Fragmentation studies of sartans by electrospray ionization mass spectrometry

Sartans and related analogues with 5‐oxo‐l, 2, 4‐oxadiazole ring and tetrazole ring are investigated in detail using collision‐induced dissociation (CID) method in positive ion mode by electrospray ionization tandem mass spectrometry (ESI‐MSⁿ). It is found that the protonated sartans and related analogues tend to form the N‐substituted‐3‐substituted phenanthridin‐6‐amine ion which has a large conjugative structure. The possible fragmentation pathways were proposed for the first time, and the key structure of product ions was confirmed by high resolution tandem mass spectrometry and theoretical calculation. It is very helpful for understanding the intriguing roles of sartans analogues in fragmentation reactions and enriching the knowledge of the gas‐phase chemistry of the oxadiazole and tetrazole ring. Copyright © 2017 John Wiley & Sons, Ltd.     

Fragmentation of deprotonated cyclic dipeptides by electrospray ionization mass spectrometry

The fragmentation pathways of deprotonated cyclic dipeptides have been studied by electrospray ionization multi‐stage mass spectrometry (ESI‐MSⁿ) in negative mode. The results showed that the fragmentation pathways of deprotonated cyclic dipeptides depended significantly on the different substituents, the side chains of amino acid residues at the diketopiperazine ring. In the spectra of deprotonated cyclic dipeptides, the ion [M? H? substituent radical]^? was firstly observed in the ESI mode. The characteristic fragment ions [M? H? substituent radical]^? and [M? H? (substituent? H)]^? could be used as the symbols of particular cyclic dipeptides. The hydrogen/deuterium (H/D) exchange experiment, the high‐resolution mass spectrometry (Q‐TOF) and theoretical calculations were used to rationalize the proposed fragmentation pathways and to verify the differences between the fragmentation pathways. The relative Gibbs free energies (ΔG) of the product ions and possible fragmentation pathways were estimated using the B3LYP/6–31++G(d, p) model. The results have some potential applications in the structural elucidation and interpretation of the mass spectra of homologous compounds and will enrich the gas‐phase ESI‐MS ion chemistry of cyclic dipeptides. Copyright © 2009 John Wiley & Sons, Ltd.     

Negative electrospray ionization tandem mass spectrometric investigation of ent-kaurane diterpenoids from the genus Isodon

ent-Kaurane diterpenoids are a class of natural compounds isolated from genus Isodon, which have been found to have important bioactivities. Negative electrospray ionization tandem mass spectrometry ((-)ESI-MS(n)) was used to investigate the fragmentation pattern of C-20-nonoxygenated ent-kauranes and two subtypes of C-20-oxygenated ent-kauranes by using an ion trap instrument and accurate mass measurement on an ESI-Q-time-of-flight (TOF) mass spectrometer. The analysis revealed that loss of CH(2)O or CO(2) is the predominant process for 7, 20-epoxy and 7, 20 : 14, 20-diepoxy subgroup of C-20-oxygenated ent-kauranes. In addition, compounds of C-20-nonoxygenated ent-kauranes with a conserved core structure but different substituent groups, such as a hydroxyl, aldehyde, carboxyl, and acetyl moiety, resulted in diagnostic product ions through losses of H(2)O, CO, CO(2), and AcOH, respectively. This work clearly demonstrates the utility of tandem mass spectrometry for studies on the rationalization of the diagnostic fragmentation of a series of compounds from two main types of the ent-kaurane diterpenoids.     

