[Analytical Method for Melengestrol Acetate in Livestock Products Using LC-MS/MS].

The present study verified that it is possible to analyze melengesterol acetate using the existing multi-residue method. Melengestrol acetate was extracted from livestock products using acidic acetonitrile acidified with acetic acid in the presence of n-hexane and anhydrous sodium sulfate. The crude extracts were cleaned up using an octadecylsilanized silica gel cartridge column. Separation by HPLC was performed using an octadecylsilanized silica gel column with linear gradient elution of 0.1 vol% formic acid and acetonitrile containing 0.1 vol% formic acid. For the determination of the analyte, tandem mass spectrometry with positive ion electrospray ionization was used. In recovery tests using four livestock products fortified with maximum residue limits levels of melengestrol acetate (0.001-0.02 mg/kg), the truenesses ranged from 82% to 100%, and the repeatabilities for the entire procedure ranged from 0.5 RSD% to 5.6 RSD%. In recovery tests using 11 livestock products fortified with 0.0005 mg/kg of melengestrol acetate, the truenesses ranged from 88% to 99%, and the repeatabilities ranged from 1.3 RSD% to 5.4 RSD%. The limit of quantification for melengestrol acetate in livestock products was 0.0005 mg/kg.

Determination of Cyanide and Cyanoglycosides in Sweetened Bean Paste by HPLC with Fluorescence Detection.

It is necessary to evaluate the efficiency of reduction for cyanide and cyanoglycosides during the manufacturing process from raw material beans to sweetened bean paste in a food hygiene control system from the viewpoint of food safety. Analytical methods for cyanide and cyanoglycoside determination in sweetened bean paste by HPLC with fluorescence detection were developed. In analysis of collection time of free cyanide in the free cyanide assay, the recovery was improved by extending the collection time, the recovery rate was >80% by 2 h. The accuracy, repeatability and intra-laboratory precision of the free cyanide assay were 82.3, 2.0, and 2.4%, respectively. The method for cyanoglycoside analysis was evaluated by 5 repeated spiked recovery experiments at a concentration of 10 ppm. The accuracy, repeatability and intra-laboratory precision of the cyanoglycoside method were 82.2, 1.9, and 3.4%, respectively. These analytical methods will enable the analysis of cyanide and cyanoglycosides in sweetened bean paste without using steam distillation method in the pretreatment.

[Method Improvement for Quantitative Analysis of Chlorophyll Degradation Compounds, Including Pheophorbide, in Chlorella Products].

An official analytical method for chlorophyll degradation compounds, including pheophorbide, in chlorella products, is described in notification Kanshoku No. 99 (May 8, 1981). However, this method has several operational issues, such as the formation of emulsion during liquid-liquid partitioning. Additionally, impurities present in the reagents (sodium sulfate decahydrate or anhydrous sodium sulfate) used to prepare saturated sodium sulfate solution can degrade pheophorbide and other related compounds, resulting in a significant decrease in analytical values. In this study, we thoroughly examined each step of the official method to enhance the operability and develop an alternative method that eliminates the need for saturated sodium sulfate solution. The developed method was evaluated for pheophorbide a and pyropheophorbide a at 100 mg%. Satisfactory analytical performance was achieved with trueness of 100% for pheophorbide a and 90% for pyropheophorbide a, and relative standard deviations of intra- and inter-day precision below 5% for both compounds. The proposed method is considered suitable for regulatory analysis of chlorophyll degradation compounds and would be useful for quality control of chlorella products.

[Estimation of the Distribution of Dietary Exposure to Lead Using Two-Dimensional Monte Carlo Simulation: An Attempt to Apply to Total Diet Samples].

