Biomolecular analysis and cancer diagnostics by negative mode probe electrospray ionization.

We have examined several combinations of solvents and probes with the aim of optimizing the ionization conditions for biomolecules e.g., proteins, peptides and lipids by negative mode probe electrospray ionization mass spectrometry (PESI-MS). With the data presented in this study, negative-mode PESI-MS can be considered as a potential tool for biomolecular analysis and cancer diagnostics because of its simplicity in instrumental configuration. A sharper sampling probe was found to be better for obtaining high quality mass spectra because it can generate stable electrospray without the occurrence of gas breakdown. Although the best conditions may depend on each sample, aqueous organic solvent solutions, especially isopropanol-H(2)O (1/1) with a pH of ≥7, are shown to be preferable for negative-mode PESI-MS, which was successfully applied to colon cancer diagnosis.

Solid probe assisted nanoelectrospray ionization mass spectrometry for biological tissue diagnostics.

To perform remote and direct sampling for mass spectrometry, solid probe assisted nanoelectrospray ionization (SPA-nanoESI) has been newly developed. After capturing the sample on the tip of the needle by sticking it to the biological tissue, the needle was inserted into the solvent-preloaded nanoESI capillary from the backside. NanoESI gave abundant ion signals for human kidney tissues and the liver of a living mouse. The method is easy to operate and versatile because any biological specimen could be sampled away from the mass spectrometer. Minimal invasiveness is another merit of this method.

Differentiation between sulfoaildenafil and its analogs.

An analog of aildenafil, which is a potent and highly selective inhibitor of phosphodiesterase 5, was found in a dietary supplement marketed for enhancement of sexual function. The compound was isolated by silica gel column chromatography, and its structure was identified by means of 13C-NMR spectrometry, 1H-NMR spectrometry, high-resolution MS, and X-ray structure determination. The compound was identified to be sulfoaildenafil (other names: thioaildenafil, dimethyl sildenafil thione, and thiomethisosildenafil). Sulfoaildenafil is very similar to the compound thiohomosildenafil. As it is difficult to distinguish between them by LC-photodiode array detector analysis, ultra-performance LC (UPLC)/MS, ion trap LC/MS/MS (LC/IT-MS/MS), and GC/MS were performed. The mass spectra of thiohomosildenafil by UPLC/MS and LC/IT-MS/MS showed mass fragments of m/z 58, 72, and 355, and the mass spectrum by GC/MS showed mass fragments of m/z 56, 72, and 420. Some of these fragments had low intensities, but they were useful for distinguishing between the two compounds. The relationship between aildenafil (other names: dimethylsildenafil and methisosildenafil) and homosildenafil is similar to that between sulfoaildenafil and thiohomosildenafil. Therefore, these compounds were also examined.

Effects of pH and temperature on the composition of polar lipids in Thermoplasma acidophilum HO-62.

Thermoplasma acidophilum HO-62 was grown at different pHs and temperatures, and its polar lipid compositions were determined. Although the number of cyclopentane rings in the caldarchaeol moiety increased when T. acidophilum was cultured at high temperature, the number decreased at low pHs. Glycolipids, phosphoglycolipids, and phospholipids were analyzed by high-performance liquid chromatography with an evaporative light-scattering detector. The amount of caldarchaeol with more than two sugar units on one side increased under low-pH and high-temperature conditions. The amounts of glycolipids increased and those of phosphoglycolipids decreased under these conditions. The proton permeability of the liposomes obtained from the phosphoglycolipids that contained two or more sugar units was lower than that of the liposomes obtained from the phosphoglycolipids that contained one sugar unit. From these results, we propose the hypothesis that T. acidophilum adapts to low pHs and high temperatures by extending sugar chains on their cell surfaces, as well as by varying the number of cyclopentane rings.

Prebiotic origin of glycolytic metabolism: histidine and cysteine can produce acetyl CoA from glucose via reactions homologous to non-phosphorylated Entner-Doudoroff pathway.

