Species Identification of Pufferfish Products Using RAPD Analysis.

A simple and rapid method was developed to identify the source species of pufferfish products. Randomly amplified polymorphic DNA (RAPD) analysis was applied to identify 8 species of pufferfish. Commercial kits were used for DNA extraction and amplification. Simultaneous identification was possible by polyacrylamide gel electrophoresis of PCR products. Two primers were chosen based on the result of pre-examination with 40 primers, and the PCR conditions were optimized. Characteristic RAPD patterns were obtained for each pufferfish species. The developed method was applied to identify the source species of 26 pufferfish products. The results suggest that the developed method would be useful for verification of the labeled species of pufferfish products.

[Determination method of ultra-high-intensity sweetener, advantame, in processed foods by HPLC and LC-MS/MS].

A simple method using HPLC and LC-MS/MS was developed for the determination of ultra-high-intensity sweetener, advantame, in processed foods. Advantame was extracted by dialysis, and cleaned up on a Sep-Pak Plus C18 cartridge, then determined by HPLC and LC-MS/MS. The recoveries from 5 kinds of processed foods fortified at the levels of 0.001 g/kg and 0.01 g/kg were 64.1-89.9% (RSD 0.9-6.9%) by HPLC and 68.8-99.9% (RSD 0.8-4.9%) by LC-MS/MS. The quantitation limit was 0.0004 g/kg by HPLC and 0.00004 g/kg by LC-MS/MS.

Observed distribution of radiocaesium contamination in shiitake lots and variability of test results.

Shiitake mushrooms (Lentinula edodes) cultivated on bed-log are known to accumulate radiocaesium. Since the Fukushima-Diichi nuclear power plant accident (2011), the violation rate has been higher for log-cultivated shiitake than that for agricultural products or other foodstuffs. When testing shiitake mushrooms for radionuclide contamination, the validation of the sampling plan can be severely compromised by the heterogeneous contamination within shiitake lots. Currently, few data are available on the statistical properties of the radiocaesium contamination of log-cultivated shiitake. In this paper, shiitake lots contaminated by radiocaesium were identified and the distribution of the radiocaesium concentration within the lots investigated. The risk of misclassifying shiitake lots was predicted from the operating characteristic curve generated from Monte Carlo simulations and the performance of various sampling plans was evaluated. This study provides useful information for deciding on an acceptable level of misclassification risk.

[Determination method of linamarin in cassava products and beans by ultra high performance liquid chromatography with tandem mass spectrometry].

A rapid and simple method using ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was developed for the determination of linamarin in cassava products and beans containing cyanogen compounds. Linamarin was extracted with acetonitrile-water (3 : 1) cleaned up using an amino solid-phase extraction cartridge, and then determined by UHPLC-MS/MS. The recoveries from cassava fortified at the levels of 10 µg/g and 100 µg/g were 96.1% and 95.3%, respectively, and their relative standard deviations were 2.6% and 1.4%, respectively. The recoveries from tapioca fortified at the levels of 1 µg/g, 10 µg/g and 100 µg/g were 81.1%, 91.9% and 97.1%, respectively, and their relative standard deviations were 3.3%, 5.4% and 2.1%, respectively. The contents of linamarin in 14 cassava, 9 tapioca and 4 butter bean samples were surveyed by this method, and linamarin was detected in the range of 0.1-245 µg/g in 11 cassava, 0.1-0.5 µg/g in 5 tapioca and 4,950-5,590 µg/g in 4 butter bean samples. The quantitation limit was 0.1 µg/g and the detection limit was 0.03 µg/g.

[Examination of radioactive contamination in foods].

Following the Fukushima nuclear plant accident in Mar. 2011, the examination of radioactive contamination in foods is being carried out in Nagoya. During the period between 30 Mar. 2011 and 31 Oct. 2012, a total of 300 food samples were collected and the concentrations of radioactive nuclides were determined by means of γ-ray spectrometry using a high-purity germanium semiconductor detector. The results of analysis indicate that the concentrations of radioactive iodine (I) and cesium (Cs) were below the regulatory limits. Radioactive I ((131)I) was detected in 7 samples which belonged to the categories of green and yellow vegetables and other vegetables. Radioactive Cs ((134)Cs and (137)Cs) was detected in 60 samples which belonged to the categories of rice and its processed products, potatoes and its processed products, nuts and seeds, green and yellow vegetables, other vegetables, fruits, mushrooms, fishes and shellfishes, processed sea foods, meat, milk and dairy products and other beverages.

