Development and validation of a specific and sensitive liquid chromatography tandem mass spectrometry method for determination of tetrodotoxin in human urine and its pharmacokinetic study

Tetrodotoxin (TTX) exhibits the therapeutic potential in blocking pain and in low doses can safely relieve severe pain. The urinary excretion profiles of TTX in humans have not been reported due to the extremely low lethal dose. In this study, a rapid and specific method based on protein precipitation coupled to liquid chromatography tandem mass spectrometry was developed to determine the level of TTX in human urine samples. 11-Deoxytetrodotoxin was used as an internal standard (IS). Multiple reaction monitoring mode was used for quantification using target fragment ions m/z 320.0 → 162.1 for TTX and m/z 304.0 → 176.0 for 11-deoxyTTX. The separation of analytes was achieved on a hydrophilic interaction liquid chromatography column (250 × 4.6 mm, 5.0 μm). The mobile phase consisted of 5 mM ammonium formate in water (pH = 4.50) and 5 mM ammonium formate in acetonitrile (pH = 4.50). The flow rate was set at 0.80 mL/min in a gradient condition. Calibration plots were linear throughout the range 0.986–98.6 ng/mL of TTX in human urine. The intra-assay accuracies and precisions were within the acceptable range. The method was successfully applied to a urinary excretion study after intravenous administration of TTX to healthy volunteers. The developed method will be helpful for future pharmacological studies of TTX.     

Separation of five diketopiperazines from the marine fungus Alternaria alternate HK‐25 by high‐speed counter‐current chromatography

High‐speed counter‐current chromatography was applied to the separation of five diketoperazines from the marine Alternaria alternate HK‐25 for the first time using one‐step elution method with a pair of two‐phase solvent systems composed of petroleum ether/ethyl acetate/methanol/water (5.5:11:5:7, v/v). Where 151.6 mg of crude sample yielded five diketoperazines, 12,13‐dihydroxy‐fumitremorgin C ( 1 ), gliotoxin ( 2 ), demethoxyfum itremorgin C ( 3 ), bisdethiobis(methylthio)gliotoxin ( 4 ), fumitremorgin C ( 5 ), and the purities of all compounds were above 94% as determined by high‐performance liquid chromatography. The structures of these compounds were identified by ¹H and ¹³C NMR spectroscopy. These results showed that high‐speed counter‐current chromatography can provide a feasible way for highly effective preparation of marine natural products, which ensured the supple of numerous samples for drug development.     

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL⁻¹ for urine and 20 ng mL⁻¹ for plasma. The limit of detection and limit of quantification were 0.13 ng mL⁻¹ and 2.5 ng mL⁻¹, respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL⁻¹, but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng μmol⁻¹ creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.     

Bioactive metabolites of the marine actinobacterium <Emphasis Type="Italic">Streptomyces</Emphasis> sp. KMM 7210

New 3-(4-hydroxybenzyl)piperazine-2,5-dione, together with the known N-[2-(4-hydroxy-phenyl)ethyl]acetamide (N-acetyltyramine), was isolated for the first time from the marine actinobacterium Streptomyces sp. The chemical structures of these compounds were determined by NMR spectroscopy and mass spectrometry. The cytotoxic activities of the compounds were estimated from their effects on sperm and eggs of the sea urchin Strongylocentrotus intermedius. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 807–809, April, 2007.     

Fatty Acids from the Sponge <Emphasis Type="Italic">Tedania dirhaphis</Emphasis>

The fatty acid (FA) composition of total lipids from the marine sponge Tedania dirhaphis from the Sea of Okhotsk was studied. GC and GC-MS identified 50 acids, particular attention being paid to components with 14–22 C atoms. Acids 16-Me-19:0, 10,14-Me₂-15:1(Δ6), 18:1(Δ6), 18:1(Δ8), and 22:1(Δ16) were observed and identified for the first time in sponges. The main FA in lipids from T. dirhaphis was 28:3(Δ5,9,21), the relative content of which reached 63.3%.__________Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 233–236, May–June, 2005.     