Structural characterization of isoprenylated flavonoids from Kushen by electrospray ionization multistage tandem mass spectrometry

Eighteen isoprenylated flavonoids (8 flavanones, 3 flavanols, and 7 chalcones) isolated from Kushen or synthesized were studied by positive and negative ion electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). Plausible fragmentation patterns were obtained by comparing their MS(n) spectra with each other, which were further supported by high-resolution MS data and two model compounds. It was shown that the 2'-OH group would make the C-ring of flavonoids studied more labile through a six-membered mechanism, resulting in base peaks of (1,3)A+ (positive mode) and (1,4)A(-) (negative mode). In addition, the 2'-OH is also responsible for the neutral loss of water in (+)ESI/MS(2) of flavanones. The neutral loss of water (or methanol) in (-)ESI/MS(2) of flavanols was elucidated by a E2 elimination mechanism. Different relative abundances (RA) of (1,3)A(+) and S(+) in (+)ESI/MS(2) spectra were used to discriminate flavanones with their open-ring products, chalcones, since the equilibrium for flavanone<-->chalcone isomerization in ESI ion source could not be obtained in positive mode.     

Fast profiling of chemical constituents in Yiqing Capsule by ultra-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry

An ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC-ESI-MS(n)) has been developed for structural characterization and identification of multi-constituents in Yiqing Capsule, a well-known combined herbal remedy prepared from the extract mixtures of Rhizoma Coptidis, Radix et Rhizoma Rhei and Radix Scutellariae. The UPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6 mm×50 mm, 1.8 μm) and gradient elution of 0.1% formic acid solution and acetonitrile in 16 min. Based on their retention times and mass spectra in comparison with the data from standards or references, a total of 29 compounds including 3 phenolic acids and 4 anthraquinones from Radix et Rhizoma Rhei, 8 alkaloids from Rhizoma Coptidis and 14 flavonoids from Radix Scutellariae were unambiguously identified or tentatively characterized in the complex system. The MS data and fragmentation information of two isomers of feruloylquinic acid were first reported in Radix et Rhizoma Rhei and in Yiqing Capsules. This study is expected to be accepted as an effective and reliable pattern for comprehensive and systematic characterization of this commonly used Chinese herbal preparation.     

Fragmentation of 2‐aroylbenzofuran derivatives by electrospray ionization tandem mass spectrometry

We investigated the gas‐phase fragmentation reactions of a series of 2‐aroylbenzofuran derivatives by electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The most intense fragment ions were the acylium ions m/z 105 and [M+H–C₆H₆]⁺, which originated directly from the precursor ion as a result of 2 competitive hydrogen rearrangements. Eliminations of CO and CO₂ from [M+H–C₆H₆]⁺ were also common fragmentation processes to all the analyzed compounds. In addition, eliminations of the radicals •Br and •Cl were diagnostic for halogen atoms at aromatic ring A, whereas eliminations of •CH₃ and CH₂O were useful to identify the methoxyl group attached to this same ring. We used thermochemical data, obtained at the B3LYP/6‐31+G(d) level of theory, to rationalize the fragmentation pathways and to elucidate the formation of E , which involved simultaneous elimination of 2 CO molecules from B .     

The investigation of substituent effects on the fragmentation pathways of pentacoordinated phenoxyspirophosphoranes by ESI‐MSn

The fragmentation pathways of pentacoordinated phenoxyspirophosphoranes were investigated in the positive mode by electrospray ionization multistage mass spectrometry. The results demonstrate that the sodium adducts of the title compounds undergo two competitive fragmentation pathways, and the fragmentation patterns are heavily dependent on the various substituent patterns at the phenolic group. An electron‐withdrawing substituent at the ortho‐position always results in the removal of a corresponding phenol analogue, while cleavage by spiroring opening becomes the predominant fragmentation pathway if an electron‐donating substituent is at the phenolic group. The substituent effects on the competitive fragmentation pathways were further elucidated by theoretical calculations, single crystal structure analysis, and high‐resolution mass spectrometry. The results contribute to the understanding of the gas‐phase fragmentation reactions and the structure identification of spirophosphorane analogues by electrospray ionization multistage mass spectrometry.     

Fragmentation studies of synthetic 2-acylamino-1,4-naphthoquinones by electrospray ionization mass spectrometry

We report here the fragmentation mechanism for five 2-acylamino-1,4-naphthoquinone derivatives using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analyses were performed on a low-resolution, triple-quadrupole mass spectrometer. Fragmentation pathways for protonated molecular derivatives 2-acylamino-1,4-naphthoquinone [M+H]+ are proposed on the basis of theoretical calculations. There is evidence that the nitrogen atom is the protonation site, based on the nucleophilic atomic indices.     

