Novel simultaneous analysis of 18 types of glycosaminoglycan-derived disaccharides using 4-aminobenzoic acid ethyl ester derivatization by HPLC with fluorescence detection.

Glycosaminoglycans (GAGs), including hyaluronic acid (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), heparan sulfate (HS)/heparin (HP), and keratan sulfate (KS), play pivotal roles in living organisms. Generally, GAGs are analyzed after enzymatic digestion into unsaturated or saturated disaccharides. Due to high structural similarity between disaccharides, however, separation during analysis is challenging. Additionally, little is known about the structures of GAGs and their functional relationships. Elucidating the function of GAGs requires highly sensitive quantitative analytical methods. We developed a method for the simultaneous analysis of 18 types of disaccharides derived from HA (1 type), CS/DS (7 types), HS/HP (8 types), and KS (2 types) potentially detectable in analyses of human urine. The simple method involves HPLC separation with fluorescence detection following derivatization of GAG-derived disaccharides using 4-aminobenzoic acid ethyl ester (ABEE) as a pre-labeling agent and 2-picoline borane as a reductant. The ABEE derivatization reaction can be performed under aqueous conditions, and excess derivatization reagents can be easily, rapidly, and safely removed. This method enables highly sensitive simultaneous analysis of the 18 abovementioned types of GAG-derived disaccharides using HPLC with fluorescence detection in small amounts of urine (1 mL) in a single run. The versatile method described here could be applied to the analysis of GAGs in other biological samples.

Development of an Analytical Method for Unsaturated Disaccharides from Heparan Sulfate Using Dansylhydrazine Derivatization and High-Performance Liquid Chromatography with Fluorescence Detection.

Glycosaminoglycans (GAGs), such as heparan sulfate (HS), play essential roles in living organisms. Understanding the functionality of HS and its involvement in disease progression necessitates the sensitive and quantitative detection of HS-derived unsaturated disaccharides. Conventionally, fluorescence derivatization precedes the HPLC analysis of these disaccharides. However, the presence of excess unreacted derivatization reagents can inhibit rapid and sensitive analysis in chromatographic determinations. In this study, we describe analytical methods that use dansylhydrazine as a derivatization agent for the detection and determination of HS-derived unsaturated disaccharides using HPLC. In addition, we have developed a straightforward method for removing excess unreacted reagent using a MonoSpin NH2 column. This method may be employed to remove excess pre-labeling reagents, thereby facilitating the analysis of HS-derived unsaturated disaccharides with satisfactory reproducibility.

[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.

[Implementation and Evaluation of Risk Communication Regarding Residual Pesticides].

In this study, a public seminar on risk communication methods was conducted to raise awareness and disseminate accurate knowledge about residual pesticides to consumers. Additionally, surveys on consumer awareness were conducted on the attendees before and after the seminar to evaluate its effectiveness. Responses were obtained from 84 participants. The paired t-test was used to analyze the changes in awareness before and after the seminar. The results showed significant improvements in "trust in the government" and "understanding of residual pesticides." Furthermore, step-wise multiple regression analysis was performed to explore the factors influencing satisfaction with the risk communication seminar, and the item "understanding of the safety of residual pesticides in food" was extracted. Understanding food safety is a crucial concern in daily life for consumers. To enable consumers to have an accurate understanding of food risks and make appropriate judgments, it is essential to continue implementing risk communication and conveying information about food safety and security in the future.

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.

Elucidation of Degradation Behavior of Hydroxy Group-Containing Benzodiazepines in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (III).

The degradation behavior of three benzodiazepines (BZPs)-lormetazepam (LMZ), lorazepam, and oxazepam-with hydroxy groups on the diazepine ring in artificial gastric juice and the effect of storage pH conditions on drug degradability were monitored using an LC/photodiode array detector (PDA) to estimate their pharmacokinetics in the stomach. Although the three BZPs degraded in artificial gastric juice, none could be restored, despite increasing the storage pH, implying that the degradation reaction was irreversible. As for LMZ, we discussed the physicochemical parameters, such as the activation energy and activation entropy involved in the degradation reaction as well as the reaction kinetics; one of the degradation products was isolated and purified for structural analysis. In the LMZ degradation experiment, peaks corresponding to degradation products, (A) and (B), were detected through the LC/PDA measurements. Regarding the degradation behavior, we hypothesized that LMZ was degraded into (B) via (A), where (A) was an intermediate and (B) was the final product. Although the isolation of degradation product (A) was challenging, degradation product (B) could be isolated and was confirmed to be "methanone, [5-chloro-2-(methylamino)phenyl](2-chlorophenyl)-" based on structure determination using various instrumental analyses. The compound exhibited axis asymmetry as determined using single-crystal X-ray structure analysis. Because the formation of degradation product (B) was irreversible, it would be prudent to target the final degradation product (B) and LMZ for identification when detecting LMZ in human stomach contents, such as during forensic dissection.

