[Proteome Analysis for Identification of the Origin of Biological Foreign Substances].

We have developed a method for determination of the species of origin of biological foreign substances using proteomics analysis technology. That is, the amino acid sequence of the tryptic digested product of the protein extracted from the foreign substance is determined by high-resolution LC-MS, and the amino acid sequence is collated with a public protein database to determine the origin species of the foreign substance. As a result of testing meat (beef, pork, chicken) and egg (chicken, quail) from known origin as simulated foreign substances, we were able to find species-specific amino acid sequences for each species, suggesting that the developed method is useful for species discrimination of a foreign substances that have been heat-treated with retort, this method is potentially useful to complement DNA analysis, for determination of the species of origin of foreign substances.

[Development of a Method for Estimating the Time Point of Hair Contamination in Retort Pouch Foods].

DNA from hair that has undergone retort pouch sterilization shows considerably more fragmentation. Assessing the extent of DNA fragmentation using a PCR assay could therefore be applied to infer the sterilization history of a contaminated food sample. Such assessments could make it possible to determine whether a food sample had been contaminated by hair during the production process. To determine the extent of retort pouch sterilization, primers specific for the detection of human DNA were designed to give an amplification product of approx 500 bp. Hair was subjected to retort sterilization as a model of contamination, and PCR was performed using the extracted DNA as a template and the primer set for determining retort pouch sterilization. The results showed that no DNA amplification occurred in retort pouch samples, whereas amplification was observed in samples that were unheated, heated in hot water, or heated in a microwave oven. The present method was thus able to specifically detect thermal decomposition of DNA from hair that had undergone retort sterilization, and will be useful for determining whether hair discovered in a retort pouch was responsible for contamination.

[Simultaneous LC-MS/MS Determination of 18 Plant Toxins in Beverages].

Assuming the intentional adulteration of beverages with plant toxins, an LC-MS/MS method for the simultaneous determination of 18 plant toxins (lycorine, galantamine, ricinine, scopolamine, gelsemine, atropine, colchicine, α-solanine, jervine, α-chaconine, veratramine, mesaconitine, digoxin, protoveratrine A, aconitine, hypaconitine, oleandrin, and digitoxin) was developed. As analytical samples, beer, distilled spirits, blend tea, ready-to-drink (RTD) coffee, and fermented milk drink were selected. The extraction and purification of the analytes were performed using modified QuEChERS method. Method validation in terms of intra-day precision, accuracy, and extraction recovery obtained satisfactory results. The calibration curves for the analytes were linear from 5 to 200 ng/mL (r>0.990), which enabled the determination of toxins in even trace amounts.

Minimization of carryover for high-throughput liquid chromatography with tandem mass spectrometry analysis of 14 mycotoxins in corn grits.

A method for the simultaneous analysis of 14 mycotoxins with the minimization of carryover was developed. Our verification experiments suggested that the carryover occurred due to the chelation of fumonisins with the metal. To wash the fumonisins from the metal, the inner surface of the injection needle was rinsed with 10 mM trisodium citrate and 1% formic acid in water/methanol/acetonitrile/isopropanol after each injection, and the analysis was performed on a metal-free Mastro C18 column. This approach remarkably minimized the carryover of fumonisins. Fourteen mycotoxins in samples were extracted with 2% acetic acid in water/acetonitrile and a quick, easy, cheap, effective, rugged, and safe extraction kit, purified on a MultiSep 229 Ochra, and then quantified by liquid chromatography with tandem mass spectrometry. Determinations performed using this method produced a linearity greater than 0.99 and recoveries ranging from 72.6 to 117.4%, with good intraday precision from 4.0 to 12.4%, and interday precision from 6.5 to 17.0%. The limits of detection ranged from 0.01 to 0.71 µg/kg, demonstrating that a highly sensitive method for the simultaneous analysis of mycotoxins over a wide range of concentrations was achieved with minimal carryover. When 12 samples of commercially available corn grits were analyzed with this method, deoxynivalenol, fumonisin B1, fumonisin B2, fumonisin B3, and zearalenone were present most frequently.

Fate of mycotoxins during beer brewing and fermentation.

