Mass Spectral Fragmentation Patterns Research Articles

Characterizing the electrospray ionization mass spectral fragmentation pattern of indole derivatives synthesized from 2‐keto glycosides

Four indole derivatives synthesized from 2-keto glycosides were analyzed by electrospray ionization tandem mass spectrometry (ESI-MSn) with low-energy collision-induced dissociation to establish a general structural elucidation of indole derivatives. Their fragmentation pathways are proposed on the basis of the MSn studies and deuterium-labeled experiments. Indole derivatives undergo complicated gas-phase rearrangements in addition to simple bond cleavages. A rearrangement, which involves a contraction of the six-membered ring, was observed and a mechanism was proposed. The observations may have some potential applications in the interpretation of the mass spectra of indole derivatives.

An Elegant Synthesis of Quino[2,3‐a]carbazoles and Their Antibacterial Studies.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Fresh organically grown ginger ( Zingiber officinale): composition and effects on LPS-induced PGE 2 production

Gas chromatography in conjunction with mass spectrometry, a technique previously employed to analyze non-volatile pungent components of ginger extracts modified to trimethylsilyl derivatives, was applied successfully for the first time to analyze unmodified partially purified fractions from the dichloromethane extracts of organically grown samples of fresh Chinese white and Japanese yellow varieties of ginger, Zingiber officinale Roscoe (Zingiberaceae). This analysis resulted in the detection of 20 hitherto unknown natural products and 31 compounds previously reported as ginger constituents. These include paradols, dihydroparadols, gingerols, acetyl derivatives of gingerols, shogaols, 3-dihydroshogaols, gingerdiols, mono- and diacetyl derivatives of gingerdiols, 1-dehydrogingerdiones, diarylheptanoids, and methyl ether derivatives of some of these compounds. The thermal degradation of gingerols to gingerone, shogaols, and related compounds was demonstrated. The major constituent in the two varieties was [6]-gingerol, a chemical marker for Z. officinale. Mass spectral fragmentation patterns for all the compounds are described and interpreted. Anti-inflammatory activities of silica gel chromatography fractions were tested using an in vitro PGE 2 assay. Most of the fractions containing gingerols and/or gingerol derivatives showed excellent inhibition of LPS-induced PGE 2 production.

Biodegradation of alachlor by soil streptomycetes.

Streptomycetes resistant to the herbicide alachlor [2-chloro-2',6'-diethyl- N-(methoxymethyl) acetanilide] were used in degradation assays to characterize the products of alachlor biodegradation. Of six strains tested, Streptomyces sp. LS166, LS177, and LS182 were able to grow at an alachlor concentration of 144 mg l(-1) and degraded approximately 60-75% of the alachlor in 14 days, as evaluated by high performance liquid chromatography. The alachlor biodegradation products were identified by gas chromatography-mass spectrometry based on mass spectral data and fragmentation patterns. All compounds detected in these assays were similar for all streptomycetes strains tested, and involved dechlorination with subsequent N-dealkylation and cyclization of the remaining N-substituent with one of the ethyl groups to produce indole and quinoline derivatives. The enzymatic pathway used by Streptomyces sp. LS182 did not generate DEA (2',6'-diethylaniline), a carcinogenic derivative of alachlor reported in other studies. Given the high degradation rates observed here, the Streptomyces strains tested may be useful in the degradation/detoxification processes of alachlor.

A new abscisic acid catabolic pathway.

