MSn Data Research Articles

A novel neutral loss/product ion scan-incorporated integral approach for the untargeted characterization and comparison of the carboxyl-free ginsenosides from Panax ginseng, Panax quinquefolius, and Panax notoginseng

Differentiated composition in precursor ions for different subclasses of ginsenosides in the negative electrospray-ionization mode has been reported, which lays a foundation for the sorted and untargeted identification of ginsenosides. Carboxyl-free ginsenosides simultaneously from Panax ginseng, P. quinquefolius, and P. notoginseng, were comprehensively characterized and statistically compared. A neutral loss/product ion scan (NL-PIS) incorporated untargeted profiling approach, coupled to ultra-high performance liquid chromatography, was developed on a linear ion-trap/Orbitrap mass spectrometer for characterizing carboxyl-free ginsenosides. It incorporated in-source fragmentation (ISF) full scan-MS1, mass tag-MS2, and product ion scan-MS3. Sixty batches of ginseng samples were analyzed by metabolomics workflows for the discovery of ginsenoside markers. Using formic acid (FA) as the additive, carboxyl-free ginsenosides (protopanaxadiol-type, protopanaxatriol-type, and octillol-type) gave predominant FA-adducts, while rich deprotonated molecules were observed for carboxyl-containing ginsenosides (oleanolic acid-type and malonylated) when source-induced dissociation (SID) was set at 0 V. Based on the NL transition [M+FA‒H]− > [M−H]− and the characteristic sapogenin product ions, a NL-PIS approach was established. It took advantage of the efficient full-information acquisition of ISF-MS1 (SID: 50 V), the high specificity of mass tag (NL: 46.0055 Da)-induced MS2 fragmentation, and the substructure fragmentation of product ion scan-MS3. We could characterize 216 carboxyl-free ginsenosides, and 21 thereof were potentially diagnostic for the species differentiation. Conclusively, sorted and untargeted characterization of the carboxyl-free ginsenosides was achieved by the established NL-PIS approach. In contrast to the conventional NL or PIS-based survey scan strategies, the high-accuracy MSn data obtained can enable more reliable identification of ginsenosides.

Separation and Characterization of New Components and Impurities in Leucomycin by Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Combined with Ion Trap/Time-of-Flight Mass Spectrometry

In this study, nine new components and six impurities in leucomycin were discovered. A method was developed for the separation and characterization of new components and impurities in leucomycin by multiple heart-cutting two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry in both positive and negative electrospray ionization modes. With this method, a non-volatile buffer solution was used as mobile phase in the first-dimensional system for good separation. Eluent of each peak from the first-dimensional system was trapped by a switching valve and sent to the liquid chromatography-mass spectrometry system using a volatile mobile phase. The complete fragmentation patterns of the new components and degradation impurities were deduced based on MSn data. The structures of nine new components in leucomycin were deduced as unsaturated ketone in the 16-membered ring of leucomycin. The structures of six impurities were characterized for the first time, four of which were acid degradation products, and the other two were process impurities. The correlation between impurities and the purification process of leucomycin was also studied. The degradation impurities were produced during purification of leucomycin fermentation broth, which requires a low-pH environment. Based on the characterization of impurities, this study not only revealed the mechanism of impurity production, thus providing guidance to pharmaceutical companies for manufacturing process improvement and impurity reduction, but also provided a scientific basis for further improvement of official monographs in pharmacopoeias.

TLC-electrostatic field induced spray ionization-MS analysis of diverse structural skeletons and its coupling with TLC bioautography for characterization of lipase inhibitory components in American ginseng

Natural compounds with diverse structural skeletons have different ionization efficiencies and different mass spectrometry (MS) responses. Some key factors influencing the electrostatic field induced spray ionization (EFISI)-MS for a variety of natural compounds have been optimized and improved. Fifteen reference substances representing ten well-known skeletons of natural products including alkaloids, flavonoids, phenolic acids, lignans, coumarins, anthraquinones, monoterpenoids, sesquiterpenoids, diterpenoids and triterpenoids, were selected and investigated for their EFISI-MSn responses on TLC plates using a dot-blot test. The optimized ionization conditions for these compounds in the positive ion mode were achieved, together with their limits of detection. In addition, to avoid the limitation of some compounds being difficultly ionized in the positive ion mode, the negative ion mode of the TLC-EFISI-MS method was developed and optimized for the first time. By coupling with a TLC bioautographic assay, nine lipase inhibitory components in American ginseng (Panax quinquefolium roots) have been successfully identified/ tentatively identified in situ by their EFISI-MSn data and further confirmed by comparisons of their Rf values and MSn data with those of reference substances. These lipase inhibitory compounds were 24(S)-pseudo-ginsenoside F11, ginsenosides Rg1, Re, XVII, Rc, Rb2/Rb3, Rb1, Ro and malonyl-ginsenoside Rb1. This is the first report that the TLC-EFISI-MSn method is adapted to a broad-spectrum analysis of structural skeletons present in herbal medicines.

