Electrospray Ionization Multistage Mass Spectrometry Research Articles

Mulberrofuran G and Isomulberrofuran G from Morus alba L.: Anti-hepatitis B Virus Activity and Mass Spectrometric Fragmentation

Mulberrofuran G (1) and isomulberrofuran G (2), a pair of isomeric Diels-Alder-type adducts, were isolated from the root bark of Morus alba L. Isomulberrofuran G (2) as a new IIB-type Diels-Alder-type adduct, was elucidated by extensive (1)H, (13)C, and two-dimensional (2D) nuclear magnetic resonance (NMR) and mass spectrometry (MS) spectroscopic analyses. A fragmentation study on compounds 1 and 2 was performed by high-resolution electrospray ionization (ESI) multistage tandem mass spectrometry linked with ion-trap (IT) and time-of-flight (TOF) mass analyzers (ESI-MS(n)/IT-TOF) in negative mode, which resulted in obviously different fragmentations. In the MS(2) experiments, the characteristic ions at m/z 451 and 439 could be revealed as their respective diagnostic ions. Mulberrofuran G (1) showed moderate activity, inhibiting hepatitis B virus (HBV) DNA replication with the IC(50) value of 3.99 μM, according to the anti-HBV assay on the HepG 2.2.15 cell line in vitro.

Fragmentation mechanism of product ions from protonated proline-containing tripeptides in electrospray ionization mass spectrometry

The dissociation chemistry of primary fragment ions from the protonated proline-containing tripeptides glycylprolylglycine, prolylglycylglycine, and prolylprolylglycine was investigated by electrospray ionization multi-stage mass spectrometry. Calculations showed the a2 ions generated from b2 ions were cyclic, which is energetically more favorable than the linear form. The prolyl residue in the structure affected the energy hypersurface of the dissociation reaction from the b2 ion to the a2 ion. In the fragmentation of a2 ions, the iminium-imine complex corresponding to loss of CO from the a2 ion was suggested to be an ion-neutral complex (INC). The a1 ion was generated from direct separation of this INC, and the internal iminium ion, which was absent in PGG, was generated from another INC that was formed from the first INC via proton-bridged complex-mediated intramolecular proton transfer. Although these intermediates are unstable, their existence is supported by experiments and density functional theory calculations.

Degradability studies of poly(l-lactide) after multi-reprocessing experiments in extruder

Poly(l-lactide), PLLA, belongs to the most widely used biodegradable polyesters. These polymers have attracted great attention in recent years due to their excellent material properties, which give a rise to potential applications in various commercial areas. Previously, PLLA reprocessing in a co-rotating twin-screw extruder was used as a model test for material recycling. It may be assumed basing on this study, that PLLA technological wastes may be suitable for reuse, for example, as an additive for neat PLLA. In order to verify such suitability of reprocessed PLLA its degradability under industrial composting conditions was studied at the static composting open-air pile. Incubation of this material in water at 70 °C (abiotic conditions) was also conducted. The progress of the degradation process was monitored by surface changes and measurements of sample weight loss and changes in the PLLA molecular weight. The molecular level structure of the water soluble degradation products of PLLA samples was determined by electrospray ionization multistage mass spectrometry (ESI-MSn). Lactic acid and its oligomers terminated by hydroxyl and carboxyl end groups were identified as hydrolytic degradation products. The obtained results indicated that in selected environments (industrial composting and incubation under abiotic conditions) hydrolytic degradation via random ester bond scission occurs preferentially. It was demonstrated, that multi-reprocessing of PLLA did not significantly affect the rate of degradation and only slightly affected the disintegration progress of the PLLA samples.

