[Validation Procedure for a Multi-Element Analytical Method Aiming at Dietary Intake Estimation].

Total diet study (TDS) is a useful way to estimate the dietary intake of hazardous and chemical substances. Regarding the analysis performed in TDS, international guidelines published by the World Health Organization, recommend selecting and confirming the validity of suitable analytical methods to achieve the purpose of TDS. However, concrete procedures and/or approaches for confirming the validity of suitable methods have yet to be established. In the present study, we aimed to develop samples, referred to as SEMPs; Samples to estimate methods performance, that can be used to evaluate the performance of the analytical methods applied to the composite samples prepared in TDS. The concentrations of 14 kinds of elements, including hazardous substances such as arsenic, lead, and cadmium, in SEMPs were measured for use in the validation of a multi-element analytical method for estimating dietary intake. After examining the appropriate amount of relevant elements added to the samples, we established a performance evaluation procedure by repeatedly analyzing five fortified and non-fortified SEMPs each.

[Development of a Rapid Determination Method for Strontium-90 in Foods Using a Strontium Extraction Column for Use during Emergency Events].

To ensure food safety during emergency events such as nuclear disasters, we developed a practical rapid determination method for strontium-90 (Sr-90) in foods. Purification of Sr from foods was simplified using a commercial Sr-extraction column. We also reduced the waiting time to achieve radiative equilibrium between Sr-90 and Y-90. Finally, we developed a rapid determination method for Sr-90 that can be completed in about a week. Using the new method, stable Sr recoveries exceeded 85%. The trueness of the method ranged from 109 to 115% and the detection limit of Sr-90 was estimated to be 0.07 Bq/kg fresh weight according to a performance evaluation using standard materials. Sr-90 radioactivity concentrations in food samples determined by the new method were highly correlated and nearly equal to concentrations determined by the conventional method. The present study suggests that the new method offers highly sensitive and rapid detection of Sr-90 which are necessary attributes for food tests during emergency events.

[Concentration of Radioactive Cesium in Domestic Foods Collected from the Japanese Market in Fiscal Years 2014-2016].

Between fiscal years 2014 and 2016, we surveyed the concentration of radioactive cesium in commercial foods produced in areas where there is a risk of radiation contamination due to the Fukushima Daiichi nuclear disaster. The number of samples with a concentration of radioactive cesium that exceeded the regulatory limit （100 Bq/kg for general foods） was 9 out of 1,516 （0.6％） in fiscal 2014, 12 out of 900 （1.3％） in fiscal 2015, and 10 out of 654 （1.5％） in fiscal 2016. Even though some samples were expected to be contaminated with radioactive cesium, because wild mushrooms and edible wild plants were intentionally included in this survey, the percentage of samples that exceeded the regulatory limit was only around 1％. The surveillance results confirmed that the pre-shipment food monitoring conducted by local governments was properly and efficiently performed, although continuous monitoring of the concentration of radioactive cesium in cultivated and wild mushrooms, edible wild plants, and wild animal meats is still required.

Simultaneous determination of seven anticoagulant rodenticides in agricultural products by gel permeation chromatography and liquid chromatography-tandem mass spectrometry.

A sensitive and reliable method for the simultaneous determination of hydroxycoumarin-type (brodifacoum, bromadiolone, coumatetralyl, and warfarin) and indandione-type (chlorophacinone, diphacinone, and pindone) rodenticides in agricultural products by gel permeation chromatography (GPC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The procedure involved extraction of the rodenticides from samples with acetone, followed by liquid-liquid partitioning with hexane/ethyl acetate (1:1, v/v) and 10% sodium chloride aqueous solution, then cleanup using GPC, and finally, analysis using LC-MS/MS. High recoveries from the GPC column were obtained for all rodenticides tested using a mobile phase of acetone/cyclohexane/triethylamine (400:1600:1, v/v/v). An ODS column, which contains low levels of metal impurities, gave satisfactory peak shapes for both hydroxycoumarin- and indandione-type rodenticides in the LC-MS/MS separation. The average recoveries of rodenticides from eight agricultural foods (apple, eggplant, cabbage, orange, potato, tomato, brown rice, and soybean) fortified at 0.0005-0.001 mg/kg ranged from 76 to 116%, except for bromadiolone in orange (53%) and diphacinone in soybean (54%), and the relative standard deviations ranged from 1 to 16%. The proposed method effectively removed interfering components, such as pigments and lipids, and showed high selectivity. In addition, the matrix effects were negligible for most of the rodenticide/food combinations. The results suggest that the proposed method is reliable and suitable for determining hydroxycoumarin- and indandione-type rodenticides in agricultural products.