Characterization of a new component and impurities in josamycin by trap-free two-dimensional liquid chromatography coupled to ion trap time-of-flight mass spectrometry.

The toxicities of the impurities of a drug will affect the clinical effects and cause potential health risk; therefore, it is essential to study profiles of the impurities. In this study, a new structural type of component and two acid degradation impurities in josamycin were discovered and characterized for the further improvement of official monographs in pharmacopoeias. The component and acid degradation impurities in josamycin were separated and preliminary characterized by trap-free two-dimensional liquid chromatography coupled to high-resolution ion trap time-of-flight mass spectrometry (2D LC/IT-TOF MS) in both positive and negative electrospray ionization mode. The eluent of each peak from the first dimensional chromatographic system was trapped by a switching valve and subsequently transferred to the second dimensional chromatographic system, which was connected to the mass spectrometer. Full scan MS was firstly conducted to obtain the exact m/z values of the molecules. Then LC/MS/MS and LC/MS/MS/MS experiments were performed on the compounds of interest. A new structural type of component, which was named as josamycin A, and two acid degradation impuritiess, which were identified as impurity I and impurity II, were discovered in josamycin. Their structures and fragmentation pattern were deduced according to MSn data. Furthermore, josamycin A was synthesized and impurity I was separated by preparative HPLC. The structures of josamycin A and the impurities were confirmed by 1 H NMR and 13 C NMR data. Josamycin A was produced when the hydroxyl group on the macrolide of josamycin was oxidized into a carbonyl group. Impurity I and impurity II were produced by the loss of one molecule of acetyl mycaminose from josamycin and josamycin A, respectively. Compared with josamycin, the experimental results showed that josamycin A had a higher antibacterial activity with similar cytotoxicity, while impurity I had no antibacterial activity but a higher cytotoxicity. As a result, the control of impurity I is significant.

O uso de ferramenta síncrona na coleta de dados na pesquisa em educação online

The article describes the features of MSN, shows the history of how it was developed and its possibilities of use in online education. Reports the experience lived through an interview with a teacher researcher in technology education making use of MSN. It presents difficulties in this collection. Brings considerations regarding the importance and limitations of the MSN data collection of online search.

Assessing Fucosyltransferase Activity via MSn

Fucosyltransferases add fucose residues to oligosaccharides. Resulting fucosylated epitopes include the common core fucosylation of N‐glycans, as well as important ligands such as Lewis structures and H antigens. Many of these structural motifs can be found on N‐linked glycans, O‐linked glycans, and glycosphingolipids. In complex biological samples, multiple fucosyltransferases, as well as multiple substrate specificities of individual fucosyltransferases, can result in multiple isomeric forms within individual glycan compositions. Intact mass analysis alone will not distinguish these isomers. Chromatographic separation may or may not resolve these structures, and, will not provide direct structure identification.Typically, MS/MS analysis of fucosylated N‐linked glycans can distinguish core fucosylation from antennal fucosylation. Finer details such as distinguishing Lewis structures from H antigens is a more difficult problem, as fucosylated lactosamine antennae fragments are produced from both structures. Disassembling these structural fragments to a further stage, MS3, in an ion trap, can easily distinguish Lewis from H antigen structures, providing direct empirical evidence of structure.We demonstrate MSn data from a variety of samples. Fucosyltransferase activity was assessed through transfection of fucosyltransferase (3,4,5,6,7, and 9) genes into mesenchymal stem cells, with comparison to non‐transfected cells as a control. MCF‐7 and MDA‐MB‐231 cultured cells were also interrogated for fucosylation isomers. Glycophorin C was also interrogated, with fucosylation on polylactosamine structures.For cultured cells, glycolipids were extracted via solvent extraction. Proteins were enzymatically digested, followed by enzymatic release of N‐linked glycans. Released N‐glycans were separated from O‐glycopeptides; O‐glycans were released by reductive b‐elimination. Purified glycan and glycosphingolipid fractions were permethylated. Glycan samples were directly infused into the mass spectrometer via nano‐ESI. Precursor ion selection was made manually.Fucosyltransferase‐transfected mesenchymal stem cells were interrogated for synthesis of Lewis X structures, with data shown to indicate that different fucosyltransferases show differing preferences for synthesizing Lewis X vs. sialylated Lewis X.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Feruloyloacetone can be the main curcumin transformation product