We report the discovery of a new hydroxylated abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism in Brassica napus siliques. This metabolite reveals a previously unknown catabolic pathway for ABA in which the 9'-methyl group of ABA is oxidized. Analogs of (+)-ABA deuterated at the 8'-carbon atom and at both the 8'- and 9'-carbon atoms were fed to green siliques, and extracts containing the deuterated oxidized metabolites were analyzed to determine the position of ABA hydroxylation. The results indicated that hydroxylation of ABA had occurred at the 9'-methyl group, as well as at the 7'- and 8'-methyl groups. The chromatographic characteristics and mass spectral fragmentation patterns of the new ABA metabolite were compared with those of synthetic 9'-hydroxy ABA (9'-OH ABA), in both open and cyclized forms. The new compound isolated from plant extracts was identified as the cyclized form of 9'-OH ABA, which we have named neophaseic acid (neoPA). The proton nuclear magnetic resonance spectrum of pure neoPA isolated from immature seeds of B. napus was identical to that of the authentic synthetic compound. ABA and neoPA levels were high in young seeds and lower in older seeds. The open form (2Z,4E)-5-[(1R,6S)-1-Hydroxy-6-hydroxymethyl-2,6-dimethyl-4-oxo-cyclohex-2-enyl]-3-methyl-penta-2,4-dienoic acid, but not neoPA, exhibited ABA-like bioactivity in inhibiting Arabidopsis seed germination and in inducing gene expression in B. napus microspore-derived embryos. NeoPA was also detected in fruits of orange (Citrus sinensis) and tomato (Lycopersicon esculentum), in Arabidopsis, and in chickpea (Cicer arietinum), as well as in drought-stressed barley (Hordeum vulgare) and B. napus seedlings.

Analytical techniques for sesquiterpene emission rate studies in vegetation enclosure experiments

Sesquiterpene (SQT) compounds (C 15 H 24 ) and their oxygenated alcohol and ketone derivatives are biogenic volatile organic compounds that have been identified in emissions from vegetation. SQT emission rates and landscape flux estimates are highly uncertain. Reliable ambient flux measurements have not been possible because of low-ambient concentrations, rapid atmospheric reactions (prohibiting ambient tower flux measurements), and analytical challenges and uncertainties that stem from the low volatility of SQT. Standards from an in situ capillary diffusion system with 18 SQT compounds and four other organic compounds (geranylacetone, 1,3,5-tri-isopropylbenzene, diphenylmethane, nonylbenzene) were used to thoroughly investigate experimental procedures for SQT emission rate studies by vegetation enclosure techniques. Recovery rates in tubing materials, sampling bags, leaf cuvettes, on six solid adsorbent materials (Tenax TA, Tenax GR, Carbotrap, Carbotrap C, Unibeads, Glass Beads) for gas chromatography analysis, and gas chromatography retention indices and mass spectral fragmentation patterns were determined. SQT compounds were found to exhibit a high degree of stickiness to all materials tested. However, the non-oxygenated SQT can be recovered in enclosure experiments for quantitative emission rate determination after careful consideration of the analytical conditions. It is utmost important to allow sufficient purging and equilibration times for all materials in contact with the sample air. In contrast oxygenated SQT were irreversibly lost in enclosure experiments which made their quantitative measurement prohibitive. Results for the other organic compounds were similar and indicate that these data mostly stem from the volatility of these compounds. Consequently, the findings of this study provide guidelines for the analysis of a wide range of volatile organic compounds in the ∼C13–C17 volatility range.

Recognition of cysteine-containing peptides through prompt fragmentation of the 4-dimethylaminophenylazophenyl-4'-maleimide derivative during analysis by MALDI-MS.

Under specified UV-MALDI conditions, the 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) derivative of cysteine-containing peptides undergoes prompt fragmentation to produce a characteristic mass spectral pattern. As reported previously by others, derivatization with chromophoric DABMI allows cysteine-containing peptides and proteins to be tracked during HPLC by absorbance at upper UV and visible wavelengths. Here, we describe methodology by which these same peptide derivatives can be recognized by their distinctive MALDI mass spectral fragmentation pattern, then mass mapped, allowing for easy, rapid identification of cysteine-containing peptides.

An approach to the proteomic analysis of a breast cancer cell line (SKBR-3).

