve was necessary for each of the ELISA examinations. The matrix of the agricultural products and pipetting skill of the lab technician both influenced the measurment results.

[Validation study on a multi-residue method for determination of pesticides in agricultural products by LC-MS/MS].

A multi-residue method for 122 pesticides in agricultural products was validated in accordance with Japanese guidelines for residual agricultural chemicals in food. The sample was extracted with acetonitrile. Co-extractives were removed by GPC-graphitized carbon column SPE, followed by silica gel/PSA cartridge column SPE. The pesticides in the test solution were determined by LC-MS/MS using scheduled MRM in combination with polarity switching. Validation tests were performed on spinach, brown rice, soybean, orange and tomato fortified at 0.01 and 0.1 µg/g. The pesticides at each level were analyzed in two samples per day on five different days. In conclusion, among 122 pesticides tested for each level, 121 pesticides (but not disulfoton) were found to meet all of the guideline criteria.

[Simultaneous determination of pesticide residues in agricultural products by LC-MS/MS].

A method for the simultaneous determination of multiple pesticide residues in agricultural products was developed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The sample was extracted with acetonitrile. Co-extractives were removed by GPC/graphitized carbon column SPE, and silica gel/PSA cartridge column SPE. Pesticides in the test solution were determined by LC-MS/MS using scheduled MRM. Recoveries of 124 pesticides from spinach, brown rice, soybean, orange and tomato were tested at the level of 0.1 µg/g, and those of 121 pesticides ranged from 70 to 120% (RSD≤15%). Pesticide residues in 239 agricultural products were investigated by this method, and residues of 49 pesticides were detected in 98 agricultural products.

[Analysis of spinosad in animal and fishery products by LC-MS].

We investigated the determination of spinosyn A and spinosyn D, the active ingredients of spinosad, in animal and fishery products by liquid chromatography with mass spectrometry (LC-MS). The sample was homogenized with 1 mol/L dipotassium hydrogenphosphate aqueous solution and extracted with acetone-n-hexane under mildly alkaline conditions. After n-hexane-acetonitrile partitioning using an EXtrelut(®) column, the extract was cleaned up on a tandem SAX/PSA mini-column, and examined by means of fragmenter-voltage-switching ESI-SIM mode LC-MS. Mean recoveries (n=5) of spinosyn A and spinosyn D from eleven kinds of fortified samples at the analyte concentration of 0.01 µg/g and 0.05 µg/g ranged from 76.1% to 93.8% (RSD≤8.7%) and from 75.1% to 104.1% (RSD≤8.6%), respectively.

[Multiresidue analysis of PCB, organochlorine pesticides and chlordanes in marine products by GC-micro ECD].

A multiresidue method using dual-injection, dual-column, and dual-micro electron capture detection gas chromatography (dual-column GC-µECD) was developed for the determination of PCB, organochlorine pesticides and chlordanes in marine products. The sample was extracted with hexane-acetone (2 : 1), and the extract was cleaned up by gel permeation chromatography(GPC)/solid-phase extraction (SPE). The GPC fraction was selectively collected, and loaded directly onto a graphitized carbon/PSA 2-layered column. After fractionation by 4% hydrated silica-gel column chromatography, each fraction was determined by dual-column GC-µECD. Recoveries of PCB, organochlorine pesticides and chlordanes were in the ranges of 84-109% (RSD ≤ 21.6%), 74-117% (RSD ≤ 14.6%) and 69-114% (RSD ≤ 12.9%), respectively. This method is superior to single chromatography for the determination of total PCB, and should be useful for monitoring of these pollutants in marine products.

Binding assay for cholera toxin based on sequestration electrochemistry using lactose labeled with an electroactive compound.