[Estimation of the intake of radioactive cesium based on analysis of total diet samples in Nagoya].

Food samples were purchased in Nagoya based on daily intake in the Tokai region, and prepared as total diet samples according to the market basket method. The contents of radioactive cesium (Cs) were determined by using a γ-ray spectrometer with a germanium semiconductor detector, and a committed effective dose was estimated. Radioactive Cs was not detected in samples collected in 2006 before the Fukushima nuclear plant accident. Radioactive Cs was detected in samples prepared in August, 2011, five months after the accident. The sources were sugar and confectioneries (3rd food group), other vegetables, seaweeds and mushrooms (8th food group) and fishes, shellfishes and processed seafoods (10th food group). Only Cs-137 was detected in samples prepared in August, 2012, one year and five months after the accident. The sources were the 8th and the 10th food groups. The estimated committed effective dose for radioactive Cs was 0.0015 mSv in 2011 and 0.00016 mSv in 2012.

[Species identification of animal hair present as a contaminant in food by PCR-APLP method].

A rapid, simple and inexpensive method was developed for identifying the species of animal hair present as a contaminant in food. A polymerase chain reaction-amplified product length polymorphism (PCR-APLP) assay was applied to identify hair from human and others (cat, dog, rabbit, rat and mouse) or livestock (pig, cattle, horse, sheep, goat and chicken). The PCR primers were designed to amplify partial sequences from the 16S rRNA gene to the NADH dehydrogenase subunit 1 (ND1) gene of mitochondrial DNA (mtDNA), which generate different length fragments for different animal species. The PCR-APLP assay utilized two PCR reaction tubes, each of which contained one universal forward primer and six species-specific reverse primers (human, etc. or livestock). Simultaneous identification was possible by agarose gel electrophoresis of PCR products. The developed method was applied to identify the source species of 52 animal hair samples. The expected amplified product length was obtained from all samples.

[Analytical method for carbamate pesticides in processed foods by LC/MS/MS].

A rapid analytical method for the simultaneous determination of carbamate pesticides in processed foods was established by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The pesticides were extracted from samples with acetonitrile using accelerated solvent extract equipment, except for the fine powder type spices, which were extracted in an ultrasonic bath. The crude extract was cleaned up with a multi-solvent GPC column (Shodex Asahipak GF-310 HQ) using acetonitrile as a mobile phase. The eluent from the column at the retention time between 13 to 18 min was concentrated under nitrogen gas and dissolved in a mixture of acetonitrile-water-0.2 mol/L ammonium formate buffer pH 6.0 (10 : 9 : 1). An aliquot was injected into the LC/MS/MS using electrospray ionization (ESI) with acquisition in the positive mode. The recoveries of 29 kinds of pesticide from dried fruits (raisin, prune and mango) and spices (turmeric, masala, sage, thyme and red pepper) fortified at levels of 0.1 and 0.01 microg/g were mostly in the range of 50 to 150% and those from soybean paste and soy sauce fortified at 0.01 microg/g were 46.9 to 122.6% (C.V. 3.8 to 37.6%), except for 4 kinds of pesticide. The determination limits (S/N> or =10) corresponded to 0.001 to 0.05 mug/g of the pesticides in red pepper.

[Simultaneous determination of veterinary drugs in livestock foods and seafoods using liquid chromatography/tandem mass spectrometry].

A simultaneous determination of veterinary drugs in livestock food and seafood using liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed. Veterinary drugs were extracted with 95% acetonitrile. The solution was passed through a Florisil column, and the solvent was replaced with phosphate buffer. The extract was charged on a Sep-Pak Plus C(18) mini-column and divided into 40% methanol eluate fraction and 70% acetonitrile eluate fraction. Test solutions were analyzed by LC/MS/MS with gradient elution. By using this method, 37 kinds of veterinary drugs were obtained with over 60% recovery, and quantitation was possible in cattle muscle, egg and fish. This method was inapplicable to 28 kinds of veterinary drugs. Although quantitation was not achieved, 42 other kinds of veterinary drugs can be screened. Since the limit of quantitation for this method is less than the provisional limit in general, it is useful as a screening method in residual analysis of veterinary drugs.