Interdisciplinary study for the evaluation of biochemical alterations on mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> exposed to a tributyltin-polluted area

An interdisciplinary approach was employed to monitor the concentration and the effects of butyltin compounds in mussels (Mytilus galloprovincialis). Tissues from animals exposed to a marine area (Vado Ligure harbour) with a high concentration of tributyltin (TBT) were analysed and compared with control samples. TBT concentrations were measured by gas chromatography–mass spectrometry and the protein pattern in gill tissues was studied by proteomic analysis. Several proteomic signatures associated with contaminant exposure were observed; spots that were significantly increased in all contaminated samples were identified by mass spectrometry as fragments of β-tubulin. The degradation of β-tubulin was then confirmed by western blot analysis with specific anti-β-tubulin antibody. The effects observed on mussel gills after exposure in the TBT-polluted area are discussed.     

Rapid Determination of Tetrodotoxin in Human Plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

A sensitive analytical method was developed to determine tetrodotoxin(TTX) in human plasma samples using protein precipitation, followed by ultra performance liquid chromatography(UPLC) analysis coupled with tandem mass spectrometry(MS/MS) using 11-deoxytetrodotoxin(11-deoxyTTX) as an internal standard. The plasma samples were prepared using protein precipitation prior to being analyzed by UPLC-MS/MS to identify TTX over a zwitterionic-hydrophilic interaction liquid chromatography column. The retention time values of TTX and 11-deoxyTTX were 4.12 and 3.67 min, respectively. TTX and 11-deoxyTTX were monitored and quantitated on the basis of their ion transitions for their respective precursor ions to their product ions(i.e., m/z 320.0→162.1 for TTX and m/z 304.0→176.0 for 11-deoxyTTX) in the multiple reaction-monitoring mode. The lower limit of quantification of this method was determined to be 0.0199 ng/mL. This method showed good linearity for plasma samples that contained TTX concentrations in the range of 0.0199-1.99 ng/mL. The specificity, precision, accuracy, matrix effect, and stability characteristics of this method were also examined. The intra-assay precision and accuracy ranged from 1.89% to 6.00% and from 92.21% to 100.00%, whereas the inter-assay precision and accuracy ranged from 0.64% to 7.75% and from 99.38% to 101.26%, respectively. This new method therefore represents a rapid, accurate, reliable, and highly sensitive method for the qualitative and quantitative analyses of a trace amount of TTX in human plasma samples.     

Purification of the 90 kDa heat shock protein (hsp90) and simultaneous purification of hsp70/hsc70, hsp90 and hsp96 from mammalian tissues and cells using thiophilic interaction chromatography

Heat shock proteins (HSPs) hsp70/hsc70, hsp90 and hsp96 were separated from mammalian cells and tissues on a gel obtained by the reaction of β‐mercaptoethanol with divinyl sulfone‐activated Sepharose CL‐6B (thiophilic gel or T‐gel). Hsp90 revealed a much higher affinity towards the T‐gel than the other HSPs. One‐step thiophilic interaction chromatography of proteins resulted in a more than 80% purity and 85% yield of hsp90. Based on this observation, a simple and efficient method for the purification of hsp90 and a procedure for the simultaneous purification of several HSPs (hsp70/hsc70, hsp90 and hsp96) using thiophilic interaction chromatography was developed. All the HSPs were recovered with a high yield and purity (90–99%). The results indicated that the thiophilic gel is a highly efficient affinity matrix for the purification of hsp90 and can be used in the protocols of purification of different HSPs from cells and tissues of various animal species. Copyright © 2009 John Wiley & Sons, Ltd.     

Fungicidal lipid-transfer peptide from <Emphasis Type="Italic">Daucus carota sativa</Emphasis> seeds

A non-specific lipid-transfer peptide (nsLTP) with fungicidal activity was isolated from Daucus carota sativa carrot seeds. Peptides were purified by a method including aqueous extraction, anion-exchange chromatography over CM-TSK-650M, and HPLC over a column of 250/8/4 Protein@Peptide C₁₈ using an acetonitrile gradient. The molecular weight of the peptide was determined as 9624 Da by mass spectrometry. The peptide was found to have fungicidal activity against the pathogenic fungus Verticillium dahliae. The partial N-terminal sequence, which was highly homologous to the N-terminal sequences of lipid-transfer peptides from seeds of rice, tobacco, and maize, was determined using Edman automated sequencing. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 371–373, July–August, 2007.     