The average dietary exposure to lead (Pb) in male and female Japanese individuals >1 year of age was estimated using 280 total diet samples representing 14 food groups from 10 areas over a two-year period. A probabilistic exposure estimation was performed using a two-dimensional Monte Carlo simulation (2D-MCS) with a Bayesian estimation that consided the uncertainty of the estimation process. The Bayesian estimation was performed using the likelihood function with cumulative distribution function between the lower and upper boundary values for no-detected values. The median dietary exposure to Pb was estimated as 5.82 µg/person/day. The 90% interval was 2.51-16.9 µg/person/day. Comparison with previously reported Pb exposure values indicates that the estimation of Pb exposure distribution using total diet samples is reasonable. The contribution to Pb exposure was highest in the order of food group 8 (light-colored vegetables, mushrooms, and seaweeds: 20.0±16.1%)>food group 1 (rice and rice products: 12.3±19.0%)>food group 10 (fish and shellfish: 10.5±13.9%). Owing to the high uncertainties of contribution ratios, it was not possible to identify dominant food groups contributing to Pb exposure. However, it was evident that the uncertainty of the estimation of Pb exposure was influenced by the uncertainty of Pb concentration than the uncertainty of food consumption rate. In particular, the effect of uncertainty from the Pb concentration of the food group 1 was 68.2%. When the margin of exposures were calculated, the estimated probabilities that a value would be <1 were 14.5% for developmental neurotoxicity to children (1-6 years old), 0.13% for blood pressure and 0.93% for kidney disease in Japanese individuals ≥1 year of age. The findings suggest that the health risk due to dietary Pb exposure is small but not negligible.

[An LC-MS/MS Analytical Method for Moenomycin A in Livestock Products].

A simple and sensitive method for the determination of moenomycin A residues in livestock products using LC-MS/MS was developed. Moenomycin A, a residual definition of flavophospholipol, was extracted from samples with a mixture of ammonium hydroxide and methanol (1 : 9, v/v) preheated at 50℃. The crude extracted solutions were evaporated and purified by liquid-liquid partitioning between a mixture of ammonium hydroxide, methanol and water (1 : 60 : 40, v/v/v) and ethyl acetate. The alkaline layer was taken, and cleaned up using a strong anion exchange (InertSep SAX) solid phase extraction cartridge. The LC separation was performed on an Inertsil C8 column with liner gradient elution using 0.3 vol% formic acid and acetonitrile containing 0.3 vol% formic acid. Moenomycin A was detected using tandem mass spectrometry with negative ion electrospray ionization. Recovery tests were conducted using three porcine samples (muscle, fat and liver) and chicken eggs. Samples were spiked with moenomycin A at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The trueness ranged from 79 to 93% and precision ranged from 0.5 to 2.8%. The limit of quantification (S/N≥10) of the developed method is 0.01 mg/kg. The developed method would thus be very useful for regulatory monitoring of flavophospholipol in livestock products.

[Development of Analytical Method and Surveillance ofGibberellic Acid in Banana, Cherry, and Kiwi Fruit].

Gibberellic acid (GA3) is commonly used as a plant growth regulator in many food crops owing to its essential signaling functions during plant growth and development. In Japan, a threshold for administrative action for GA3 content of 0.3 mg/kg applies in produce in which maximum residue limits have not been established. Although the threshold is based on previous studies, the GA3 concentrations in individual foods are still unknown. Thus, we surveyed the concentrations of GA3 in banana, cherry, and kiwi fruit on the Japanese market. We developed and validated a method for the analysis of GA3 using solid-phase extraction and LC-MS/MS in accordance with accepted criteria of trueness, repeatability, and selectivity. The limits of detection and of quantification were determined as 0.005 and 0.05 mg/kg, respectively, in all fruits. Concentrations of GA3 did not exceed 0.3 mg/kg regardless of ripeness, suggesting the reasonability of the current regulation of GA3 in banana, cherry, and kiwi fruit. These findings can support prompt administrative action on these fruits, contributing to the regulation of GA3 in Japan.

Monitoring Analysis of Perfluoroalkyl Substances and F-53B in Bottled Water, Tea and Juice Samples by LC-MS/MS.