Identification of tiagabine degradation products using liquid chromatography with electrospray ionization multistage mass spectrometry and ultra‐performance liquid chromatography/high‐resolution mass spectrometry

Tiagabine hydrochloride monohydrate drug substance (TGB) is an antiepileptic agent effective in the treatment of seizure disorders. The stability of TGB was studied and its degradation products were identified for the first time. TGB was heated in the presence of H(2)O(2). Degradation products were analyzed by liquid chromatography coupled to electrospray ionization multistage mass spectrometry (LC/ESI-MS(n)) and high-resolution mass spectrometry (HR-MS). This study showed that TGB was degraded by oxidative pathways involving attack of oxygen at different centers but mainly at the double bond of the molecule. The oxidative cascade reactions initiated by the epoxidation of the double bond of tiagabine led to dihydroxy, ketohydroxy and ketone derivatives as well as bisthiophene ketone. Nine degradation products of TGB were identified. Some diagnostic MS/MS product ions, characteristic of the piperidine or thiophene moiety, were highlighted.

Systematic screening and characterization of the major bioactive components of Poria cocos and their metabolites in rats by LC‐ESI‐MSn

Poria cocos is a well-known medicinal plant widely used in China and other East Asian countries owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of Poria cocos and their metabolites in vivo are still unclear to date. The aim of the present study was to develop a practical method based on the combined use of the liquid chromatography coupled with electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS(n) ) for the comprehensive and systematic separation and characterization of the bioactive constituents of Poria cocos extract and their metabolites in rats. Based on the proposed strategy, a total of 34 compounds were characterized from the extract of Poria cocos. Among them, eight were unambiguously identified by comparing their retention times and mass spectra with those of reference standards, and 26 were tentatively identified on the basis of their MS(n) fragmentation behaviors and molecular weight information from literatures. In vivo, seven compounds were successfully detected in rat urine whereas one was found in rat plasma. This study proposed a series of potential bioactive components and provided helpful chemical information for further research on the action mechanism of traditional Chinese medicine.

Fragmentation of pentacoordinate spirobicyclic aminoacyl‐phosphoranes (P‐AAs) by electrospray ionization tandem mass spectrometry concerning P–O and P–N bond cleavage

The fragmentation pathways of both protonated and sodiated pentacoordinate spirobicyclic aminoacylphosphoranes (P-AAs) have been studied by electrospray ionization multi-stage mass spectrometry (ESI-MS(n)) in positive mode. The possible pathways and their mechanisms are elucidated through the combination of ESI-MS/MS, isotope ((15)N and (2)H) labeling and high-resolution Fourier transform ion cyclotron resonance (FTICR)-MS/MS. The relative Gibbs free energies (ΔG) of the product ions and possible fragmentation pathways are estimated at the B3LYP/6-31 G(d) level of theory. The theoretical calculations show that both protonated and sodiated P-AAs would quickly fragment before Berry pseudorotation. For protonated P-AAs, they have different tendencies to P-O or P-N bond cleavage. For sodiated P-AAs, the P-N bond is easier to cleave and produces the tetracoordinated phosphorus ion H. These results to some extent may give a clue to the chemistry of the active sites of phosphoryl transfer enzymes and will enrich the gas-phase ESI-MS ion chemistry of pentacoordinate phosphoranes.

Qualitative and Quantitative Evaluation of Phenolic Compounds in Iris dichotoma Pall

The rhizome of Iris dichotoma Pall., a traditional Chinese herbal medicine that contains (iso) flavonoids as the main bioactive compound, has been used to treat several disorders such as inflammation, throat disorders, asthma and coughs. However, there is a lack of suitable methods for its qualitative and quantitative analysis as well as quality control. To establish an HPLC method for the simultaneous determination of phenolic constituents, the identification of characteristic metabolites in the rhizomes of I. dichotoma and to compare their contents in samples from different areas of China. HPLC coupled with diode-array detection (DAD) and electrospray ionisation multistage mass spectrometry (ESI-MS(n) ), and principal component analysis (PCA) were used for the qualitative and quantitative analysis of phenolic compounds in I. dichotoma samples. Twenty characteristic compounds were identified or tentatively characterised. A quantitative HPLC-UV method allowing the simultaneous quantification of 10 phenolic compounds was optimised and validated in terms of linearity, precision, accuracy, and limits of detection and quantification. The methodology developed proved to be effective for the identification and quantification of phenolic compounds in the rhizomes of I. dichotoma. Samples were classified into three groups according to their geographical origin based on their phenolic composition using principal component analysis.