Simultaneous determination of acidic pesticides in vegetables and fruits by liquid chromatography--tandem mass spectrometry.

A sensitive and efficient method has been developed for the simultaneous determination of 73 multi-class acidic pesticides, such as phenoxy acid and sulfonylurea herbicides, in vegetables and fruits. The sample preparation procedure was carefully optimized for the efficient removal of co-extracted matrix components. The method involves extraction of acidic pesticides with acetonitrile containing hydrochloric acid, removal of water from crude extract by salting out, and sequential cleanup by octadecylsilyl silica gel and silica gel columns. For samples containing high amounts of pigments, such as spinach, additional cleanup using a graphitized carbon column was performed prior to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Recovery tests were performed for five times for each sample of cabbage, spinach, potato, eggplant, orange, and apple fortified at 0.01 mg kg-1. Out of the 73 tested pesticides, 70 for cabbage, 67 for spinach, 69 for potato, 67 for eggplant, 64 for orange, and 70 for apple were within the range of 70-120%, with relative standard deviations below 25%. Nitenpyram and pyrasulfotole showed low recoveries for all the samples tested, probably due to low recoveries from silica gel column. The developed method effectively removed co-extracted matrix components and was highly selective, with no interfering peaks found in the chromatograms of blank samples. The overall results indicate that the developed method is suitable for the quantitative analysis of acidic pesticide residues in vegetables and fruits.

Characterization of natural aryl hydrocarbon receptor agonists from cassia seed and rosemary.

Many recent studies have suggested that activation of the aryl hydrocarbon receptor (AhR) reduces immune responses, thus suppressing allergies and autoimmune diseases. In our continuing study on natural AhR agonists in foods, we examined the influence of 37 health food materials on the AhR using a reporter gene assay, and found that aqueous ethanol extracts of cassia seed and rosemary had particularly high AhR activity. To characterize the AhR-activating substances in these samples, the chemical constituents of the respective extracts were identified. From an active ethyl acetate fraction of the cassia seed extract, eight aromatic compounds were isolated. Among these compounds, aurantio-obtusin, an anthraquinone, elicited marked AhR activation. Chromatographic separation of an active ethyl acetate fraction of the rosemary extract gave nine compounds. Among these compounds, cirsimaritin induced AhR activity at 10-10² µM, and nepitrin and homoplantagenin, which are flavone glucosides, showed marked AhR activation at 10-10³ µM.

[Quality assurance of the chemical analysis measurements of foods].

This document outlined the quality assurance of measurements in the chemical analysis practiced in the food testing in Japan. The quality required for a measurement is the confidence, but necessary degree of confidence is dependent on the intended use of the measurement. The recognition of the purpose of measurement is important in quality assurance of measurements. Once the required quality is decided, the quality of the measurement is assured by various quality assurance means. The international documents about quality assurance of measurement are introduced in this document, as well as the domestic notifications enforced in Japan. Means such as the validation of analytical method and the internal quality control are explained. The concept of the measurement uncertainty is also introduced.

Dietary Exposure of the Japanese General Population to Elements: Total Diet Study 2013-2018.