Monitoring analysis of 14 per- and polyfluoroalkyl substances (PFAS), 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate (F-53B) and dodecafluoro-3H-4,8-dioxanonanoate (ADONA) in bottled drinking water, tea and juice samples was performed using LC coupled with tandem mass spectrometry (LC-MS/MS) and solid-phase extraction (SPE). In the electrospray negative ion mode, the limit of detection and limit of quantification (LOQ) values were 0.1 to 0.8 ng/mL and 0.2 to 1.6 ng/mL, respectively. The calibration curves were linear from LOQ to 50 ng/mL (r2 > 0.999). The SPE procedure (Presep PFC-II) was utilized for sample preparation and recovery rates for three standards (35, 70 and 140 ng/L) were 80.4-118.8% with relative standard deviation (RSD) ≤ 0.6%. Using the developed method, various samples (n = 54) from Japanese markets were investigated for PFAS and F-53B contamination, and values below the LOQ were observed. It is concluded that for monitoring products in the Japanese market, our method represents a significant improvement over complex techniques for the quantification of PFAS and related compounds from various foods.

Analysis of an Impurity, N-Nitrosodimethylamine, in Valsartan Drug Substances and Associated Products Using GC-MS.

Valsartan products, commonly used to treat high blood pressure and heart failure, have been recalled in many countries due to the presence of an impurity, N-nitrosodimethylamine (NDMA), in the recalled products. We present and evaluate a GC-MS-based analytical method for the determination of NDMA levels and attempt an investigation of NDMA concentrations in valsartan drug substances and associated products. The limit of detection and limit of quantification for the method were estimated to be 0.1 and 0.5 µg/g, respectively, when testing a 0.5-g sample. A good trueness (99%) with a small relative standard deviation (1.9%) was obtained for a valsartan product spiked with NDMA at a concentration of 1.0 µg/g. Additionally, a valsartan drug substance and the associated product, which were previously determined to have NDMA contamination, were analyzed by the method. The NDMA content by our method was very close to previously determined values. Finally, six samples, including valsartan drug substances and associated, commercially available products in Japan, all of which were derived from the company implicated in the NDMA contamination, were analyzed by our method, revealing that none of these samples contained detectable concentrations of NDMA. Overall, the data indicate that the present method is reliable and useful for determination of NDMA in valsartan drug substances and associated products.

[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.

Development of Species-Specific Ah Receptor-Responsive Third Generation CALUX Cell Lines with Enhanced Responsiveness and Improved Detection Limits.

The Ah receptor (AhR)-responsive CALUX (chemically activated luciferase expression) cell bioassay is commonly used for rapid screening of samples for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), dioxin-like compounds, and AhR agonists/antagonists. By increasing the number of AhR DNA recognition sites (dioxin responsive elements), we previously generated a novel third generation (G3) recombinant AhR-responsive mouse CALUX cell line (H1L7.5c3) with a significantly enhanced response to DLCs compared to existing AhR-CALUX cell bioassays. However, the elevated background luciferase activity of these cells and the absence of comparable G3 cell lines derived from other species have limited their utility for screening purposes. Here, we describe the development and characterization of species-specific G3 recombinant AhR-responsive CALUX cell lines (rat, human, and guinea pig) that exhibit significantly improved limit of detection and dramatically increased TCDD induction response. The low background luciferase activity, low minimal detection limit (0.1 pM TCDD) and enhanced induction response of the rat G3 cell line (H4L7.5c2) over the H1L7.5c3 mouse G3 cells, identifies them as a more optimal cell line for screening purposes. The utility of the new G3 CALUX cell lines were demonstrated by screening sediment extracts and a small chemical compound library for the presence of AhR agonists. The improved limit of detection and increased response of these new G3 CALUX cell lines will facilitate species-specific analysis of DLCs and AhR agonists in samples with low levels of contamination and/or in small sample volumes.

