Determination of lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) and domoic acid in mussels by solid-phase extraction and reversed-phase liquid chromatography with tandem mass spectrometry.

An accurate and efficient method was developed for the determination of azaspiracid shellfish toxins (azaspiracids-1, -2, and -3), neurotoxic shellfish toxins (brevetoxins-2 and -3), diarrhetic shellfish toxins (okadaic acid and dinophysistoxins-1 and -2), and the amnesic shellfish toxin (domoic acid) in mussels (Mytilus galloprovincialis). Lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) were extracted with 0.5 % acetic acid in methanol under heating at 60°C to improve the extraction efficiency of okadaic acid group toxins and then cleaned up with a C18 solid-phase extraction cartridge. Domoic acid was extracted with 50 % aqueous methanol and then cleaned up with a graphitized carbon solid-phase extraction cartridge. Lipophilic marine biotoxins and domoic acid were quantified by reversed-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The developed method had insignificant matrix effects for the nine analytes and good recoveries in the range of 79.0 % to 97.6 % at three spiking levels for all analytes except brevetoxin-2 (43.8-49.8 %). The developed method was further validated by analyzing mussel tissue certified reference materials, and good agreement was observed between certified and determined values.

Simultaneous determination of ten paralytic shellfish toxins and tetrodotoxin in scallop and short-necked clam by ion-pair solid-phase extraction and hydrophilic interaction chromatography with tandem mass spectrometry.

Paralytic shellfish toxins and tetrodotoxin (puffer-fish toxin), the latter of which was recently found in bivalves from Europe, Japan, and New Zealand, are potent neurotoxins. A simple and effective clean-up procedure was developed for the simultaneous determination of ten paralytic shellfish toxins (gonyautoxins 1-6, decarbamoylgonyautoxins 2 and 3, and N-sulfocarbamoylgonyautoxins 2 and 3) and tetrodotoxin in the scallop, Mizuhopecten (Patinopecten) yessoensis, and the short-necked clam, Ruditapes philippinarum. To reduce matrix effects, 1% aqueous acetic acid extracts of the bivalves were cleaned up by ion-pair solid-phase extraction using a graphite carbon cartridge with tridecafluoroheptanoic acid as the volatile ion-pair reagent, followed by fourfold dilution. The ten paralytic shellfish toxins and tetrodotoxin were then separated on a hydrophilic interaction chromatography column and quantified by tandem mass spectrometry. The limits of detection and the limits of quantification for the ten PSTs ranged from 0.09 to 13.0 µg saxitoxin equivalents/kg and from 0.26 to 39.4 µg saxitoxin equivalents/kg, respectively. The limit of detection and the limit of quantification for tetrodotoxin ranged from 27.4 to 27.9 µg/kg and from 83.1 to 84.4 µg/kg, respectively. The proposed method yielded minimal matrix effects for the 11 analytes, thus allowing their quantification by simple external calibration. The proposed method also gave good mean recoveries of the 11 analytes ranging from 75.7 to 96.2% with relative standard deviations less than 16% at three fortification levels for the ten paralytic shellfish toxins (total concentrations of 277, 554, and 1107 µg saxitoxin equivalents/kg) and tetrodotoxin (100, 200, and 400 µg/kg) in the two bivalve samples. Finally, the proposed method was applied for the determination of the ten paralytic shellfish toxins and tetrodotoxin in scallop and short-necked clam samples.

A Case of Perilymphatic Fistula with Inner Ear Anomaly Diagnosed Preoperatively by the Cochlin-Tomoprotein Detection Test.

We present a case of perilymphatic fistula (PLF) with inner ear anomalies having sudden, progressive sensorineural hearing loss and describe the fistula repair surgeries. We focus on the diagnosis methods of PLF and clinical course of PLF with inner ear anomaly. The cochlin-tomoprotein (CTP) detection test is very useful for the surgeons to encourage the earlier operation to sudden hearing loss cases. It is also helpful to define the diagnosis of PLF after operation. We could not get the good result as to hearing from the fistula repair surgery mainly because surgery was held 1 month after the onset. The results of the case, as well as recommendations of other reports, suggest that patients with sudden sensorineural hearing loss and PLF may need repair surgery within at most 2 weeks from the onset. We describe how to diagnose PLF more accurately using CTP detection combined with intraoperative findings.

Simultaneous determination of eight underivatized biogenic amines in salted mackerel fillet by ion-pair solid-phase extraction and volatile ion-pair reversed-phase liquid chromatography-tandem mass spectrometry.