Some countries have conducted a total diet study (TDS) focused on the estimation of specific trace elements. Although some results of a Japanese TDS examining trace elements were published, there have been no reports of a nationwide TDS across Japan over a multi-year period to estimate the level of exposure to multiple elements. In the present study, a TDS using a market basket approach was performed to estimate the dietary exposure levels of the general population of Japan to 15 elements, including aluminum (Al), total arsenic (tAs), boron (B), barium (Ba), cadmium (Cd), cobalt (Co), chromium (Cr), total mercury (THg), molybdenum (Mo), nickel (Ni), lead (Pb), antimony (Sb), selenium (Se), tin (Sn), and uranium (U). Samples prepared in eight regions across Japan over a 6-year period were analyzed using validated methods. The robust mean exposure estimates for Al, tAs, B, Ba, Cd, Co, Cr, THg, Mo, Ni, Pb, Sb, Se, Sn, and U were 48, 4.2, 29, 8.6, 0.35, 0.17, 0.49, 0.14, 4.2, 2.8, 0.15, 0.022, 1.8, 0.10, and 0.021 µg/kg body weight/day, respectively. Although the variability in exposure estimates varied greatly from element to element, the relative standard deviations calculated from the robust means and robust standard deviations were ≤ 50% for all elements except Sn. Compared against the health-based guidance values, none of the robust and precise estimates obtained for the target elements would be associated with urgent health risk concern. In addition, the estimated exposure levels were generally in agreement with previously reported estimates, indicating that health risks associated with exposure to these elements have not changed markedly nationwide in Japan in recent years.

Estimation of dietary intake of 90Sr in Japan after the Fukushima Daiichi Nuclear Power Plant accident: market basket study, 2013-2018.

Radionuclide contamination in foods has been a public concern in Japan after the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident. To estimate time and regional trends of daily intake and annual committed effective dose of strontium-90 (90Sr) after the accident, we analysed Market basket samples using a low background 2π gas-flow counter. Samples were collected from six regions, once a year from 2013 to 2018. There appeared to be little variation in estimated daily intake and annual committed effective dose of 90Sr across the time periods and regions. The estimated maximum annual committed effective dose of 90Sr was 0.00076 mSv/year, a value sufficiently lower than the intervention exemption level, 1 mSv/year, in foods in Japan. There was no noticeable difference between the range of estimated daily intake of 90Sr in this study compared with daily intake measured before the FDNPP accident. These results suggested that no obvious increase in dietary intake of 90Sr was observed after the FDNPP accident, and that the effects on commercial foods from 90Sr due to the FDNPP accident were negligible.

Aryl hydrocarbon receptor ligand activity of commercial health foods.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates toxicological effects by binding to agonists such as dioxins. We previously reported the presence of natural dioxin-like ligands in foods. To further characterise natural ligands with dioxin-like activity, we examined the influence of 50 kinds of commercial supplement and health food on the AhR, using a reporter gene assay. Some samples, prepared using soybean, sesame, or propolis as an ingredient, were revealed to show AhR-binding activity, similar to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at high concentrations. To characterise the AhR-activating substances in eight active samples, the respective extracts were subjected to fractionation with n-hexane, ethyl acetate, and water, followed by estimating their AhR activities. The n-hexane fraction of the propolis extract sample, and the ethyl acetate fractions of the other samples, showed AhR activity similar to that of TCDD, at a high concentration range. HPLC analysis of the active fractions identified isoflavones, such as daidzein and glycitein, and flavones, such as tectochrysin and chrysin, in the samples. Among these compounds, tectochrysin exhibited marked AhR activation. Flavonoids, which are characterised as natural AhR ligands, are known to have representative beneficial effects on human health. The natural AhR ligands identified in this study are known to be useful for human health. Therefore, it is considered that AhR may play a beneficial regulatory role in humans.

Probabilistic Estimation of Dietary Intake of Methylmercury from Fish in Japan.