Structural characterization and identification of biflavones in Selaginella tamariscina by liquid chromatography‐diode‐array detection/electrospray ionization tandem mass spectrometry

Biflavonoids, a special class of flavonoids, are widely distributed in gymnosperm plants and have various biological activities. They are also major bioactive ingredients in Selaginella tamariscina. In this work, we report the use of high-performance liquid chromatography with a diode-array detector (HPLC-DAD) and electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) to study the fragmentation behavior of three main types of biflavonoids using seven biflavonoid reference compounds and analyze the biflavonoids in Selaginella tamariscina. The most useful fragmentations in terms of structural identification are those involving the C-ring cleavage of biflavonoids. For amentoflavone-type biflavonoids (containing flavonoid parts I and II), fragmentation on the flavonoid part II at positions 1/3 and 0/4 are the primary pathways, whereas the chances are greater for C-ring cleavage fragmentation occurring on flavonoid part I at positions 1/3 and 1/4 for robustaflavone-type biflavonoids. However, the predominant diagnostic ions of the specific C-O-C-connected hinokiflavone-type biflavonoids are a series of ions resulting from the rupture of the connective C-O bond. Based on the fragmentation patterns of these reference compounds, 17 biflavonoids were identified in an extract of Selaginella tamariscina, three of which have not been previously reported as constituents of this plant. This study provides a powerful approach for the online structural elucidation and identification of different types of biflavonoids and positional isomers from Selaginella tamariscina and other biflavonoids distributed in related plants and prescriptions.

In vivo metabolism study of polygalic acid in rat using HPLC‐ESI‐MSn

A very simple and direct method has been established for the determination of polygalic acid and its metabolites in rat urine based on HPLC coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC-ESI-MS(n)). The rats were administered a single dose (100 mg/kg) of polygalic acid by oral gavage. The urine samples were collected and purified through a C(18) solid-phase extraction cartridge, and then these pretreated samples were injected into a reversed-phase C(18) column with a gradient elution program, whereas acetonitrile-0.5% aqueous formic acid was used as mobile phase and detected by an on-line MS/MS system. As a result, the parent drug and its four metabolites were identified and characterized in rat urine for the first time by comparing their changes in molecular mass (ΔM), retention times and full-scan MS(n) spectra with those of the parent drug. A possible metabolic pathway of polygalic acid was investigated and proposed. More importantly, the results demonstrated that the newly developed method (HPLC-ESI-MS(n)) was sensitive, simple and suitable for the determination of polygalic acid and its metabolites in biological samples.

Identification of the major constituents in Xuebijing injection by HPLC‐ESI‐MS

Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of sepsis. This is the first report concerning the identification of XBJ constituents. In addition, to evaluate XBJ's quality, partial least square discrimination analysis (PLS-DA) was performed on chemical fingerprint data. Establish an LC-MS method to identify the components in XBJ for the purpose of quality control. Compounds were separated by HPLC using a C(18) column and gradient elution of acetonitrile-methanol (60:40, v/v) and water-acetic acid (100 : 0.5, v/v) in 80 min. HPLC equipped with diode array detector (DAD) coupled with time-of-flight (TOF) tandem mass spectrometry and HPLC electrospray ionisation (ESI) multi-stage tandem ion-trap mass spectrometry (IT-MS(n) ) method was developed to analyse XBJ's major components. Both positive and negative ionisation modes were employed. Twenty-one compounds including amino acids, phenolic acids, flavonoid glycoside, terpene glycoside and phthalide were identified or tentatively characterised. Their retention times, UV and MS spectra were compared with those of authentic compounds or literature data. The score plot of PLS-DA clearly revealed variations among samples produced in different commercial batches. The analytical method developed is highly effective for the discrimination and quality control of XBJ.

