Monitoring of cyanogenic compounds behavior during the manufacturing process of sweetened bean paste.

To ensure food safety, food business operators must eliminate or reduce hazardous factors in manufacturing processes by implementing effective process controls. Since some beans are known to contain cyanogenic compounds, their distribution and use are permitted only as a raw bean paste material. Therefore, from the perspective of Hazard Analysis and Critical Control Points (HACCP), the purpose of this study is to demonstrate the validity of establishing CCPs and to determine the cyanogenic compounds in intermediate products for effectively managing hazardous substances in the manufacturing process. The previously reported method, post-column HPLC with fluorescence detection, was used for determine cyanogenic compounds in CCPs. While free cyanide ions were only detected at CCP#1, cyanoglycoside analysis was crucial throughout the manufacturing process. Results indicated a decrease in cyanoglycoside concentration as manufacturing progressed, with levels below 10 ppm in the final product. Notably, cyanoglycosides decreased significantly during the shibukiri process (soaking, boiling, and discarding water). The concentration of cyanogenic compounds in raw beans were below the regulated 500 ppm, and the concentrations in the final product were below regulated 10 ppm. In conclusion, it was found that proposed method is very useful for HACCP management to monitor the decrease of cyanide compounds in the manufacturing process.

Analysis of methamphetamine in urine by GC/MS coupled with solid-phase dispersive extraction and solid-phase derivatization.

In this study, we investigated a solid-phase dispersive extraction (SPDE) method and solid-phase derivatization method using the same solid-phase gel to extract methamphetamine (MA) from urine samples more efficiently and perform trifluoroacetic acid (TFA) derivatization for MA analysis by gas chromatography/mass spectrometry (GC/MS). N-Methylbenzylamine (NMe-BA) was added to the urine sample as a surrogate, and MA was extracted by SPDE using Oasis® HLB gel as a solid-phase agent. After drying the solid-phase gel of the SPDE, anhydrous TFA was added to the MA-absorbed HLB gel in order to derivatize MA with TFA on the solid-phase, followed by elution of the TFA derivative from this gel using ethyl acetate. As a validation of the analytical method, the limit of detection (S/N = 3) and the limit of quantification (S/N > 10) of MA were 0.002 µg/mL and 0.01 µg/mL, respectively. And the average recovery rate was 97.6-100.1%, repeatability was 5.6-10.7%, and intermediate precision was 10.1-11.4% for low (0.02 µg/mL), intermediate (0.1 µg/mL), and high (1 µg/mL) concentrations of MA added in urine. The developed pretreatment method enabled continuous extraction, cleanup, and TFA derivatization of urinary MA on the same solid-phase. Thus, urinary MA can be analyzed easily, rapidly, and with high sensitivity and accuracy.

Simultaneous determination of glyphosate, glufosinate and their metabolites in soybeans using solid-phase analytical derivatization and LC-MS/MS determination.

Glyphosate and glufosinate are the most widely used herbicides worldwide. We developed a simple and rapid analytical method for detecting glyphosate, glufosinate, and their metabolites (N-acetyl glyphosate: Gly-A, N-acetyl glufosinate: Glu-A, and 3-(hydroxymethylphosphinyl)propanoic acid: MPPA) in soybeans. The method involved extraction with water, trapping in a mini-column containing polymer-based resin with strong anion exchange groups, dehydration with acetonitrile, and solid-phase analytical derivatization at ambient temperature for 1 min using N-(tert-butyldimethylsilyl)-N-methyl trifluoroacetamide (MTBSTFA), followed by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination. This method offers a straightforward and rapid analysis, using on-solid phase dehydration and rapid derivatization at an ambient temperature with MTBSTFA, yielding reliable results for glyphosate, glufosinate, and their metabolites. The method was applied to both domestic and imported soybean samples. Glyphosate, glufosinate, and Glu-A were detected in imported feed soybeans and processed soybean meal for feed use, reflecting the current conditions of GM soybean cultivation.

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.

Elucidation of Degradation Behavior of Barbiturates in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (V).

The gastric stability of eight barbiturates (BARs) (barbital, primidone, allobarbital, phenobarbital, cyclobarbital, pentobarbital, secobarbital, and thiobutabarbital (TBB)) was examined in artificial gastric juice using LC/UV detection. Among the eight BARs, only TBB was degraded at higher temperatures. Furthermore, the degradation product of TBB was isolated, structurally analyzed, and finally identified as 5-butan-2-yl-5-ethyl-1,3-diazinane-2,4,6-trione, also known as butabarbital. The study elucidated that butabarbital was formed by substituting the sulfur atom of the carbonyl group at the 2-position of TBB with an oxygen atom under acidic condition.

Basic Principles for Setting MRLs for Pesticides in Food Commodities in Japan.