Mycotoxins are frequent contaminants of grains, and breweries need, therefore, to pay close attention to the risk of contamination in beer made from such grains as barley and corn. The fate of 14 types of mycotoxin (aflatoxins, fumonisins, ochratoxin A, patulin, trichothecenes, and zearalenone) during beer brewing was investigated in this study. Malt artificially spiked with each mycotoxin was put through the mashing, filtration, boiling and fermentation processes involved in brewing. After brewing, the levels of aflatoxins, ochratoxin A, patulin, and zearalenone were found to have decreased to less than 20% of their initial concentration. They had been adsorbed mainly to the spent grain and removed from the unhopped wort. Additionally, as zearalenone was known, patulin was metabolized to the less toxic compound during the fermentation process. The risk of carry-over to beer was therefore reduced for half of the mycotoxins studied. However, attention still needs to be paid to the risk of trichothecene contamination.

Simultaneous LC-MS/MS analysis of glyphosate, glufosinate, and their metabolic products in beer, barley tea, and their ingredients.

Glyphosate and glufosinate are non-selective herbicides that have been extensively used worldwide. Their ionic and water-soluble characteristics often make it difficult to analyze them, especially in food components. A method was developed in this study for the simultaneous analysis of glyphosate, glufosinate, and three metabolic products in beer, barley tea, and their ingredients (malt and corn). The analytical samples were extracted with H2O, purified with a strong anion-exchange solid-phase extraction (SPE) cartridge, and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an anion-exchange high-performance liquid chromatography (HPLC) column. This method enabled a rapid and sensitive analysis [limit of quantification (LOQ) = 10 µg/kg] of the herbicides to be achieved.

Asymmetric total synthesis of (-)-stenine and 9a-epi-stenine.

A route for the asymmetric synthesis of (-)-stenine, a member of the Stemona alkaloid family used as folk medicine in Asian countries, is described. The key features of the sequence employed include stereoselective transformations on a cyclohexane ring controlled by a chiral auxiliary unit and an intramolecular Mitsunobu reaction to construct the perhydroindole ring system. By using an intermediate in the route to (-)-stenine, an asymmetric synthesis of 9a-epi-stenine was also executed. The C(9a) stereocenter in 9a-epi-stenine was installed by using a Staudinger/aza-Wittig reaction of a keto-azide precursor followed by reduction of the resulting imine. The results of this effort demonstrate the applicability of the chiral auxiliary based strategy to the preparation of naturally occurring alkaloids that contain highly functionalized cyclohexane cores.

The fate of mycotoxins during the distillation process of barley shochu, a distilled alcoholic beverage.

Mycotoxins are frequent contaminants of grains and critical risk substances for brewers. Fermented barley mash contaminated artificially with 13 representative mycotoxins was distilled with small-scale apparatuses to elucidate the possibility of mycotoxin transfer from mash to distillates. None of these were detected in the distillates. The distillation process can effectively reduce the contamination risk posed by mycotoxins in distilled alcoholic beverages.

Fate of pesticides in a distilled spirit of barley shochu during the distillation process.

Moromi (the fermented mash) of "mugi shochu" that had been artificially contaminated with pesticides was distilled to elucidate the fate of pesticides in the distillation process. The pesticides residing in the distillate were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of the analyzed pesticides (249 compounds), 89% were not detected in the distillate, showing that the distillation process minimized the risk of pesticide contamination.

Identification of Acrylamide Adducts Generated during Storage of Canned Milk Coffee.

Since coffee is a significant contributor to the consumption of acrylamide, its reduction is required. Acrylamide is produced during the roasting of coffee beans, but the roasting process is an essential step in determining the taste of coffee. Acrylamide content in coffee has been suggested to decrease by reacting with proteins and/or other substances during storage, but details are unknown. Investigation of acrylamide adducts may contribute to a strategy for acrylamide reduction in coffee. In this study, a stable isotope labeling technique, combined with high-resolution mass spectrometry, allows the identification of acrylamide adducts (3-hydroxypyridine-acrylamide and pyridine-acrylamide) in canned milk coffee. Other acrylamide adducts derived from milk coffee proteins, Lys-acrylic acid and CysSO2-acrylic acid, were identified. During a 4-month storage period, the formation of these four adducts was found to reduce the total content of acrylamide by 75.3% in canned milk coffee. Therefore, endogenous proteins can be used in acrylamide reduction.

Discovery of novel arylpyrazole series as potent and selective opioid receptor-like 1 (ORL1) antagonists.