Identification of Amino Acid Phosphorodiamidates of Antiviral Nucleosides Using Electrospray Ionisation Tandem Mass Spectrometry

Several amino acid phosphorodiamidate derivatives of d4T as anti-HIV prodrugs were synthesized and investigated using electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). A novel methyl group migration in gas phase was observed in ESI-MS(2) of the sodium adducts of amino acid methyl ester of phosphorodiamidates of 2',3'-didehydro-2',3'-dideoxythymidine (d4T). The proposed structures of the rearrangement ions were confirmed by high resolution tandem mass spectrometry. A possible mechanism involving the pentacoordinate phosphoric-carboxylic phosphate anhydride was proposed, in which a seven-membered ring intermediate was formed by coordination with the metal ion between the phosphoryl group and carbonyl oxygen atom. Thus, the intrinsic properties of phosphoryl group might be the key factors responsible for this migration.

A novel methyl migration to the phosphoryl group with the formation of cyclic aminoacylphosphoramidates in electrospray ionization tandem mass spectra of amino acid ester phosphoramidates of antiviral nucleosides

The mass spectral behavior of amino acid methyl ester phosphoramidate derivatives have been investigated using electrospray ionization multistage mass spectrometry (ESI-MS(n)) and moderate theoretical calculations at the B3LYP/6-31G(d) level. A novel methyl group migration to the phosphoryl group with the formation of the intermediate cyclic aminoacylphosphoramidate was found. The proposed structures of the rearrangement ions were confirmed by high-resolution tandem mass spectrometry. A possible mechanism involving the pentacoordinate phosphoric-carboxylic phosphate anhydride was proposed, in which the metal ion coordination with the phosphoryl and carbonyl groups and the intrinsic properties of phosphoryl group might be the key factors responsible for this novel migration.

Fragmentation studies of pentacoordinated bisaminoacylspirophosphoranes by negative electrospray ionization mass spectrometry

Fragmentation pathways of a series of pentacoordinated bisaminoacylspirophosphoranes were elucidated by electrospray ionization multistage mass spectrometry (ESI-MS(n)) in negative mode. The deprotonated ions of pentacoordinated bisaminoacylspirophosphoranes tend to eliminate a corresponding amino acid to form base peak. The hydrogen/deuterium exchange experiment, the high-resolution mass spectrometry, (13)C stable isotope labeling experiment and theoretical calculations were used to rationalize the proposed fragmentation pathways and to verify the differences between the fragmentation pathways. The results indicate that the negative molecular ions of pentacoordinated bisaminoacylspirophosphoranes dissociate through its open-chain tricoordinated tautomers. The relative Gibbs free energies (ΔG) of the product ions and proposed fragmentation pathways were estimated using the B3LYP/6-31 + + G(d, p) model. The results have some potential applications in the identification structures of similar spirophosphorane compounds by ESI-MS(n).

Quality Evaluation of Potentilla discolor by High‐performance Liquid Chromatography Coupled with Diode Array Detection and Electrospray Ionisation Tandem Mass Spectrometry

The whole herb of Potentilla discolor has long been used for treatment of diarrhoea, malaria, haemoptysis and haematemesis in clinical applications. However, until now, there has been no literature regarding to the quality control of it. To develop a simple and accurate HPLC-DAD (diode array detection)-ESI/MS(n) (electrospray ionisation multistage mass spectrometry) method for the identification of constituents in P. discolor and simultaneous quantification of its marker compounds. Separations were performed on a Shimpack C₁₈ column by gradient elution using acetonitrile:0.1% formic acid. The identification of constituents in P. discolor were achieved by HPLC-DAD-ESI/MS(n) while six flavonoids and five triterpenoids were determined by HPLC-DAD at 360 and 210 nm, repectively. A total of 23 compounds including 13 flavonoids and 10 triterpenoids were identified or tentatively characterised. A quantitative HPLC-DAD method allowing the simultaneously quantification of 11 marker compounds was optimised and validated for linearity, precision, accuracy, and limits of detection and quantification. The method was successfully applied to the quantification of 11 marker compounds in 10 samples of P. discolor collected from different provinces of China. This developed method was validated as simple, precise and accurate, and could be used for effectively and comprehensibly evaluating the quality of P. discolor.