C5–C10 Directly Bonded Tetrodotoxin Analogues: Possible Biosynthetic Precursors of Tetrodotoxin From Newts

The identification of novel tetrodotoxin (TTX, 1 ) analogues would significantly contribute to the elucidation of its biosynthetic pathway. In this study, the first C5–C10 directly bonded TTX analogues, 4,9‐anhydro‐10‐hemiketal‐5‐deoxyTTX ( 2 ) and 4,9‐anhydro‐8‐epi‐10‐hemiketal‐5,6,11‐trideoxyTTX ( 3 ), were found in the newt Cynops ensicauda popei by using a screening method involving HILIC‐LC–MS/MS focused on the fragment ions of TTX analogues, and their structures were elucidated by spectroscopic methods. Compound 2 was detected in a wide range of newt species, and the 2 and TTX contents of 22 newt specimens were correlated (r_s=0.88). Based on these results and its structural features, 2 was predicted to serve as a precursor of TTX that would be directly converted into 4,9‐anhydroTTX ( 4 ) by Baeyer–Villiger‐like oxidation or via 4,9‐anhydro‐5‐deoxyTTX formed by cleavage of the C5–C10 bond. The bicyclic carbon skeletons of 2 and 3 suggested a possible monoterpene origin for TTX.     

Determination of avoparcin in animal tissues and milk using LC‐ESI‐MS/MS and tandem‐SPE

A highly sensitive and selective method using LC‐ESI‐MS/MS and tandem‐SPE was developed to detect trace amounts of avoparcin (AV) antibiotics in animal tissues and milk. Data acquisition using MS/MS was achieved by applying multiple reaction monitoring of the product ions of [M + 3H]³⁺ and the major product ions of AV‐α and ‐β at m/z 637 → 86/113/130 and m/z 649 → 86/113/130 in ESI(+) mode. The calculated instrumental LODs were 3 ng/mL. The sample preparation was described that the extraction using 5% TFA and the tandem‐SPE with an ion‐exchange (SAX) and InertSep C18‐A cartridge clean‐up enable us to determine AV in samples. Ion suppression was decreased by concentration rates of each sample solution. These SPE concentration levels could be used to detect quantities of 5 ppb (milk), 10 ppb (beef), and 25 ppb (chicken muscle and liver). The matrix matching calibration graphs obtained for both AV‐α (r >0.996) and ‐β (r >0.998) from animal tissues and milk were linear over the calibration ranges. AV recovery from samples was higher than 73.3% and the RSD was less than 12.0% (n = 5).     

Development of an efficient method for the preparative isolation and purification of chlorophyll a from a marine dinoflagellate Amphidinium carterae by high-speed counter-current chromatography coupled with reversed-phase high-performance liquid chromatography

In our research into chlorophylls of marine dinoflagellates, chlorophyll a was separated rapidly from the hexane extract of Amphidinium carterae in three steps. The first step was silica gel column chromatography, where elution was performed with 0–50% ethyl acetate in n-hexane. The second was high-speed counter-current chromatography using a two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (5:5:5:1, v/v), and the third step was preparative reversed-phase high-performance liquid chromatography using a solvent system of acetone–water (89:11, v/v). HPLC analysis showed that the purity of chlorophyll a from the second step was over 83%, and after the third it was over 99%. Thirty milligrams of chlorophyll a was isolated from a crude sample of 250 mg of chlorophylls, and its structure was identified by analyzing its MS, ¹H NMR and ¹³C NMR spectra.     

