LC-MS based simultaneous profiling of adrenal hormones of steroids, catecholamines, and metanephrines.

Metabolic changes in adrenocortical steroids and medullary catecholamines characterize adrenal tumors, but they are measured using different analytical protocols. To increase bioanalytical validity while maintaining sample homogeneity, LC-MS-based profiling of 29 cortical steroids and 6 medullary amines, including catecholamines and metanephrines, in a single run was developed. Alkyloxycarbonylation with isobutyl chloroformate was employed together with our comprehensive steroid assay, and all adrenal hormones were separated on a reversed-phase C18 column (50 × 2.1 mm, 1.9 µm) at a flow rate of 0.3 ml/min. The lower limits of quantification for all analytes ranged from 0.1 to 2.0 ng/ml, with extraction recoveries of 58.5%-109.5%, while the imprecision and accuracy were 1.6%-14.8% and 89.2%-114.9%, respectively. The validated LC-MS assay was applied to serum samples obtained from 60 patients with adrenal Cushing syndrome, primary aldosteronism, and pheochromocytoma/paraganglioma (PPGL). In addition to the characteristic metabolic changes in glucocorticoids, mineralocorticoids, catecholamines, and metanephrine, the molecular ratios of dehydroepiandrosterone sulfate and 20α-dihydrocortisol indicated Cushing syndrome and primary aldosteronism (P < 0.01 for all compounds), respectively. Moreover, the interactive molecular ratios of 11-deoxycortisol with normetanephrine, metanephrine, norepinephrine, and epinephrine (P < 0.01 all compounds) were proposed to characterize the metabolic features of PPGL. Novel LC-MS-based quantitative profiling of steroids, catecholamines, and metanephrines in human serum was successfully established and characterized metabolic features of individual adrenal tumors that could be used for clinical purposes.

The genome insights of Streptomyces lannensis T1317-0309 reveals actinomycin D production.

The members of Streptomyces have been identified as a major source of antimicrobial agents with broad spectrum. This study is mainly focused on bioactivity-guided isolation and characterization of bioactive molecule from strain Streptomyces sp. T1317-0309 and its whole-genome sequence analysis for possible isolation of novel natural products. Strain Streptomyces sp. T1317-0309 showed 100% sequence similarity with strain Streptomyces lannensis TA4-8T consisting 10, 453,255 bp of genome with 5 scaffolds and 69.9 mol% G + C content. The genome analyses revealed a total of 17 putative biosynthetic gene clusters (BGCs) responsible for various secondary metabolites including actinomycin, bacteriocin, ectoine, melanin, terpene, siderophore, betalactone, NRPS, T2PKS, and T3PKS. The BGC and bioactivity-guided purification of ethyl acetate extract of strain T1317-0309 showed the great potency of antimicrobial activities against various gram-positive multi-drug resistant human pathogens including MRSA. The BGC-predicted bioactive secondary metabolite was identified by various NMR analyses and confirmed as actinomycin D. In addition, this study reveals the first genome study of Streptomyces lannensis as a novel source for actinomycin D.

Evaluation and analysis of volatile organic compounds and formaldehyde emission of building products in accordance with legal standards: A statistical experimental study.

Building materials have been developed mainly for thermal performance, strength, low energy consumption, and aesthetics. Consequently, large amounts of chemicals have been added to building products, resulting in the release of abundant pollutants that adversely affect human health. In particular, pollutants from the materials used to build modern dwellings can cause sick house syndrome, which leads to health resilience problems and diseases. In this study, more than 100 investigations were conducted annually from 2004 to 2017 by using the 20 L small chamber method to analyze the contents of formaldehyde (HCHO) and total volatile organic compounds (TVOC) released from 2780 building products in total. High emissions were released by some building components with raw materials containing hazardous chemicals. However, since the 2004 enactment of a legal standard for the regulation of emissions of harmful substances in building products, the pollutant emissions have tended to decrease over the years. As a result of the experiment, all 2780 building materials met the legal standard on average. Therefore, legal restrictions on the release of hazardous materials from building products have achieved reductions in pollutant emissions.

Effective Soil Extraction Method for Cultivating Previously Uncultured Soil Bacteria.