Determination and confirmation of five phenolic antioxidants in foods by LC/MS and GC/MS.

Identification and determination of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) by means of LC/MS and GC/MS were examined. These five phenolic antioxidants were detected as their pseudo-molecular ions [M-H]- by LC/MS using a Shim-pack FC-ODS column with drying gas. Moreover, BHA, BHT and TBHQ were detected based on their mass fragment ions by GC/MS. Decomposition of TBHQ, NDGA and PG during analysis could be prevented by the addition of L-ascorbic acid (AsA) to the extraction solvent. All five antioxidants were extracted from nikuman, olive oils, peanut butter, pasta sauce and chewing gum with a mixture of acetonitrile-2-propanol-ethanol (2:1:1) containing 0.1% AsA (AsA mixture), which had been cooled in a freezer and filtered. One part filtrate and 5 parts water were mixed and placed on a Mega-Bond Elut C18 cartridge, except in the case of chewing gum. Lipids in foods were removed on a C18 cartridge by washing with 5 mL of 5% acetic acid, and antioxidants were eluted with 5 mL of AsA mixture. The antioxidants spiked into nikuman, olive oil, peanut butter, pasta sauce and chewing gum were successfully identified and their concentrations determined by LC/MS, and GC/MS with good recoveries.

[Determination of dl-alpha-tocopherol acetate and dl-alpha-tocopherol in foods by HPLC using post-column photochemical reaction].

A reliable analytical method for the simultaneous determination of dl-alpha-tocopherol acetate and dl-alpha-tocopherol in foods was established by HPLC using post-column photochemical reaction with UV and fluorescence detection. For low-fat food such as fruit juice and vegetable sauce, the tocopherols were extracted with methanol containing 0.1% ascorbic acid and the extract solution was injected into the HPLC. For fatty foods such as butter and margarine, the tocopherols were extracted with a mixed solvent of acetonitrile-2-propanol (9:1) containing ascorbic acid. The extract was cleaned up using a Sep Pak plus C18 cartridge and the eluent from the cartridge was injected into the HPLC. The peaks corresponding to tocopherols on the chromatogram were confirmed by comparing their UV spectra with those of the standard mixture at lamp-on and lamp-off of the photochemical reactor. The recoveries of tocopherols from low-fat foods (orange juice and barbecue sauce) fortified at levels of 10 and 100 microg/kg each were 88.3 to 105.8% (RSD 0.5 to 6.0%) and those from the fatty foods (peanut butter and margarine) fortified at 100 microg/kg each were 57.1 to 88.3% (RSD 3.0 to 6.4%). The determination limits corresponded to 10 microg/kg of the tocopherols in the low-fat foods and 20 microg/kg in the fatty foods.

[Determination of acrylamide in processed foods by column-switching HPLC with UV detection].

A simple and convenient analytical method for the determination of acrylamide in processed foods was established. Acrylamide was extracted with water in an ultrasonic bath. The extract was passed through an OASIS HLB cartridge and the eluate was injected into the HPLC system using a column-switching technique. The HPLC system consisted of two pumps, two 6-port-2-position valves, two columns and a UV detector. At first, the sample solution was chromatographed on an ODS column with a mobile phase of water, then the flow of the mobile phase was switched using a 6-port-2-position valve, and the acrylamide peak fraction was introduced into an aqueous gel permeation column (analytical column). The fraction was chromatographed again on the analytical column with a mobile phase of water, and the eluate was monitored with a UV detector (205 nm). The recoveries of acrylamide from potato chips, fried potato, croquette and instant noodle fortified at levels of 50 to 1,000 micrograms/kg were 93.1 to 101.5% and the coefficient of variation was 1.5 to 5.2%. The detection limit corresponded to 10 micrograms/kg in processed foods. Forty-six samples, potato chips (11), fried potato (10), croquette (20) and instant noodle (5), were analyzed by this method. The acrylamide level was 67-4,499 micrograms/kg for potato chips, 125-1,183 micrograms/kg for fried potato, nd-255 micrograms/kg for croquette and nd-151 micrograms/kg for instant noodle.