Conversion of glucose to pyruvate via reactions homologous to the non-phosphorylated Entner-Doudoroff (non-P ED) pathway could be achieved in the presence of two amino acid catalysts, cysteine and histidine: cystine oxidizes glucose to gluconic acid by the reaction homologous to glucose dehydrogenase and histidine changes gluconic acid to 2-keto-3-deoxy gluconic acid, then to pyruvate by the reaction homologous to gluconic acid dehydratase and 2-keto-3-deoxy gluconate aldolase, respectively. Pyruvate can be converted to acetyl CoA by the reaction with CoA, TPP and FAD in the presence of cysteine and histidine, which resembles pyruvate dehydrogenase reaction. It was found that gluconic acid dehydration alone is non-specific, in contrast to other reactions. The non-P ED pathway is used by some extreme thermophiles in bacteria and archaea, usually thought as the oldest among the contemporary organisms. This study suggests the possible contribution of amino acid to the origin of the glycolytic pathway.

Effect on both aglycone and sugar moiety towards Phase II metabolism of anthocyanins.

The effect of sugar moiety on anthocyanin metabolism was studied using anthocyanidin 3-rutinosides (cyanidin 3-O-rutinoside (Cy3R) and delphinidin 3-O-rutinoside (Dp3R)) and 3-O-glucosides (delphinidin 3-O-glucoside (Dp3G)). O-methylated Cy3R and Dp3R were detected in rat blood plasma after oral administration of Cy3R and Dp3R (100mg/kg body weight). On the basis of HPLC retention time and UV-visible spectra together with the data of our previous studies on the hydrophobic metabolites of anthocyanidin 3-O-glucosides, it was concluded that both 3'- and 4'-O-methyl Cy3R were metabolites of Cy3R. On the other hand, only 4'-O-methyl Dp3R was detected as hydrophobic metabolite of Dp3R. A group of hydrophilic metabolites was also detected in rat blood plasma after oral administration of anthocyanins (Dp3G, Cy3R and Dp3R) and their structures were determined to be extended glucuronides and their O-methyl analogues by tandem MS analysis. The amounts of extended glucuronides of Dp3G, Cy3R and Dp3R were less than those of cyanidin 3-O-glucoside (Cy3G) reported in our previous study. On the other hand, anthocyanidin-glucuronides (both cyanidin-glucuronide and delphinidin-glucuronide) were not detected after oral administration of Cy3R, Dp3R and Dp3G. These results indicated that both the type of sugar moiety and stability of aglycone largely affected phase II metabolism of anthocyanins, and also indicated that the type of sugar moiety did not affect the O-methylation metabolism but affected glucuronyl conjugation in both liver and small intestine.

Isolation and characterization of bacteria from the copepod Pseudocaligus fugu ectoparasitic on the panther puffer Takifugu pardalis with the emphasis on TTX.

A total of 50 bacterial isolates was obtained from the copepod Pseudocaligus fugu, which is a common parasite, collected from the body surface of the panther puffer Takifugu pardalis. On the basis of colony characteristics, these bacterial isolates were grouped into six types, of which only two (Types-I and -II) showed a high affinity for adhesion to the carapace of the banana shrimp Penaeus merguiensis. These two types of adhesive bacteria were identified through 16S rRNA sequence analysis as Shewanella woodyi (Type-I) and Roseobacter sp. (Type-II). Representative isolates of these two adhesive bacteria were examined for tetrodotoxin (TTX) production by high-performance liquid chromatography (HPLC)-fluorometric system, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). It was rather unexpectedly revealed that TTX and anhydroTTX were present in the supernatant of culture of the Type-II isolate Roseobacter sp.

Rapid Analysis of Ingredients in Cream Using Ultrasonic Mist-Direct Analysis in Real-Time Time-of-Flight Mass Spectrometry.

A novel method for the simultaneous detection of ingredients in pharmaceutical applications such as creams and lotions was developed. An ultrasonic atomizer has been used to produce a mist containing ingredients. The analyte molecules in the mist can be ionized by using direct analysis in real time (DART) at lower temperature than traditionally used, and we thus solved the problem of normal DART-MS measurement using a high-temperature gas. Thereby, molecular-related ions of heat-unstable components and nonvolatile components became detectable. The deprotonated molecular ion of glycyrrhizic acid (m/z 821), which is unstable at high temperatures, was detected without pyrolysis by ultrasonic mist-DART-MS using unheated helium gas, although it was not detected by normal DART-MS using heated helium gas. The cationized molecular ions of derivatives of polyethylene glycol fatty acid monoesters, which are nonvolatile compounds, were also detected as m/z peaks observed from 800 to 2300. Although the protonated molecular ion of tocopherol acetate was not detected in ionization by ultrasonic mist, it was detected by ultrasonic mist-DART-MS even in the emulsion. It was not necessary to dissolve a sample completely to detect its ions. This method enabled us to obtain the composition of pharmaceutical applications simply and rapidly. Graphical Abstract ᅟ.