A simple electrochemical binding assay for cholera toxin (CT) was developed using lactose labeled with daunomycin as an electroactive compound. The labeled lactose (LL) was determined with high sensitivity by adsorptive stripping voltammetry (AdSV). The electrochemical behaviors of LL at glassy carbon (GC), plastic formed carbon (PFC) and carbon nanotubes paste (CNTP) electrode were investigated. The CNTP electrode showed the greatest accumulation capacity for LL. The assay for CT based on the sequestration electrochemistry was demonstrated. The binding event of the LL to CT was detected by the decrease in the electrochemical response of daunomycin as an electroactive label without a separation process to remove the free LL from the one bound with CT before any measurements can be made. The detection limit of the CT assay using the CNTP electrode was 0.5 nM (42 ng mL(-1)).

[Analysis of acephate, methamidophos and omethoate in animal and fishery products, and honey by LC-MS].

We studied the simultaneous determination of acephate, methamidophos, and omethoate in animal and fishery products, their processed foods, and honey by means of liquid chromatography coupled with mass spectrometry (LC-MS). The sample was extracted with ethyl acetate in the presence of anhydrous Na(2)SO(4) (and diatomaceous earth for honey). An aliquot of the crude sample extract was loaded into the GPC system, and the pesticide fraction was selectively collected. The extract was cleaned up on a PSA mini-column, and determined by a column-switching ESI-SIM mode LC-MS. Mean recoveries (2 replicates x 5 days) of compounds from eleven kinds of samples, except honey, fortified at the analyte concentration of 0.05 microg/g were from 71.4% to 98.4%. The repeatability relative standard deviation values were < or =12.5%, and the intermediate reproducibility relative standard deviation values were < or =14.1%. In honey, the recoveries were improved to 97.6-98.6% by using highly purified surrogates.

[Analysis of demeton-S-methyl, oxydemeton-methyl and demeton-S-methylsulfone in agricultural products by LC-MS].

We studied the simultaneous determination of demeton-S-methyl, oxydemeton-methyl, and their oxide demeton-S-methylsulfone, in agricultural products by liquid chromatography coupled with mass spectrometry (LC-MS). The sample homogenized with antioxidants L-ascorbic acid and butylhydroxytoluene was extracted with acetone. An aliquot of the crude extract was reextracted with ethyl acetate by using an EXtrelut column. After hexane/acetonitrile partitioning lipid-rich samples such as cereals, the extract was cleaned up on a PSA column or tandem graphitized carbon/PSA column, and determined by ESI-SIM mode LC-MS. Average recoveries (n=5) of compounds from ten kinds of samples fortified at the analyte concentration of 0.05 microg/g were from 73.8% to 102.5%, and the relative standard deviations were

[Study on the method of setting limits for pesticides residues under the positive list system based on the data for pesticide residues in vegetables and fruits collected in Aichi Prefecture (Fiscal Years 2001-2005)].

Based on the data for pesticide residues in vegetables and fruits collected in Aichi prefecture (fiscal years 2001-2005), we selected groups of foods and pesticides that would allow efficient and effective inspection under the positive list system. Statistical analyses were done to examine the rates of detection of pesticides and the numbers of kinds of pesticides detected in samples of domestic vegetables, domestic fruits, imported vegetables, and imported fruits. The rate of detection of pesticides has decreased gradually in domestic vegetables. The number of different kinds of pesticides detected in each sample was significant higher in domestic fruits. Data for previous years were reassessed in terms of the present maximum residue limits (MRL), and classified as relative value to the MRL. The proportion of pesticides detected at levels that exceeded the MRLs showed a decreasing tendency. In addition, we were able to identify combinations of pesticides and agricultural commodities in which the MRLs were more likely to be exceeded.

[Multiresidue analysis of pesticides in agricultural products by GC/MS coupled with database software].