Characterization and identification of iridoid glucosides,flavonoids and anthraquinones in Hedyotis diffusa by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

The multiple bioactive constituents in Hedyotis diffusa Willd. (H. diffusa) were extracted and characterized by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MSⁿ). The optimized separation condition was obtained using an Agilent ZorBax SB‐C₁₈ column (4.6×150 mm, 5 μm) and gradient elution with water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), under which baseline separation for the majority of compounds was achieved. Among the compounds detected, 14 iridoid glucosides, 10 flavonoids, 7 anthraquinones, 1 coumarin and 1 triterpene were unambiguously identified or tentatively characterized based on their retention times and mass spectra in comparison with the data from standards or references. The fragmentation behavior for different types of constituents was also investigated, which could contribute to the elucidation of these constituents in H. diffusa. The present study reveals that even more iridoid glycosides were found in H. diffusa than hitherto assumed. The occurrence of two iridoid glucosides and five flavonoids in particular has not yet been described. This paper marks the first report on the structural characterization of chemical compounds in H. diffusa by a developed HPLC‐ESI‐MSⁿ method.     

Qualitative and quantitative analyses of Epimedium wushanense by high-performance liquid chromatography coupled with diode array detection and electrospray ionization tandem mass spectrometry

A new HPLC-DAD-ESI-MS(n) method was developed for rapid separation, characterization and quantitation of flavonoids in Epimedium wushanense, a popular Chinese herbal medicine. For qualitative identification, a total of 37 compounds were characterized from the underground and aerial parts of E. wushanense. Among them, 28 compounds were prenylated flavonoids, and 23 were confirmed by comparing with reference standards. For quantitative analysis, 12 major flavonoids including kaempferol glycosides, desmethylicaritin glycosides, and icaritin glycosides were simultaneously determined by HPLC/UV. Samples were separated on a Waters Symmetry C(18) column at 35 °C eluted with a gradient three-component mobile phase of acetonitrile, methanol, and water containing 0.03% v/v formic acid. All the flavonoids showed good linearity (r(2) ≥0.9997). The recoveries varied from 92.6 to 106.1% at three concentration levels. This method was applied to the determination of 20 samples of different geographical sources, harvesting time, and plant parts. Contents of the predominant flavonoid, epimedin C, ranged from 1.4 to 5.1% in aerial parts and 1.0 to 2.8% in underground parts. The methods established in this paper were simple and reliable and could be used for the quality control of E. wushanense.     

Quality evaluation of Hpericum japomicum by using high‐performance liquid chromatography coupled with photodiode array detector and electrospray ionization tandem mass spectrometry

A high‐performance liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry (HPLC‐PAD‐ESI‐MSⁿ) method was developed to evaluate the quality of Hpericum japomicum through establishing chromatographic fingerprint and simultaneous determination of seven phenolic compounds. The analysis was achieved on an Ultimate XB‐C₁₈ analytical column (250 mm × 4.6 mm i.d., 5 µm) using an aqueous solution of acetic acid (pH 3.8) and methanol as the mobile phase. Ten samples of H. japomicum from various habitats were investigated and the correlation coefficients of similarity were determined from the HPLC fingerprints. By using an online ESI‐MSⁿ, 20 common peaks in chromatographic fingerprints were identified as phenols, including flavones and their glycosides, flavonones and their glucosides, flavanols, xanthones, phloroglucinols, phenyl propanoids and chromones. Based on the above study, seven phenols which are considered to be major constituents in H. japomicum, including 3,4‐dihydroxybenzoic acid (1), taxfolin‐7‐O‐α‐l ‐rhamnoside (7), 7‐dihydroxy‐2‐(1‐methylpropyl)chromone‐8‐β‐d ‐glucoside (8), isoquercitrin (14), quercitrin (16), quercetin‐7‐O‐α‐l‐ rhamnoside (18) and quercetin (19) were quantified by the validated HPLC‐PAD method. This developed method by combination of chromatographic fingerprint and quantification analysis could be applied to control the quality of H. japomicum. Copyright © 2009 John Wiley & Sons, Ltd.     

