2-氨基-3-硝基吡啶和2-氨基-5-硝基吡啶的EI/MS/MS分析

2 氨基 3 硝基吡啶和2 氨基 5 硝基吡啶的一级质谱图接近,单纯通过一级质谱图较难区分这两种异构体,利用离子阱质谱的串联质谱技术对2 氨基 3 硝基吡啶和2 氨基 5 硝基吡啶在离子阱内以He作碰撞气进行碰撞诱导裂解,所得的二级质谱图表明,两者之间存在明显的差别,可用于2 氨基 3 硝基吡啶和2 氨基 5 硝基吡啶的鉴别。     

Electron ionisation induced fragmentation of fused norbornene analogues containing SiMe2 or GeMe2 and oxygen bridges. Migration of SiMe2 and GeMe2 groups

The main electron ionisation induced fragmentation processes of fused norbornene analogues containing SiMe2 or GeMe2 and oxygen bridges, as well as their dependence on substitution, were investigated using mass (MS) and tandem mass (MS/MS) spectrometric analysis. Formation of the rearrangement ions of m/z 176 in the mass spectra of fused norbornene analogues containing SiMe2 and m/z 222 in the mass spectrum of norbornene analogues containing GeMe2 provides firm evidence for the migration of a SiMe2 and GeMe2 bridge, respectively.     

A fully automated nanoelectrospray tandem mass spectrometric method for analysis of Caco-2 samples

Caco-2 cells offer a means to rapidly screen permeability of drug candidates, allowing pharmaceutical companies to eliminate candidates unable to cross the intestinal barrier early in the discovery process. This screening process is typically performed by conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS), which can require time-consuming method development. An alternative to LC/MS/MS, automated nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS), is introduced. This novel approach requires an off-line ZipTip desalting step followed by automated nanoESI-MS/MS, using the NanoMate 100 and ESI Chip. In addition to reduced method development time, automated nanoESI-MS/MS also offers no carry-over between samples, low sample consumption, and ease-of-use as compared with conventional pulled-capillary nanoelectrospray. Furthermore, the infusion system described has the potential to be high-throughput. A comparison of Caco-2 samples analyzed both by LC/MS/MS and by automated nanoESI-MS/MS is presented. The permeability and recovery data of the two compounds analyzed in this study obtained from conventional LC/MS/MS and by automated nanoESI-MS/MS were in excellent agreement.     

Development and validation of an on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry method for the simultaneous determination of prostaglandins E(2) and F(2alpha) and 13,14-dihydro-15-keto prostaglandin F(2alpha) levels in human plasma

We developed and validated an on-line reverse-phase two-dimensional LC/MS/MS (2D-LC/MS/MS) system for simultaneous determination of the levels of prostaglandin (PG) E(2) as well as PGF(2alpha) and its metabolite 13,14-dihydro-15-keto PGF(2alpha) (F(2alpha)-M) in human plasma. Analytes were extracted by a three-step solid-phase extraction. Samples were then analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system is composed of two reverse-phase analytical columns with a trapping column linking the two analytical columns. While an acidic buffer was used for both separation dimensions, differing organic solvents were employed for each dimension: methanol for the first and acetonitrile for the second to increase resolving power. The 2D-LC/MS/MS method was highly selective and sensitive with a significantly lower limit of quantitation (0.5 pg/mL for PGE(2) and 2.5 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively). Linearity of the 2D-LC/MS/MS system was demonstrated for the calibration ranges of 0.5-50 pg/mL for PGE(2) and 2.5-500 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively. Acceptable precision and accuracy were obtained throughout the calibration curve ranges. This highly selective and sensitive method was successfully utilized to determine the endogenous levels of PGE(2), PGF(2alpha), and F(2alpha)-M in plasma samples from six (four male and two female) normal volunteers. The mean concentrations for each analyte were 0.755 pg/mL for PGE(2), 5.70 pg/mL for PGF(2alpha) and 9.48 pg/mL for F(2alpha)-M.     

Characteristic fragmentation behavior of the analogs of endomorphin-2 with phenylglycine in position 3 or 4 by ESI-FT tandem mass spectrometry

A series of analogs of endomorphin-2 （EM-2） with phenylglycine （Phg） in position 3 or 4 were synthesized. In electrospray ionization Fourier transform ion cyclotron resonance （ESI-Fr-ICR） MS/MS spectra of these compounds, some b, y, a, and internal ions were observed and slight mass differences between the calculated and observed results are obtained. Their sequences were derived successfully. However, the MS/MS patterns of these analogs with Dphg and Lphg were very similar. It is hard to distinguish them by MS/MS spectra. Moreover, if the third position was substituted by phenylglycine （L or D）, a rearrangement could occur in MS/MS experiment to lose proline residue.     