[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.

[Pharmaceutical Ingredients Detected in Dietary Supplements Purchased by Chiba Prefecture (Fiscal Years 2014-2021)].

In the pharmaceutical ingredients contamination testing of 702 commercial dietary supplement products, during fiscal years 2014-2021, 14 pharmaceutical ingredients, barrenwort, leaf axils of senna, and small leaf of senna were detected in 28 products. Screening and confirmation of the pharmaceutical ingredients in the products were performed by ultra-high performance liquid chromatography with photodiode array detector and ultra-high performance liquid chromatography-quadrupole-kingdon trap mass spectrometry, respectively. In particular, leaf axils and small leaf of senna were identified by stereomicroscopy and scanning electron microscopy. Furthermore, we found several pharmaceutical ingredients that exceeded the daily medicated dosage; therefore, it is important to prevent the distribution of such products to prevent the occurrence of health hazard. For that reason, it is necessary to continue the sample purchasing and testing systems to monitor the distribution of products containing pharmaceutical ingredients.

Synthesis of Norgestomet and its 17ß-isomer and evaluation of their agonistic activities against progesterone receptor.

Norgestomet is a synthetic progesterone derivative applied in veterinary medicine to control estrus and ovulation in cattle. Norgestomet has been widely used in the livestock industry to promote the synchronization of estrus in cattle and increase pregnancy rates. However, highly reproducible synthetic methods for Norgestomet have been rarely reported. Here, we described a method for the synthesis of Norgestomet and performed quantitative NMR analysis to determine the purity of the products. Moreover, the agonistic activity of the synthesized compounds against progesterone receptors (PRs) was evaluated using an alkaline phosphatase assay. We synthesized Norgestomet with 97.9% purity that exhibited agonistic activity against PR with EC50 values of 4.5 nM. We also synthesized the 17ß-isomer of Norgestomet with 92.7% purity that did not exhibit any PR agonistic activity. The proposed synthetic route of Norgestomet can facilitate the assessment of residual Norgestomet in foods.

Characterization of Components in Natural Products for the Evaluation of Existing Food Additives in Japan.

In Japan, existing food additives are those included in the List of Existing Food Additives specified in the Supplementary Provisions to the Law Concerning Amendments to the Food Sanitation Law and Nutrition Improvement Law. Most of the currently available food additives are natural extracts containing various ingredients. However, the characteristic and active components of existing food additives are not always properly defined due to poor characterization of the constituents of the respective raw materials. For that reason, the characteristic components of existing food additives from natural extracts have been evaluated using various methods and reported. Here we review examples of our research on the characterization of marker constituents of existing food additives from natural products.

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.

[Evaluation of a Risk Communication Program for Pesticide Residues].

To promote and raise the awareness of accurate knowledge on pesticide residues, the symposium program on risk communication on pesticide residues was held by the broadcasted online style. The risk communication program was statistically evaluated using a pre- and post-program online questionnaire survey. We had the questionnaire answers of the 105 valid participants. The analysis of post-program questionnaires shows that the risk communication program was effective in terms of levels of understanding and interest. Pre-program risk perception or awareness of safety assessments was significantly and positively correlated with awareness for establishing standard values of pesticide residues by the analysis of pre-program questionnaires. Risk perception after the program was significantly higher than before the program, suggesting that risk perception increased due to the program by analysis the same question between pre- and post-program questionnaires. Multiple regression analysis suggests that the participants with higher pre-program awareness of safety assessments or pre-program awareness for establishing standard values appeared to have higher levels of understanding and post-program risk perception.