Curcumin is a phenolic compound produced by some plants, among which Curcuma longa is the reachest in this principal curcuminoid. At elevated temperature curcumin degrades to trans-6-(4′-hydroxy-3′-methoxyphenyl)-2,4-dioxo-5-hexenal, vanillin, ferulic acid and feruloylmethane, however, the formation of feruloyloacetone ((5E)-6-(4-hydroxy-3-methoxyphenyl)hex-5-ene-2,4-dione) in the curcumin degradation process has not been reported yet. As results from experiments, even 28.8% or 20.6% of the degraded curcumin is transformed to feruloyloacetone during 2 h heating of alkaline or acidic curcumin solution, respectively. The structure of the identified feruloyloacetone was confirmed by MSn, HRMS and NMR data. The presented results are important for food processing as feruloyloacetone is formed during food products preparation and its biological activity has not been fully recognized.

Gas-phase fragmentation reactions of protonated benzofuran- and dihydrobenzofuran-type neolignans investigated by accurate-mass electrospray ionization tandem mass spectrometry.

We have investigated gas-phase fragmentation reactions of protonated benzofuran neolignans (BNs) and dihydrobenzofuran neolignans (DBNs) by accurate-mass electrospray ionization tandem and multiple-stage (MSn ) mass spectrometry combined with thermochemical data estimated by Computational Chemistry. Most of the protonated compounds fragment into product ions B ([M+H-MeOH]+ ), C ([B-MeOH]+ ), D ([C-CO]+ ), and E ([D-CO]+ ) upon collision-induced dissociation (CID). However, we identified a series of diagnostic ions and associated them with specific structural features. In the case of compounds displaying an acetoxy group at C-4, product ion C produces diagnostic ions K ([C-C2 H2 O]+ ), L ([K-CO]+ ), and P ([L-CO]+ ). Formation of product ions H ([D-H2 O]+ ) and M ([H-CO]+ ) is associated with the hydroxyl group at C-3 and C-3', whereas product ions N ([D-MeOH]+ ) and O ([N-MeOH]+ ) indicate a methoxyl group at the same positions. Finally, product ions F ([A-C2 H2 O]+ ), Q ([A-C3 H6 O2 ]+ ), I ([A-C6 H6 O]+ ), and J ([I-MeOH]+ ) for DBNs and product ion G ([B-C2 H2 O]+ ) for BNs diagnose a saturated bond between C-7' and C-8'. We used these structure-fragmentation relationships in combination with deuterium exchange experiments, MSn data, and Computational Chemistry to elucidate the gas-phase fragmentation pathways of these compounds. These results could help to elucidate DBN and BN metabolites in in vivo and in vitro studies on the basis of electrospray ionization ESI-CID-MS/MS data only.

Separation and Characterization of Unknown Impurities and Isomers in Cefminox Sodium and Study of the Forming Mechanisms of Impurities by Liquid Chromatography Coupled with Ion Trap/Time-Of-Flight Mass Spectrometry.

Thirteen unknown impurities and isomers in cefminox sodium were separated and characterized by liquid chromatography coupled with high-resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF-MS) with the positive mode of electrospray ionization (ESI) method. New HPLC-gradient elution method was developed for the detection of impurities in cefminox sodium. And the ESI ion trap multiple-stage tandem mass spectrometry had been applied successfully to the direct investigation of impurities and isomers in cefminox sodium. The fragmentation patterns and structural assignment of these impurities were studied. Full scan liquid chromatography-mass spectrometry (LC-MS) was first performed to obtain the m/z value of the protonated molecules and formulas of all detected peaks, LC-MSn (n = 1-6) were then carried out on the compounds of interest. Structures of 13 degradation products in cefminox sodium were deduced based on the high-resolution MSn (n = 1-6) data, assisted by the UV spectra and stress testing. And the forming mechanisms of degradation products in cefminox were also studied. The method of LC-IT-TOF-MSn (n = 1-6) was worthy of widespread use and application for the further improvement of official monographs in pharmacopoeias with the advantages of stability and repeatability.

