Characteristic Fragmentation Behavior Research Articles

Investigation of the Influence of Charge State and Collision Energy on Oligonucleotide Fragmentation by Tandem Mass Spectrometry.

Due to the increasing pharmaceutical interest of oligonucleotides, for example in antisense therapy and vaccines, their analytical characterization is of fundamental importance due to their complex structure. For this purpose, mass spectrometry is a viable tool for structural studies of nucleic acids. Structural information regarding the primary sequence of a nucleic acid can reliably be gained via tandem mass spectrometry (MSMS) fragmentation. In this work, we present the characteristic fragmentation behavior of short-chain oligonucleotides (15-35 nucleotides) with respect to the collision-induced dissociation (CID) voltage used. The relationship and influence of the length of the oligonucleotide and its charge state is also discussed. The results presented here can be helpful for estimating the required fragmentation energies of short-chain oligonucleotides and their sequencing.

Rapid Identification of 3,6'-Disinapoyl Sucrose Metabolites in Alzheimer's Disease Model Mice Using UHPLC-Orbitrap Mass Spectrometry.

Alzheimer’s disease (AD) is a degenerative disease of the central nervous system characterized by the progressive impairment of neural activity. Studies have shown that 3,6′-disinapoyl sucrose (DISS) can alleviate the pathological symptoms of AD through the activation of the cAMP/CREB/BDNF signaling pathway. However, the exact biochemical mechanisms of action of DISS are not clear. This study explores metabolism of DISS in an AD mouse model, induced by the microinjection of a lentiviral expression plasmid of the APPswe695 gene into CA1 of the hippocampus. After gavage administration of DISS (200 mg/kg), the kidneys, livers, brains, plasma, urine, and feces were collected for UHPLC–Orbitrap mass spectrometry analysis. Twenty metabolites, including the prototype drug of DISS, were positively or tentatively identified based on accurate mass measurements, characteristic fragmentation behaviors, and retention times. Thus, the metabolic pathways of DISS in AD mice were preliminarily elucidated through the identification of metabolites, such as ester bond cleavage, demethoxylation, demethylation, and sinapic acid-related products. Furthermore, differences in the in vivo distribution of several metabolites were observed between the model and sham control groups. These findings can provide a valuable reference for the pharmacological mechanisms and biosafety of DISS.

Evaluating the performance of an ETD-cleavable cross-linking strategy for elucidating protein structures

Chemical cross-linking is a powerful strategy for elucidating the structures of protein or protein complexes. The distance constraints obtained from cross-linked peptides represent the three-dimensional structures of the protein complexes. Unfortunately, structural analysis using cross-linking approach demands a significant amount of data to elucidate protein structures. This requires the development of several cleavable cross-linkers with different range of spacer chains. An Electron Transfer Dissociation (ETD) tandem mass spectrometry cleavable bond hydrazone was reported. Its fragmentation with conjugated peptides showed promise for the development of a new ETD cleavable cross-linker. However, no cross-linker was developed utilizing this ETD cleavable bond. For the first time, we attempted to develop an ETD cleavable cross-linker utilizing a hydrazone bond. We overcome the pitfall for the synthesis of this cross-linker and an easy synthesis scheme is reported. In this report, we evaluated the performance of this cross-linker called Hydrazone Incorporated ETD cleavable cross-linker (HI-ETD-XL) in model peptides and proteins. The characteristic fragmentation behavior of HI-ETD-XL during electron transfer dissociation and subsequent sequence identification of the peptide fragment ions by tandem mass spectrometry allowed the identification of cross-linked peptides unambiguously. We believe the availability of this ETD cleavable cross-linker will advance structural proteomics research significantly. SignificanceMany cellular processes rely on the structural dynamics of protein complexes. The detailed knowledge of the structure and dynamics of protein complexes is crucial for understanding their biological functions and regulations. However, most of the structure of these multiprotein entities remain uncharacterized and sometimes is very challenging to reveal with biophysical techniques alone. Chemical cross-linking combined with mass spectrometry (MS) has proven to be a dependable strategy in structural proteomics field. However, data complexity and false identifications are significant hindrances for unambiguous identification of cross-linked peptides. Confident identifications demand structural studies with cross-linkers with different properties and variable spacer chain lengths. This new ETD cleavable cross-linking workflow will provide additional confidence to overcome these drawbacks and allow us to pinpoint cross-linked peptides confidently.

