First paralytic shellfish poison (PSP) infestation of bivalves due to toxic dinoflagellate Alexandrium tamiyavanichii, in the southeast coasts of the Seto Inland Sea, Japan

The mussel Mytilus edulis and the cultured ark shell Anadara broughtonii in the southeast coasts of the Seto Inland Sea were contaminated with paralytic shellfish poison (PSP) following the appearance of the dinoflagellate Alexandrium tamiyavanichii in early December 1999. A. tamiyavanichii plankton collected around the Straits of Naruto on December 3, 1999 showed PSP toxicity, of which 83 mol% was accounted for by GTX2, GTX3 and GTX4. Its specific toxicity was 112.5 fmol/cell, and one MU was equivalent to 7,200 cells. Toxicity values at the beginning of toxification were 4.7 MU/g for the ark shell and 7.3 MU/g for the mussel. In the former, the value remained at almost 4 MU/g, resulting in prohibition of marketing for about two months. In the latter, it sharply decreased to less than 4 MU/g. These bivalves collected during the toxification period were dissected into five tissues, mantle, adductor muscle, hepatopancreas, gills and "others", and submitted to high-performance liquid chromatography (HPLC). The cultured ark shell accumulated GTX2, GTX3 and STX as major components and GTX1, GTX4, GTX5, neoSTX, dcSTX and PX1-3 (C1-C3) as minor ones. The amount of GTX3 decreased with time, while STX tended to increase. At the early stage of PSP toxification, toxins were accumulated in the gills and "others", most of which were quickly detoxified. On the other hand, PSP of the toxified mussel consisted of GTX4 as a main component, and GTX1, GTX2, GTX3, GTX5, STX and PX1-2 (C1-C2) as minor ones. Its toxin composition pattern was similar to that of the ingested causative plankton. Its total toxin decreased soon after disappearance of the dinoflagellate. During the decrease of toxicity, PSP tended to be retained in the hepatopancreas, resulting in accumulation of 50 mol% of total toxin.     

Simultaneous determination of five antioxidants in food by HPLC with fluorescence detection

An HPLC method with fluorescence detection was developed for the determination of propyl gallate, nordihydroguaiaretic acid, butylated hydroxyanisole (2- and 3-tert-butyl-4-hydroxyanisole), tert-butylhydroquinone and octyl gallate in edible oils and foods. The antioxidants in edible oil were isolated directly with acetonitrile saturated with n-hexane. The antioxidants in food were extracted with ethyl acetate and the extract was concentrated under vacuum. They were isolated from the residue with acetonitrile saturated with n-hexane. The acetonitrile layer was centrifuged at 5,000 rpm for 10 min. The HPLC separation was performed on a Symmetry C18 column (3.5 microns, 4.6 mm i.d. x 150 mm) using a mixture of 5% acetic acid-acetonitrile-methanol (4:3:3, v/v/v) as the mobile phase and monitored by using a fluorescence detector with time programming. Sample peaks were identified by comparison of the fluorescence spectra with those of antioxidant standards. Average recoveries of fortified antioxidants at 100 micrograms/g were 72.1-99.6%. Coefficients of variation were 0.7-7.2%.     

Contents of barbaloin-related compounds in aloe drinks and their change during storage

The contents of barbaloin (BA), isoBA, aloin-dimers A, B, C, D and aloe-emodin (AE) in aloe drinks were investigated. BA and isoBA were detected in 30 of the 31 samples at the levels of 120-570 micrograms/mL and 120-580 micrograms/mL, respectively. Aloin-dimers A, B, C and D were detected in 8 of the 31 samples at the levels of 12-38 micrograms/mL, 13-39 micrograms/mL, 11-36 micrograms/mL and 16-69 micrograms/mL, respectively. AE was detected in all of the 31 samples at the levels of 0.03-1.3 micrograms/mL. When aloe drinks were stored for 4 weeks at 5 degrees C after opening the bottle, decrease of BA and isoBA, and increase of AE and aloin-dimers A, B, C and D were observed in most cases. However, in a few aloe drinks, all of BA, isoBA, aloin-dimers A, B, C, D and AE decreased. In these drinks, the existence of aloin-trimer was elucidated by LC/MS analyses. These data suggested that BA in aloe drinks is converted to the dimer and then to the trimer during storage.     

Study on the determination of pesticide residues in crops by ion-trap GC/MS/MS

Ion-trap GC/MS/MS was evaluated for the multi-residue determination of pesticides in agricultural products. Matrices were extracted from samples (spinach, carrot, onion and brown rice) with acetone and submitted to gel permeation chromatography, followed by a clean-up step through a graphite carbon cartridge. Thirty-five pesticides were added to either matrix, and analyzed by GC/MS/MS. Detection limits of pesticides by GC/MS/MS was almost the same as those by GC/MS (SIM). Coefficients of variation of peak area in 5 measurements of each pesticide at 0.1 microgram/mL or 0.05 microgram/mL with or without matrices were mostly acceptable, though those of 20 pesticides out of 35 were higher than 10% at a concentration of 0.02 microgram/mL. It was indicated that matrix artifacts, which interfere with GC/MS-Scan analysis, could be eliminated in some cases by using GC/MS/MS.     