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.

Development of a processing factor prediction model for pesticides in processed tomato foods using elastic net regularization.

A novel regularized elastic net regression model was developed to predict processing factor (PF) for pesticide residues, which represents a change in the residue levels during food processing. The PF values for tomato juice, wet pomace and dry pomace in the evaluations and reports published by the Joint FAO/WHO Meeting on Pesticide Residues significantly correlated with the physicochemical properties of pesticides, and subsequently the correlation was observed in the present tomato processing study. The elastic net regression model predicted the PF values using the physicochemical properties as predictor variables for both training and test data within a 2-fold range for 80-100% of the pesticides tested in the tomato processing study while overcoming multicollinearity. These results suggest that the PF values are predictable at a certain degree of accuracy from the unique sets of physicochemical properties of pesticides using the developed model based on a processing study with representative pesticides.

Polychlorinated Biphenyl Concentrations and Estimated Intakes in Fish Oil Supplements on the Japanese Market.

Polychlorinated biphenyls (PCBs) are synthetic organic contaminants that are widespread in the environment. There are 209 PCB congeners. Fish oil produced from marine fish is widely used as a health supplement. PCB contamination of fish oil is of concern. We determined the concentrations of all 209 PCB congeners in commercially available fish oil supplements from Japan and estimated PCB intakes for humans consuming the supplements. We determined the concentrations of non-dioxin-like PCBs separately. The total PCB concentrations in 37 fish oil supplements purchased in Japan were 0.024-19 ng/g whole weight, and the non-dioxin-like PCB concentration range was also 0.024-19 ng/g whole weight. The total PCB intakes calculated for a 50 kg human consuming the supplements were 0.039-51 ng/day (0.00078-1.0 ng/(kg body weight per day)) and the non-dioxin-like PCB intake range was also 0.039-51 ng/day (0.00078-1.0 ng/(kg body weight per day)). The total PCB intakes were much lower than the tolerable daily intake of 20 ng/(kg body weight per day) recommended by the WHO. The results indicated that PCBs in the fish oil supplements pose acceptable risks to humans consuming the fish oil supplements daily.

Measuring linguistics of the wokototen chart made inductively by deciphering kunten materials.

In this paper, we focus on wokototen markings, which are a system of kunten annotations used to facilitate the reading of classical Chinese documents by Japanese readers. Using digitized data, we performed basic measurements of wokototen by using a chart that summarizes the wokototen markings of actual kunten materials described by Hiroshi Tsukishima, and we quantitatively clarified their characteristics. Kunten materials are classical Chinese books with annotations, called kunten, on the Chinese text. The wokototen is a type of kunten. In ancient East Asian countries, kunten systems were developed as a way of directly annotating Chinese documents so that they could be read and understood by non-native readers. For this reason, kunten materials and kunten are treated as historical sources for linguistic and historical research. The shape and position of a wokototen marking determines what kind of reading it indicates. The results of our basic survey quantitatively show that almost all the wokototen charts in actual kunten materials contain particles represented by "te", "ni", and "wo", the most common shapes of wokototen are dots and shapes that can be written with a single stroke, such as ｜, ─, and ＼, and that the most common places to find these markings are to the right of characters in the horizontal direction and below characters in the vertical direction.

Radiocesium Concentration in Commercially-Available Foods Produced in Japan: 2017-2019.