Dietary intake of methylmercury from fish was estimated via Monte Carlo simulation using data for methylmercury concentrations in 210 fish samples and data regarding fish consumption extracted from the Japanese National Health and Nutrition Survey. The fish analyzed were classified into 5 groups according to categories used in the survey. The distribution of consumption of fish from each group was used without fitting to statistical distributions. A log-normal distribution was fitted to the distribution of methylmercury concentration in each fish group. Two random numbers that followed these distributions were generated, and a trial value was calculated by multiplying these random numbers. The trial value was divided by the body weight (50 kg) to arrive at an estimate of dietary methylmercury intake. A total of 100,000 Monte Carlo simulation iterations were performed. The estimated mean daily intake of methylmercury was 0.093 µg/kg body weight (bw)/day. This value is well below the tolerable daily intake of 0.292 µg/kg bw/day calculated from the tolerable weekly intake (2.0 µg/kg bw/week) established by the Food Safety Commission of Japan. The probability that the daily intake of methylmercury exceeds the tolerable daily intake was 7.6%. As there are no data regarding fish consumption for consecutive days, estimation of the weekly intake of methylmercury is a subject for future studies.

Concentrations of Polycyclic Aromatic Hydrocarbons in Smoked Foods in Japan.

ABSTRACT: We evaluated the performance of a gas chromatography-tandem mass spectrometry method for quantifying 16 polycyclic aromatic hydrocarbons (PAHs), which the Scientific Committee on Food and the Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Food Additives have considered to be of concern to human health and used the method to determine the PAH concentrations in smoked foods. Eighty-seven samples of smoked fish, smoked meat, smoked eggs, dried bonito flakes, and dried bonito-related soup-stock products (disposable powder packets for infusion, instant bouillons, and liquids) were purchased in Japan to analyze their content of the 16 PAHs. Because of the low certainty of some results, the analytical values for some PAHs (e.g., benzo[c]fluorene, chrysene, and dibenzo[a,h]pyrene) are given for informational purposes only. The highest median concentrations of benzo[a]pyrene and the sum of all the 16 PAHs (29 and 760 µg/kg, respectively) were found in the disposable powder packets, followed by dried bonito flakes (24 and 512 µg/kg, respectively), and instant bouillons (11 and 227 µg/kg, respectively). These concentrations were much higher than those in the other products tested. We also investigated the percentages of the PAHs transferred from dried bonito flakes and a disposable powder packet to soup stocks commonly prepared at home. These were extremely low (<4%), even though they contained relatively high concentrations of the PAHs. Finally, the intake of benzo[a]pyrene and the sum of the intakes of four PAHs, as a marker proposed by the European Food Safety Authority, were estimated based on the data from a Japanese food consumption survey and the mean concentrations found in smoked fish and smoked fish products. These estimates suggest intakes of PAHs pose a low concern for consumer health.

Continuous Estimation of Annual Committed Effective Dose of Radioactive Cesium by Market Basket Study in Japan from 2013 to 2019 after Fukushima Daiichi Nuclear Power Plant Accident.

Radionuclide contamination in foods has been a great concern after the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident. To estimate time trends of daily intake and annual committed effective dose of radionuclides after the accident, radioactive cesium (r-Cs; 134Cs and 137Cs) and potassium-40 (40K) in market basket (MB) samples prepared at 6-month intervals in periods from September 2013 to March 2019 in 15 regions of Japan were analyzed using γ-ray spectrometry. The annual committed effective dose of r-Cs, calculated at non-detected radionuclide levels assumed to be half the limit of detection (LOD), appeared to decrease gradually in 11 regions close to the FDNPP that were more likely to be affected by the accident. Differences in doses among the 15 regions were large just after the accident, but gradually decreased. In particular, 134Cs has not been detected in any MB sample in any region since September 2018, and annual committed effective dose from 134Cs in all regions was mostly constant at around 0.3 µSv/year (given the respective LODs). The maximum annual committed effective dose of r-Cs in this study was decreased from 2.7 µSv/year in September 2013 to 1.0 µSv/year in March 2019. In contrast, the range of annual committed effective dose of 40K varied from approximately 150 to 200 µSv/year during that time frame and did not change much throughout the period of this study. Although annual committed effective doses of r-Cs in regions close to the FDNPP appeared to be higher than in regions far from the FDNPP, doses in all regions are remaining at a much lower levels than the intervention exemption level, 1 mSv/year, in foods in Japan.