Structure analysis of triterpene saponins in Polygala tenuifolia by electrospray ionization ion trap multiple-stage mass spectrometry

Eighteen different triterpene saponins isolated from Polygala tenuifolia were investigated by electrospray ionization ion trap multiple-stage mass spectrometry (ESI-ITMS(n)) in positive and negative ion modes. MS(1)-MS(3)/MS(4) spectra of the both modes were analyzed, and they all gave fragments in line and shared common fragmentation patterns. Key fragments from MS(n) spectra of both the modes and their proposed fragmentation pathways were constructed with examples illustrated for the formation of characteristic fragments in the saponins. Two special fragmentation patterns were proposed: (1) the formation of fragments by cleavage of CH(2)O from Delta(12)-14alpha-CH(2)OH of the oleanene-type saponin aglycone in both positive and negative MS(n) (n > or = 2) modes; (2) the occurrence of fragments by cleavage of CO(2) and 3-glucose as the characteristic structure feature of 23-COOH at the oleanene-type saponin aglycones coupled with 3-Glc substitutes in the negative MS(n) (n > or = 2) modes. Peak intensities in MS(n) spectra were also correlated with structural features and fragmentation preferences of the investigated saponins, which are discussed in detail. In general, fragments formed predominantly by cleavages of glycosidic bonds in the positive mode, while selective cleavages of acyl bonds preceded that of glycosidic bonds in negative MS(n) (n > or = 2) mode, both of which could well be applied to the structural analysis of these saponins. Interpretation of MS(n) spectra presented here provided diagnostic key fragment ions important for the structural elucidation of saponins in P.tenuifolia.     

Studies on lignan constituents from Schisandra chinensis (Turcz.) Baill. fruits using high-performance liquid chromatography/electrospray ionization multiple-stage tandem mass spectrometry

A reversed-phase high-performance liquid chromatography-diode array detector-electrospray ionization multiple-stage tandem mass spectrometry (RP-HPLC-DAD-ESI-MS(n)) method has been developed for the detection and analysis of lignan constituents in the methanol extract from the fruits of Schisandra chinensis (Turcz.) Baill. RP-HPLC-DAD-ESI-MS(n) and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MS(n)) have been applied to investigate the characteristic product ions of four lignan reference compounds. Then, the logical fragmentation pathways of the lignans have been proposed. By comparing the retention time (t(R)) of HPLC, the ESI-MS(n) data and the structures of analyzed compounds with the data of reference compounds and in the literature, 11 peaks in HPLC have been unambiguously identified and another 5 peaks have been tentatively identified or deduced. Also, in the present paper, the extracted ion chromatograms (EIC) have been used to analyze the lignan isomers. The experimental results demonstrate that RP-HPLC-DAD-ESI-MS(n) is a specific and useful method for the identification of the lignan constituents and their isomers.     

Evaluation of relative DNA binding affinities of anthrapyrazoles by electrospray ionization mass spectrometry

Binding interactions of a new series of anthrapyrazoles (APs) with DNA were evaluated by electrospray ionization mass spectrometry (ESI-MS). Relative binding affinities were estimated from the ESI-MS data based on the fraction of bound DNA for DNA/anthrapyrazole mixtures, and they show a correlation to the shift in melting point of the DNA measured from a previous study. Minimal sequence specificity was observed for the series of anthrapyrazoles. Upon collisionally activated dissociation of the duplex/anthrapyrazole complexes, typically ejection of the ligand was the dominant pathway for most of the complexes. However, for complexes containing AP2 or mitoxantrone, strand separation with the ligand remaining on one of the single strands was observed, indicative of a different binding mode or stronger binding.     