Here, a new medium, named intensive soil extract medium (ISEM), based on new soil extract (NSE) using 80% methanol, was used to efficiently isolate previously uncultured bacteria and new taxonomic candidates, which accounted for 49% and 55% of the total isolates examined (n = 258), respectively. The new isolates were affiliated with seven phyla (Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, and Bacteroidetes). The result of chemical analysis showed that NSE included more diverse components of low-molecular-weight organic substances than two conventional soil extracts made using distilled water. Cultivation of previously uncultured bacteria is expected to extend knowledge through the discovery of new phenotypic, physiological, and functional properties and even roles of unknown genes.IMPORTANCE Both metagenomics and single-cell sequencing can detect unknown genes from uncultured microbial strains in environments, and either method may find the significant potential metabolites and roles of these strains. However, such gene/genome-based techniques do not allow detailed investigations that are possible with cultures. To solve this problem, various approaches for cultivation of uncultured bacteria have been developed, but there are still difficulties in maintaining pure cultures by subculture.

Effective and sensitive determination of eleven disinfection byproducts in drinking water by DLLME and GC-MS.

The aim of this study was to optimize the dispersive liquid-liquid microextraction (DLLME) parameters for simultaneous analysis through DLLME-gas chromatography-mass spectrometry (GC-MS) of six iodo-trihalomethanes, four haloacetonitriles, and one halonitromethane, which are residual disinfection products found in drinking water. Eleven disinfection by-product (DBPs) remaining in aqueous samples were extracted and concentrated using a simple, rapid, and environmentally friendly DLLME method, and then analyzed simultaneously by GC-MS. The optimized DLLME parameters were a sample volume of 5 mL, 100 µL of dichloromethane as the extraction solvent, 1 mL of methanol as the dispersion solvent, an extraction time of 60 s, and 1.5 g of sodium chloride for the salting out effect. The enrichment factor values obtained using the established DLLME-GC-MS method were 19.8-141.5, and the limit of detection and limit of quantification were 0.22-1.19 µg/L and 0.75-3.98 µg/L, respectively. The calibration curves had correlation coefficients (r2) of 0.9958-0.9992 in the concentration range of 0.5-40 µg/L, and they exhibited good linearity in quantitative analysis. This new method could be useful for analyzing eleven DBPs that remain in drinking water.

Rapid Quantification of N-Methyl-2-pyrrolidone in Polymer Matrices by Thermal Desorption-GC/MS.

Analysis of a residual solvent in polymeric materials has become an important issue due to the increased regulations and standards for its use. N-Methyl-2-pyrrolidone (NMP) is a solvent widely used in many industries and restricted as one of the chemicals under EU REACH regulations due to its potential harmful effects. In this study, thermal desorption-gas chromatography/mass spectrometry (TD-GC/MS) is applied for the quantitative analysis of NMP with the use of a polymer-coated sample cup. By using the polymer-coated sample cup, the vaporization of NMP was prevented during waiting time before TD-GC/MS analysis. The calibration curve for the TD method showed good linearity (correlation coefficient, r2 = 0.9998) and precision values (below 5.3% RSD). NMP recovery rates in different polymer matrices (PS, PMMA and PVC) were in the range of 98.8 to 106.6% with RSD values below 5.0%. The quantification result (600 mg NMP/kg PVC) for the blind NMP carrying sample in a PVC matrix by TD-GC/MS was higher than that (532 mg NMP/kg PVC) by solvent extraction-GC/MS method.

Description of Novosphingobiumflavum sp. nov., isolated from soil.

A novel yellow bacterial strain, designated UCM-28T, was isolated from forest soil in Gyeonggi-Do, South Korea. The isolated strain was Gram-stain-negative, aerobic, non-spore-forming, non-motile and rod-shaped, and grew at 10-37 °C, pH 5.5-9 and with 0-1 % NaCl. It could reduce nitrate to nitrite and hydrolyse aesculin. We determined the taxonomic position of strain UCM-28T; based on the 16S rRNA gene sequence, the strain belongs to the genus Novosphingobium. The bacterium showed the highest similarity to Novosphingobiumpiscinae SLH-16T (98.9 %), Novosphingobium rhizosphaerae JM-1T (97.7 %), Novosphingobium taihuense T3-B9T (97.2 %), Novosphingobium subterraneum DSM 12447T (97.1 %), Novosphingobium aromaticivorans DSM 12444T (97.1 %) and Novosphingobium capsulatum GIFU 11526T (96.7 %). Phylogenic trees also confirmed that strain UCM-28T is most closely related to Novosphingobiumpiscinae SLH-16T and others, and is positioned within the genus Novosphingobium. The DNA relatedness of strain UCM-28T with its references was in the range of 20.9-35.2 %. The polar lipid profile revealed diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, phosphatidylcholine, phosphatidylmonomethylethanolamine, six unidentified polar lipids and two unknown glycolipids. The major quinone was ubiquinone Q-10, and the major polyamine was spermidine. The DNA G+C content was 63.5 mol%. The major fatty acids included (>10 %) summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) (46.3 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) (24.9 %) and C14 : 0 2-OH (11.8 %). Based on the phylogenetic and phenotypic data, strain UCM-28T should be classified within the genus Novosphingobium as a representative of a novel species, named Novosphingobium flavum sp. nov. The type strain is UCM-28T (=KACC 18571T=NBRC 111647T).