Determination of aflatoxins in peanuts, maize feed and whole milk by HPLC-MS2 and MS3 tandem mass spectrometry

A method is presented for the detection of six toxins of fungal origin in various foods: peanuts, a sample of maize feed and whole milk. Extraction and HPLC-APCI-MS detection were discussed. The performances of MS/MS, MS3 and SRM detection modes were checked and best conditions of sensitivity were obtained in MS/MS mode.     

Analysis of sugar epimers using mass spectrometry: N-acetyllactosamine-6,6'-disulfate and the 2'-epimer

Galbeta1-4GlcNAc-6,6'-disulfate and 2'-epimer corresponding to Galbeta1- 4ManNAc-6,6'-disulfate were distinguished by mass spectrometry by utilizing fast atom bombardment (FAB), electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) methods. As for the steric information, negative-ion ESI mass spectrometry/mass spectrometry (MS/MS) provides the most extensive data, but FAB MS/MS also reveals detailed structural information of interest in our case, where MALDI MS is not yet fully equipped with post-source decay.     

Synthesis and Electrospray Ionization Mass Spectra of N-（1,3, 2-Dioxaphosphorinan-2-ylmethyl）thiophosphoramidates

N-（1,3,2-Dioxaphosphorinan-2-ylmethyl）thiophosphoramidates were synthesized and determined by NMR spectra and positive ion electrospray ionization mass spectrometry （ESI-MS） in conjunction with tandem mass spectrometry （MS/MS）. The fragmentation pathways were investigated. The results show that these characteristic ions in ESI mass spectra are useful in the structural determination of N-（1,3,2-dioxaphosphorinan-2-ylmethyl）- thiophosphoramidates.     

High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1

The inhibition potential of drugs towards five major human hepatic cytochrome P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated via cassette dosing of the five probe substrates (coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96-well plate format. After microsomal incubations had been terminated with formic acid, the five marker metabolites (7-hydroxycoumarin, 1'-hydroxymidazolam, 4-hydroxytolbutamide, dextrorphan, and 6-hydroxychlorzoxazone) were simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS). Several advantages resulted from the use of a short C(18) guard cartridge (4 mm in length) for DI-GCE/MS/MS, including minimal sample preparation, fast online extraction, short analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an inter-day accuracy range from -8.7 - 7.4% with a precision less than 8.3% for the quantification of all the marker metabolites. The inhibition assay for the five CYP isozymes was evaluated using their known selective inhibitors via individual and cassette dosing of the probe substrates. The IC(50) values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement with the reported values. In addition, the validated assay was used to evaluate the inhibitory potential of 23 generic drugs (randomly selected) towards the five CYP isozymes. The results suggest the integration of the cassette dosing strategy and the DI-GCE/MS/MS method can provide a reliable in vitro approach to screening the inhibitory potential of new chemical entities, with maximal throughput and cost-effectiveness, in support of drug discovery and development.     

An unusual cleavage reaction of a peptide observed during dithiotreitol and tris(2-carboxyethyl)phosphine reduction: application to sequencing of HpTx2 spider toxin using nanospray tandem mass spectrometry

A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem mass spectrometry (MS/MS). This 30-residue toxin possesses a highly knotted structure with cystines arranged in close proximity. The low-energy collision-induced dissociation MS/MS spectrum of the [M+4H](4+) ion permitted characterization of the C-terminal sequence of HpTx2 up to Cys(26) that is involved in a disulfide bridge. Chemical pre-treatment with DTT or TCEP was then investigated, and it was found that an unexpected cleavage reaction of HpTx2 gave two smaller peptides which were completely sequenced by MS/MS experiments using a Qq-TOF mass spectrometer. This unusual hydrolysis reaction facilitated the determination of the complete sequence of the HpTx2 toxin.     