This report describes the profiling of proteins in a sample prepared by laser capture microdissection (LCM) from a breast cancer cell line (SKBR-3). This experimental approach serves as a model system for proteomic studies on selected tissue samples and for studies of specific cell types. The captured cells were isolated in a dehydrated and reduced state and solubilized with a denaturing buffer. After dilution the protein mixture was digested with trypsin and the resulting peptide mixture was fractionated by reversed phase HPLC (RPLC) and analyzed on an ion trap mass spectrometer. A key part of this study is the combination of the LCM process with an extraction/digestion procedure that allowed effective solubilization of a significant part of the cellular sample in a single step. The identity of the peptides was determined by tandem mass spectrometry measurements in which the resulting spectra were compared with genomic and proteomic databases and protein identifications were made. While only peptides with a high probability assignment were used, the interpretation of mass spectral fragmentation patterns were also confirmed by manual interpretation of the spectra. Also, for the more abundant proteins the initial protein assignment from the best match peptide was strengthened by the observation of additional confirmatory peptide identifications. Another selection criteria was correlation of the mass spectrometric studies with clinical and genomic studies of potential cancer markers in tumor samples. This proteomic study allowed identification of the following proteins: human receptor protein kinase HER-2 or ERBB-2 and related kinases HER-3 and HER-4, the gene products from breast cancer type I and II susceptibility genes and cytoskeletal components such as cytokeratins 8, 18 and 19. Other proteins include fibroblast growth factor receptor variants (FGFR-2&4) and T-lymphoma invasion and metastasis inducing protein 1 (TIAM1). In addition several nonreceptor protein kinases YES, FAK and JAK-1 and 3 were identified. Since the study was performed on a limited number of cells (approximately 10,000) it raises the possibility of such studies being performed on individual patient samples prepared by needle biopsy.

Characterizing the electrospray-ionization mass spectral fragmentation pattern of enzymatically derived hyaluronic acid oligomers

Oligosaccharides derived from hyaluronic acid by action of bovine testicular hyaluronidase (BTH) (hyaluronate 4-glycanohydrolase, E.C. 3.2.1.35) were characterized by mass spectrometry (MS) with electrospray-ionization mass spectrometry (ESIMS) and compared with results obtained by matrix-assisted laser desorption/ionization. Both oligomers with an odd number and even number of sugar units with molecular masses up to 8 kDa were observed in the ESI spectra. However, the generation of odd-numbered oligomers is not consistent with the regiospecificity of the enzyme and with the MALDI results, which indicated even-numbered oligomers exclusively. In addition, a third method of characterization, high-performance anion-exchange chromatography (HPAEC), showed only even-numbered oligomers. Relative intensities of the odd-numbered oligomers demonstrated in ESIMS a cone-voltage dependence suggesting the odd-numbered oligomers resulted from collisional activation. In order to achieve results by ESI that mirror results from other techniques, the cone voltage must be kept low and precisely controlled. This study displays the usefulness and possible vulnerabilities of ESIMS when utilized for carbohydrate analysis without corroborating data from other methods of analysis.

Two Distinct Conformers of the Cyclic Heptapeptide Phakellistatin 2 Isolated from the Fijian Marine Sponge Stylotella aurantium.

AbstractFor Abstract see ChemInform Abstract in Full Text.

In vivo biotransformation of 17 alpha-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry.

The in vivo phase I biotransformation of 17 alpha-methyltestosterone in the horse leads to the formation of a complex mixture of regio- and stereoisomeric C(20)O(2), C(20)O(3) and C(20)O(4) metabolites, excreted in urine as glucuronide and sulphate phase II conjugates. The major pathways of in vivo metabolism are the reduction of the A-ring (di- and tetrahydro), epimerisation at C-17 and oxidations mainly at C-6 and C-16. Some phase I metabolites have been identified previously by positive ion electron ionisation capillary gas chromatography/mass spectrometry (GC/EI + MS) mainly from the characteristic fragmentation patterns of their methyloxime-trimethylsilyl ether (MO-TMS), enol-TMS or TMS ether derivatives. Following oral administration of 17 alpha-methyltestosterone to two castrated thoroughbred male horses, the glucuronic acid conjugates excreted in post-administration urine samples were selectively hydrolysed by E. coli beta-glucuronidase enzymes. Unconjugated metabolites and the steroid aglycones obtained after enzymatic deconjugation were isolated from urine by solid-phase extraction, derivatised as MO-TMS ethers and analysed by GC/EI + MS. In addition to some of the known metabolites previously identified from the characteristic mass spectral fragmentation patterns of 17 alpha-methyl steroids, some isobaric compounds exhibiting a diagnostic loss of 103 mass units from the molecular ions with subsequent losses of trimethylsilanol or methoxy groups and an absence of the classical D-ring fragment ion were detected. From an interpretation of their mass spectra, these compounds were identified as 17-hydroxymethyl metabolites, formed in vivo in the horse by oxidation of the 17-methyl moiety of 17 alpha-methyltestosterone. This study reports on the GC/EI + MS identification of these novel 17-hydroxymethyl C(20)O(3) and C(20)O(4) metabolites of 17 alpha-methyltestosterone excreted in thoroughbred horse urine.