Comparative study of thermal desorption and solvent extraction-gas chromatography-mass spectrometric analysis for the quantification of phthalates in polymers.

For the quantitative analysis of phthalates in polymers, a thermal desorption (TD)-GC-MS method was compared with solvent extraction (SE)-GC-MS methods which require the long pretreatment procedures using large amount of harmful organic solvents. Calibration curves of TD-GC-MS showed good linearity (r(2)>0.9997) and low method detection limit (<30mg/kg with 9.0% RSD). Quantification results for three kinds of test phthalate polymer samples (test PTPSs) showed an RSD below 7.4% and acceptable recoveries (78.3-117.4%) as in the standard method of International Electrotechnical Commission. Even in a sample with a high concentration of phthalates (PTPS #3), the method also showed good recovery with low RSD values. The TD-GC-MS results were comparable with those results by SE-GC-MS methods, indicating that TD-GC-MS method also can be used for the quantification of phthalates in polymers. The average recovery (92-103%) and RSD (<20%) values obtained from international inter-laboratory study for TD-GC-MS performed in six laboratories also indicated that TD-GC-MS can be used as an international standard method for the quantification of phthalates in polymers.

Occurrence and ecological hazard assessment of selected veterinary medicines in livestock wastewater treatment plants.

The occurrence of some veterinary medicines in the livestock wastewater plants (WWTPs) was investigated. This investigation represented the occurrence of veterinary medicines to treat in the livestock WWTPs or be discharged into the water system in Korea since the sampling sites were widely distributed across the nation and samples were collected from the 11 livestock WWTPs. Nine antibiotics, two analgesics, and two disinfectants occurred in the livestock wastewater plants (WWTPs). From 11 livestock WWTP influents, chlortetracycline, oxytetracycline, acetylsalicylic acid, and disinfectants frequently occurred with the high concentrations. Meanwhile, sulfamethoxazole, erythromycin-H2O, and trimethoprim did not occur during sampling periods. The values for log Kow of each chemical showed a high correlation with the number of hydrogen bonding acceptors and were important parameters to estimate and understand the biodegradability and toxicity of a compound in the environment. The biodegradability of each compound was proportional to the hydrophilicity of each compound and the toxicity was proportional to the number of hydrogen bonding acceptors of each compound. The expected introductory concentration (EIC), predicted exposure concentration (PEC), and hazard quotient showed that the livestock WWTP effluents were hazardous to ecosystems.

Simultaneous measurement of serotonin, dopamine and their metabolites in mouse brain extracts by high-performance liquid chromatography with mass spectrometry following derivatization with ethyl chloroformate.

In order to measure the levels of serotonin (5-hydroxyltryptamine, 5-HT), dopamine (DA), 3,4-hydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) simultaneously, an effective derivatization method followed by high-performance liquid chromatography (HPLC) coupled to electrochemical ionization mass spectrometry was used. The derivatization reaction of biological samples with ethyl chloroformate occurred rapidly at room temperature in aqueous conditions, and the resulting derivatives were isocratically separated with good selectivity using a C18 reversed-phase column within 30 min. The study results showed that the new derivatization procedure offers an excellent means of simultaneous determination of 5-HT, DA and their metabolites in mouse brain homogenates, which are important in a number of physiological and behavioral functions.

Electronic effect on the kinetics of the diaza-cope rearrangement.

Hammett plot reveals that there is a significant electronic effect on the rate of resonance assisted hydrogen bond (RAHB) directed diaza-Cope rearrangement reaction with a rho value of 1.6. DFT computation shows that the rearrangement reaction becomes thermodynamically more favorable for the substrates with electron withdrawing substituents. A substrate with the nitro substituent (1a) reacts about 50-fold faster than that with the methoxy substituent (1g).

Gas chromatograph-mass spectrometric method for the determination of carvedilol and its metabolites in human urine.

