Study of Cyclization of Chelating Compounds Using Electrospray Ionization Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used for the study of cyclization of organic chelating compounds (chelators). Four chelating compounds were studed: Symmetrical ethylenediaminediacetic acid (s-EDDA), Unsymmetrical ethylenediaminediacetic acid (u-EDDA), N-(2-hydroxyethyl) ethylenediaminetriacetic acid (HEDTA), and N-(2-hydroxyethyl)iminodiacetic acid (HEIDA). The chelators were cyclized with treatments of acids and heating. The open and cyclized form of the chelators were semi-quantified by both positive and negative ion modes ESI-MS. The kinetics of chelator cyclization was studied as a function of reaction temperature and the pH of the matrix. The cyclization of s-EDDA was found to be a pseudo-first order reaction in s-EDDA and overall second order. The cyclizations of HEIDA and HEDTA are reversible reactions. Higher temperature and lower pH favors cyclization.     

Bilirubin degradation by uncoupled cytochrome P450. Comparison with a chemical oxidation system and characterization of the products by high-performance liquid chromatography/electrospray ionization mass spectrometry

Bilirubin is a protective antioxidant; however, when its conjugation and excretion are impaired, as in neonatal and hereditary jaundice, bilirubin accumulates and may cause severe neurotoxicity. Degradation of bilirubin takes place (a) on interaction with oxidative free radicals and (b) when cytochrome P450 (CYP) enzymes are uncoupled by polyhalogenated substrate analogues. The products of pathways (a) and (b) above have now been characterized by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the mechanisms of fragmentation in part clarified. Oxidation of bilirubin by uncoupled CYP1A5 and by a Fe-EDTA/H2O2 system produced both biliverdin and an identical profile of dipyrrolic fragments, as detected by positive ESI-MS. A similar profile of oxidation products was found from mesobilirubin, all showing the expected increase in mass, thus providing direct evidence for fragmentation at the central methene bridge of the tetrapyrroles. Two degradation products, also detected by negative ESI-MS, were characterized as dipyrroles retaining the central bridge carbon, with one or two oxygen atom(s) bound (probably as the aldehyde and hydroperoxide derivatives). Ions compatible with propentdyopents and bilifuscins were also detected, but here the assignment was less certain. It is concluded that the first step in the oxidation of bilirubin may be hydrogen abstraction at the central methene bridge. This is followed either by loss of another hydrogen to give biliverdin, or by oxygen binding and fragmentation. Fe-EDTA/H2O2 and uncoupled CYP(Fe=O) may both initiate the reaction, the latter in an attempt to reduce the ferryl oxygen to water. These studies shed light on the CYP uncoupling mechanism and are of potential significance for the therapy of severe jaundice.     

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.     

Fragmentation study of short-chain products derived from oxidation of diacylphosphatidylcholines by electrospray tandem mass spectrometry: identification of novel short-chain products

Lineloyl-palmitoyl (PLPC) and arachidonoyl-palmitoyl (PAPC) phosphatidylcholine were oxidized under Fenton reaction conditions (H2O2 and Fe2+), and the short-chain products formed were identified by electrospray ionization mass spectrometry (ESI-MS). The short-chain products resulted from beta-cleavage of oxygen-centered radicals and comprised aldehydes, hydroxyaldehydes and dicarboxylic acids that yielded both [MH]+ and [MNa]+ ions. The fragmentation of the [MH]+ and [MNa]+ ions of the peroxidation products was studied by tandem mass spectrometry (MS/MS). The MS/MS spectra of both ions showed ions resulting from characteristic losses of glycerophosphatidylcholine. Other product ions, resulting from C-C cleavages occurring in the vicinity of the functional group, and fragmentations involving the hydroxy groups, were the most informative since they allowed us to obtain structural information relating to the sn-2 acyl residue. Both fragmentation pathways are due to charge-remote fragmentation occurring by a 1,4-hydrogen elimination mechanism and/or by homolytic cleavage. Furthermore, the fragmentation pathway of some ions observed in the ESI-MS spectrum was not consistent with the fragmentation behavior expected for some of the short-chain species identified in the literature and allowed the reassignment of the ions as different structures. Isobaric ions were observed in the ESI-MS spectra of both oxidized phospholipids, and were differentiated based on distinct fragmentation. The detailed knowledge of lipid peroxidation degradation products is of major importance and should be very valuable in providing new markers for oxidative stress signaling and for disease states monitoring.     