We investigated the concentration of radioactive cesium (r-Cs: 134Cs and 137Cs) in commercially-available foods to confirm the effectiveness of pre-shipment radioactive material inspections mainly conducted by local governments. We focused on selected production areas and foods with high probability of r-Cs detection. To this end, we evaluated 715, 685, and 683 samples using scintillation spectrometer and high-purity germanium γ-spectrometer in fiscal years 2017, 2018, and 2019, respectively. The results accounted for 9 samples (1.3%), 10 samples (1.5%), and 5 samples (0.7%) for each fiscal year exceeded the standard limit of radioactive material (100 Bq/kg as r-Cs concentration for general foods). Although we selected and evaluated foods with high probability of r-Cs detection, percentage of samples exceeding the standard limit in each fiscal year was very low, less than 2% to be exact. This suggests that food management system, including pre-shipment inspections, were effectively functioning. In addition, samples exceeding the standard limit were bound to edible wild plants and wild mushrooms, and log-cultivated mushrooms. The former is consider to be difficult for cultivation/feeding control, and the latter was know to be parts of foods greatly affected by radioactive materials. This suggests that the concentration of r-Cs in these items remains at relatively high levels. In contrast, r-Cs was not detected in items with controalble cultivation/feeding. Based on these observations, it is better to be inspected on more difficult-to-cotrol cultivation/feeding items, in order to achieve further streamlining and improving of inspection efficiency. Our results indicate that r-Cs concentration in commercially-available foods of easy-to cultivation/feeding control, such as general vegetables, fruits, and meat, have been well-controlled in Japan, however, difficult-to-cultivation/feeding control items need to be more paid attention to r-Cs concentrations.

Comprehensive analysis of a decade of cumulative radiocesium testing data for foodstuffs throughout Japan after the 2011 Fukushima Daiichi Nuclear Power Plant accident.

The unexpected accident at the Fukushima Daiichi Nuclear Power Station in Japan, which occurred on March 11th, 2011, after the Great East Japan Earthquake and tsunami struck the north-eastern coast of Japan, released radionuclides into the environment. Today, because of the amounts of radionuclides released and their relatively long half-life, the levels of radiocesium contaminating foodstuffs remain a significant food safety concern. Foodstuffs in Japan have been sampled and monitored for 134,137Cs since the accident. More than 2.5 million samples of foodstuffs have been examined with the results reported monthly during each Japanese fiscal year (FY, from April 1st to March 31st) from 2012 to 2021. A total of 5,695 samples of foodstuffs within the "general foodstuffs" category collected during this whole period and 13 foodstuffs within the "drinking water including soft drinks containing tea as a raw material" category sampled in FY 2012 were found to exceed the Japanese maximum permitted level (JML) set at 100 and 10 Bq/kg, respectively. No samples from the "milk and infant foodstuffs" category exceeded the JML (50 Bq/kg). The annual proportions of foodstuffs exceeding the JML in the "general foodstuffs" category varied between 0.37% and 2.57%, and were highest in FY 2012. The 134,137Cs concentration for more than 99% of the foodstuffs monitored and reported has been low and not exceeding the JML in recent years, except for those foodstuffs that are difficult to cultivate, feed or manage, such as wild mushrooms, plants, animals and fish. The monitoring data for foodstuffs show the current status of food safety risks from 134,137Cs contamination, particularly for cultured and aquaculture foodstuffs on the market in Japan.

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.

Chemical identification of an active component and putative neural mechanism for repellent effect of a native ant's odor on invasive species.

The invasive Argentine ants (Linepithema humile) and the red imported fire ants (Solenopsis invicta) constitute a worldwide threat, causing severe disruption to ecological systems and harming human welfare. In view of the limited success of current pest control measures, we propose here to employ repellents as means to mitigate the effect of these species. We demonstrate that cuticular hydrocarbons (CHCs) used as nestmate-recognition pheromone in the Japanese carpenter ant (Camponotus japonicus), and particularly its (Z)-9-tricosene component, induced vigorous olfactory response and intense aversion in these invasive species. (Z)-9-Tricosene, when given to their antennae, caused indiscriminate glomerular activation of antennal lobe (AL) regions, creating neural disarray and leading to aversive behavior. Considering the putative massive central neural effect, we suggest that the appropriate use of certain CHCs of native ants can facilitate aversive withdrawal of invasive ants.

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.