Detection of tetrodotoxin (TTX) from two copepods infecting the grass puffer Takifugu niphobles: TTX attracting the parasites?

In May 2002, two parasitic copepods, Pseudocaligus fugu and Taeniacanthus sp., were collected from the body surface and gill of the grass puffer Takifugu niphobles, respectively, in Takehara city, Hiroshima Prefecture, faced with Seto Inland Sea located in the western part of Japan. To them was added 5 ml of 0.1% acetic acid, then the suspension was subjected to ultrasonic disruption with an ultrasonicator for 10 min. The resulting mixture was heated in a boiling water bath for 10 min, and then centrifuged. The supernatant was concentrated under reduced pressure, and loaded on to a Sep-Pak plus C18 Environmental Cartridge (Waters). The unbound fraction was analyzed by HPLC and gas chromatography-mass spectrometry (GC-MS) for tetrodotoxin (TTX). It was rather unexpectedly revealed from these results that this fraction was comprised of TTX and its analogues. As far as we know, this is the first record to show the existence of TTX in the copepods. In addition, relationships between the more and less than the average number of the two parasites and the toxicity of its skin mucus of the host were examined by student's t-test. In P. fugu, the average number per host was 13.9, and those are 520.7 (n=9) and 269.0 MU/g (n=22), respectively. A highly significant difference between them was detected at p-value 0.0011. In contrast, as for Taeniacanthus sp., the average number was 2.7, and those were 338.0 (n=14) and 345.5 MU/g (n=17), respectively. No significant difference was detected in Taeniacanthus sp. The high host-specificity of P. fugu on the toxic puffer and the present bioassay of its skin mucus suggest a possibility that TTXs may attract the parasite.

Bioavailability and tissue distribution of anthocyanins in bilberry (Vaccinium myrtillus L.) extract in rats.

To clarify how structural diversity of anthocyanins relates to their in vivo function, bioavailability was precisely studied in rats using bilberry (Vaccinium myrtillus L.) extract (Bilberon 25) as an anthocyanin source that contains 15 different anthocyanins. The bilberry extract was orally or intravenously administered to rats, and the plasma levels of each anthocyanin were determined by high-performance liquid chromatography. As the result, all anthocyanins except peonidin 3-O-alpha-L-arabinoside were detectable in the blood plasma. The plasma concentration of anthocyanins as a whole reached the maximum level of 1.2 microM at 15 min after oral administration of 400 mg/kg bilberry extract (153.2 mg/kg as anthocyanins) and then decreased with time. Uptake and decay profiles of each anthocyanin in the plasma were almost the same for all anthocyanins except a few with their maximum after 30 min. Among the anthocyanins carrying the same aglycone, the plasma level after 15 min of oral administration was as follows: galactoside > glucoside > arabinoside. Plasma clearance of anthocyanins after intravenous administration clearly showed that arabinoside disappeared more rapidly than glucoside and galactoside. On the other hand, when anthocyanins carrying the same sugar moiety were compared, the half disappearance time of plasma anthocyanins was in the following order: delphinidin > cyanidin > petunidin = peonidin > malvidin. The bioavailability of anthocyanins was in the range of 0.61-1.82% and was 0.93% as the anthocyanin mixture. The bioavailability of anthocyanins carrying the same aglycone was in the following order: Galactoside showed the highest followed by glucoside and arabinoside for cyanidin and delphinidin, but arabinoside and galactoside showed a higher bioavailability than glucoside for petunidin and malvidin. Anthocyanins recovered in urine and bile during the first 4 h after intravenous administration were only 30.8 and 13.4%, respectively. Anthocyanin profiles in tissues were quite different from those in blood plasma. The major anthocyanins distributed in liver and kidney were the O-methyl anthocyanins such as peonidin, malvidin, and other O-methyl anthocyanins derived from delphinidin, cyanidin, and petunidin-glycosides.