Gas chromatography/mass spectrometric study of non-commercial C-4-substituted 1,4-dihydropyridines and their oxidized derivatives.

A gas chromatography/mass spectrometry (GC/MS) method for the qualitative and quantitative determination of the calcium-channel antagonists C-4-substituted 1,4-dihydropyridines, and their corresponding N-ethyl derivatives, is presented. Also, the electrochemical oxidation and the reactivity of the compounds with alkyl radicals derived from 2,2'-azobis-(2-amidinopropane) were monitored by GC/MS. Mass spectral fragmentation patterns for the C-4-substituted 1,4-dihydropy-ridine parent drugs were significantly different from those of their oxidation products, generated either by electrochemical oxidation or by reaction with alkyl radicals. However, for N-ethyl-1,4-dihydropyridine compounds it was not possible to detect the final products (pyridinium salts) using these experimental conditions.

Two distinct conformers of the cyclic heptapeptide phakellistatin 2 isolated from the fijian marine sponge stylotellaaurantium.

The isolation, structure determination, and solution conformation of two conformers of the cyclic heptapeptide phakellistatin 2 (cyclo-[Phe1-cis-Pro2-Ile3-Ile4-cis-Pro5-Tyr6-cis-Pro7]) isolated from the Fijian marine sponge Stylotella aurantium are reported. The conformers can be isolated separately by HPLC and are stable in methanol solution over a period of weeks as determined by NMR. Their NMR spectra and mass spectral fragmentation patterns differ significantly. Their solution conformations were determined by NOE-restrained molecular dynamics calculations and indicated that the two conformers had different folds, hydrogen bonding patterns, and solvent accessible surfaces. These factors may contribute to the independent stability of the two conformers, and may explain the variable biological activity previously reported for phakellistatin 2.

Isolation of (R)-(+)-Pulegone from the European Pennyroyal Mint, Mentha Pulegium

Students obtain, via steam distillation, pennyroyal essential oil that is analyzed by capillary gas chromatography—mass spectrometry. TLC experiments establish conditions for preparative scale purification of the major oil component via flash column chromatography. The terpene obtained, (R)-(+)-pulegone, is characterized spectroscopically, employing at a minimum mass spectral molecular ion and fragmentation patterns, IR, and 300-MHz 1H NMR. Optionally, 13C and 2-D correlation NMR spectra can be utilized to enable unambiguous assignment of all C and H resonances. The project has been successfully incorporated into our upper-level advanced organic chemistry laboratory. The experiment provides opportunities for instruction in and experience with a wide variety of chromatographic and spectroscopic methods. Further, it centers on a plant and natural product well suited for the discussion of contemporary health care issues surrounding nontraditional/alternative medicine and herbal remedies.

Isolation and multiresidue detection of macrolide endectocides present in animal matrices.

A simple, rapid, and sensitive method for isolation and detection of macrolide endectocides (moxidectin, doramectin, selamectin, ivermectin, and eprinomectin) in animal sera and liver is described. Fortified sera or homogenized liver samples were treated with sodium chloride followed by organic solvent extraction. No additional steps were required prior to analysis. Separation of analytes and collection of mass information was achieved by liquid chromatography/mass spectrometry with positive atmospheric pressure chemical ionization set in selected ion monitoring mode with each sample analysis complete in 15 minutes. Presence of each compound was confirmed based on 2 separate extracted ion profiles. Detection of avermectins and moxidectin in a range of working standards was achieved at 10, 50, and 100 ppb. Quantitation of these compounds in fortified samples was based on standard calibration curves with R2 > 0.99. Detection limits of 10 ppb for ivermectin, moxidectin, and doramectin, 50 ppb for selamectin, and 100 ppb for eprinomectin were achieved in spiked sera. Recoveries of avermectins and moxidectin in 500 ppb fortified sera fell between 61 and 89% (+/-5.7-15.7). Analysis of fortified liver gave comparable results with recovery of selamectin of 83-91% +/- 18.3. A complete mass spectral fragmentation pattern of selamectin and affordable screening method for 6 macrolide endectocides are reported. Method comparison for salt treatment and solid-phase extraction of fortified samples is discussed.