Saponins of Glycine max Merill.: Isolation and further characterization by NMR and ESI-MS/MS

Legumes play a pivotal role in the traditional diets of many regions throughout the world. Soybeans are unique among the legumes because they are a concentrated source of soysaponins. Soysaponins are basically triterpene glycosides and comprise a hydrophobic aglycone linked to one or more hydrophilic mono- or oligosaccharide moieties [1]. Chemical and health effects of soysaponins are numerous and suggest a number of biological properties [2]. As a continuation of our work on the chemical elucidation of bioactive markers from plants, ten soysaponins were isolated from Glycine max. All saponins were isolated from saponin concentrate of said plant by using CHCl3-MeOH-H2O lower layer in an almost isocratic way through a gravity column. All isolated compounds were characterized by NMR. A number of constituents and different possibilities of sugar chain composition and attachment cause greater diversity of soysaponin structures. In order to determine the structures of isolated compounds, ten soysaponins were analyzed under positive and negative mode respectively, by electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) coupled with an ion-trap analyzer. In positive mode, the sodium adduct molecular ions [M+Na]+ and [M+2Na-H]+ were easily detected. However, β-D-glucopyranosiduronic acid required negative mode to detect [M-H]-, [M-H-sugar(s)]- and [aglycone]-. The proposed fragmentation pathway is useful for structural characterization of soysaponins.

LC-Multistage Mass Spectrometry for the Characterization of Poly(Butylene Adipate-co-Butylene Terephthalate) Copolyester

A comprehensive study using liquid chromatography electrospray ionization multistage mass spectrometry (LC-ESI MS(n)) was performed to get detailed structural information on poly(butylene adipate-co-butylene terephthalate) co-polyester and its product of partial degradation. LC-MS and LC-MS(n) identified the existence of cyclic structures in the original samples that disappear completely during the degradation. The occurrence of methanol transesterification in the degradation process was confirmed. MS(2) on the first (13)C isotope peak helped to determine the elemental composition of the fragments and facilitated end group determination. The method can be used to provide an alternative for high mass accuracy MS(2) experiments. Sequence information was also revealed for certain copolymeric structures.

An Isotope (18O, 15N, and 2H) Technique to Investigate the Metal Ion Interactions Between the Phosphoryl Group and Amino Acid Side Chains by Electrospray Ionization Mass Spectrometry

Cationic metal ion-coordinated N-diisopropyloxyphosphoryl dipeptides (DIPP-dipeptides) were analyzed by electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). Two novel rearrangement reactions with hydroxyl oxygen or carbonyl oxygen migrations were observed in ESI-MS/MS of the metallic adducts of DIPP-dipeptides, but not for the corresponding protonated DIPP-dipeptides. The possible oxygen migration mechanisms were elucidated through a combination of MS/MS experiments, isotope ((18)O, (15)N, and (2)H) labeling, accurate mass measurements, and density functional theory (DFT) calculations at the B3LYP/6-31 G(d) level. It was found that lithium and sodium cations catalyze the carbonyl oxygen migration more efficiently than does potassium and participation through a cyclic phosphoryl intermediate. In addition, dipeptides having a C-terminal hydroxyl or aromatic amino acid residue show a more favorable rearrangement through carbonyl oxygen migration, which may be due to metal cation stabilization by the donation of lone pair of the hydroxyl oxygen or aromatic π-electrons of the C-terminal amino acid residue, respectively. It was further shown that the metal ions, namely lithium, sodium, and potassium cations, could play a novel directing role for the migration of hydroxyl or carbonyl oxygen in the gas phase. This discovery suggests that interactions between phosphorylated biomolecules and proteins might involve the assistance of metal ions to coordinate the phosphoryl oxygen and protein side chains to achieve molecular recognition.