Extended glucuronidation is another major path of cyanidin 3-O-beta-D-glucopyranoside metabolism in rats.

We previously determined that five rather hydrophobic metabolites appeared in blood plasma after oral administration of cyanidin 3-O-beta-D-glucopyranoside, but a group of hydrophilic metabolites still remained unidentified. In the present study, 12 hydrophilic metabolites found were collected from urine and plasma samples by high-performance liquid chromatography (HPLC) and then analyzed by tandem MS spectrometry. From the MS spectra, four metabolites out of 12 were assigned as glucuronides of cyanidin 3-O-beta-D-glucopyranoside and six out of 12 were glucuronides of the primary metabolites of cyanidin 3-O-beta-D-glucopyranoside (O-methyl cyanidin 3-O-beta-D-glucopyranoside). Extended glucuronides of cyanidin 3-O-beta-D-glucopyranoside and O-methyl cyanidin 3-O-beta-D-glucopyranoside showed their maximum plasma concentrations at 15 and 60 min (or 30 min) after oral administration, respectively. Their maximum plasma concentrations ranged from 15 to 70 nM. From the profile of urinary-excreted anthocyanins after intravenous administration, it was deduced that extended glucuronides of cyanidin 3-O-beta-D-glucopyranoside and O-methyl cyanidin 3-O-beta-D-glucopyranoside were mainly produced in the liver rather than by intestinal flora. The area under the plasma concentration curve was 0.25 micromol min/L for extended glucuronides of cyanidin 3-O-beta-D-glucopyranoside and 0.14 micromol min/L for O-methyl cyanidin 3-O-beta-D-glucopyranoside, respectively, when evaluated as cyanidin 3-O-beta-D-glucopyranoside equivalent, indicating that extended glucuronidation is a critical pathway in cyanidin 3-O-beta-D-glucopyranoside metabolism in rats.

Nasunin from eggplant consists of cis-trans isomers of delphinidin 3-[4-(p-coumaroyl)-L-rhamnosyl (1-->6)glucopyranoside]-5-glucopyranoside.

Two major anthocyanins were isolated from the acidified methanolic extract of eggplant (Solanum melongena L.) by column chromatography and preparative high-performance liquid chromatography. These anthocyanins were interconvertible under room light illumination condition. By means of tandem time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy, their structures were identified and elucidated as delphinidin 3-[4-(cis-p-coumaroyl)-l-rhamnosyl(1-->6)glucopyranoside]-5-glucopyranoside (compound 1) and delphinidin 3-[4-(trans-p-coumaroyl)-l-rhamnosyl-(1-->6)glucopyranoside]-5-glucopyranoside (compound 2), respectively. The results indicated that nasunin comprised cis and trans isomers of the p-coumaric acid moiety in its structure.

Metabolic pathway of cyanidin 3-O-beta-D-glucopyranoside in rats.

For better understanding of the physiological function of anthocyanins, the absorption and metabolism of cyanidin 3-O-beta-D-glucopyranoside (Cy3G), which is one of the major anthocyanins in colored food materials, were precisely investigated. Combining two modalities newly developed, that is, highly sensitive semi-micro-HPLC and vein cannulation, Cy3G and its four major metabolites (M1-M4) were detected in the blood plasma of rats after oral administration of Cy3G (100 mg/kg of body mass). The plasma concentration of Cy3G reached its maximum at 15 min after the ingestion. Metabolite 2 (M2) and metabolite 3 (M3) showed their maximum plasma levels at 15 and 30 min, respectively, whereas metabolite 1 (M1) and metabolite 4 (M4) showed their maximum levels at 60 and 120 min, respectively. The maximum plasma concentrations of the four metabolites were in the following order: M3 (21 nM) > M4 (20 nM) > M1 (8.5 nM) > M2 (5 nM). When Cy3G was directly injected into the neck vein, only M2 and M3 were detected in the plasma, indicating that both M1 and M4 were produced during absorption from the gastrointestinal tract. Tandem MS analysis of the metabolites showed that M2 and M3 were monomethylated Cy3G, while M1 and M4 were glucuronides of Cy and methylated Cy, respectively. M3 was assigned as peonidin 3-O-beta-D-glucopyranoside (Pn3G) from the comparison of the retention time of authentic Pn3G.