The synthesis and biological evaluation of new potent opioid receptor-like 1 antagonists are presented. A structure-activity relationship (SAR) study of arylpyrazole lead compound 1 obtained from library screening identified compound 31, (1S,3R)-N-{[1-(3-chloropyridin-2-yl)-5-(5-fluoro-6-methylpyridin-3-yl)-4-methyl-1H-pyrazol-3-yl]methyl}-3-fluorocyclopentanamine, which exhibits high intrinsic potency and selectivity against other opioid receptors and hERG potassium channel.

Discovery and biological profile of isoindolinone derivatives as novel metabotropic glutamate receptor 1 antagonists: a potential treatment for psychotic disorders.

We describe here the discovery and biological profile of a series of isoindolinone derivatives as developed mGluR1 antagonists. Our combined strategy of rapid parallel synthesis and conventional medicinal optimization successfully led to N-cyclopropyl 22 and N-isopropyl isoindolinone analogs 21 and 23 with improved in vivo DMPK profiles. Moreover the most advanced analog 23 showed an oral antipsychotic-like effect at a dose of 1mg/kg in an animal model.

Discovery and in vitro and in vivo profiles of 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide as novel class of an orally active metabotropic glutamate receptor 1 (mGluR1) antagonist.

We identified 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide 27 as a potent mGluR1 antagonist. The compound possessed excellent subtype selectivity and good PK profile in rats. It also demonstrated relatively potent antipsychotic-like effects in several animal models. Suitable for development as a PET tracer, compound 27 would have great potential for elucidation of mGluR1 functions in human.

A highly sensitive determination method for acrylamide in beverages, grains, and confectioneries by supercritical fluid chromatography tandem mass spectrometry.

Acrylamide (AA) analysis is an important topic in food safety. However, it is difficult to rapidly and accurately analyze low concentrations of AA with currently available methods. In the present study, we introduce a highly sensitive method that enables the determination of AA in beverages, grains, and confectioneries by supercritical fluid chromatography tandem mass spectrometry (SFC/MS/MS). The sensitivity of the SFC/MS/MS technique is 11-times higher than that obtained by ultra-high performance liquid chromatography tandem mass spectrometry. We demonstrated that the highly sensitive SFC/MS/MS method was able to quantify low concentrations of AA in beverages (i.e., roasted barley tea and coffee) extracts at less than 10 µg kg-1 level without solid-phase purification. Furthermore, the simplification of the sample preparation procedure provided an improvement in data acquisition time (60 samples per 12 h). In conclusion, the developed analytical system is a potentially useful tool for practical AA determination.

Identification of novel benzimidazole series of potent and selective ORL1 antagonists.

Structure-activity studies on benzimidazole lead 1 obtained from library screening led to the discovery of potent and selective ORL1 antagonist 28, 5-chloro-2-[(1-ethyl-1-methylpropyl)thio]-6-[4-(2-hydroxyethyl)piperazin-1-yl]-1H-benzimidazole, which is structurally distinct from conventional non-peptide antagonists known to date.

Discovery and biological profile of 4-(1-aryltriazol-4-yl)-tetrahydropyridines as an orally active new class of metabotropic glutamate receptor 1 antagonist.

We describe here the discovery and the structure-activity relationship (SAR) of a series of 4-(1-Aryltriazol-4-yl)-tetrahydropyridines as novel mGluR1 antagonists. Our extensive chemical modification of lead compound 2 successfully led to fluoropyridine analogs 7j and 1 with improved in vivo antagonistic activities. Among the evaluated compounds, chemically stable urea analog 1 showed oral antagonistic activity at dose ranges of 10-30mg/kg in an animal model.

Development of an analytical method for polycyclic aromatic hydrocarbons in coffee beverages and dark beer using novel high-sensitivity technique of supercritical fluid chromatography/mass spectrometry.

Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic substances that are mainly generated during heating in food; therefore, the European Union (EU) has regulated the amount of benzo[a]pyrene and PAH4 in various types of food. In addition, the Scientific Committee on Food of the EU and the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives have recommended that 16 PAHs should be monitored. Since coffee beverages and dark beer are roasted during manufacture, monitoring these 16 PAHs is of great importance. On the other hand, supercritical fluid chromatography (SFC) is a separation method that has garnered attention in recent years as a complement for liquid and gas chromatography. Therefore, we developed a rapid high-sensitivity analytical method for the above-mentioned 16 PAHs in coffee beverages and dark beer involving supercritical fluid chromatography/atmospheric pressure chemical ionization-mass spectrometry (SFC/APCI-MS) and simple sample preparation. In this study, we developed a novel analytical technique that increased the sensitivity of MS detection by varying the back-pressure in SFC depending on the elution of PAHs. In addition, analysis of commercially available coffee and dark beer samples in Japan showed that the risk of containing the 16 PAHs may be low.