Qualitative Analysis and Simultaneous Quantification of Phenolic Compounds in the Aerial Parts of Salvia miltiorrhiza by HPLC‐DAD and ESI/MSn

The increasing demands of roots and rhizomes of Salvia miltiorrhiza almost exhausted the wild Salvia sources in China. However, the content and composition of phenolic acids in the aerial parts of the plant and their potential to be used as a substitute has not been explored. To evaluate the potential of the aerial parts of Salvia miltiorrhiza as new natural sources of phenolic acids. HPLC coupled with diode array detection (DAD) and electrospray ionization multistage mass spectrometry (ESI/MS(n) ) has been used for qualitative and quantitative analysis of phenolic compounds. A total of 38 phenolic compounds were identified or tentatively characterized. A quantitative HPLC-DAD method allowing the simultaneously quantification of six phenolic acids was optimized and validated for linearity, precision, accuracy, and limits of detection and quantification. Calibration curves showed good linear regression (r² > 0.9991) within test ranges; the recoveries ranged between 95.64 and 101.67% and the RSDs were less than 3.01%. The developed methods have been proved to be effective for the identification and quantification of phenolic acids in S. miltiorrhiza. The results obtained suggest that the aerial parts of the plant could be used as an alternative source of sage phenolics.

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MS n ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MS n detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.

New features on the fragmentation patterns of homoisoflavonoids in Ophiopogon japonicus by high‐performance liquid chromatography/diode‐array detection/electrospray ionization with multi‐stage tandem mass spectrometry

Homoisoflavonoids, a special class of flavonoids, are mainly distributed in the Liliaceae family and have various biological activities. Previously, very little research has been reported on the gas-phase fragmentation patterns of homoisoflavonoids by electrospray ionization mass spectrometry. In this paper, we report the use of high-performance liquid chromatography with a diode-array detector (HPLC-DAD) and electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) to study the fragmentation behavior of 11 homoisoflavonoid standards and to analyze homoisoflavonoids in Ophiopogon japonicus. In total, 28 homoisoflavonoids (including seven novel constituents) were characterized. The deprotonated [M--H](-) molecules of the homoisoflavonoids containing a saturated C2--C3 bond afforded the A or B product ion (base peak) according to whether the B-ring was substituted with a hydroxyl group. For the homoisoflavonoids containing a C-2-C-3 double bond, the product ions (A or C ion) were created from the precursor [M-H](-) ion as the base peak when the B-ring was substituted with a hydroxyl group. The homoisoflavonoids carrying a formyl group in the A-ring readily eliminated one molecule of CO to form the product ion [M + H-CO](-) (base peak) irrespective whether the C-2-C-3 bond was saturated or not. This product ion afforded the [M-H-CO-B-ring--CH(2) + H](-) ion by cleavage of the C3-C9 bond. This latter product ion always appeared in tandem mass (MS/MS) spectra of type I homoisoflavonoids. The common features of flavonoids observed during the gas-phase fragmentation mechanisms were the loss of the following groups: 15 Da (CH(3)), 18 Da (H(2)O), 28 Da (CO), 44 Da (CO(2)) and 46 Da (CH(2)O(2)). A retro-Diels-Alder (RDA)-like cleavage was also observed for the homoisoflavonoids. The different gas-phase fragmentation routes were characterized for the deprotonated molecules obtained from the various homoisoflavonoids and collision-induced dissociation (CID) fragmentation differences were noted for the different locations of the various substituents. In conclusion, we can say that this study allowed us to structurally elucidate and identify homoisoflavonoids distributed in related plants and their complex prescriptions.