Absorption and metabolism of delphinidin 3-O-beta-D-glucopyranoside in rats.

The absorption and metabolism of delphinidin 3-O-beta-d-glucopyranoside (Dp3G), which is the most potent antioxidant among the blueberry anthocyanins, were studied in rats. Dp3G rapidly appeared in the blood plasma within 15 min of oral administration (100 mg/kg body wt). The plasma level of absorbed Dp3G showed two peaks at 15 and 60 min after ingestion and then decreased time-dependently. However, the plasma level was maintained at approximately 30 nmol/l even after 4 h. Besides the Dp3G peak, a single major metabolite peak was detected by HPLC in the blood plasma obtained at 15 min. MS and NMR spectroscopy clarified that the chemical structure of the metabolite was 4'-O-methyl delphinidin 3-O-beta-d-glucopyranoside (methylation of the 4'-OH on the delphinidin B-ring). The present finding of this unique metabolite in anthocyanin metabolism strongly suggests that methylation of the 4'-OH on the flavonoid B-ring is a common metabolic pathway for flavonoids that carry the pyrogallol structure on the B-ring, as the same type of metabolite has been reported for other flavonoids such as epigallocatechin, but not for flavonoids carrying the catechol structure.

Paralytic toxicity in the ribbon worm Cephalothrix species (Nemertea) in Hiroshima Bay, Hiroshima Prefecture, Japan and the isolation of tetrodotoxin as a main component of its toxins.

Paralytic toxicity of ribbon worms ("himomushi" in Japanese), identified as undescribed species of the genus Cephalothrix, found on the surface of the shells of cultured oysters in Hiroshima Bay, Hiroshima Prefecture was examined between April 1998 and December 2001. The toxicity study showed that all of specimens were found to contain toxins with strong paralytic action in mice; the highest toxicity (as tetrodotoxin, TTX) was 25,590 mouse units (MU) per gram for whole body throughout the monitoring period. The main toxic component of this himomushi toxin (HMT) was isolated from a pooled specimen (390 g; total toxicity 2,897,000MU) by a method that consisted of treatment with activated charcoal, chromatography on Bio-Gel P-2 and Bio-Rex 70 (H+ form), and finally crystallization from an acidified methanolic solution. The recrystallized toxin showed a specific toxicity of 3520MU/mg. This toxin showed (M+H)+ and (M+H-H(2)O)+ ion peaks at m/z 320 and 302, respectively, by electrospray ionization-mass spectrometry (ESI-MS). The absorption band at 3353, 3235, 1666, 1612 and 1076 cm(-1) were observed in infrared spectrum of this toxin. This spectrum was indistinguishable from that of TTX. The 1H-NMR spectrum for the recrystallized toxin was the same as that for TTX. The pair of doublets centered at 2.33 (J=10.0Hz) and 5.48 ppm (J=10.0Hz) which are characteristic of TTX, were shown to be coupled by double irradiation. Furthermore, by gas chromatography-mass spectrometry (GC-MS) of the alkali-hydrolyzate of this toxin indicated the presence of quinazoline skeleton (C9-base) specific to TTX.

Absorption and metabolism of delphinidin 3-O-beta-D-glucoside in rats.

Anthocyanins, kind of flavonoids (FL) found in plants and vegetables, are known to have varieties of physiological functions. In the present study, we examined absorption and metabolism of delphinidin 3-O-beta-D-glucoside (Dp3G) in rats. Dp3G appeared in the plasma at 15 min after oral administration as an intact glucosidic form. The plasma level also showed another peak at 60 min. One metabolite peak was detected in the plasma and the structure was assigned as 4'-O-methyl Dp3G (MDp3G) by NMR and MS. The metabolite was also identified in several tissues as a major metabolite especially in the liver. No 3'-O-methyl Dp3G was detected in any tissues, therefore, 4'-OH methylation is the main path of Dp3G metabolism in rats. This finding generalized the metabolic formation of FL having pyrogallol B ring because it has been reported that FL having catechol structure produced 3'-O-methyl-derivatives, but FL having pyrogallol structure produced 4'-O-methyl-derivatives.