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).     

Ring contraction reactions of 2,3-dihydro-4H-1,3-oxazin-4-ones under electrospray ionization conditions

The mass spectral behavior of 2,3-dihydro-4H-1,3-oxazin-4-ones has been investigated using electrospray ionization multi-stage mass spectrometry (ESI-MS(n)). All compounds showed a predominant retro-Diels-Alder (RDA) fragmentation pathway, and a novel ring contraction reaction by loss of isocynates was also found. The fragmentation mechanisms proposed for 4H-1,3-oxazin-4-ones are supported by ESI-MS/MS/MS spectra.     

Fragmentation patterns of novel dispirocyclopiperazinium dibromides with strong analgesic activity under electrospray ionization tandem mass spectrometry conditions

The fragmentation patterns of a series of dispirocyclopiperazinium dibromides with strong analgesic activity are analyzed by positive ion electrospray ionization mass spectrometry in conjunction with tandem mass spectrometry (ESI-MS(n)). Instead of the parent molecular ions, the fragment ions [M-Br](+) are detected as characteristic double peaks and usually the base peaks. Meanwhile, the fragment ions [M-2Br](2+) are unique for this series of diquaternary ammonium dibromides and show high intensity. Besides the common fragmentation patterns around the carbonyl group, elimination of hydrogen bromide from [M--Br](+) ions is another important pathway. Following the elimination, an interesting rearrangement takes place in the unsaturated spirocyclopiperazine and transforms it into a dihydropyrrole structure, whose fragmentations are similar to its precursor ion in the succeeding steps.     

Determination of binding sites in carboplatin-bound cytochrome c using electrospray ionization mass spectrometry and tandem mass spectrometry

Interaction of carboplatin with cytochrome c (Cyt. c) has been investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). ESI-MS studies revealed that the ring-opened adducts of carboplatin with Cyt. c were formed in the stoichiometric ratio of 1:1 and 2:1 at pH 5.0 and 37 degrees C and in the stoichiometric ratio of 1:1 only at pH 7.0 and 37 degrees C. It was also found that Cyt. c could be cleaved by carboplatin at pH 2.5 and 50 degrees C. The cleaved fragments of Cyt. c were determined by ESI-MS and MS/MS analysis to be Glu66 approximately Met80, Ac-Gly01 approximately Met65, Glu66 approximately Glu104, Ac-Gly01 approximately Met80 and Ile81 approximately Glu104. The carboplatin prefers to anchor to Met65 first, then to Met80. To further confirm the binding site of Met, AcMet-Gly was used as the model molecule to investigate its interaction with carboplatin and its hydrolysis reaction. On the basis of species detected during the reaction monitored by ESI-MS, a possible pathway of the cleavage reaction was proposed.     

淫羊藿苷的电喷雾质谱研究

研究了淫羊藿苷的电喷雾质谱行为.结果表明,该化合物在正、负离子模式下均可得到较好的质谱信息,在负离子模式下,采用H/D交换方法,证明淫羊藿苷易形成加合2个水分子的准分子离子.利用电喷雾碰撞诱导解离方法,分别阐明了该化合物在正、负离子模式下的电喷雾质谱碎裂规律.     

Electrospray ionization tandem mass spectrometric characterization of the new functional oligo(ether-ester)s structure

The oligomeric products obtained by the polymerization of monomethacrylates of oligoethylene glycols possessing up to four oxyethylene units were examined by electrospray ionization mass spectrometry (ESI-MS). The oligomerization initiated by sodium and potassium hydrides proceeded via Michael-type addition of hydroxyl groups to carbon-carbon double bonds. Extended reaction time caused gelation, especially in the case of the more reactive potassium hydride. This study was focused on the structural assignments of the oligomeric products by use of ion trap multistage mass spectrometry (ESI-MS(2)). Precise structural characterization of the individual oligomers was based on fragmentation data.     