Analysis of 27 β-Blockers and Metabolites in Milk Powder by High Performance Liquid Chromatography Coupled to Quadrupole Orbitrap High-Resolution Mass Spectrometry

This paper presents an application of high performance liquid chromatography coupled with quadrupole orbitrap high-resolution mass spectrometry (HPLC-Q-Orbitrap HRMS) for the analysis of 27 β-blockers and metabolites in milk powder. Homogenized milk power samples were extracted by acetonitrile and purified by using Oasis PRiME HLB solid-phase extraction cartridges. The Ascentis® C8 chromatographic column was used to separate the analytes. The quantification was achieved by using matrix-matched standard calibration curves with carazolol-d7 and propranolol-d7 as the internal standards. The results show an exceptional linear relationship with the concentrations of analytes over wide concentration ranges (0.5–500 μg kg−1) as all the fitting coefficients of determination r2 are > 0.995. All the limits of detection (LODs) and quantitation (LOQs) values were within the respective range of 0.2–1.5 μg kg−1 and 0.5–5.0 μg kg−1. Overall average recoveries were able to reach 66.1–100.4% with the intra- and inter-day variability under 10%. This method has been successfully applied to the screening of β-blockers and metabolites in commercial milk powders. At the same time, the corresponding characteristic fragmentation behavior of the 27 compounds was explored. The characteristic product ions were determined and applied to the actual samples screening.

Characterization and Analysis of 2-(2-Phenylethyl)-chromone Derivatives from Agarwood (Aquilaria crassna) by Artificial Holing for Different Times.

A total of fifty-six chromones, including seven 5,6,7,8-tetrahydro-2-(2-phenylethyl)-chromones (THPECs), five 5,6-epoxy-2-(2-phenylethyl)chromones (EPECs), seven 5,6:7,8-diepoxy-2-(2-phenylethyl)chromones (DEPECs) and thirty-seven 2-(2-phenylethyl)chromones of the flidersia type (FTPECs), were characterized by HPLC/DAD/ESI/MS/MS in three agarwood samples (from Aquilaria crassna) induced by artificial holing with different holing times. The characteristic fragmentation behavior of DEPECs and EPECs, and the methods to distinguish these four types of chromones by MS analysis were described for the first time. In addition, it was found that the relative contents of DEPECs and EPECs were down-regulated, while the relative contents of THPECs and FTPECs were up-regulated for the samples from two, four and five years of the agarwood formation time. However, the relative contents of six most widespread and abundant FTPECs presented roughly upward based on the formation time. These results could be referenced to distinguish different agarwood samples collected from different formation time.

Structural characterization and identification of flavonoid aglycones in three Glycyrrhiza species by liquid chromatography with photodiode array detection and quadrupole time-of-flight mass spectrometry.

Flavonoids, including flavones, isoflavones, flavanones, chalcones, and isoflavans, have long been recognized as the main active ingredients in licorice. A method combining liquid chromatography with photodiode array detection and quadrupole time-of-flight mass spectrometry was developed to characterize components in three Glycyrrhiza species, and to expound the characteristic fragmentation behaviors in the positive ion mode. Based on the fragmentation patterns of reference compounds, a total of 39 compounds, including 37 flavonoid aglycones and two coumestans, were identified or tentatively identified. Besides, some common features, such as H2 O, CO, and CH2 O2 losses, together with retro-Diels-Alder fragmentation, were observed in these compounds. Furthermore, diagnostic fragmentations of C-ring cleavages and UV absorption on the skeleton groups were observed to structurally characterize flavonoid aglycones. In addition, typical losses of different substituent groups were detected: Neutral losses of 56 (C4 H8 ) and 68 Da (C5 H8 ) were yielded from a prenyl chain; neutral losses of 42 (C3 H6 ), 54 (C4 H6 ), and 70 Da (C4 H6 O) were generated by a pyran ring. Particularly, neutral losses of 18 (H2 O), 16 (CH4 ), 112 (C8 H16 ), and 98 Da (C7 H14 ) predicted a hydroxyl, a methoxyl, double prenyl chains, and a prenyl chain with a pyran ring, respectively.