The Committee on Pesticides and Veterinary Drugs of the Food Sanitation Council under the Pharmaceutical Affairs and Food Sanitation Council set the maximum residue limits (MRLs) for residual pesticides, veterinary drugs, and feed additives in food commodities according to the basic principles for establishing MRLs for pesticides in food commodities in Japan. The basic principles consist of the following seven concepts: 1. Outline of setting Japanese MRLs for pesticide residue in food commodities; 2. Preparation of draft MRLs for pesticides in livestock commodities; 3. Preparation of draft MRLs for pesticides in fish and shellfish; 4. Technical guideline for setting MRLs for pesticides, etc., in honey; 5. Methods of setting standards for chemical substances used as pesticides in the past that are now detected as contaminants; 6. Concept of setting MRLs for pesticides at an extremely low level; and 7. Commodity groups and representative commodities regarding MRLs based on international harmonization. The present paper introduces and explains the basic principles for establishing MRLs for pesticides, veterinary drugs, and feed additives in food commodities.

Egg protein exposure estimation in risk assessment for Japanese food allergy labeling.

To assess the risk of food allergies in foods processed under the Japanese food labeling system, estimating exposure to hidden allergens is necessary. We assessed exposure to egg protein in foods processed according to the Japanese food labeling system. First, we estimated the concentration distribution of egg protein by Bayesian methods using data from the literature and the measurement of food products with precautional declarations in the labeling margin. We then estimated the food-intake portion-size distribution under two scenarios: soft drink consumption as an example of single, high-intake consumption, and confections, which are frequently consumed by children, as a realistic example of low-intake consumption. Finally, we estimated the distribution of unexpected intake of egg proteins in the form of single consumption. The mean exposure to egg protein under the high-intake scenario was estimated to be 0.0164 mg for 1-15-year-olds, 0.0171 mg for 4-15-year-olds, 0.0181 mg for 7-15-year-olds, and ≥0.0188 mg for 16-year-olds. The mean exposure to egg protein under the low-intake scenario was estimated to be 0.0018 mg for 1-15-year-olds, 0.0019 mg for 4-15-year-olds, 0.0020 mg for 7-15-year-olds, and ≥0.0022 mg for 16-year-olds. Compared to the reference dose of 2.0 mg proposed by the Joint the Food and Agriculture Organization (FAO)/World Health Organization (WHO) Expert Committee, the risk of onset of food allergies due to egg protein contamination from foods without egg labeling is considered to be extremely low under the current Japanese food labeling system.

Development of a rapid and reliable method to simultaneously detect seven food allergens in processed foods using LC-MS/MS.

Rapid analysis of multiple food allergens is required to confirm the appropriateness of food allergen labelling in processed foods. This study aimed to develop a rapid and reliable method to simultaneously detect trace amounts of seven food allergenic proteins (wheat, buckwheat, milk, egg, crustacean, peanut, and walnut) in processed foods using LC-MS/MS. Suspension-trapping (S-Trap) columns and on-line automated solid-phase extraction were used to improve the complex and time-consuming pretreatment process previously required for allergen analysis using LC-MS/MS. The developed method enabled the simultaneous detection of selected marker peptides for specific proteins derived from seven food ingredients in five types of incurred samples amended with trace amounts of allergenic proteins. The limit of detection values of the method for each protein were estimated to be <1 mg/kg. The developed analytical approach is considered an effective screening method for confirming food allergen labelling on a wide range of processed foods.

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.

[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.

Myosin Light Chain Phosphatase Plays an Important Role in Cardiac Fibrosis in a Model of Mineralocorticoid Receptor-Associated Hypertension.

BACKGROUND: Myosin phosphatase targeting subunit 2 (MYPT2) is an important subunit of cardiac MLC (myosin light chain) phosphatase, which plays a crucial role in regulating the phosphorylation of MLC to phospho-MLC (p-MLC). A recent study demonstrated mineralocorticoid receptor-related hypertension is associated with RhoA/Rho-associated kinase/MYPT1 signaling upregulation in smooth muscle cells. Our purpose is to investigate the effect of MYPT2 on cardiac function and fibrosis in mineralocorticoid receptor-related hypertension. METHODS AND RESULTS: HL-1 murine cardiomyocytes were incubated with different concentrations or durations of aldosterone. After 24-hour stimulation, aldosterone increased CTGF (connective tissue growth factor) and MYPT2 and decreased p-MLC in a dose-dependent manner. MYPT2 knockdown decreased CTGF. Cardiac-specific MYPT2-knockout (c-MYPT2-/-) mice exhibited decreased type 1 phosphatase catalytic subunit ß and increased p-MLC. A disease model of mouse was induced by subcutaneous aldosterone and 8% NaCl food for 4 weeks after uninephrectomy. Blood pressure elevation and left ventricular hypertrophy were observed in both c-MYPT2-/- and MYPT2+/+ mice, with no difference in heart weights or nuclear localization of mineralocorticoid receptor in cardiomyocytes. However, c-MYPT2-/- mice had higher ejection fraction and fractional shortening on echocardiography after aldosterone treatment. Histopathology revealed less fibrosis, reduced CTGF, and increased p-MLC in c-MYPT2-/- mice. Basal global radial strain and global longitudinal strain were higher in c-MYPT2-/- than in MYPT2+/+ mice. After aldosterone treatment, both global radial strain and global longitudinal strain remained higher in c-MYPT2-/- mice compared with MYPT2+/+ mice. CONCLUSIONS: Cardiac-specific MYPT2 knockout leads to decreased myosin light chain phosphatase and increased p-MLC. MYPT2 deletion prevented cardiac fibrosis and dysfunction in a model of mineralocorticoid receptor-associated hypertension.