Orally administered apple procyanidins protect against experimental inflammatory bowel disease in mice.

Apple procyanidins (ACT) is a natural biologically active compound extracted from apple. Our recent studies have shown that ACT ameliorates the symptoms of atopic dermatitis and inhibits food-allergen-induced oral sensitization. The aim of this study was to investigate the potential protective effect and mechanism of action of ACT in a murine model of inflammatory bowel disease. We investigated the preventive effects of ACT in experimental models of colitis induced by dextran sulfate sodium (DSS) or oxazolone. Oral administration of ACT before DSS treatment attenuated the DSS-induced mortality rate and decreased body weight loss. ACT also prevented the body weight loss associated with oxazolone-induced colitis. Next we examined the effect of ACT on intraepithelial lymphocytes (IEL), which is a major T cell population in the intestine. Oral administration of ACT increased the proportions of TCRgammadelta and TCRalphabeta-CD8alphaalpha T cells in IEL and suppressed interferon gamma synthesis in stimulated IEL. In addition, ACT inhibited phorbol 12-myristate 13-acetate-induced secretion of interleukin 8 (IL-8) in intestinal epithelial cells. The combined anti-inflammatory and immunomodulatory effects of ACT on intestinal epithelial cells and IEL suggest that it may be an effective oral preventive agent for inflammatory bowel diseases.

Optimization of capillary liquid chromatography with electrochemical detection for determining femtogram levels of baicalin and baicalein on the basis of the FUMI theory.

Capillary liquid chromatography with electrochemical detection (CLC-ECD) was developed for determining the femtogram levels of baicalin and baicalein. The CLC-ECD system and the experimental conditions were optimized based on the precision (=information content) phi and efficiency (=information content/time) theta, which were calculated from a relative standard deviation (R.S.D.) by a chemometric tool called the FUMI theory. When CLC-ECD was established using a capillary column (Inertsil ODS-3, 150 mm x 0.2 mm i.d.), a sample injector fitted with a 0.2 microL injection loop, an applied potential of +650 mV vs. Ag/AgCl, and a flow rate at 1.8 microL/min, baicalin and baicalein were determined at femtogram levels. Moreover, the present method was validated using a chemometric tool and a conventional method. Since the FUMI theory makes it possible to predict R.S.D. without repetitive measurements, the chemometric tool saves considerable amounts of chemicals and experimental time, and was found to be useful for the optimization of conditions and validation for determination by CLC-ECD. The present method was applied to the analysis of Japanese Pharmacopoeia Scutellaria Root and Scutellaria baicalensis Georgi for determining baicalin and baicalein.

Detection limit of measurement of pharmaceuticals labeled with short-lived isotopes in HPLC with flow-through gamma-counter.

This paper proposes a method for estimating the detection limit, which is defined as 3.3 times the standard deviation (S.D.) of blank measurements under the situations where the repetition of measurement is difficult or impossible because of a short half-life of radioactivity. The FUMI theory, which can estimate an S.D. value without repetition in various instrumental analyses, is adopted and proved here to be available in a radio-HPLC system as well. (99m)Tc-ECD (T(1/2)=360.6 min) that is a lipophilic compound for the diagnosis of regional brain perfusion is taken as an example.

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the determination of crustacean protein in processed foods.

The labeling of foods containing material derived from crustaceans such as shrimp and crab is to become mandatory in Japan because of increases in the number of allergy patients. To ensure proper labeling, 2 novel sandwich enzyme-linked immunosorbent assay (ELISA) kits for the determination of crustacean protein in processed foods, the N kit (Nissui Pharmaceutical Co., Ltd, Ibaraki, Japan) and the M kit (Maruha Nichiro Holdings, Inc., Ibaraki, Japan), have been developed. Five types of model processed foods containing 10 and/or 11.9 microg/g crustacean soluble protein were prepared for interlaboratory evaluation of the performance of these kits. The N kit displayed a relatively high level of reproducibility relative standard deviation (interlaboratory precision; 4.0-8.4% RSDR) and sufficient recovery (65-86%) for all the model processed foods. The M kit displayed sufficient reproducibility (17.6-20.5% RSDR) and a reasonably high level of recovery (82-103%). The repeatability relative standard deviation (RSDr) values regarding the detection of crustacean proteins in the 5 model foods were mostly < 5.1% RSDr for the N kit and 9.9% RSDr for the M kit. In conclusion, the results of this interlaboratory evaluation suggest that both these ELISA kits would be very useful for detecting crustacean protein in processed foods.