A rapid method for chemical fingerprint analysis of Pan Panax notoginseng powders by ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

A method coupling ultra-performance liquid chromatography (UPLC) with quadrupole time-of-flight mass spectrometer (Qtof MS) using the electrospray ionization (ESI) source was developed for the identification of the major saponins from Panax notoginseng powder (PNP). Ten different PNP samples were analyzed and evaluated for their quality by similarity evaluation and principle component analysis (PCA). Based on the accurate mass, summarized characteristic fragmentation behaviors, retention times of different types of saponins, related botanical biogenesis, and reported chromatographic behavior of saponins, fifty-one common peaks were effectively separated and identified, including 28 protopanaxadiol saponins and 18 protopanaxatriol saponins. Simultaneously, 15 significant discrepancy compounds were identified from the disqualified PNP samples. The established UPLC/Qtof MS fingerprint method was successfully applied for profiling and identifying the major saponins of PNP, providing a fast quality evaluation tool for distinguishing the authentic PNP and the adulterated products.

Qualitative and quantitative analysis of amorfrutins, novel antidiabetic dietary natural products, by HPLC

Context: Initially isolated from fruits of Amorpha fruticosa L. (Fabaceae), amorfrutins are promising antidiabetic natural products as selective peroxisome proliferator-activated receptor γ-agonists.Objective: The objective of this study is to develop a sensitive and convenient HPLC method to analysis amorfrutins in plant materials derived from genera Amorpha and Glycyrrhiza.Materials and methods: The reference compounds were isolated from fruits of A. fruticosa and characterized by UV, HR-ESI-MS, and NMR spectrometric techniques. Three amorfrutins were analyzed by HPLC on a Hypersil BDS C18 column (Waters Corporation, Milford, MA) within a gradient elution of acetonitrile and 0.2% glacial acetic acid. HR-ESI-MS spectra were acquired on a quadrupole-time-of-flight (Q-TOF) mass spectrometer.Results: Three amorfrutins showed good linearity (r2 > 0.999) in the ranges of 4.2–84.5, 4.5–90.2, and 4.6–92.7 μg/mL. The amorfrutins were only detected in fruits of A. fruticosa with the contents of 2-carboxy-3,5-dihydroxy-4-geranylbibenzyl, amorfrutin A, and amorfrutin B in the ranges of 1.31–7.43, 0.54–3.52, and 0.64–4.63 mg/g, respectively. No amorfrutin was detected in fruits of Amorpha canescens Pursh and roots of Glycyrrhiza uralensis Fisch. (Fabaceae), Glycyrrhiza inflate Bat., or Glycyrrhiza glabra L.Discussion and conclusion: A novel HPLC method was developed, validated, and applied to identify and quantify amorfrutins in complex botanic matrixes and the characteristic MS fragmentation behaviors of amorfrutins were revealed for the first time. The analytical method presented in this study could be used for the quality control of related plant materials and amorfrutin-based nutraceuticals.

Detection and characterization of triamcinolone acetonide metabolites in human urine by liquid chromatography/tandem mass spectrometry after intramuscular administration.

Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. intramuscular, IM), whereas they are allowed using other ways of administration. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of triamcinolone acetonide (TA), one of the most used glucocorticosteroids, was studied using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). Urine samples obtained after IM administration of TA were analyzed using two sample treatments: (a) hydrolysis with β-glucuronidase enzymes and liquid-liquid extraction under alkaline conditions, and (b) liquid-liquid extraction under acidic conditions. The extracts were analyzed by LC/MS/MS. TA, commercially available metabolites (6β-hydroxytriamcinolone acetonide, 6β-OH-TA, and triamcinolone), and their C20-reduced derivatives showed characteristic fragmentation behavior. Besides common product ions and neutral losses for corticosteroids containing fluorine, additional characteristic neutral losses (58 Da, loss of acetone; 44 Da, loss of acetaldehyde) were observed in positive electrospray ionization. Based on that behavior, two complementary approaches were applied to detect TA metabolites: (a) open detection by precursor ion and neutral loss scan methods and (b) targeted detection by selected reaction monitoring methods (SRM) containing theoretical ion transitions of the potential metabolites. Two main compounds, TA and 6β-OH-TA, and nine minor potential metabolites, were detected by open screening methods. Using SRM, two additional metabolites were detected. Some of the metabolites were characterized using reference standards and, for the rest of metabolites, feasible structures were proposed based on mass spectrometric data. Metabolites resulting from hydroxylation in C-6, oxidation of the 11-hydroxyl group, reduction of the Δ(4) double bond and oxidation of the side chain were detected. Some of them have not been previously described. Excretion profiles of the detected metabolites after IM administration are presented.

Characteristic fragmentation behavior of tobacco‐specific N‐nitrosamines using electrospray ionization multistage tandem mass spectrometry incorporating deuterium labeling

Tobacco-specific N-nitrosamines (TSNAs) mainly include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT) that are formed from tobacco alkaloids during the curing process and contained in tobacco and tobacco smoke. They are linked with carcinogenesis. Analytical methods for quality control of products and determination of their metabolites are therefore of great importance. The characteristic fragmentation behaviors of tobacco-specific TSNAs have been studied by electrospray ionization multistage tandem mass spectrometry. The deutero-labeled TSNA compounds have also been employed to clarify the fragmentation mechanism, which further confirms the proposed fragmentation patterns. Detailed analysis of the resultant fragments shows there are two different kinds of fragmentation patterns with the general fragment backbone of pyrrolidine or piperidine rings. In one route, pyrrolidine or piperidine rings undergo direct fragmentation and form some stable intermediates without affecting the parent rings. The other, however, involves ring opening and then ring closure at the pyridine-2 carbon atom to form multi-membered rings. This characteristic fragmentation behavior therefore provides useful information on identification of TSNAs that may be used to monitor such kinds of compound in the biological metabolism.

Characterization and Determination of 2-(2-Phenylethyl)chromones in Agarwood by GC-MS

Agarwood is the fragrant resinous heartwood obtained from certain trees in the genus Aquilaria belonging to the family Thymelaeaceae. 2-(2-Phenylethyl)chromones and characteristic sesquiterpenes are the main classes of aromatic compounds isolated from agarwood. Although there are many sesquiterpenes, relatively few 2-(2-phenylethyl)chromones have been determined in agarwood by GC-MS. After analysis of the MS spectra of eighteen 2-(2-phenylethyl)chromone derivatives isolated from agarwood and identified by NMR spectroscopy, together with the reported MS data and characteristic of structures of 2-(2-phenylethyl)chromones, the MS characterization, fragmentation patterns and characteristic fragment peaks for the compounds were deduced and a table summarizing MS characterization of 2-(2-phenylethyl)chromones in agarwood is presented. All the 2-(2-phenylethyl)chromones previously reported in agarwood are substituted by methoxy or/and hydroxy groups, except for one compound. Due to the fact they all possess the same basic skeleton (molecular weight: 250) and similar substituent groups (methoxy or hydroxy groups), a formula (30m + 16n = MW − 250) is provided to calculate the number of methoxy (m) or hydroxy (n) groups according to molecular ion peak or molecular weight (MW). We deduced that the characteristic fragmentation behaviors of the 2-(2-phenylethyl)chromones are the cleavages of the CH2-CH2 bond between chromone moiety and phenyl moiety. Thus, characteristic fragment ions, such as m/z 91 [C7H7], 107 [C7H6+OH], 121 [C7H6+OCH3], 137 [C7H5+OH+OCH3] are formed by different substituted benzyl moieties, while characteristic fragment ions such as m/z 160 [C10H8O2], 176 [C10H7O2+OH], 190 [C10H7O2+OCH3], 220 [C10H6O2+OCH3×2] are formed by different substituted chromone moieties. Furthermore, rules regarding to the relationship between the positions of hydroxy or methoxy groups and the relative abundances of benzyl and chromone fragment ions have been deduced. Elucidation of how the positions of hydroxy or methoxy groups affect the relative abundances of benzyl and chromone fragment peaks is also provided. Fifteen unidentified compounds of an artificial agarwood sample analyzed by GC-MS, were preliminary determined as 2-(2-phenylethyl)chromones by analysis of their MS characterization and by comparison of their MS spectra with those of 18 standard compounds or 2-(2-phenylethyl)chromones reported in literature according to the above-mentioned methods and rules. This report will be helpful for the analysis and structural elucidation of 2-(2-phenylethyl)chromones in agarwood by GC-MS, and provides fast and reliable characterization of the quality of agarwood.