Role of opposite charges in protein electrospray ionization mass spectrometry

The conformation dependence of protein spectra recorded by electrospray ionization mass spectrometry (ESI-MS) is an interesting and useful phenomenon, whose origin is still the object of debate. Different mechanisms have been invoked in the attempt to explain the lower charge state of folded versus unfolded protein ions in ESI-MS, such as electrostatic repulsions, solvent accessibility, charge availability, and native-like interactions. In this work we try to subject to direct experimental test the hypothesis that conformation-dependent neutralization of charges with polarity opposite to the net charge of the protein ion could play a critical role in such an effect. We present results of time-of-flight nano-ESI-MS on the peptide angiotensin II, indicating that negative charges of carboxylate groups can contribute to spectra recorded in positive-ion mode when stabilized by favorable electrostatic interactions, which is the central assumption of our hypothesis. Comparison of horse and spermwhale myoglobin (Mb) shows that changing the total number of basic residues within a given three-dimensional structure shifts the charge-state distribution (CSD) of the folded protein in positive-ion mode. This result appears to be in contrast to models in which electrostatic repulsions or availability of charges in the ESI droplets represent the limiting factor for the ionization of folded protein ions in ESI-MS. At the same time, it suggests a role of acidic residues in conformational effects in positive-ion mode. Furthermore, an attempt is made to rationalize those cases in which, in contrast, the main charge state observed in ESI-MS under non-denaturing conditions deviates considerably from the net charge expected on the basis of the amino-acid composition. These cases usually correspond to proteins with quite balanced content in basic and acidic residues, suggesting that this might be a factor influencing their charging behavior in ESI-MS. Experiments on mutants of ribonuclease Sa (RNase Sa) reveal that progressively reducing the excess of acidic residues, replacing them by lysine, causes almost no shift in the spectrum of the folded protein in negative-ion mode. Analogously, variants with an excess of three or five basic residues give similar spectra in positive-ion mode. These results indicate a lower limit to the extent of ionization observable by ESI-MS (6- or 8+ in the case of RNase Sa in water). Below such limit of net charge, changes in the relative amount of ionizable side chains do not affect the qualitative features of the observed CSDs. A progressive loss of signal intensity caused by the mutations in negative-ion mode suggests that low charge states might also be counterselected, even within the m/z range theoretically accessible to the instrument.     

Formation and recognition of G-quadruplex in promoter of c-myb oncogene by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) is used to study the formation of G-quadruplex by d(GGAGGAGGAGGA) which locates at the promoter region of c-myb gene. In addition, a natural small molecule, dehydrocorydaline from a Chinese herb, is found to have the highest binding affinity with the G-quadruplex in nine natural small molecules studied, and the binding selectivity of this natural molecule toward the c-myb G-quadruplex with respect to corresponding duplex DNA is significantly higher than that of the broad-spectrum G-quadruplex-ligand TMPyP4. The result from ESI-MS indicates that the gas-phase kinetic stability of the G-quadruplex can be enhanced by binding of dehydrocorydaline. To further investigate the binding properties of dehydrocorydaline to the G-quadruplex, Autodock3 is used to calculate the docked sites and docked energies of small molecules binding to the G-quadruplex and the result shows that the docked energy of dehydrocorydaline is the biggest in the nine small molecules used, consistent with the result from ESI-MS.     

Metal-ligand solution equilibria studied by electrospray ionization mass spectrometry: effect of instrumental parameters

Electrospray ionization mass spectrometry (ESI-MS) is being increasingly employed in the study of metal-ligand equilibria in aqueous solution. In the present work, the ESI-MS spectral changes due to different settings of the following instrumental parameters are analyzed: the solution flow rate (F(S)), the nebulizer gas flow rate (F(G)), the sprayer potential (E), and the temperature of the entrance capillary (T). Twenty-eight spectra were obtained for each of six samples containing aluminum(III) and 2,3-dihydroxypyridine at various pH, in the absence or in the presence of a buffer and of sodium ions. Among the considered instrumental parameters, T produced the largest effects on the ionic intensities. F(S) and F(G) affected the ESI-MS spectra to a lower extent than T. In the investigated conditions E had the weakest effects on the spectra.The correlations observed between the ionic intensities and these instrumental parameters were interpreted considering the presence of three kinds of perturbations occurring in the ESI-MS ion source: formation of some dimers in the droplets, different transfer efficiencies from the droplets to the gas phase for different complexes (according to their surface activity), and subsequent partial thermal decomposition of the dimers and of one of the monomeric complexes in the gas phase. Our results show that the evaluation of the effects produced in the ESI-MS spectra by a change of instrumental parameters can allow to identify the perturbations occurring when metal-ligand solutions are studied by ESI-MS.     