Characterization of solution stress degradation products of aliskiren and prediction of their physicochemical and ADMET properties

Forced degradation studies on aliskiren were carried out according to ICH and WHO guidelines. Six degradation products were formed in total in the solution state. Their separation among themselves and from the drug was successfully achieved on a C-18 column utilizing acetonitrile and phosphate buffer (pH 3.0) in the mobile phase, which was run in a gradient mode. To characterize them, a complete mass fragmentation pathway of the drug was first established with the help of MS/TOF and MSn data. This was followed by LC-MS/TOF studies on the degradation products. Some of the degradation products were also isolated and subjected to 1D (1H, 13C and DEPT-135) and 2D (COSY, HSQC and HMBC) NMR studies for confirmation of their structures. An interesting observation was hydrolysis followed by cyclization in case of three degradation products. Also, acetonitrile was found to react with aliskiren, leading to formation of a pseudo degradation product. Additionally, comparative ADMET properties of the drug and degradation products were established using ADMET Predictor™.

Separation and characterization of allergenic polymerized impurities from cephalosporin for injection by trap free two-dimensional high performance size exclusion chromatography × reversed phase liquid chromatography coupled with ion trap time-of-flight mass spectrometry

As requested by regulatory authorities, polymerized impurities are an important issue of quality control. In this study, we presented the utilization of a trap-free two-dimensional chromatography, which was consisted by a high performance size exclusion chromatography (HPSEC) and a reversed-phase liquid chromatography (RP-LC) coupled to ion trap time-of-flight mass spectrometry with positive mode of electrospray ionization, to separate and characterize ten allergenic impurities in ceftazidime for injection, cefazolin sodium for injection, cefoperazone sodium and sulbactam sodium for injection and cefamandole nafate for injection. An effective method for characterizing the polymerized impurities in β-lactam antibiotics was established on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension, the column was the Xtimate SEC-120 analytical column (7.8 mm × 30 cm, 5 μm) and the flow rate was 1.0 mL min−1 with gradient elution using 0.005 mol L−1 phosphate buffer solution at pH 7.0 and acetonitrile as mobile phase. In the second dimension, the analytical column was ZORBAX SB-C18 (4.6 × 150 mm, 3.5 μm) using ammonium formate solution (10 mM) and ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase at a flow rate of 0.4 mL min−1. Eluent associate with each peak separated in the first dimension was trapped by a 20 μL quantitative loop and then transferred (via a six-port valve) into the second dimension system with volatile mobile phase. Through the multiple heart-cutting 2D-LC approach and online desalting technique, the problem of incompatibility between non-volatile mobile phase and mass spectrometry was solved completely. The fragmentation behaviors of ten allergenic impurities were studied. The structures of ten allergenic impurities in cephalosporin drug substance were deduced based on the HPLC–MSn data, in which four impurities were polymerized impurities. The forming factors of polymerized impurity in cephalosporins were also studied.

Simultaneous Analysis of Saccharides between Fresh and Processed Radix Rehmanniae by HPLC and UHPLC-LTQ-Orbitrap-MS with Multivariate Statistical Analysis.

Radix Rehmanniae (RR) is a kind of herb which is widely used in the clinical and food processing industry. There are four forms of RR used in traditional Chinese medicine practice, which include fresh RR (FRR), raw RR (RRR), processed RR (PRR), and another processed RR (APRR), in which the APRR was processed by nine cycles of repeated steaming and drying. There are a large number of saccharides in RR. However, the differences in content were shown by different processing methods. In this study, an effective method using high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry (LC-MS) coupled with multivariate statistical analysis to rapidly distinguish different RR samples and validate the proposed chemical conversion mechanism. The datasets of the content of saccharides were subjected to principal component analysis (PCA) and one-way analysis of variance. The results showed that there different changes occurred in the contents of saccharides corresponding to the different processing methods, in which the contents of monosaccharides—namely arabinose, glucose, mannose, and galactose—had an increasing trend or remained relatively stable. However, the contents of fructose and oligosaccharides, including manninotriose, melibiose, sucrose, and raffinose, first increased and then reduced, or gradually decreased, yet the content of stachyose gradually decreased. The MSn data indicated that manninotriose, melibiose, and some monosaccharides were produced by the hydrolysis of oligosaccharides. In addition, the fragmentation pathways of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization of monosaccharides were also found that its glycosidic bond was first broken and subsequently its inside ring broke, and the characteristic fragment ions were produced at m/z 511.22, 493.20, 373.16, and 175.08 in the PMP derivatization of monosaccharides. In conclusion, this study illustrates the change and chemical conversion mechanism of saccharides by processing in RR samples which might play a key role in further application of RR.

