Shizuyo Horiyama

Molecularly imprinted polymers for simultaneous determination of antiepileptics in river water samples by liquid chromatography-tandem mass spectrometry.

A restricted access media-molecularly imprinted polymer (RAM-MIP) for cyclobarbital has been developed for selective extraction of antiepileptics in river water samples. The RAM-MIP was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method followed by a surface modification technique. The RAM-MIP for cyclobarbital showed molecular recognition abilities for phenobarbital, amobarbital and phenytoin as well as cyclobarbital. Thus, selective analysis of antiepileptics in river water samples was attained with RAM-MIP extraction followed by column-switching liquid chromatography-tandem mass spectrometry. The concentrations of phenobarbital and phenytoin in river water samples were about 15 and 4 ng/L, respectively, while that of amobarbital was below the limit of quantitation.

Analysis of heptakis(2,6-di-O-methyl)-β-cyclodextrin by thin-layer, high-performance liquid and gas chromatography and mass spectrometry

Abstract Methods for the analysis and isolation of heptakis(2,6-di-O-methyl)-β-cyclodextrin (CD) by thin-layer and high-performance liquid chromatography (HPLC) were investigated. By using these methods a commercial and two synthetic samples of heptakis(2,6-di-O-methyl)-β-CD were analysed and it was found that they contained two major and at least four minor components, of which the two major and one minor components were isolated by semi-preparative HPLC. Careful fragmentation analyses, which consisted of successive hydrolysis, reduction, acetylation and characterization of the partially methylated d -glucitol peracetates by gas chromatography—mass spectrometry and determination of molecular weights by fast-atom bombardment mass spectrometry indicated that these compounds were heptakis(2,6-di-O-methyl)-β-CD, hexakis(2,6-di-O-methyl)-mono(2,3,6-tri-O-methyl)-β-CD and pentakis(2,6-di-O-methyl)-bis(2,3,6-tri-O-methyl)-β-CD.

Separation of cyclic (1→2)-β-D-glucans (cyclosophoraoses) produced by agrobacterium and rhizobium, and determination of their degree of polymerization by high-performance liquid chromatography

Conditions for the separation of the eight components contained in cyclic (1→2)-β-D-glucans (cyclosophoraoses) from Agrobacterium and Rhizobium were investigated by high-performance liquid chromatography on a chemically modified amine column (μBondapak CH) with mixtures of acetonitrile and water as eluent, and on a reversed-phase column (Dextro-Pak cartridge) with methanol—water (4:96) as eluent. Although retention on the amine column was related to molecular mass, that on the reversed-phase column differed from it, and was probably related to the solubility in water. The degree of polymerization of each cyclosophoraose was determined by high-performance liquid chromatography of its partial hydrolysate. Sophoro-oligomers having degrees of polymerization up to 24 were well resolved on a different amine column (Finepak SIL NH2, 10 μm) within 27 min by using a simple isocratic elution of acetonitrile-water (55:45). On hydrolysis under the appropriate conditions the longest straight chain sophoro-oligomer was present in sufficient amount to be able to recognize it in the partial hydrolysate; the distinguishing last peaks in the chromatograms of the eight cyclosophoraose hydrolysates could be detected clearly as the 17th–24th peaks.

Simultaneous determination of non-steroidal anti-inflammatory drugs in river water samples by liquid chromatography–tandem mass spectrometry using molecularly imprinted polymers as a pretreatment column

A restricted access media-molecularly imprinted polymer (RAM-MIP) for flufenamic acid has been developed for the simultaneous determination of non-steroidal anti-inflammatory drugs (NSAIDs) in river water samples. The RAM-MIP was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method followed by a surface modification technique. The RAM-MIP for flufenamic acid showed excellent molecular recognition abilities for flufenamic acid and mefenamic acid, and moderate molecular recognition abilities for indomethacin, etodolac and ketoprofen. The simultaneous determination of NSAIDs (mefenamic acid, indomethacin, etodolac and ketoprofen) in river water samples was carried out by LC-MS/MS using the RAM-MIP for flufenamic acid as a pretreatment column. The concentrations of mefenamic acid, indomethacin and etodolac in river water samples were determined to be 0.4, 0.7 and 0.3ng/L, respectively, while ketoprofen was below the limit of quantitation.