In-source fragmentation and accurate mass analysis of multiclass flavonoid conjugates by electrospray ionization time-of-flight mass spectrometry

In-source collision-induced dissociation (CID) fragmentation features of multiclass flavonoid glycoconjugates were examined using liquid chromatography electrospray time-of-flight mass spectrometry. Systematic experiments were performed to search for optimal conditions for in-source fragmentation in both positive and negative ion modes. The objective of the study was to attain uniformly appropriate conditions for a wide range of analytes independently of the aglycone, the attached sugar part and the type of bond between the aglycone and the glycan moieties (O- or C-glycosides). Studied substances included representatives of flavonols, flavones, flavanones and anthocyanins and, regarding their glycan parts, mono-, di- and triglycosides with varying distribution of carbohydrate moieties (di-O-glycosides, O-diglycosides, O,C-diglycosides). The breakdown properties of the analytes along with the abundances of the characteristic diagnostic ions required for structural elucidation of complex flavonoid derivatives were evaluated. An optimized value was found for the instrument parameter (fragmentor voltage) affecting the in-source CID fragmentation of the analytes [230 V (ESI+) and 330 V (ESI-)]. Thus, appropriate performance in terms of both highly sensitive full-scan acquisition and fragmentation information was obtained for all the investigated flavonoids. In addition, singularities in the abundance of selected diagnostic ions (e.g. Y(0), Y(1) and Y*) due to variations in the interglycosidic linkage (rutinoside-neohesperidoside) in the glycan part were found and are also evaluated and discussed in detail. The combination of in-source CID fragmentation with high mass accuracy MS detection establishes a working basis for the development of versatile and useful LC-MS methods for wide-scope screening, non-targeted detection and tentative identification of flavonoid derivatives.     

Characterization of aluminum species with nitrate, perchlorate and sulfate ions in the positive and negative ion mode by electrospray ionization mass spectrometry

The application of electrospray ionization mass spectrometry (ESI-MS) for aluminum speciation in the positive and negative ion modes was discussed. Aluminum nitrate, perchlorate and sulfate solutions were measured by ESI-MS. In the positive ion mode, aluminum species containing anions (Al-L; L=NO3, ClO4 and SO4) were identified, while [Al(OH)2(H2O)n]+ (n=2-4) were the main species. The affinity of the anions with Al3+ estimated by ESI-MS was consistent with the hardness of the anions (hard and soft acids and bases principle) and the results from 27Al nuclear magnetic resonance studies. This indicates that the results observed from the positive ion mode preserved the chemical state of aluminum in the solution. In the negative ion mode, [Al(OH)4-nLn]- (n=0-2, L=NO3, ClO4) were the main species, which were considered to be converted from positive aluminum species, [Al(OH)(H2O)n]+ (n=2-4), by the successive addition of anions. Anions did not only attach to one aluminum ion but also bridged two aluminum ions. In Al2(SO4)3 solution, the behavior of SO4(2-) in the negative ion mode differed from that of NO3- and ClO4-. This may reflect the affinity of SO4(2-) with Al3+ in the solution or in the mass spectrometer or in both. Finally, detection mechanisms for the aluminum species in the solution are proposed for both the positive and negative ion modes. It is shown that ESI-MS can be used to observe the interaction between Al3+ and anions. We show the importance of the interpretation of the results by ESI-MS for obtaining new information of the metal species in the solution.