Evaluation of a nitric acid-based partial-digestion method for selective determination of inorganic arsenic in rice.

Arsenic (As) uptake in human occurs via the food chain mainly. The Joint FAO/WHO Expert Committee on Food Additives has established the provisional tolerable weekly intake level for As as an inorganic As (iAs) value, because iAs in food is much more toxic than organic As. In this study, we studied an acid based partial-digestion method for the complete extraction of arsenicals from rice. HPLC/ICP-MS was used to determine the concentration of iAs selectively. The conditions adopted to extract arsenicals from a 0.5 g of finely ground rice sample were addition of 2 mL of 0.15 mol/L nitric acid and heating at 80 degrees C for 2 hr. The LOD and LOQ for iAs were 0.0024 and 0.0079 mg/kg dry weight, respectively. Recovery studies showed good accuracy. When the method was applied to ten short-grain brown rice samples, the iAs concentrations were 0.108-0.227 mg/kg dry weight and the total As concentrations were 0.118-0.260 mg/kg dry weight. Although dimethylarsinic acid was also detected in most samples, the percentage of iAs content in total As content was 62.2-96.3%. Thus, iAs was the principal As species in the short-grain brown rice samples tested.

Dioxin concentrations in dietary supplements containing animal oil on the Japanese market between 2007 and 2014.

We determined the concentrations of dioxins (polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls) in 46 dietary supplement products, containing the oil of fish, marine mammals, or egg yolk, on the Japanese market between 2007 and 2014. Dioxins were detected in 43 of the 46 products tested at concentrations from 0.00015 to 67 pg TEQ/g. The highest concentration of dioxins was found in a shark liver oil product which varied insignificantly in five batches collected over a two-year period. The dioxin intakes from these five batches reached 2.3-2.8 pg TEQ/kg bw/day, or 58%-70%, respectively, of the Japanese tolerable daily intake (TDI) of 4 pg TEQ/kg bw/day. However, the dioxin intakes from most of the other products tested were less than 5% of the TDI. Although rare, supplements based on animal oils may contain relatively high concentrations of dioxins, leading to a substantial increase in dioxin intakes.

Dietary intake of dioxins in Japan in 2016 with time trends since 1998.

Total diet samples collected from seven regions throughout Japan in 2016 were analysed for polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls (DL-PCBs), known collectively as dioxins. This led to estimates of the latest dietary intake of these contaminants for the general Japanese population (≥1 year old). The average daily intake of dioxins for a person weighing 50 kg, calculated at non-detected congener concentrations assumed to be equal to zero, was estimated to be 0.54 pg TEQ (toxic equivalents) kg-1 body weight (bw) day-1. This value is well below the tolerable daily intake of 4 pg TEQ kg-1 bw day-1 for dioxins in Japan. The average intake was highest from fish and shellfish, followed by meat and eggs. The TEQ contribution of the fish and shellfish group to the total dietary TEQs was significant (89%). The DL-PCBs accounted for about 67% of the dioxin intake. The latest dioxin intake level was compared with previous estimates from total diet study results obtained annually since 1998 to determine the time trends in the dietary intake of dioxins in Japan. Overall, the average dioxin intake appeared to be decreasing gradually during the period of study. The previous average intakes of dioxins ranged from 0.58 to 1.9 pg TEQ kg-1 bw day-1. The latest average intake was the lowest since 1998 and was about one-third of the average intake in 1998. This decreasing trend in the dietary intake of dioxins was mainly influenced by the decreased dioxin intakes from two food groups, fish and shellfish, and meat and eggs.