Synthesis and tandem mass spectrometry of chlorinated triacylglycerols

The incorporation of 9,10-dichlorooctadecanoyl groups using enzyme-catalyzed acylation and protecting group strategies yielded specific regioisomers of di- and tetrachlorinated triacylglycerols. Hexachloro- and hexabromotriacylglycerols were synthesized by addition of chlorine or bromine to tri-(cis-9-octadecenoyl)glycerol. Upon electrospray ionization and tandem mass spectrometry, the sodium adduct ions of all compounds containing a 9,10-dichlorooctadecanoyl group readily lost two molecules of HCl when subjected to collision-induced dissociation. A mechanism describing sequential HCl losses and the formation of a conjugated diene is proposed for the loss of both vicinal chlorine atoms from an alkyl chain. This characteristic fragmentation behavior and the availability of characterized standards will facilitate the development of quantitative analytical methods for the determination of chlorinated triacylglycerols in lipid mixtures isolated from marine and other biological sources.

SEPARATION AND CHARACTERIZATION OF STEROIDAL SAPONINS IN PARIS PLLYPHYLLA BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY COUPLED WITH TIME-OF-FLIGHT MASS SPECTROMETRY AND ION TRAP MASS SPECTROMETRY

Steroidal saponins are the major bioactive constituents in Paris Polyphylla. In the present study, we developed a novel analysis method for separation and characterization of steroidal saponins in Paris Polyphylla extract using high-performance liquid chromatography electrospray ionization time of flight mass spectrometry (HPLC/ESITOFMS) and ion trap mass spectrometry (HPLC/ITMS). A total of 30 steroidal saponins in Paris Polyphylla extract were identified effectively according to their accurate mass measurements, molecular formulae, and various characteristic fragmentation behaviors. It is concluded that the established analysis method is sensitive, rapid, and practical. Additionally, it will provide help for further quality control and pharmacological mechanism study of Paris Polyphylla.

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS(3) analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

Chemical correlation between Gegen Qinlian dispensing granule and its four raw herbs by LC fingerprint

Gegen Qinlian dispensing granule, a favorite composite formula, is a combination of Radix Puerariae Lobatae, Radix Scutellariae, Rhizoma Coptidis and Radix et Rhizoma Glycyrrhizae Praeparata cum Melle. To develop a method to overall evaluate correlation between the formula and its four raw herbs, LC fingerprints of the formula and its raw herbs were developed and LC-DAD-MS was employed to identify the components in the formula fingerprint. According to the characteristic fragmentation behavior of known flavonoids, alkaloids and saponins isolated from the four raw herbs as well as retention time, UV and MS data of detected compounds, a total of 23 constituents in the formula fingerprint were structurally characterized. Chemical correlation between the formula and the four crude herbs was evaluated qualitatively and quantitatively by the developed LC fingerprints. The results showed that 8 components in the formula fingerprint were addressed to Radix Puerariae Lobatae, 11 to Radix Scutellariae, 7 to Rhizoma Coptidis, 3 to Radix et Rhizoma Glycyrrhizae Praeparata cum Melle, and that the relative area ratios of the common peaks in the formula vary slightly in comparison with corresponding ratios in its crude herbs, demonstrating the chemical constituents in the formula have patterns similar to those in its crude herbs.