We evaluated a multiresidue method for determination of pesticides in agricultural products by SCAN mode GC/MS coupled with three kinds of database for 253 pesticides: relative retention time, mass spectra and calibration curve (SCAN method). Twenty-six pesticides, a total of 131 pesticides were detected in samples by the SCAN method. The detection results agreed closely with those of the SIM mode GC/MS method using calibration standards (SIM method). The ratios of the SCAN method to the SIM method ranged from 0.3 to 3.1 with SD values of 0.63. It was judged that the SCAN method could be applied to the screening analysis of pesticide residues in agricultural products, provided that the sample preparation method makes it possible to effectively remove sample matrixes with minimal loss of analytes.

[Multiresidue analysis of pesticides in animal and fishery products by NCI mode GC/MS and dual-column GC-micro ECD].

A sensitive and quantitative multiresidue method using NCI mode GC/MS and GC-micro ECD for determining pesticides in animal and fishery products was established. The crude sample extract obtained by acetone-hexane extraction for solid samples or acetonitrile extraction for liquid samples was cleaned up with a GPC/SPE system. The first GPC pesticide fraction containing lipids and pigments was selectively collected, and loaded directly onto a graphitized carbon/PSA 2-layered column. After the second GPC pesticide fraction was collected, the 2-layered column was eluted with acetone-hexane (3 : 7). The combined eluate was subjected to NCI-SIM/Scan mode GC/MS for semi-quantification. After fractionation by Florisil cartridge column SPE, each fraction was subjected to dual-column GC-micro ECD for quantification. Average recoveries (n=5) of pesticides, except for chlorothalonil and some others, from fortified samples ranged from 76.8% to 107.9% with RSD values of <9.7%.

[Analytical method for methoprene in foods using HPLC].

We studied the determination of methoprene in foods by high-performance liquid chromatography (HPLC). The sample was extracted with acetonitrile and the extract was salted out by adding sodium chloride, allowing the acetonitrile layer to separate. The acetonitrile solution was washed with hexane saturated with acetonitrile, cleaned up on a Florisil column and determined by HPLC. The recovery of methoprene from spiked samples was 74.6-82.8%. In an evaluation of this method by 6 analytical laboratories, mean recoveries from spiked samples ranged from 79.4% to 84.6%. Repeatability relative standard deviation values were 2.3-8.8% and reproducibility relative standard deviation values were 8.8-23.6%. The detection limits were 0.001-0.02 microg/g and below the detection limit of the Notified Analytical Method.

Determination of spinosad in vegetables and fruits by high-performance liquid chromatography with UV and mass spectrometric detection after gel permeation chromatography and solid-phase extraction cleanup on a 2-layered column.

A simple and reliable method was developed for the determination of spinosyns A and D, the active ingredients of spinosad, in vegetables and fruits, by high-performance liquid chromatography with UV detection (HPLC-UV) and confirmation by mass spectrometry (MS); the method uses selected gel permeation chromatography (GPC) and a 2-layered column for solid-phase extraction system. An aliquot of the crude sample extract obtained by acetonitrile extraction is loaded into the GPC system. The fraction containing spinosyns A and D is selectively collected and loaded directly onto a 2-layered column consisting of graphitized carbon (upper layer) and cyclohexyl-bonded silica gel (lower layer). After the column is washed with the GPC mobile phase acetone-cyclohexane (3 + 7), the column is eluted with acetonitrile containing 2% triethylamine. The eluate is used for HPLC-UV/MS analysis. Average recoveries from fortified cabbage, green perilla, fig, and strawberry at analyte concentrations of 0.05 and 0.25 microg/g were >85%, and the relative standard deviations were <9%. The detection limits for spinosyns A and D in green perilla were 0.005 microg/g by UV detection and 0.001 microg/g by MS detection.

[Multiresidue analysis of pesticides in agricultural products by GC-ECD after GPC and graphitized carbon column cleanup].