Spin Trapping Studies on Radical Cations of Styrene

AbstractThe radical cat ions of styrene (π‐type), generated by photosensitized electron‐transfer of 1,4‐dicyanobenzene (DCB), using 2‐methyl‐2‐nitrosopropane (MNP) as spin trap ping agents and nucleophiles as the co‐reactant were investigated. The spin‐trapped nitroxide radicals were subjected to HPLC‐ESR and mass spectroscopic studies. Long‐range hfsc were obtained by resolution enhancement. Mass spectral fragmentation pat terns sup port the regioselective formation of the trapped nitroxides, which is derived from the anti‐Markovnikov addition of the methanol to the β‐position of the styrene cationradicals.

Gas chromatographic analysis of diels‐alder adducts of geometrical and positional isomers of conjugated linoleic acid

AbstractThis research demonstrates the gas chromatographic analysis of the 4‐methyl‐1,2,4‐triazoline‐3,5‐dione (MTAD) adducts derived from standards of cis,trans‐9,11‐octadecadienoic acid, trans,trans‐9,11‐octadecadienoic acid, and cis,cis‐9,11‐octadecadienoic acid. Methyl cis,trans‐9,11‐octadecadienoate and methyl trans,trans‐9,11‐octadecadienoate formed Diels‐Alder addition products with MTAD to produce adducts with similar mass spectral fragmentation patterns but different retention times determined by gas chromatography/ mass spectrometry. Methyl cis,cis‐9,11‐octadecadienoate reacted slowly and produced two adducts with similar fragmentation patterns and different retention times. These results were comparable to those reported for an analogous series of conjugated hexadienes. Based on hexadiene reactions, methyl cis,trans‐9,11‐octadecadienoate produced a trans adduct as a major product while methyl trans,trans‐9,11‐octadecadienoate formed a cis adduct. Methyl cis,cis‐9,11‐octadecadienoate reacted slowly under the conditions used leaving mostly unreacted material. Of the adducts observed from this isomer, a major trans adduct and a minor cis adduct were formed.

Chiral Recognition of O-Phosphoserine by Mass Spectrometry.

Identification and quantification of the enantiomeric excess of O-phosphoserine (OP Ser) residues from post-translational modified proteins is now possible by the application of a very reproducible and sensitive mass spectrometric method. This technique is based on the different mass-spectral fragmentation patterns of the diastereomeric cluster ions generated in water/methanol solutions of the analyte (OP SerD/L ) with a suitable chiral compound (OP ThrL ) by collision-induced dissociation (CID; see mass spectra).

Mass Spectral Fragmentation Patterns of Deuterated Butyl and Ethyl Acetates: An Easy Microscale Isotopic Labeling Experiment

We have developed an experimentally uncomplicated microscale second-semester organic lab exercise that illustrates the use of isotopic labeling, in this case deuteration, to help confirm and interpret mass spectral fragmentation patterns in butyl acetate and ethyl acetate. Preparation and gas chromatographic-mass spectrometric (GC-MS) analysis of unlabeled and labeled esters is followed by pooling of student data for interpretation and write-up.

Chiral discrimination of monofunctional alcohols and amines in the gas phase

A methodology has been developed for enantiodiscriminating chiral monoalcohols and monoamines by mass spectrometry. The approach is based on the generation of supersonically expanded complexes of these molecules with suitable chromophores, i.e. R-(+)-1-phenyl-ethanol (ER) or R-(+)-1-phenyl-1-propanol (PR). The jet-cooled diastereomeric complexes, otherwise elusive at room temperature, have been ionized by one-color resonant two-photon absorption (R2PI) and their fragmentation pattern analyzed by time-of-flight (TOF) spectrometry. Enantiodifferentiation of the chiral monoalcohols and monoamines is based on: (1) the different spectral shifts of the band origin of their molecular complexes relative to that of the bare chromophore (Δ) and (2) the different mass spectral fragmentation patterns of the jet-cooled diastereomeric adducts. Detection of stable aggregates of methane, n-butane, and other simple molecules with the selected chromophores suggests that the R2PI/TOF method can be a potential tool for enantiodifferentiating chiral hydrocarbons in the gas phase.