Determination of Phosphatidylcholine in Shrimp by High-Resolution Mass Spectrometry

The molecular species of phosphatidylcholine from freshwater sources (Macrobranchium nipponense and Macrobranchium rosenbergii) and marine sources (Euphausia superba and Penaeus chinesis) were characterized by high-resolution mass spectrometry. The tandem secondary mass spectrometry (MS/MS) fragmentation allowed for the identification of fatty acyl residues of phosphatidylcholine molecular species. (16:0–18:1)Phosphatidylcholine was the main phosphatidylcholine molecular species determined in all shrimp samples, especially in E. superba. Macrobranchium rosenbergii phosphatidylcholine was particularly rich in (16:0–20:5)phosphatidylcholine and (16:0–22:6)phosphatidylcholine. The proportion of the two molecular species was next to the phosphatidylcholine of E. superba. Therefore, M. rosenbergii appears to be a potential freshwater source for the supplementation of polyunsaturated fatty acids, particularly eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6). This approach may be used as an efficient method for the identification of natural phosphatidylcholine sources from the broad range of plant, animal, and marine origins.     

Phytase Production by a Marine Yeast <Emphasis Type="Italic">Kodamea ohmeri</Emphasis> BG3

The marine yeast strain Kodamea ohmeri BG3 isolated from the gut of a marine fish (Hexagrammes otakii) was found to secrete a large amount of phytase into the medium. The crude phytase produced by this marine yeast showed the highest activity at pH 5.0 and 65 °C. The optimal medium for phytase production contained oat 10.0 g/l, ammonium sulfate 15.0 g/l, glucose 30 g/l, and NaCl 20.0 g/l, while the optimal cultivation conditions for phytase production were pH 5.0, a temperature of 28 °C, and a shaking speed of 170 rpm. Under the optimal conditions, over 557.9 mU/ml of phytase activity was produced within 72 h of fermentation at the shake flask level. This is a very high level of phytase activity produced by yeasts. We think that the medium and process for phytase production by the marine yeast strain were very simple, and such marine yeast from the gut of natural marine fish may have a potential application in the maricultural industry and marine environmental protection. The results demonstrate that phytate was actively degraded by the crude phytase within a short period.     

Bioaccumulation study of acrylates in algae (<Emphasis Type="Italic">Chlorella kessleri</Emphasis>) by Py-GC and Py-GC/MS

Acrylate monomers methylmethacrylate (MMA) and cyclohexylmethacrylate (CHMA) bioaccumulation has been determined in aquatic organism, algae (Chlorella kessleri). Algae were collected in amount of 0.4 mg and directly injected to the pyrolytical cell. In algae bodies accumulated monomers were analysed by pyrolysis gas chromatography (Py-GC) and pyrolysis gas chromatography coupled with mass spectrometry (Py-GC/MS). Traces of the accumulated monomers in algae body can be determined after 1-, 2-, 3-weeks of incubation. Maximum content of MMA was determined after 3-week of experiment, contrariwise in the case of CHMA after 2-week exposition. Relationship with pyrolysis temperature has also been studied.     

Physiological aspects involved in production of xylanolytic enzymes by deep-sea hyperthermophilic archaeon Pyrodictium abyssi

Xylanases (EC3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. The hyperthermophilic archaeon Pyrodictium abyssi, which was originally isolated from marine hot abyssal sites, grows optimally at 97°C and is a prospective source of highly thermostable xylanase. Its endoxylanase was shown to be highly thermostable (over 100 m in at 105°C) and active even at 110°C. The growth of the deep-sea archaeon P. abyssi was investigated using different culture techniques. Among the carbohydrates used, beech wood xylan, birch wood glucuronoxylan and the arabinoxylan from oats pelt appeared to be good inducers for endoxylanase and β-xylosidase production. The highest production of arabinofuranosidase, however, was detected in the cell extracts after growth on xylose and pyruvate, indicating that the intermediate of the tricarboxylic acid cycle acted as a nonrepressing carbon source for the production of thi enzyme. Electron microscopic studies did not show a significant difference in the cell surface (e.g., xylanosomes) when P. abyssi cells were grown on different carbohydrates. The main kinetic parameters of the organism have been determined. The cell yield was shown to be very low owing to incomplete substrate utilization, but a very high maximal specific growth rate was determined (μ_max=0.0195) at 90°C and pH 6.0. We also give information on the problems that arise during the fermentation of this hyperthermophilic archaeon at elevated temperatures.     

