A real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event, MON87701. First, a standard plasmid for MON87701 quantification was constructed. The conversion factor (Cf) required to calculate the amount of genetically modified organism (GMO) was experimentally determined for a real-time PCR instrument. The determined Cf for the real-time PCR instrument was 1.24. For the evaluation of the developed method, a blind test was carried out in an inter-laboratory trial. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined biases and the RSDr values were less than 30 and 13%, respectively, at all evaluated concentrations. The limit of quantitation of the method was 0.5%, and the developed method would thus be applicable for practical analyses for the detection and quantification of MON87701.
Lac dye and cochineal extract contain laccaic acids and carminic acid as the main pigments, respectively. Both laccaic acids and carminic acid are anthraquinone derivatives. 4-Aminocarminic acid (acid-stable carmine), an illegal colorant, has been detected in several processed foods. 4-Aminocarminic acid is obtained by heating cochineal extract (carminic acid) in ammonia solution. We attempted to prepare ammonia-treated lac dye and to identify the structures of the main pigment components. Ammonia-treated lac dye showed acid stability similar to that of 4-aminocarminic acid. The structures of the main pigments in ammonia-treated lac dye were analyzed using LC/MS. One of the main pigments was isolated and identified as 4-aminolaccaic acid C using various NMR techniques, including 2D-INADEQUATE. These results indicated that ammonia-treatment of lac dye results in the generation of 4-aminolaccaic acids.
A method for the determination of diphenylamine in agricultural products was developed. Diphenylamine was extracted with acetonitrile from a sample under an acidic condition, passed through a C18 cartridge column, re-extracted with n-hexane, cleaned up on a PSA cartridge column, determined by HPLC with fluorescence detector and confirmed by liquid chromatography with tandem mass spectrometry. Average recoveries (n=5) from brown rice, corn, soybeans, potato, cabbage, eggplant, spinach, orange, apple and green tea were in the range from 76.7 to 94.9%, and the relative standard deviations were from 0.6 to 5.8% at concentrations equal to the maximum residue limits (MRLs). The quantification limits were 0.01 mg/kg, which is the uniform limit in the positive list system for agricultural chemical residues in food in Japan.
The effect of pH of the clean-up process in the analysis of 11 permitted tar dyes in high protein food was investigated by using a handmade polyamide column (PA column) and Oasis HLB. Boiled fish paste spiked with the 11 dyes was extracted with appropriate solvents and the pH of the extract was adjusted to 3.0–7.0 in increments of 0.5, followed by clean-up with the PA column. At pH 3.0–5.5, precipitate formed in the extract clogged the column, and the recoveries of R3, R104 and R105 were 26–68%. At pH 6.0–7.0, clogging was not observed and the recoveries of the 3 dyes were somewhat higher, at 38–79%. The recoveries of other dyes were more than 80% at pH 3.0–7.0. With Oasis HLB, column loading was conducted at pH 11.0, and the recoveries of the 3 dyes improved to 70–83%. In conclusion, all 11 dyes could be cleaned-up with the PA column and Oasis HLB and the recoveries exceeded 70%.
We surveyed the presence of allergenic substances such as egg, milk, wheat, buckwheat, peanut, shrimp and crab in foods produced or distributed in Chiba Prefecture during the fiscal years 2004–2014. Six hundred and ninety-five samples that did not display the target allergenic substances were screened by using ELISA. Three percent of the samples (21/695 samples) were found to be positive. Wheat had the highest positivity rate of 9.7% (10/103 samples). Among the samples tested, advisory labeling was displayed in the case of 109 samples. Among these samples, 77.1%(84/109 samples) contained less than 1.0 μg/g of the specified allergen. Out of the 21 samples that tested positive in the screening-test, 16 did not display the advisory labeling.
The Japanese Food Sanitation Law sets a limit on the migration level of caprolactam for food-contacting nylon products. Here, we carried out an interlaboratory study in twenty laboratories to evaluate the performance of the official GC-FID test method and a GC-MS method as an alternative test method to the official method. Each laboratory quantified caprolactam in three test solutions in 20% ethanol as blind duplicates using GC-FID or GC-MS. The official method (GC-FID with absolute calibration) gave trueness, repeatability (RSDr) and reproducibility (RSDr) values of 96–97%, 3.3–5.4% and 4.0–6.7%, respectively. These values met the target criteria (trueness: 80–110%, RSDr: 10%, RSDr: 25%). The performance of the method was further improved by the introduction of heptalactam as an internal standard. As for GC-MS method, some values of the RSDr exceeded 10% when absolute calibration was used. However, when an internal standard was introduced, the trueness, RSDr and RSDr of GC-MS method were all acceptable at 94–96%, 2.0–4.4% and 7.0–9.4%, respectively. Therefore, GC-MS with an internal standard is available as an alternative test method to the official method.