Primary and secondary locations of charge sites in angiotensin II (M + 2H)2+ ions formed by electrospray ionization

High-energy tandem mass spectrometry and molecular dynamics calculations are used to determine the locations of charge in metastably decomposing (M + 2H)2+ ions of human angiotensin II. Charge-separation reactions provide critical information regarding charge sites in multiple charged ions. The most probable kinetic energy released (Tm.p.) from these decompositions are obtained using kinetic energy release distributions (KERDs) in conjunction with MS/MS (MS2), MS/MS/MS (MS3), and MS/MS/MS/MS (MS4) experiments. The most abundant singly and doubly charged product ions arise from precursor ion structures in which one proton is located on the arginine (Arg) side chain and the other proton is located on a distal peptide backbone carbonyl oxygen. The MS3 KERD experiments show unequivocally that neither the N-terminal amine nor the aspartic acid (Asp) side chain are sites of protonation. In the gas phase, protonation of the less basic peptide backbone instead of the more proximal and basic histidine (His) side chain is favored as a result of reduced coulomb repulsion between the two charge sites. The singly and doubly charged product ions of lesser abundance arise from precursor ion structures in which one proton is located on the Arg side chain and the other on the His side chain. This is demonstrated in the MS3 and MS4 mass-analyzed ion kinetic energy spectrometry experiments. Interestingly, (b7" + OH)2+ product ions, like the (M + 2H)2+ ions of angiotensin II, are observed to have at least two different decomposing structures in which charge sites have a primary and secondary location.     

Structural characterization of 2-aminobenzamide-derivatized oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight tandem mass spectrometer

Oligosaccharides were derivatized by reductive amination using 2-aminobenzamide (2-AB) and analyzed by matrix-assisted laser desorption/ionization two-stage time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2-AB-derivatized oligosaccharides. A systematic study was conducted on a series of 2-AB-derivatized oligosaccharides to allow rationalization of the fragmentation processes. The MALDI-TOF/TOF-MS/MS spectra of the [M+Na]+ ions of 2-AB-derivatized oligosaccharides were dominated by glycosidic cleavages. These fragments originating both from the reducing and the non-reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, the MALDI-TOF/TOF-MS/MS spectra were also characterized by abundant cross-ring fragments which are very informative on the linkages of the monosaccharide residues constituting these oligosaccharides. MALDI-TOF/TOF-MS/MS analysis of 2-AB-derivatized oligosaccharides, by providing structural information at the low-picomole level, appears to be a powerful tool for carbohydrate structural analysis.     

Development and in-house validation of a robust and sensitive solid-phase extraction liquid chromatography/tandem mass spectrometry method for the quantitative determination of aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods

A sensitive and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods. Samples were extracted with a mixture of acetonitrile/water (84:16, v/v) and cleaned up through a polymeric solid-phase extraction column. Detection and quantification of the nine mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS), using fully (13)C-isotope-labelled mycotoxins as internal standards. The method was validated in-house for five different cereal processed products, namely barley, oat and durum wheat flours, rye- and wheat-based crisp bread. Recoveries and repeatability of the whole analytical procedure were evaluated at contamination levels encompassing the EU maximum permitted levels for each tested mycotoxin. Recoveries ranged from 89 to 108% for deoxynivalenol, from 73 to 114% for aflatoxins, from 85 to 114% for T-2 and HT-2 toxins, from 64 to 97% for zearalenone, from 74 to 102% for ochratoxin A. Relative standard deviations were less than 16% for all tested mycotoxins and matrices. Limits of detection (signal-to-noise ratio 3:1) ranged from 0.1 to 59.2 μg/kg. The trueness of the results obtained by the proposed method was demonstrated by analysis of reference materials for aflatoxins, deoxynivalenol, zearalenone. The use of inexpensive clean-up cartridges and the increasing availability of less expensive LC/MS/MS instrumentation strengthen the potential of the proposed method for its effective application for reliable routine analysis to assess compliance of tested cereal products with current regulation.     

双电荷离子[C12H12N2O]2+和[C12H12N2S]2+的气相单分子分解反应研究

The unimolecular dissociation reactions of doubly charged ions were reported,which resulted from a tandem mass spectrometer and a reversed geometry double focusing mass spectrometer by electron impact.Mass analyzed ion kinetic energy spectrometry(MIKES) was used to obtain the kinetic energy releases in charge separation reactions of doubly charged ions.The intercharge distances between the two charges at transition states can be calculated from the kinetic energy releases.Transition structures of unimolecular dissociation reactions were infered from MIKES and MS/MS.     