Determination of sucralose in foods by HPLC using pre-column derivatization

The development of a sensitive pre-column derivatization high-performance liquid chromatography (HPLC) method for determination of sucralose is reported. Sucralose is converted into a strongly ultraviolet (UV)-absorbing derivative, possessing strong absorption at 260 nm, by treatment with p-nitrobenzoyl chloride (PNBCl). Homogenized samples were dialyzed and washed with a Bond Elut ENV cartridge, then the eluate was evaporated to dryness and the residue was derivatized. Subsequently, the sucralose derivative was purified with hexane-ethyl actate (9:1) in a silica cartridge, and then the sucralose derivative was eluted with acetone. HPLC was performed on a phenyl column, using acetonitrile-water (73:27) as a mobile phase with UV detection (260 nm). The calibration curve was linear in the range of 1 microgram/mL to 50 micrograms/mL of sucralose. The recoveries of sucralose from eight kinds of foods spiked at the levels of 0.20 and 0.05 g/kg of sucralose were more than 76.2% with SD values in the range from 0.90% to 4.31%. The quantitative limit of the developed method was 0.005 g/kg for sucralose in samples.     

Toxicity and tetramine contents of salivary glands from carnivorous gastropods

Salivary glands from 29 species of marine carnivorous gastropods in nine families were examined for lethal activity against mice and tetramine content. Mouse lethality was assayed by intravenous injection of buffer extracts into mice, and was detected in 14 species. Heat-stability tests confirmed that toxins in four species were thermolabile, while those in eight species were thermostable. Based on the tetramine contents determined by the colorimetric method using methanolic extracts, the thermostable toxins in seven species (Neptunea eulimatalamellosa, N. vinosa, N. arthritica, N. bulbacea, N. intersculpta f. pribiloffensis, N. intersculpta f. frater pilsbry and Hemifusus tuba) were considered to be tetramine contained at high levels (more than 900 micrograms/g salivary gland), but that in one species (Buccinum opisthoplectum) appeared to be a low-molecular-weight compound differing from tetramine. It is interesting that one (Hemifusus tuba) of the seven species containing high amounts of tetramine belongs to the family Melongenidae, although the other six Neptunea species are members of the family Buccinidae, as expected from previous studies.     

An investigation of the pH-activity relationships of Cex, a family 10 xylanase from Cellulomonas fimi: xylan inhibition and the influence of nitro-substituted aryl-beta-D-xylobiosides on xylanase activity

The kinetic parameters of Cex, a family 10 xylanase from Cellulomonas fimi, were determined at various pH levels using soluble birchwood xylan (BWX) as a natural polymeric substrate along with three other synthetic aryl-beta-D-xylobioside substrates. Using BWX, a high level of substrate inhibition was observed which increased with decreasing pH. In contrast, typical Michaelis-Menten-type profiles were obtained using the three aryl-beta-D-xylobiosides as substrates. The k(cat) values determined using o-nitrophenyl-beta-D-xylobioside did not change as the pH increased, whereas the k(cat) values obtained with BWX, phenyl-beta-D-xylobioside and p-nitrophenyl-beta-D-xylobioside decreased, suggesting that the presence of an ortho nitro group affects the activity displayed by Cex. These differences were not observed with XynB from Clostridium stercorarium F9, a member of the same family of xylanases as Cex. These results indicate that a careful evaluation is required when employing substituted aryl-beta-D-xylobiosides in the characterization of xylanases.     

Kinetic resolution of an indan derivative using Bacillus sp. SUI-12: synthesis of a key intermediate of the melatonin receptor agonist TAK-375

The chiral indan derivative (S)-2 (2-[(8S)-1,6,7,8-tetrahydro-2H-indeno[5,4-b]furan-8-yl]ethyl-amine) was synthesized by enzyme-catalyzed asymmetric hydrolysis of the racemic acetamide 1 (N-[2-(1,6,7,8-tetrahydro-2H-indeno[5,4-b]furan-8-yl)ethyl]acetamide). The reaction was carried out using Bacillus sp. SUI-12 screened for the ability to hydrolyze 1 to give (S)-2 with high enantioselectivity. In a scaled-up experiment, a low reaction rate was observed. However, by changing the culture medium and the reaction conditions, it became possible to run the reaction to 40% conversion on a 10-g or more scale, obtaining (S)-2 at >;99% enantiomeric excess (ee). The (S)-2 obtained was available for the synthesis of the melatonin receptor agonist TAK-375 (N-[2-[(8S)-1,6,7,8-tetrahydro-2H-indeno[5,4-b]furan-8-yl]ethyl]propanamide).