A sensitive and efficient method was developed for the determination of carvedilol and its metabolites in human urine by gas chromatography-mass spectrometry (GC-MS). Urine samples were hydrolyzed with beta-glucuronidase/arylsulfatase (from Helix pomatia) and the target compounds were extracted with liquid-liquid extraction. The extracts were completely derivatized with MSTFA and MBTFA and analyzed by GC-MS using an Ultra-2 column. The linearity of the assay ranges were 0.75-75 ngmL(-1) for carvedilol and o-desmethyl carvedilol (o-DMC), and 3.0-75 ngmL(-1) for 4-hydroxyphenyl carvedilol (4-HPC) and 5-hydroxyphenyl carvedilol (5-HPC). The absolute recovery of carvedilol and its metabolites added to a blank urine sample was 80.1-97.8%. The limits of detection (LOD) and quantitation (LOQ) of carvedilol and o-DMC were 0.30 and 0.75 ngmL(-1), and its of 4-HPC and 5-HPC were 0.75 and 3.0 ngmL(-1), respectively. The reproducibilities were 1.86-11.5% for the intra-day assay, and 0.70-1.71% for the inter-day assay precision and the degree of inaccuracy was -3.0 to 3.9% at the concentration of 75 ngmL(-1). The proposed GC-MS method was effective for the determination of carvedilol and its three metabolites in human urine.

Absorption, distribution, metabolism, and excretion of CKD-732, a novel antiangiogenic fumagillin derivative, in rats, mice, and dogs.

The pharmacokinetics of CKD-732 (6-O-4-[dimethyl-aminoethoxy)cinnamoyl]-fumagillol x hemioxalate) was investigated in male SD rats and beagle dogs after bolus intravenous administration. The parent compound and metabolites obtained from in vitro and in vivo samples were determined by LC/MS. The main metabolite was isolated and identified as an N-oxide form of CKD-732 by NMR and LC/MS/MS. CKD-732 was metabolized into either M11 or others by rapid hydroxylation, demethylation, and hydrolysis. The blood level following the intravenous route declined in first-order kinetics with T(1/2)beta values of 0.72 to approximately 0.78 h for CKD-732 and 0.92 to approximately 1.09 h for M11 in rats at a dose of 7.5 to approximately 30 mg/kg. In dogs, T(1/2)beta values of CKD-732 and M11 were 1.54 and 1.79 h, respectively. Moreover, AUC values increased dose dependently for CKD-732 and M11 in rats and dogs. The CLtot and Vdss did not change significantly with increasing dose, indicating linear pharmacokinetic patterns. The excretion patterns through the urine, bile, and feces were also examined in the animals. The total amount excreted in urine, bile, and feces was 2.13% for CKD-732 and 1.29% for M11 in rats, and 1.58% for CKD-732 and 2.28% for M11 in dogs.

Analysis of benzene ethylamine derivatives in urine using the programmable dynamic liquid-phase microextraction (LPME) device.

An automated LPME device for a dynamic LPME method was manufactured and its extraction efficiency was tested using spiked urine samples. The developed home-made LPME device was a programmable automated syringe dispenser to overcome deteriorating precision and difficulties in manually manipulating the plunger repeatedly. To establish the optimum parameters for benzene ethylamines, the effects of sampling volume, solvent volume, pH, salt-effect, choice of solvents, plunger speed, and number of samplings were investigated. Good repeatabilities for the extraction of mephentermine, ephedrine, methoxyphenamine, selegiline, and bupropion were obtained and the RSD values were 2.4, 1.9, 1.3, 1.6 and 1.5% at a concentration of 3 microg mL(-1) in spiked urine samples, respectively. The limit of detection was below 0.05 microg mL(-1) for the investigated drugs. This developed device for LPME analysis gave good validation results and improved convenience.

The identification of in vitro metabolites of CKD-732 by liquid chromatography/tandem mass spectrometry.

The structural elucidation of fourteen metabolites of CKD-732, formed in vitro with rat liver microsomes, was performed using high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS). To identify proposed structures of the metabolites, the product ion mass spectra of the protonated molecules ([M + H]+), the retention times on reversed-phase HPLC, and UV-Vis spectra were utilized. Characteristic product ions for the identification of CKD-732 metabolites were observed at m/z 231, 236, and 252. The fragment ions at m/z 236 and 252 indicated the unchanged form and the N-oxide of the dimethylaminoethoxycinnamoyl group, respectively. The ion at m/z 231 indicated the presence of the hydroxylated form of the fumagillol group. The N-oxide of CKD-732, which was detected at m/z 515 and eluted later than CKD-732 in the reversed-phase HPLC system, was measured as a major metabolite. Three cis-trans isomers were also found.

Mass spectrometric analysis of cyclofenil and its metabolites in human urine.

Using gas chromatography/electron impact-mass spectrometry (GC/EI-MS) and high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), the structures of cyclofenil metabolites in human urine have been assigned. The hydroxyl metabolites liberated from the glucuronide conjugates after acid hydrolysis were characterized as the trimethylsilyl (O-TMS) derivatives using GC/MS. The conjugate glucuronide forms were detected without hydrolysis by HPLC/MS. Cyclofenil was not observed in urine. Tentative structures for the two metabolites are proposed.