Quantum chemical exploration of formaldehyde clusters (H2CO)n (n = 2–4)

Global exploration of equilibrium structures and interconversion pathways on the quantum chemical potential energy surface (PES) is performed for (H₂CO)n (n = 2–4) by using the Scaled Hypersphere Search‐Anharmonic Downward Distortion Following (SHS‐ADDF) method. Density functional theoretical (DFT) calculations with empirical dispersion corrections (D3) yielded comparable results for formaldehyde dimer in comparison with recent detailed studies at CCSD(T) levels. Based on DFT‐D3 calculations, trimer and tetramer structures and their stabilities were studied. For tetramer, a highly symmetrical S₄ structure was found as the most stable form in good accordance with experimentally determined tetramer unit in the formaldehyde crystal. © 2018 Wiley Periodicals, Inc.     

Preparation of a monolithic column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction for capillary liquid chromatography

A monolithic capillary column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in‐situ copolymerization of a homemade monomer N,N‐dimethyl‐N‐acryloxyundecyl‐N‐(3‐sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed‐mode stationary phase. It was demonstrated that the mixed‐mode stationary phase displayed typic dual retention mechanisms of reversed‐phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.     

Solvent quality as reflected in concentration‐ and temperature‐dependent Flory–Huggins interaction parameters

Flory–Huggins interaction parameters (χ) between poly(dimethylsiloxane) (weight‐average molecular weight = 152 kg/mol) and various solvents (methyl ethyl ketone, toluene and n‐octane) were determined as a function of composition and temperature with vapor‐pressure measurements. These data, complemented by independent information for dilute and very concentrated solutions, serve as the basis for a discussion of solvent quality via different theoretical relations. Regardless of polymer concentration, the χ values fall from methyl ethyl ketone via toluene to n‐octane, the ketone being the worst solvent and the hydrocarbon being the best solvent. The variation of χ with composition and temperature is complex. Within the range of moderate polymer concentrations, the influences of composition decrease with increasing solvent quality. Additional effects become noticeable at the ends of the composition scale. The enthalpy parts (χ_H) and entropy parts (χ_S) of the Flory–Huggins interaction parameter, obtained from χ(T), vary considerably with composition and change their sign in some cases; these constituents of the Flory–Huggins interaction parameter do not permit a direct assessment of solvent quality. A clear‐cut picture is, however, regained with a comparison of the interdependence of χ_S and χ_H. The elimination of explicit concentration influences re‐establishes the order in the solvent quality setup via χ. © 2001 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 39: 651–662, 2001     

Overexpression of an Endochitinase Gene (<Emphasis Type="Italic">ThEn-42</Emphasis>) in <Emphasis Type="Italic">Trichoderma atroviride</Emphasis> for Increased Production of Antifungal Enzymes and Enhanced Antagonist Action Against Pathogenic Fungi

Trichoderma is one of the most promising biocontrol agents against plant fungal diseases. In this study, a transgenic strain of Trichoderma atroviride was characterized. The transgenic strain contains an endochitinase gene (ThEn-42) driven by the cellulase promoter cbh1 of T. reesei for overexpression of ThEn-42. The culture filtrates of the transformant and the parental strain grown in eight different media were evaluated for chitinase and antifungal enzyme production based on activity gels, protein profiles, and antifungal activities. Results demonstrated that chitinases are important components and synergistic interactions play a key role in the antagonistic action of T. atroviride. Moreover, altering medium nutrient concentration and composition led to enhanced production of antifungal enzymes, a potential strategy for mass production. Two of the culture filtrates contained almost pure endochitinase, and could be excellent commercial sources for this enzyme. Several culture filtrates were highly antifungal. Two filtrates were so effective in biocontrol of a fungal pathogen, Penicillium digitatum, that they not only inhibited spore germination but destroyed the spores completely when 20 μl of culture filtrate (corresponding to approximately 104 μg of total protein) was applied in a total volume of 150 μl (approximately 0.7 mg protein ml⁻¹).