Electrospray ionization for analysis of platelet-activating factor.

Platelet-activating factor (PAF) was analyzed by electrospray-ionization mass spectrometry (ESI-MS) using a single quadrupole mass spectrometer. The positive-ion spectrum was dominated by an ion corresponding to a sodiated molecule when a low potential difference between the capillary exit (nozzle) and the skimmer was employed, but when the capillary exit voltage was increased, fragmentation of PAF was observed. Initial fragmentation involved the loss of the elements of trimethylamine from the sodiated molecule to yield [M+Na-59]+. An intense ion at m/z 147, generated by the loss of trimethylamine from the sodiated phosphocholine portion of the molecule was also detected, along with a lower intensity ion at m/z 184 which is representative of a protonated phosphocholine moiety. With negative-ion detection the major molecular species was [M+Cl]-. Interpretation of the mass spectral fragments was verified by ESI tandem mass spectrometry on a triple-quadrupole tandem mass spectrometer.     

Quantitative detection of N(7)-(2-hydroxyethyl)guanine adducts in DNA using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

High-performance liquid chromatography (HPLC) was combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) to develop a sensitive and selective method for the quantitative measurement of N(7)-(2-hydroxyethyl)guanine (N(7)-HEG) adducts in DNA obtained from ethylene oxide-exposed biological samples. Selected reaction monitoring (SRM) was used as the detection mode while the fragmentation product ion at m/z 152 generated from the precursor protonated N(7)-HEG (m/z 196) was monitored. The detection limits for N(7)-HEG were estimated by twofold serial dilution and determined to be 4 fmol in neat standard solution and 16 fmol when a matrix effect is considered. When the mass spectrometer was operated in the selected ion monitoring mode using only the first quadrupole (without MS/MS function), the detection limits increased to 128 fmol and 1 pmol (when matrix effect is considered), respectively. A good linear correlation (R(2) = 0.999) was observed for signal intensities obtained by injecting 16 fmol--33 pmol of N(7)-HEG into the HPLC/ESI-MS/MS system. Hep G2 cells were incubated for 8 h with medium containing various concentrations of ethylene oxide (ranging from 0.05 to 5.0 mM). A dose-response relationship was established, indicating that the adduct formation increases with the exposure level. The method shows potential, although the detection limit needs to be lowered for practical applications, for use in monitoring N(7)-HEG formation in other biological systems.     

The mechanism of the Stille reaction investigated by electrospray ionization mass spectrometry

On-line monitoring of Stille reactions was performed via direct infusion electrospray ionization mass spectrometry (ESI-MS) and its tandem version (ESI-MS/MS). When operated in the positive ion mode, ESI(+)-MS was able to transfer, directly from solution to the gas phase, the species involved in all main steps of a Stille reaction, that is, the catalytically active palladium species Pd(PPh3)2, in its molecular ion form as well as the key cationic Pd(II) intermediates, including cyclic IPd-(CH2CH)Sn species. When searching for anionic species, ESI(-)-MS monitoring showed I- as the only anion detectable in the reaction medium. A detailed catalytic cycle for a Stille reaction was elaborated in which reaction intermediates and the previously elusive catalytically active Pd(0) species are shown in association with the respective ionic species intercepted by ESI-MS and further characterized by ESI-MS/MS.     

Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation

A rapid, sensitive and selective method involving reversed-phase liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) was employed for determination of commercial ceramides in cosmetics for quality control of the product formulation. Using this LC/ESI-MS technique, simultaneous separation and characterization of ceramides and an impurity substance were possible. Informative fragmentation patterns were obtained by employing LC/ESI-MS in both positive and negative ionization modes to identify the structures of both sphingoid base and N-acyl chains of ceramides, and also of an impurity. The combination of positive and negative mass spectra can be used for unambiguous confirmation of ceramides and for characterization of unknown species. In-source collision-induced fragmentation resulted in characteristic product anions for the ceramides containing a phytosphingosine moiety at m/z 267, 255 and 225, and for those with a sphingosine moiety at m/z 263 and 237, regardless of the length of the fatty acyl chains. The detection limit was about 0.5 pmol in selected-ion monitoring mode. Quantification using internal standards showed good linearity and a relative standard deviation of 4%. These ceramides were more sensitively detected in positive than in negative ion mode.     