A Comprehensive, Open-source Platform for Mass Spectrometry-based Glycoproteomics Data Analysis

Glycosylation is among the most abundant and diverse protein post-translational modifications (PTMs) identified to date. The structural analysis of this PTM is challenging because of the diverse monosaccharides which are not conserved among organisms, the branched nature of glycans, their isomeric structures, and heterogeneity in the glycan distribution at a given site. Glycoproteomics experiments have adopted the traditional high-throughput LC-MSn proteomics workflow to analyze site-specific glycosylation. However, comprehensive computational platforms for data analyses are scarce. To address this limitation, we present a comprehensive, open-source, modular software for glycoproteomics data analysis called GlycoPAT (GlycoProteomics Analysis Toolbox; freely available from www.VirtualGlycome.org/glycopat). The program includes three major advances: (1) "SmallGlyPep," a minimal linear representation of glycopeptides for MSn data analysis. This format allows facile serial fragmentation of both the peptide backbone and PTM at one or more locations. (2) A novel scoring scheme based on calculation of the "Ensemble Score (ES)," a measure that scores and rank-orders MS/MS spectrum for N- and O-linked glycopeptides using cross-correlation and probability based analyses. (3) A false discovery rate (FDR) calculation scheme where decoy glycopeptides are created by simultaneously scrambling the amino acid sequence and by introducing artificial monosaccharides by perturbing the original sugar mass. Parallel computing facilities and user-friendly GUIs (Graphical User Interfaces) are also provided. GlycoPAT is used to catalogue site-specific glycosylation on simple glycoproteins, standard protein mixtures and human plasma cryoprecipitate samples in three common MS/MS fragmentation modes: CID, HCD and ETD. It is also used to identify 960 unique glycopeptides in cell lysates from prostate cancer cells. The results show that the simultaneous consideration of peptide and glycan fragmentation is necessary for high quality MSn spectrum annotation in CID and HCD fragmentation modes. Additionally, they confirm the suitability of GlycoPAT to analyze shotgun glycoproteomics data.

Transformation products elucidation of forchlorfenuron in postharvest kiwifruit by time-of-flight mass spectrometry

Forchlorfenuron (1-(2-chloro-4-pyridyl)-3-phenylurea, FCF) is a plant growth regulator, being extensively used for increasing kiwifruit size. The toxicological properties of its may persist in their transformation products (TPs) or even higher toxicity than FCF. TPs elucidation of FCF in postharvest kiwifruit (Actinidia chinensis, Chinese gooseberry) by the liquid chromatography ionization hybrid ion trap and time-of-flight mass spectrometry (LC-ESI-IT-TOF/MS) in positive mode was the objective of the present study. Fifteen days after full bloom, kiwifruits were dipped for 5s with high dosage FCF solution (60 mg/L), so that sufficient peaks could be detected. The chemical structure of unknown TPs was analyzed in combination of functions of LCMS-IT-TOF, such as high-accurate MSn, formula predictor, metabolite structural analysis software MetID Solution, profiling solution metabolomics software, and neutral loss, characteristic isotopic patterns of chlorine, the fragmentation pattern and retention time of standard substances, nitrogen rule, chemical components of kiwifruit. Total 17 TPs were detected via comparisons of their accurate MSn data of commercial analytical standards and synthesized standards with high purity, such as 4-amino-2-chloropyridine, phenylurea, 2-hydroxy-FCF, 1-(2-chloro-6-((3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) pyridin-4-yl)-3-phenylurea, 1, 3-bis (2-chloropyridin-4-yl) urea, 1,3-diphenylurea, 1-(2-chloropyridin-4-yl)urea, FCF-2-O-β-D-glucoside, and so on. The major transformation pathways of FCF in kiwifruit were biochemical and photochemical cleavage pathway. The experimental results indicate that LCMS-IT-TOF is powerful and effective tool for identification of FCF TPs.