Preparation of cyclosophoraose-A and its complex-forming ability

Cyclosophoraoses (CySs) are unbranched cyclic (1→2)-β-D-glu-cans produced by many strains of Agrobacterium and Rhizobium. Pure CyS-A, the group member having the smallest molecular size (degree of polymerization 17), was efficiently prepared by liquid chromatography using charcoal and ODS columns from the culture fluid of the mutant strain RA-12 from R. phaseoli AHU 1133. The complex-forming ability of CyS-A was estimated from its enhancement of the solubilities of slightly soluble guest molecules in water using methods [I], [II], and [III]. In [I], an aqueous solution of CyS-A was shaken with the guest molecule, while, in [II], it was shaken with an acetone solution of the guest compound. In method [III], freeze-dried CyS-A powder was stirred with an acetone solution of the guest compound. The CyS-A cavity is thought to be able to accommodate three-dimensionally extended guest molecules, e.g., indomethacin. Method [II] was the best for obtaining CyS-A inclusion complexes, while method [III] would be recommended if the guest molecule is labile in the presence of water. Crystalline CyS-A inclusion complexes have not been obtained, but CyS-A complexes are expected to greatly enhance the solubilities of slightly soluble or insoluble guest molecules in water, because CyS-A is much more soluble than β-cyclodextrin. Method [II] or [III] may afford a useful means of obtaining oily drug, e.g., vitamin E and K1, in an amorphous state.

Prostaglandin E2-mediated Stimulation of Mucus Synthesis and Secretion by Rhein Anthrone, the Active Metabolite of Sennosides A and B, in the Mouse Colon

Abstract— Rhein anthrone, the active metabolite of sennosides A and B, stimulated PGE2 release into the mouse colonic lumen. At 6.24 mg kg−1, it decreased net water and Na+ absorption significantly in the case of water, but could not reverse the net absorption in mouse ligated colon, although it enhanced net K+ secretion. Pretreatment with indomethacin diminished the effects of rhein anthrone except on K+ net secretion. Rhein anthrone or PGE2 markedly stimulated mucus secretion and synthesis in mouse ligated colon. The enhanced mucus secretion and synthesis induced by rhein anthrone were significantly suppressed by pretreatment with indomethacin. Our results have shown that the colonic secretion of water and electrolytes mediated by PGE2 is partly involved in the rhein anthrone‐induced diarrhoea but that in mice, the mucoid diarrhoea induced by rhein anthrone results mainly from PGE2‐mediated mucus synthesis and secretion in the colon.

Intracellular Metabolism of α,β-Unsaturated Carbonyl Compounds, Acrolein, Crotonaldehyde and Methyl Vinyl Ketone, Active Toxicants in Cigarette Smoke: Participation of Glutathione Conjugation Ability and Aldehyde-Ketone Sensitive Reductase Activity.

The major toxicants in cigarette smoke, α,β-unsaturated aldehydes, such as acrolein (ACR) and crotonaldehyde (CA), and α,β-unsaturated ketone, methyl vinyl ketone (MVK), are known to form Michael-type adducts with glutathione (GSH) and consequently cause intracellular GSH depletion, which is involved in cigarette smoke-induced cytotoxicity. We have previously clarified that exposure to cigarette smoke extract (CSE) of a mouse melanoma cell culture medium causes rapid reduction of intracellular GSH levels, and that the GSH-MVK adduct can be detected by LC/MS analysis while the GSH-CA adduct is hardly detected. In the present study, to clarify why the GSH-CA adduct is difficult to detect in the cell medium, we conducted detailed investigation of the structures of the reaction products of ACR, CA, MVK and CSE in the GSH solution or the cell culture medium. The mass spectra indicated that in the presence of the cells, the GSH-CA and GSH-ACR adducts were almost not detected while their corresponding alcohols were detected. On the other hand, both the GSH-MVK adducts and their reduced products were detected. In the absence of the cells, the reaction of GSH with all α,β-unsaturated carbonyls produced only their corresponding adducts. These results show that the GSH adducts of α,β-unsaturated aldehydes, CA and ACR, are quickly reduced by certain intracellular carbonyl reductase(s) and excreted from the cells, unlike the GSH adduct of α,β-unsaturated ketone, MVK. Such a difference in reactivity to the carbonyl reductase might be related to differences in the cytotoxicity of α,β-unsaturated aldehydes and ketones.