Binding sites of [Ru(bpy)2(H2O)2](BF4)2 with sulfur- and histidine-containing peptides studied by electrospray ionization mass spectrometry and tandem mass spectrometry

The composition and binding sites of cis-[Ru(II)(bpy)2]2+-bound sulfur-containing peptides of Met-Arg-Phe-Ala, glutathione and oxidized glutathione, and also histidine-containing peptide of oxidized insulin B chain, were investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). The composition of Ru(II)-containing peptides was precisely determined by ESI-MS, zoom scan and simulation of isotope distribution patterns. MS/MS analysis shows that, in sulfur-containing peptides, the Ru(II) complex prefers to anchor to a carboxyl group, although some other potential binding sites of thiol, thioether and N-terminal amino groups present in these peptides, and in oxidized insulin B chain, Ru(II) first anchors to His10, then either to the hydroxyl group of Thr27 or to the carboxyl group of Ala30. Its secondary structure and microenvironment surrounding the potential binding sites may affect the binding ability of cis-[Ru(II)(bpy)2]2+ to oxidized insulin B chain.     

Electrospray ionization ion trap mass spectrometry for structural characterization of oligosaccharides derivatized with 2-aminobenzamide

The use of electrospray ionization (ESI) quadrupole ion trap mass spectrometry and reversed-phase high-performance liquid chromatography (HPLC) for the characterization of 2-aminobenzamide (2AB)-labeled oligosaccharides and N-linked protein oligosaccharide mixtures is described. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2AB-derivatized oligosaccharides. Under collision-induced dissociation, sodiated molecular species generated in the ESI mode yield simple and predictable mass spectra. Tandem mass spectrometry (MS/MS) experiments with orders higher than two offer a number of ways to enhance MS/MS spectra and to derive information not present in MS and MS2 spectra. Information on composition, sequence, branching and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds and from weak cross-ring cleavage products. Reversed-phase HPLC and derivatization by reductive amination using 2-aminobenzamide were finally applied to characterize a glycan pool enzymatically released from glycoproteins.     

Mass spectrometry for the characterization of unsulfated chondroitin oligosaccharides from 2-mers to 16-mers. Comparison with hyaluronic acid oligomers

This study reports for the first time the complete liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) analyses performed in negative ion mode of saturated unsulfated chondroitin oligosaccharides up to 16-mers and comparison with hyaluronic acid (HA) oligomers differing only in the nature of the hexosamine residue. MS/MS of the chondroitin disaccharide on the singly charged precursor at m/z 396.1 afforded a glycosidic cleavage C1 product ion at m/z 192.9. In the tetrasaccharide, C2 (m/z 396.0) and C3 (m/z 572.0) product anions were generated by glycosidic cleavage. A C5 [M-2H]2- product ion at m/z 475.1 was generated by the glycosidic cleavage of the hexasaccharide, and a C7 ion (m/z 664.6, charge state of -2) was produced from the octasaccharide. The same fragmentation pattern of deprotonated oligomers was observed for the largest oligosaccharides, from 10- to 16-mers. There has been no previous report of MS/MS spectra for unsulfated chondroitin oligomers of these sizes. Unsulfated saturated chondroitin oligosaccharides with x-mer units and larger than a tetrasaccharide dissociate to almost exclusively form CX-1-type ions. Saturated HA oligomers also afforded the same fragmentation pattern as deprotonated oligomers by ESI-MS and MS/MS analyses. Thus, under the experimental conditions used in the current study, we were unable to distinguish between unsulfated chondroitin and HA.     

Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.     

The application of 2-D dual nanoscale liquid chromatography and triple quadrupole-linear ion trap system for the identification of proteins

2-D nanoscale LC combined with a triple quadrupole-linear ion trap mass spectrometer was applied to the analysis of a complex peptide mixture. A 2-D dual nanoscale LC-MS/MS system was compared to a conventional one. Peptides were separated with a strong cation exchange (SCX) microcolumn in the first dimension and two C18 nanocolumns were used as second dimension. MS experiments were performed using information-dependent data acquisition, where two precursor ions were selected from an enhanced MS (EMS) or an enhanced multicharged ion (EMC) as survey scan. The major benefit of EMC instead of EMS was a two-fold reduction of the data file and a 15% increase of characterized proteins. The advantage of the 2-D dual nanoscale LC-MS/MS system versus the conventional 2-D nanoscale LC-MS/MS system was reflected in the significant increase of peptides which were successfully identified within the same time frame. The first factor contributing to this increase was that the mass spectrometer was collecting twice the number of relevant MS/MS data. The second factor is the use of twice the number of SCX salt fractions in the first dimension, allowing a better sample fractionation, thereby reducing the number of peptides transferred to the second chromatographic dimension per salt fraction.