A multiple-dimension liquid chromatography coupled with mass spectrometry data strategy for the rapid discovery and identification of unknown compounds from a Chinese herbal formula (Er-xian decoction)

It is very important to rapidly discover and identify the multiple components of traditional Chinese medicine (TCM) formula. High performance liquid chromatography with high resolution tandem mass spectrometry (HPLC-HRMS/MS) has been widely used to analyze TCM formula and contains multiple-dimension data including retention time (RT), high resolution mass (HRMS), multiple-stage mass spectrometric (MSn), and isotope intensity distribution (IID) data. So it is very necessary to exploit a useful strategy to utilize multiple-dimension data to rapidly probe structural information and identify chemical compounds. In this study, a new strategy to initiatively use the multiple-dimension LC–MS data has been developed to discover and identify unknown compounds of TCM in many styles. The strategy guarantees the fast discovery of candidate structural information and provides efficient structure clues for identification. The strategy contains four steps in sequence: (1) to discover potential compounds and obtain sub-structure information by the mass spectral tree similarity filter (MTSF) technique, based on HRMS and MSn data; (2) to classify potential compounds into known chemical classes by discriminant analysis (DA) on the basis of RT and HRMS data; (3) to hit the candidate structural information of compounds by intersection sub-structure between MTSF and DA (M,D-INSS); (4) to annotate and confirm candidate structures by IID data. This strategy allowed for the high exclusion efficiency (greater than 41%) of irrelevant ions in er-xian decoction (EXD) while providing accurate structural information of 553 potential compounds and identifying 66 candidates, therefore accelerating and simplifying the discovery and identification of unknown compounds in TCM formula.

Separation and characterization of unknown impurities in cefonicid sodium by trap-free two-dimensional liquid chromatography combined with ion trap time-of-flight mass spectrometry.

Seven unknown antibiotic impurities in cefonicid sodium were separated and characterized by a trap-free two-dimensional liquid chromatography coupled to high-resolution ion-trap time-of-flight mass spectrometry (2D-LC/IT-TOF MS) using both positive and negative modes of electrospray ionization. Trap-free 2D-LC and an online demineralization technique made it possible to characterize cefonicid sodium under the conditions of the official standard, and the TIC chromatogram obtained by LC/MS was in conformity with the LC chromatogram obtained by the official analytical method in the peak sequence of impurities. In the first dimension, the column was a GRACE Alltima C18 (250mm×4.6mm, 5μm), and the gradient elution used 0.02mol·L-1 ammonium dihydrogen phosphate solution and methanol as mobile phase. In the second dimension, the analytical column was a Shimadzu Shim-pack GISS C18 (50mm×2.1mm, 1.9μm) with 10mmol·L-1 ammonium formate solution and methanol as mobile phase. Full scan LC/MS was first executed to obtain the exact m/z values of the molecules. Then LC/MS2 and LC-MS3 experiments were performed on the compounds of interest. The structures of seven unknown degradation products in cefonicid sodium were deduced based on the high-resolution MSn data using both positive and negative mode. The problem of incompatibility between the non-volatile salt mobile phase and mass spectrometry was solved completely by multidimensional heart-cutting approaches and an online demineralization technique, which is worthy of widespread use and application for the advantages of stability and repeatability.

Evaluating Nitrogen-Containing Biosynthetic Products Produced by Saltwater Culturing of Several California Littoral Zone Gram-Negative Bacteria.

The biosynthetic potential of marine-sediment-derived Gram-negative bacteria is poorly understood. Sampling of California near-shore marine environments afforded isolation of numerous Gram-negative bacteria in the Proteobacteria and Bacteriodetes phyla, which were grown in the laboratory to provide extracts whose metabolites were identified by comparative analyses of LC-mass spectrometry and MSn data. Overall, we developed an assemblage of seven bacterial strains grown in five different media types designed to coax out unique secondary metabolite production as a function of varying culture conditions. The changes in metabolite production patterns were tracked using the GNPS MS2 fragmentation pattern analysis tool. A variety of nitrogen-rich metabolites were visualized from the different strains grown in different media, and strikingly, all of the strains examined produced the same new, proton-atom-deficient compound, 1-methyl-4-methylthio-β-carboline (1), C13H12N2S. Scale-up liquid culture of Achromobacter spanius (order: Burkholderiales; class: Betaproteobacteria) provided material for the final structure elucidation. The methods successfully combined in this work should stimulate future studies of molecules from marine-derived Gram-negative bacteria.

Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-HPSEC × LC-IT-TOF MS

Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and characterized by a trap-free two-dimensional high performance size exclusion chromatography (HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion trap/time-of-flight mass spectrometry (2D-HPSEC×LC-IT-TOF MS) with positive and negative modes of electrospray ionization method. Separation and characterization the allergic polymerized impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8mm×30cm, 5μm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second dimension analytical column was ZORBAX SB-C18 (4.6×150mm, 3.5μm) with ammonium formate solution (10mM) and ammonium formate (8mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase. Structures of eleven unknown impurities were deduced based on the high resolution MSn data with both positive and negative modes, in which nine impurities were polymerized impurities. The forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely by multidimensional heart-cutting approaches and online demineralization technique, which was worthy of widespread use and application for the advantages of stability and repeatability.

Antibacterial secondary metabolites from an endophytic fungus, Fusarium solani JK10

Extensive chemical investigation of the endophytic fungus, Fusarium solani JK10, harbored in the root of the Ghanaian medicinal plant Chlorophora regia, using the OSMAC (One Strain Many Compounds) approach resulted in the isolation of seven new 7–desmethyl fusarin C derivatives (1–7), together with five known compounds (8–12). The structures of the new compounds were elucidated by analysis of their spectroscopic data including 1D, 2D NMR, HRESI–MSn and IR data. The relative configuration of compounds 1/2 was deduced by comparison of their experimental electronic circular dichroism (ECD) and optical rotation data with those reported in literature. The absolute configuration of solaniol (10), a known compound with undefined absolute stereochemistry, was established for the first time by X–ray diffraction analysis of a single–crystal structure using Cu–Kα radiation. The antibacterial activities of the crude fungal extract and the compounds isolated from the fungus were evaluated against some clinically important bacterial strains such as Staphylococcus aureus and Bacillus subtilis, as well as an environmental strain of Escherichia coli and the soil bacterium Acinetobacter sp. BD4. Compounds 3/4 and 6 exhibited antibacterial efficacies against the soil bacterium Acinetobacter sp., comparable to the reference standard streptomycin. All the tested compounds (1–9) demonstrated antibacterial activity against the environmental strain of E. coli, whereas no antibacterial activity was observed against S. aureus and B. subtilis. The antibacterial activity of the isolated compounds typically against E. coli and Acinetobacter sp. provides further insight into the possible involvement of root-borne endophytes in chemical defense of their host plants in selected ecological niches.

Metabolic profiling of five flavonoids from Dragon's Blood in human liver microsomes using high-performance liquid chromatography coupled with high resolution mass spectrometry.

Although much is known about the pharmacological activities of Dragon's Blood (DB, a traditional Chinese herb), its metabolism in human liver microsomes (HLMs) and the cytochrome P450 (CYP) enzymes has not been studied. This study aims to identify the metabolic profile of five flavonoids (loureirin A, loureirin B, loureirin C, 7,4'-dihydroxyflavone and 5,7,4'-trihydroxyflavanone) from DB in HLMs as well as the CYP enzymes that are involved in the metabolism of them. High-resolution mass spectrometry was used to characterize the structures of their metabolites and 10 cDNA-expressed CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) were used to verify which isozymes mediate in the metabolism of the metabolites. Totally, 29 metabolites including 10 metabolites of loureirin A, 10 metabolites of loureirin B, 4 metabolites of loureirin C, 2 metabolites of 7,4'-dihydroxyflavone and 3 metabolites of 5,7,4'-trihydroxyflavanone were elucidated and identified on the basis of the high-resolution MSn data. The metabolic profile of the five flavonoids in HLMs involved hydroxylation, oxidation and demethylation. Among them, hydroxylation was the predominant biotransformation of the five flavonoids in HLMs, occurring in combination with other metabolic reactions. Assay with recombinant P450s revealed that CYP2C9 and CYP2C19 played an important role in the hydroxylation of flavonoids in HLMs. To the best of our knowledge, this is the first in vitro evaluation of the metabolic profile of loureirin A, loureirin B, loureirin C, 7,4'-dihydroxyflavone and 5,7,4'-trihydroxyflavanone in HLMs.