Cluster composition distributions of pure ethanol: influence of water and ion-molecule reactions revealed by liquid-ionization tandem mass spectrometry.

Studies of clusters in condensed phase at atmospheric pressure are very important for understanding the properties and structures of liquids. Liquid-ionization (LPI) mass spectrometry is useful to study hydrogen-bonded clusters at the liquid surface and in a gas phase. An improved ion source connected to a tandem mass spectrometer provides detailed information about clusters. Mass spectra of pure ethanol (99.5%) observed by the first mass analyzer (Q1) showed neat ethanol cluster ions (C2H5OH) m H(+) with m up to 10 and hydrate ions (C2H5OH) m (H2O) n H(+) with m larger than 7 and n=1, such as those with m-n=8-1 and 9-1. When the flow rate of ethanol (liquid) was increased, large ethanol cluster ions with m larger than 25 were observed by the second mass analyzer (Q3). It is interesting to note that neat ethanol cluster ions are more abundant than corresponding (with the same m) hydrate ions (n=1), and major hydrate ions contain only one molecule of water. Results indicate that ion-molecule reactions occur between Q1 and Q3, because such mass spectra have never been observed by Q1. Various results indicate that neat ethanol clusters exist at the liquid surface and are ionized to give cluster ions.

Structural elucidation and biological fate of two glucuronyl metabolites of pelargonidin 3-O-ß-D-glucopyranoside in rats.

A high proportion of pelargonidin 3-O-ß-D-glucopyranoside (Pg3G) is metabolized to glucuronides and excreted in mammal urine after ingestion of strawberry fruit, suggesting that these metabolites play important functional roles in vivo. The aim of the present study was to elucidate the structures and determine the biological fate of the two dominant metabolites of Pg3G in rats to enable an accurate discussion of the biological properties of anthocyanins. Authentic Pg3G was orally administered to rats. One pelargonidin monoglucuronide and three Pg3G-monoglucuronides (glucuronides of the glucoside) were identified together with intact Pg3G in both blood plasma and urine samples. The structures of the two dominant metabolites were elucidated as pelargonidin 3-O-ß-D-glucuronide (Pg3GlcA) and pelargonidin 3-O-ß-D-glucuronyl-(1→2)-ß-D-glucoside by means of (1)H and (13)C nuclear magnetic resonance spectroscopy and heteronuclear multiple-bond connective spectroscopy. The bioavailability of Pg3G in its intact form was 0.31% of the orally administered dose, and 0.65% was absorbed in the Pg3GlcA form.

Trace level detection of explosives in solution using leidenfrost phenomenon assisted thermal desorption ambient mass spectrometry.

The present paper demonstrates the detection of explosives in solution using thermal desorption technique at a temperature higher than Leidenfrost temperature of the solvent in combination with low temperature plasma (LTP) ionization. Leidenfrost temperature of a solvent is the temperature above which the solvent droplet starts levitation instead of splashing when placed on a hot metallic surface. During this desorption process, slow and gentle solvent evaporation takes place, which leads to the pre-concentration of less-volatile explosive molecules in the droplet and the explosive molecules are released at the last moment of droplet evaporation. The limits of detection for explosives studied by using this thermal desorption LTP ionization method varied in a range of 1 to 10 parts per billion (ppb) using a droplet volume of 20 µL (absolute sample amount 90-630 fmol). As LTP ionization method was applied and ion-molecule reactions took place in ambient atmosphere, various ion-molecule adduct species like [M+NO2](-), [M+NO3](-), [M+HCO3](-), [M+HCO4](-) were generated together with [M-H](-) peak. Each peak was unambiguously identified using 'Exactive Orbitrap' mass spectrometer in negative ionization mode within 3 ppm deviation compared to its exact mass. This newly developed technique was successfully applied to detect four explosives contained in the pond water and soil sample with minor sample pre-treatment and the explosives were detected with ppb levels. The present method is simple, rapid and can detect trace levels of explosives with high specificity from solutions.