Development and Testing of an Individual Kernel Detection System for Genetically Modified Soybean Events in Non-identity-preserved Soybean Samples.

A genetically modified (GM) soybean kernel detection system using combination of DNA preparation from individual soybean kernels and event-specific real-time PCR was developed to simultaneously identify GM soybean events authorized for food after safety assessments in Japan. Over 100 kernels in the non-identity-preserved soybean samples imported from the United States of America (two U.S.A. lots) and Brazil (one lot) were randomly selected and examined. In total, 98 and 96% of the two independent U.S.A. lots, and 100% of the Brazilian lot contained GM soybean kernels. Herbicide-tolerant events, MON89788 (trade name Genuity® Roundup Ready 2 Yield™), GTS 40-3-2 (trade name Roundup Ready™ soybean) and A2704-12 (trade name Liberty Link® soybean), were detected similarly in both U.S.A. lots. In the Brazilian lot, in addition to GTS 40-3-2, a stacked GM event, MON87701 × MON89788, having insect-resistance and herbicide-tolerance, was detected. There were no unauthorized GM soybeans comingled, and the ratio of GM soybean events detected was consistent with statistical reports on the cultivated GM soybean events in both countries.

[Analytical Method for Fipronil and Fipronil Sulfone in Livestock Products Using LC-MS/MS].

A rapid and sensitive method for the simultaneous determination of fipronil and fipronil sulfone (metabolite B) in livestock products was developed. The analytes were extracted from samples with acidic acetonitrile. The crude extracts were subjected to clean-up step using neutral alumina cartridge column. The HPLC separation was performed on a C18 column with isocratic elution of acetonitrile and ammonium formate solution. For the determination of the analytes, a tandem mass spectrometry with negative ion electrospray ionization was used. In the recovery tests using 6 livestock products fortified with MRLs levels of analytes, the truenesses for fipronil and fipronil sulfone were 95 to 115 and 94 to 101% with the repeatabilities of 0.8 to 4.1 and 0.9 to 5.1 RSD%, respectively. The limits of quantification for both analytes were estimated to be 0.001 mg/kg. The developed method is considered suitable for regulatory analysis of fipronil and fipronil sulfone.

[Surveillance of Chromium (VI) Concentrations in Mineral Water Products in 2018].

We used ion chromatography with post column derivatization to measure chromium (VI) concentrations in 155 mineral water (MW) products purchased from markets in Japan in 2018. Chromium (VI) recovery in spiked samples of the 155 MW products ranged from 95 to 106%, suggesting that this method is well suited for ensuring that MW products meet the requisite health and food safety standards. Chromium (VI) levels were above detection limits in 54 of the MW products, giving a total detection rate of 35%. The minimum and maximum chromium (VI) concentrations in all of the MW products were 0.0001 mg/L and 0.045 mg/L, respectively. The median chromium (VI) concentration was 0.0003 mg/L, with most MW products having chromium (VI) concentrations in the range of 0.0001 to 0.0002 mg/L. None of the MW products had chromium (VI) concentrations that exceeded the current standard value (0.05 mg/L) for MW in Japan.

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.

[Changes in Status of Plasticizers Used in Polyvinyl Chloride Toys].

Analytical survey of plasticizers used in about 500 polyvinyl chloride (PVC) toys in Japanese market was performed in 2014. The results obtained were compared with those obtained in 2009. Fifteen types of plasticizers including di (2-ethylhexyl) terephthalate (DEHTP) were detected. These plasticizers were also detected in the products in 2009, suggesting that the types of plasticizers used in PVC toys have not been changed. Among these detected plasticizers, the detection ratios of DEHTP were 60.3 and 73.7% in both designated and not-designated toys, respectively, both of which are the highest. These ratios are over 20-points higher than those in 2009. The six types of phthalic acid esters (PAEs) prohibited to use for the designated toys in Japan were not detected in designated toys used in this study. However, four types of PAEs, including three types of prohibited PAEs, were detected in not-designated toys with the detection ratios from 2.8 to 15.5%. These ratios are lower than those in 2009. The content levels of plasticizers used in the toys are lower than those in 2009. These results show that the main plasticizer used in PVC toys is DEHTP, and that the usage levels of plasticizers have been decreased.