[Validation study on a multi-residue analysis of pesticides in agricultural products by using phosphoric acid treatment and GC-MS/MS].

A rapid method for multi-residue determination of pesticides in agricultural products was validated. The sample was cut into pieces and placed into a mixer cup containing half weight amount of 10% phosphoric acid in order to suppress degradation of easily degraded pesticides, represented by captan, and then homogenized. Pesticides in the phosphoric acid-treated sample were extracted with acetonitrile using a homogenizer, followed by salting out with anhydrous magnesium sulfate and sodium chloride. The extract was cleaned up on a C18 and graphite carbon black/PSA mini-cartridge column. Some pesticides gave tailing peaks, but these peaks became sharp and symmetrical when polyethylene glycol (PEG) 300 was added to the test solution. Recovery tests were performed on nine kinds of agricultural products (brown rice, soybean, spinach, cabbage, potato, orange, apple, strawberry, and Japanese pear) fortified with 170 pesticides at 0.01 and 0.1 µg/g. Each concentration of pesticide residue was extracted from 2 samples on 5 separate days. The trueness of the method for 147-164 pesticides in each sample was 70-120% with satisfactory repeatability and within-run reproducibility. This method is expected to useful for multi-residue analysis of pesticides in agricultural products.

[Determination of residual pesticides in processed foods manufactured from livestock foods and seafoods using ion trap GC/MS].

A simultaneous method using iontrap gas chromatography/mass spectrometry (GC/MS) was developed for the determination of pesticide residues in four processed foods (frozen Chinese dumpling, eel kabayaki, corned beef and retort curry). Pesticide residues were extracted from samples with ethyl acetate-cyclohexane (1:1) in the presence of anhydrous sodium sulfate. The extract was concentrated and the residue was dissolved in n-hexane. The lipids in the extract were removed by acetonitrile-n-hexane partitioning, following which the acetonitrile layer was cleaned up using a C(18) mini-cartridge column and a graphite carbon/PSA silica (GCB/PSA) mini-cartridge column. The limits of quantification of compounds in 4 processed foods were below 0.01 microg/g. The recoveries of 292 compounds spiked at 0.1 microg/g in 4 kinds of processed foods, and 210 to 262 pesticides showed acceptable recoveries of 70-120% with low repeatability (15%) and intermediate precision (<20%) only at the 0.1 microg/g spiked level. This method is expected to be useful for multi-residue analysis of pesticide residues in processed foods manufactured using livestock and seafoods as the main raw materials.

[Detection of wheat as an allergenic substance in models of processed foods by a nested PCR methods].

A polymerase chain reaction (PCR) method for verifying the allergen labeling of foods (i.e., the presence of wheat, buckwheat, or peanut) was adopted as the official Japanese identification test by the Ministry of Health, Labour and Welfare of Japan in 2002. We have verified the wheat labeling of several commercial food items by using the adopted PCR method. The study has revealed that some foods with positive results in the screening test yielded negative results in the identification test. When the result of the screening test disagrees with that of the identification test, the validation of food labeling is remarkably difficult. Therefore, we developed a nested PCR method with high sensitivity and specificity and employed this method in our routine testing as necessary. In this study, we examined 11 types of models of processed foods containing 10 microg/g wheat protein by using the adopted PCR and nested PCR methods; these samples were prepared by various processes and had varying physical properties. The adopted and nested PCR methods enabled the detection of wheat in 8 and 10 types of food models, respectively. The reasons for the failure in detecting the food allergens include DNA fragmentation due to the processing of food and the presence of DNA from other sources in the extracted DNA. In both PCR methods, an increase in the amount of template DNA in the PCR mixture enabled the detection of wheat DNA in all the food samples, but an excessive increase in the amount of template DNA hindered PCR amplification. These results indicate that an increase in the amount of template DNA increases the efficiency of the detection of allergens in processed foods by conventional PCR. Further investigation is needed to remove factors that inhibit PCR amplification of the extracted DNA.

[Detection of wheat as an allergenic substance in food by a nested PCR method].

A nested PCR method was developed for the detection of DNAs extracted from allergenic substances (here, wheat) in food. Because of DNA fragmentation, detection of wheat-specific DNA extracted from food, such as retort pouch food, is very difficult. Therefore, to improve the sensitivity of detection, a nested PCR primer pair (Wtr01NE2-5' and Wtr10NE5-3': amplicon size 97 bp) was newly designed within the region of the PCR products amplified by the official Japanese primer pair (Wtr01-5' and Wtr10-3'; amplicon size 141 bp) for wheat. Genomic DNAs of seven kinds of commercial processed foods containing wheat, wheat flour and three kinds of wheat flours pressure-heated at 100, 121 and 131 degrees C were extracted with a commercial ion-exchange type kit by modifying the Japanese official method. The nested PCR method involved two PCR procedures. First, PCR was performed by varying both the PCR reagents and cycling conditions of the Japanese official method. Second, PCR was performed using the first PCR products diluted 200-fold with TE buffer. The Japanese official method enabled detection of only four of the seven kinds of foods and three of the four kinds of flours (one sample was just a trace), while the nested PCR method detected all seven foods and all four flours. Investigation of the detectability of the four kinds of wheat flours depending on the size of the amplified fragment using five primer pairs showed that its size must be kept to less than approximately 100 bp. The nested PCR method significantly improved the sensitivity of detection of wheat-specific DNA.

[Detection of allergenic substances in foods by a multiplex PCR method].

A multiplex PCR (M-PCR) method was developed for the detection of DNAs of plant and three allergenic substances (wheat, buckwheat, and peanut) in foods. Genomic DNAs were extracted from allergenic substances with a commercial ion-exchange type kit. Four primer pairs suitable for the specific detection of plant DNA were designed to establish a M-PCR method detecting simultaneously the specific DNAs of plant and allergenic substances. Our four designed primer pairs and the primer pair described in the Japanese official method were applied to the specific detection of plant DNA. A primer pair of Plant01-5' and Plant01-3' (amplicon size; 161 bp) was the most suitable for the specific detection of plant DNA. M-PCR was performed to detect the specific DNAs of allergenic substances using four primer pairs, a pair of Plant01-5' and Plant01-3', and three pairs for allergenic components described in the Japanese official method. The four specific PCR bands were simultaneously amplified from genomic DNAs of allergenic substances. The proposed method is simple, rapid and inexpensive.