An improved Simon reaction method to discriminate between methamphetamine and false-positive substances.

The conventional methamphetamine (MA) detection method using the Simon reaction can be affected by false positives owing to compounds similar to aliphatic secondary amines. In this study, we examined the new Simon reaction to improve the qualitative accuracy of MA detection to discriminate substances that give false positives in a conventional Simon reaction. After the conventional Simon reaction for MA and false positives (N-isopropylbenzylamine (NIP-BA), N-methylbenzylamine (NMe-BA), L-proline (Pro), and L-hydroxyproline (HYP)), which are colored blue, di-tert-butyl dicarbonate (t-Boc) reagent was added, and color tone changes were observed. When t-Boc was added to the false positives (NIP-BA, NMe-BA, Pro, and HYP), the colors of MA, Pro, and HYP changed to purple; NIP-BA changed to blue; and NMe-BA changed to light pink after 3 min. These results suggested that MA can be differentiated from NIP-BA and NMe-BA. Furthermore, the solid-phase chromogenic method was examined, and it was confirmed that MA could be differentiated from Pro and HYP. The method developed in this study should increase the accuracy of MA appraisal at crime scenes and contribute to the reduction of misclassifications arising from false-positive substances.

A reliable enzyme-linked immunosorbent assay for the determining of sesame proteins in raw food ingredients and in processed foods.

Sesame is a frequent cause of adverse food reactions in allergic patients. We developed a novel sandwich enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies and a unique extraction buffer for the detection and quantification of sesame proteins in processed foods and in raw food ingredients to clarify the validity of sesame labeling and for precautionary allergen labeling. The developed sandwich ELISA method is highly specific for sesame proteins. The limit of detection (LOD) and limit of quantification (LOQ) are 0.013 µg/g and 0.025 µg/g, respectively. The recoveries for incurred food samples, such as dressing, breads, sauce and pudding, ranged from 67 % to 81 %, while the repeatability and reproducibility coefficients of variation were less than 4.7 % and 4.5 %, respectively. The developed method has applicability for food products and is a reliable tool for the detection of hidden sesame proteins in raw food ingredients and in processed foods.

Elucidation of Degradation Behavior of Nitrazepam, a Benzodiazepine Drug, under Basic Conditions: Study on Degradability of Drugs in Stomach (IV).

This study investigates the stability of nitrazepam (NZP), a benzodiazepine drug, under basic conditions, since alkaline putrefactive amines and ammonia are produced once bodies are left to decompose for a long period postmortem after a murder involving NZP or an accidental overdose of NZP. The degradation of NZP in an aqueous alkaline solution was investigated by LC/photodiode array detector (PDA) where the NZP degradation product was isolated and purified by solid-phase extraction using Oasis® MCX, and its chemical structure was determined by LC/time-of-flight (TOF)-MS, NMR spectroscopy, and single-crystal X-ray crystallography. The results revealed that NZP was immediately degraded under basic conditions with 2-amino-5-nitrobenzophenone being an intermediate which further degraded to provide 2-hydroxy-5-nitrobenzophenone as the final degradation product. These results are expected to be useful in clinical chemistry and forensic science, such as the detection of drugs during postmortem examination and suspected addiction.

[Severe neuropsychiatric disorder developing during busulfan-containing regimen for stem cell transplantation].