A simple and accurate method was developed for the quantitative determination of eight biogenic amines (cadaverine, histamine, 2-phenylethylamine, putrescine, spermidine, spermine, tryptamine, and tyramine) in salted mackerel fillet. The eight biogenic amines in the samples were extracted with 5% trichloroacetic acid and then purified by ion-pair solid-phase extraction using a C18 cartridge and nonafluoropentanoic acid as the volatile ion-pair reagent, without the need for both derivatization and pH adjustment. Subsequently, the eight underivatized biogenic amines were separated and quantified by volatile ion-pair reversed-phase LC-MS/MS using a C18 column with the same volatile ion-pair reagent. The developed method was validated and showed good accuracy with mean recoveries of all the eight analytes in the range of 87-118% at two fortification levels (2.5 and 5 mg/kg). Finally, the proposed method was applied to the analysis of the eight biogenic amines in salted mackerel fillet samples.

Identification of the monobrominated derivative of Acid Red 52 (Food Red No. 106) in pickled vegetables.

Two unknown dyes (purple and purplish-red) were detected by TLC in two pickled vegetable (sakura-zuke daikon) samples containing Acid Red 52 (AR) and New Coccine as food colorants. HPLC with diode-array detection and LC/MS analyses suggested that the purple dye is monobrominated AR and the purplish-red dye is its N-desethyl derivative, which would be generated mainly during sample preparation. For the identification of the purple dye, a reference compound was prepared by bromination of AR followed by isolation of the monobrominated AR, the structure of which was elucidated as 4'-brominated AR (4'BrAR) by LC/ToF-MS and (1)H-NMR spectroscopy. The purple dye was confirmed as 4'BrAR by comparison of its retention time, ultraviolet-visible spectrum and mass spectrum with those of the prepared reference compound. To our knowledge, this is the first report of the detection of 4'BrAR in foods.

Bone marrow stem cell transplantation to olfactory epithelium.

OBJECTIVES: We sought to develop a new therapeutic strategy for degeneration of olfactory receptor neurons (ORNs). METHODS: We transplanted into Balb/C mice, locally by transnasal injection and systemically via the tail vain, BrdU-labeled bone marrow stem cells, also known as NRGs, which have the ability to differentiate into neural cells. Bone marrow stem cells engrafted into the olfactory epithelium were examined immunohistochemically. RESULTS: Compared with previous studies, in which bone marrow was transplanted rather than bone marrow stem cells, migration of transplanted bone marrow stem cells into the olfactory epithelium was observed earlier, and engraftment rates were significantly higher. However, migrated bone marrow stem cells were positive for GAP43 but not for olfactory marker protein. CONCLUSIONS: These results suggest that engrafted cells had differentiated into premature, but not mature, ORNs. Further experiments using autologous bone marrow stem cells in combination with various growth factors and/or neurotrophic factors should aid the development of new therapeutic methods for degenerated ORNs.

Effect of intranasal administration of basic fibroblast growth factor on olfactory epithelium.

To further study the effects of basic fibroblast growth factor (bFGF) on the olfactory epithelium, bFGF was intranasally administered twice a day for 6 weeks to 2.5-month-old and 7-month-old mice. The effects were immunohistochemically examined by using antibodies against proliferating cell nuclear antigen, olfactory marker protein, and GAP43. The number of cells positive for proliferating cell nuclear antigen in the supporting cell layer increased dramatically, and that of GAP43-positive cells, or globose basal cells, increased significantly, especially in aging mice. However, no significant changes were observed in the number of olfactory marker protein-positive cells or mature olfactory receptor neurons. These results suggest that topical application of bFGF promotes proliferation of globose basal cells and supporting cells.

Synovial sarcoma of the neck.

BACKGROUND: Synovial sarcoma is a soft tissue sarcoma rarely seen in the head and neck. Due to its rarity and morphologic variations, diagnosis is difficult in most cases. METHOD: A case of synovial sarcoma arising in the upper neck is presented. The detection of the specific chromosomal translocation t(X;18)(p11.2;q11.2) is also described. RESULT: A specific chimeric gene, the SSX-SYT fusion gene, was identified in the formalin-fixed paraffin-embedded surgical specimen using the reverse-transcription polymerase chain reaction (RT-PCR) technique. CONCLUSION: Synovial sarcoma contains a characteristic chromosomal translocation, which serves as a useful diagnostic tool. RT-PCR technique has enabled to detect this specific translocation not only in fresh tissues but also in archival paraffin-embedded specimens.