Characterization of steroidal saponins in saponin extract from Paris polyphylla by liquid chromatography tandem multi-stage mass spectrometry

Rhizoma Paridis saponins are bioactive steroidal saponins derived from Paris polyphylla. Optimization of the ionization process was performed with electrospray ionization tandem mass spectrometry in both positive and negative-ion modes. Negative-ion ESI was adopted for generation of the precursor deprotonated molecules to achieve the best ionization sensitivity for the analytes. Positive ionization was used to choose the most abundant fragment ion. Furthermore, according to the characteristic fragmentation behavior of known steroidal saponins isolated from this plant (polyphyllin D, formosanin C, gracillin, Paris H, Paris VII, and dioscin), 23 constituents were structurally characterized on the basis of their retention time and ESI analyses, including four pairs of naturally occurring isomers. Five of these 23 constituents were new compounds. The analytical method of LC-MS(n) in positive and negative-ion modes has been developed for the direct structural elucidation of steroid saponins of this kind in plant extracts.

Study of the characteristic fragmentation behavior of hydroquinone glycosides by electrospray ionization tandem mass spectrometry with optimization of collision energy

The fragmentation behavior of hydroquinone glycosides involving one or two sugar groups from Fraxinus sieboldiana and their analogue arbutin was investigated systematically by electrospray ionization tandem mass spectrometry in negative ion mode. The characteristic fragmentation reaction of these compounds was through the homolytic and heterolytic cleavage of the O-glycosidic bond to produce radical aglycone ion ([Y0-H]-*) and aglycone ion (Y0-), respectively. Unambiguous differentiation between the mono-O-glycoside isomers which differ in glycosylation position was achieved by comparing the relative abundance of [Y0-H]-* and Y0- ions with the optimized collision energy. In the fragmentation of 1, 4-di-O-glycosides, only the Y0- ion was produced when the first glucosyl residue was expelled. However, both the [Y0-H]-* and Y0- ions were present when the second glucosyl residue was eliminated. In addition, an interesting [Y0-2H]- ion was present in the product ion spectra of hydroquinone glycosides with methoxy group(s) substituted at C-3 or/and C-5 positions of the benzene ring. The results of this study can facilitate the rapid determination of hydroquinone glycosides in crude plant extracts and also reveal that the systematic investigation and optimization of collision energy play an important role in the differentiation of isomers which have subtle differences in structures.

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-FT-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.

Negative ion electrospray ionization mass spectrometry of nucleoside phosphoramidate monoesters: elucidation of novel rearrangement mechanisms by multistage mass spectrometry incorporating in‐source deuterium labelling

Several O-2',3'-isopropylideneuridine-O-5'-phosphoramidate monoesters were synthesized and analyzed by negative ion electrospray ionization tandem mass spectrometry (ESI-MS(n)). Two kinds of novel rearrangement reactions were observed due to the difference in the amino acid in the nucleoside phosphoramidate monoesters, and possible mechanisms were proposed. One involves a five-membered cyclic transition state. The other is formation of a stable five-membered ring intermediate by Michael addition. Results were confirmed by tandem mass spectrometry and isotopically labeled hydrogen atoms. Furthermore, the internal hydrogen exchange between active hydrogen and methyl acrylate in the heated capillary of the mass spectrometer was found. The characteristic fragmentation behavior in ESI-MS may be used to monitor this kind of compounds in the biological metabolism.