Here we describe two patients that required interruption of a busulfan (BU) containing conditioning regimen due to severe mental disorder before stem cell transplantation. The first patient was a 66-year-old man scheduled for unrelated peripheral blood stem cell transplantation with fludarabine/BU conditioning for myelodysplastic syndrome. He received 9.6 mg/kg BU and developed hallucinations that worsened the next day. BU was stopped on the final day, but the patient became comatose (grade 4). He recovered the next day. The second patient was a 69-year-old man scheduled for autologous peripheral blood stem cell transplantation with thiotepa (TT)/BU conditioning for cerebral nervous system relapse of mantle cell lymphoma. He received 12.8 mg/kg BU and developed hallucinations. His mental symptoms worsened on the next day, and thus administration was stopped on the second day of TT. His symptoms improved the next day. Both patients were over 65 years old, and their psychiatric symptoms worsened 1-2 days after the final dose of BU. Our findings suggest that BU may cause psychiatric disorders in elderly patients. When performing BU conditioning, it may be necessary to avoid azole antifungal medication and acetaminophen and to reduce the dose or perform therapeutic dose monitoring for elderly patients.

Identification of animal species by nucleic acid chromatography of hair samples and investigation of its applicability to human biological samples.

GeneFields®-Hair is a simple analysis kit that uses nucleic acid chromatography and polymerase chain reaction (PCR) to identify animal species of hair-like food contaminants. In this study, we evaluated GeneFields®-Hair as a simple and rapid method for identifying animal species from hair-like materials collected in forensic science, such as at crime scenes. The use of this kit with other human biological materials (whole blood, head dandruff, nails, saliva, oral mucosa, sebum, and urine) was also investigated. Animal body hair samples were pretreated by grinding in a buffer solution, centrifuged, and the supernatant was used for PCR. Nucleic acid chromatography of the PCR products allowed the identification of the animal species by the presence or absence of coloration on the decision line. For human biological materials, nucleic acid chromatography was performed after the appropriate pretreatment like body hair material. The determination of some animal species was difficult, even if they had a dedicated DNA Strip determination line. Furthermore, animals from the same family but different genera were sometimes detected on the same determination line. All the human biological samples were correctly identified. Smartphone photographs of the coloration of the judgment line were processed using the ImageJ software for quantitative determination.

Development of a Solid-Phase Dispersive Extraction Method for Molecularly Imprinted Polymers and LC-MS/MS for Analysis of Clenbuterol Residues in Swine Livers and Kidneys.

BACKGROUND: Clenbuterol (CLB) is approved as a veterinary drug because of its tracheal smooth muscle and uterine relaxant effects. However, if improperly administered for the purpose of fattening livestock, CLB can remain in the organs, which may pose a health hazard to humans. OBJECTIVE: We aimed to examine the combination of molecularly imprinted polymer (MIP) and solid-phase dispersive extraction (SPDE) as a pretreatment method for swine liver and kidney, which contain more coexisting impurities than muscle tissue, and attempted to construct an analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: Swine livers and kidneys were homogenized and extracted using liquid-liquid partitioning with an ethyl acetate-n-hexane (1 + 1) mixture, followed by SPDE using an MIP gel, and measured using LC-MS/MS. For LC-MS/MS, either an absolute calibration method or isotope dilution mass spectrometry (IDMS) was used. For method validation, a recovery test (additive concentrations: 0.05 and 0.5 ng/g) was conducted, and the data were analyzed using one-way analysis of variance (ANOVA). RESULTS: The recoveries (trueness), repeatability, and intermediate precision obtained using absolute calibration were similar to those obtained using IDMS. CONCLUSION: Using MIP-SPDE as a pretreatment method for CLB in swine liver and kidney samples yielded comparable results for absolute calibration and IDMS in LC-MS/MS analysis. HIGHLIGHTS: MIP-SPDE can be used as a pretreatment method to analyze CLB in swine organs with high accuracy.

Optimization of QuEChERS Extraction for Determination of Carotenoids, Polyphenols, and Sterols in Orange Juice Using Design of Experiments and Response Surface Methodology.

Several compounds with different physical properties are present in foods, biological components, and environmental samples, and there are cases in which these must be analyzed simultaneously. However, it is difficult to extract compounds with different physical properties from the same sample using a single method. In the present study, we examined the optimal conditions for the QuEChERS extraction of several kinds of compounds from orange juice using design of experiments (DoE) and response surface methodology (RSM) to determine the optimal ratio of organic solvent to sodium chloride. We determined the optimal extraction conditions, which were within the design space, using 100% tetrahydrofuran (THF) as the extraction organic solvent and NaCl:MgSO4 = 75:25 as the salt. The developed LC/MS/MS method using QuEChERS extraction achieved specific detection and precise quantification. Finally, we measured the polyphenols, sterols, and carotenoids in citrus juice using the optimized QuEChERS extraction method before LC/MS/MS analysis. Most of the analytes were quantifiable in orange juice. The optimized method achieved ease of operation, the extraction of analytes from food samples in a short time (within 30 min), minimization of analytical residues, and reliability. The DoE and RSM approach may contribute to better optimization of the extraction conditions for the lowest number of experiments.

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.