Reductive degradation of O-acyl oximes under fast-atom bombardment conditions

A degradation phenomenon of O-acyl oximes under fast-atom bombardment (FAB) conditions is described from the point of view of reactivity with the liquid matrix and irradiation time with the xenon neutral beam. Mass spectra of O-acyl oximes were compared with those of the free oxime and hydroxylamine as model compounds, by using two different matrices, m-nitrobenzyl alcohol and DTT/TG12 (a 1:2 mixture of dithiothreitol and thioglycerol). A characteristic fragment ion was observed in the FAB mass spectra of O-acyl oximes and a free oxime only when DTT/TG12 was used. Collision-activated dissociation (CAD) was used to search for the origin and to confirm the structure of the characteristic fragment ion. The CAD spectra of the molecular-related ions, [M – H]+,M+• and [M + H]+, demonstrated that this characteristic fragment ion was not derived from any of these precursor ions. The CAD spectra of the characteristic fragment ion, which originated from O-acyl oximes, were very similar to those from free oximes and these results indicated that the fragment ion had the same structure. It was concluded that the degradation, under FAB conditions, was caused by a reductive reaction of the oxime moiety (–O–N=C) in O-acyl oximes regardless of its acyl moiety and that the degradation was initiated by the thiol group(s) in the matrix. The abundance of the ion originating from reductive degradation increased with increasing irradiation time with the xenon neutral beam.

Mass Spectrometric Approaches to the Identification of Potential Ingredients in Cigarette Smoke Causing Cytotoxicity

Cigarette smoke contains many harmful chemicals that contribute to the pathogenesis of smoking-related diseases such as chronic obstructive pulmonary disease, cancer, and cardiovascular disease. Many studies have been done to identify cytotoxic chemicals in cigarette smoke and elucidate the onset of the above-mentioned diseases caused by smoking. However, definitive mechanisms for cigarette smoke toxicity remain unknown. As candidates for cytotoxic chemicals, we have recently found methyl vinyl ketone (MVK) and acetic anhydride in nicotine/tar-free cigarette smoke extract (CSE) using L-tyrosine (Tyr), an amino acid with highly reactive hydroxyl group. The presence of MVK and acetic anhydride in CSE was confirmed by gas chromatography-mass spectrometry (GC/MS). We also found new reaction products formed in B16-BL6 mouse melanoma (B16-BL6) cells treated with CSE using LC/MS. These were identified as glutathione (GSH) conjugates of α,β-unsaturated carbonyl compounds, MVK, crotonaldehyde (CA), and acrolein (ACR), by the mass value and product ion spectra of these new products. ACR and MVK are type-2 alkenes, which are well known as electron acceptors and form Michael-type adducts to nucleophilic side chain of amino acids on peptides. These α,β-unsaturated carbonyl compounds may have a key role in CSE-induced cell death.

Detoxification Mechanism of α,β-Unsaturated Carbonyl Compounds in Cigarette Smoke Observed in Sheep Erythrocytes

Highly reactive α,β-unsaturated carbonyl compounds, such as acrolein (ACR), crotonaldehyde (CA) and methyl vinyl ketone (MVK), are environmental pollutants present in high concentrations in cigarette smoke. We have previously found that these carbonyl compounds in cigarette smoke extract (CSE) react with intracellular glutathione (GSH) to produce the corresponding GSH-ACR, GSH-CA and GSH-MVK adducts via Michael addition reaction. These adducts are then further reduced to the corresponding alcohol forms by intracellular aldo-keto reductases in highly metastatic mouse melanoma (B16-BL6) cells and then excreted into the extracellular fluid. This time, we conducted a similar study using sheep erythrocytes and found analogous changes in the sheep erythrocytes after exposure to CSE as those with B16-BL6 cells. This indicates similarity of the detoxification pathways of the α,β-unsaturated carbonyl compounds in sheep blood cells and B16-BL6 cells. Also, we found that the GSH-MVK adduct was reduced by aldose reductase in a cell-free solution to generate its alcohol form, and its reduction reaction was completely suppressed by pretreatment with epalrestat, an aldose reductase inhibitor, a member of the aldo-keto reductase family. In the presence of sheep blood cells, however, reduction of the GSH-MVK adduct was partially inhibited by epalrestat. This revealed that some member of the aldo-keto reductase superfamily other than aldose reductase is involved in reduction of the GSH-MVK adduct in sheep blood. These results suggest that blood cells, mainly erythrocytes are involved in reducing the inhalation toxicity of cigarette smoke via an aldo-keto reductase pathway other than that of aldose reductase.