A rapid and sensitive analysis of dithiocarbamate fungicides using modified QuEChERS method and liquid chromatography-tandem mass spectrometry.

We herein present a method for the analysis of 10 dithiocarbamate fungicides (DTCs) in beer, fruit juice, and malt samples, where the DTCs were converted into water-soluble sodium salts in the presence of NaHCO3 prior to methylation using dimethyl sulfate. Extraction of the methylated compounds from matrices was performed using a "quick, easy, cheap, effective, rugged, and safe" (QuEChERS) method. Following a dispersive solid-phase extraction as a clean-up step, the methylated compounds were detected by liquid chromatography-tandem mass spectrometry. Performance evaluation was carried out on beer, fruit juice, and malt samples using representative compounds. Accuracies of the spiked compounds from the various matrices ranged from 92.2 to 112.6%, and the limits of quantification of propineb, mancozeb, and thiuram were <0.52, <0.55, and <6.97 µg/kg, respectively. The developed method was then applied in the determination of dithiocarbamate fungicide contents in commercial beer and fruit juice samples.

A method for simultaneous determination of 20 Fusarium toxins in cereals by high-resolution liquid chromatography-Orbitrap mass spectrometry with a pentafluorophenyl column.

A high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS) method was developed for simultaneous determination of 20 Fusarium toxins (nivalenol, fusarenon-X, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, HT-2 toxin, T-2 toxin, neosolaniol, diacetoxyscirpenol, fumonisin B1, fumonisin B2, fumonisin B3, fumonisin A1, fumonisin A2, fumonisin A3, zearalenone, α-zearalenol, ß-zearalenol, α-zearalanol, and ß-zearalanol) in cereals. The separation of 20 Fusarium toxins with good peak shapes was achieved using a pentafluorophenyl column, and Orbitrap MS was able to detect accurately from cereal matrix components within ±0.77 ppm. The samples were prepared using a QuEChERS kit for extraction and a multifunctional cartridge for purification. The linearity, repeatability, and recovery of the method were >0.9964, 0.8%-14.7%, and 71%-106%, respectively. Using this method, an analysis of 34 commercially available cereals detected the presence of deoxynivalenol, 15-acetyl deoxynivalenol, fumonisin B1, fumonisin B2, fumonisin B3, fumonisn A1, fumonisin A2, fumonisin A3, and zearalenone in corn samples with high concentration and frequency. Trichothecenes was detected from wheat samples with high frequency; in particular, the concentration of deoxynivalenol was high. Conversely, α-zearalenol, ß-zearalenol, α-zearalanol, and ß-zearalanol were not detected in any of the samples.

Identification and quantification of fumonisin A1, A2, and A3 in corn by high-resolution liquid chromatography-orbitrap mass spectrometry.

Three compounds, hypothesized as fumonisin A1 (FA1), fumonisin A2 (FA2), and fumonisin A3 (FA3), were detected in a corn sample contaminated with mycotoxins by high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS). One of them has been identified as FA1 synthesized by the acetylation of fumonisin B1 (FB1), and established a method for its quantification. Herein, we identified the two remaining compounds as FA2 and FA3, which were acetylated fumonisin B2 (FB2) and fumonisin B3 (FB3), respectively. Moreover, we examined a method for the simultaneous analysis of FA1, FA2, FA3, FB1, FB2, and FB3. The corn samples were prepared by extraction using a QuEChERS kit and purification using a multifunctional cartridge. The linearity, recovery, repeatability, limit of detection, and limit of quantification of the method were >0.99, 82.9%-104.6%, 3.7%-9.5%, 0.02-0.60 µg/kg, and 0.05-1.98 µg/kg, respectively. The simultaneous analysis of the six fumonisins revealed that FA1, FA2, and FA3 were present in all corn samples contaminated with FB1, FB2, and FB3. The results suggested that corn marketed for consumption can be considered as being contaminated with both the fumonisin B-series and with fumonisin A-series. This report presents the first identification and quantification of FA1, FA2, and FA3 in corn samples.