A multiresidue method that enables determination of many pesticides in agricultural products by GC-ECD was studied. First, 63 pesticides were selected as agrochemicals commonly used in crop protection in this country, and/or found in agricultural products over the past 6 years (April 1996-March 2002) in Aichi Prefecture. A sample was extracted with acetonitrile and the acetonitrile layer was separated by salting-out. The extract was purified on a GPC system with a graphitized carbon columns, and then by a Florisil mini-column fractionation. The test solution was subjected to one-injection, dual-column GC with dual ECD (column: Stx-CLPesticides and Stx-CLPesticides2). The detection limits of the pesticides were in a suitable range (0.0001-0.01 microg/g) for monitoring pesticide residues in agricultural products. The method was applied to 203 commercial agricultural products to demonstrate its suitability for routine analysis.

Development of a solid-phase extraction method for determination of pheophorbide a and pyropheophorbide a in health foods by liquid chromatography.

A simple solid-phase extraction (SPE) method was developed for the liquid chromatography (LC) determination of pheophorbide (Phor) a and pyropheophorbide (Pyro) a in health foods such as chlorella, spirulina, etc. The food sample was extracted with 85% (v/v) acetone. The extract was acidified with hydrochloric acid and loaded on a C18 cartridge. After washing with water, Phor a and Pyro a were eluted with the LC mobile phase. Phor a and Pyro a were separated by isocratic reversed-phase LC and quantitated by fluorescence detection. The recoveries for spiked samples of chlorella and the extract were 87.1-102.0%. Commercial health foods (chlorella, spirulina, aloe, kale, Jews mallow, and green tea leaves) were analyzed using the SPE method. The values found for Phor a and Pyro a ranged from 2 to 788 microg/g and from <1 to 24 microg/g, respectively. There was no significant difference between the SPE method and the official method in Japan (spectrophotometry after liquid-liquid extraction). The advantages of the SPE method are the short extraction times, lack of emulsions, and reduced consumption of organic solvents compared with the official method in Japan. The SPE method is considered to be useful for the screening of Phor a and Pyro a in health foods.

Multiresidue analysis of pesticides in vegetables and fruits by gas chromatography/mass spectrometry after gel permeation chromatography and graphitized carbon column cleanup.

A multiresidue method for pesticides that enables quantitative, sequential analysis of a large number of vegetable and fruit samples by gas chromatography/mass spectrometry has been developed. First, 89 important target compounds were selected for monitoring, and then the appropriate internal standards for these pesticides, 14 stable isotopically labeled pesticides (surrogates), were used. The sample was extracted with acetonitrile, and the extract was cleaned up by a salting-out step followed by redissolution in ethyl acetate. Coextractives were removed automatically by gel permeation chromatography with a graphitized carbon column, and then by use of a tandem silica-gel/PSA cartridge column. Recoveries of 82 of the 89 pesticides from fortified spinach, tomato, apple, and strawberry were within a range from 70 to 120%, and the relative standard deviation values of 80 of the 89 pesticides were <5%. The method was applied to 188 commercial vegetable and fruit samples to demonstrate its use in routine analysis.

[Simultaneous determination of organophosphorus pesticides in cruciferous vegetables by dual-column GC with pulsed FPD and FTD].

We evaluated simultaneous analytical methods for organophosphorus pesticides in cruciferous vegetables by gas chromatography (GC). Firstly, 36 pesticides were selected on the basis of monitoring data (April 1996-March 2003) in Aichi Prefecture. A sample was extracted with acetonitrile and the acetonitrile layer was separated by salting-out. The extract was cleaned up with gel permeation chromatography (GPC), and then with a tandem silica-gel/PSA mini-column. The test solution was subjected to dual-column GC equipped with a pulsed FPD (P mode, Rtx-OPPesticides column) and a FTD (Rtx-OPPesticides 2 column). Organophosphorus pesticides in such sulfur-rich matrices as cabbage and radish were determined without any serious interfering peaks on the pulsed FPD chromatograms after diluting the extracts 10- to 20-fold (0.25-0.5 mg/L of sample). The method was applied to cruciferous vegetables to demonstrate its usefulness in routine analysis.