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

Everolimus (40-O-(2-hydroxyethyl)rapamycin, Certican) is a 31-membered macrolide lactone. In lymphocytes, it inhibits the mammalian target of rapamycin (mTOR) and is used as an immunosuppressant after organ transplantation. Due to its instability in pure organic solvents and insufficient HPLC separation, NMR spectroscopy analysis of its metabolite structures is nearly impossible. Therefore, structural identification based on tandem mass spectrometry (MS/MS) and MS(n) fragmentation patterns is critical. Here, we have systematically assessed the fragmentation pattern of everolimus during liquid chromatography (LC)-electrospray ionization (ESI)-MS/MS and validated the fragment structures by (1) comparison with structurally identified derivatives (sirolimus), (2) high-resolution mass spectrometry, (3) elucidation of fragmentation pathways using ion trap mass spectrometry (up to MS(5)) and (4) H/D exchange. In comparison with the structurally related immunosuppressants tacrolimus and sirolimus, our study was complicated by the low ionization efficiency of everolimus. Detection of positive ions gave the best sensitivity, and everolimus and its fragments were mainly detected as sodium adducts. LC-ESI-MS/MS of everolimus in combination with collision-induced dissociation (CID) resulted in a complex fragmentation pattern and the structures of 53 fragments were identified. These detailed fragmentation pathways of everolimus provided the basis for structural elucidation of all everolimus metabolites generated in vivo und in vitro.     

Detection of new radiation-induced DNA lesions by liquid chromatography coupled to tandem mass spectrometry

High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) has been used to search for the formation of as yet unidentified radiation-induced DNA lesions. For that purpose, the characteristic fragmentation of most of 2'-deoxyribonucleosides that corresponds to the loss of the 2-deoxyribose moiety (loss of 116 mass units) has been utilized to specifically detect modified nucleosides. Aerated aqueous solutions of DNA were exposed to ionizing radiation, and subsequently DNA was digested to nucleosides with a cocktail of endo- and exonucleases. HPLC/ESI-MS/MS analysis of the resulting 2'-deoxyribonucleoside mixture allowed us to detect four novel DNA modifications. In a subsequent step, the sensitivity of the tandem mass spectrometer was used to search for the formation of the newly detected lesions in the DNA of gamma-irradiated cells. Thus, one of the four newly detected lesions was found to be significantly generated in cellular DNA upon exposure to ionizing radiation. In addition, the latter lesion was also shown to be present in untreated cells, indicating that the modified nucleoside could be formed endogenously.     

Analysis of oxidized epigallocatechin gallate by liquid chromatography/mass spectrometry

(-)-Epigallocatechin gallate (EGCG) of catechins changes from non-colored at around neutral pH to yellow at higher pH region in aqueous solution. The pH-dependent oxidation of EGCG was analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). LC/MS/MS analysis of EGCG and its related compounds, (-)-epicatechin gallate (ECG) and (-)-epigallocatechin (EGC), successfully elucidated the structure relationship of EGCG solution involving the color change reaction at different pH conditions. The oxidation species produced at alkaline pH was detected at a different retention time from EGCG in the chromatograms of the EGCG sample. The oxidation species was found to correspond to M+14 (where M is the molecular weight of EGCG), which has two hydrogen atoms removed and addition of one oxygen atom to the gallyl moiety in the B-ring of EGCG.     

Negative-ion electrospray ionization tandem mass spectrometry of N-phosphoryl amino acids and dipeptides

The negative-ions of N-phosphoryl amino acids were studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The negative-ion ESI-MS/MS of N-phosphoryl amino acids showed characteristic fragmentation patterns different from those observed in the corresponding positive-ion ESI-MS/MS and negative-ion fast-atom bombardment mass spectra. For negative-ion ESI-MS/MS, a unique fragmentation from the N-terminal of N-phosphoryl amino acids or peptides containing a free beta-OH or CO(2)H group was observed to yield the characteristic fragment ion (RO)(2)P(O)O(-). The ease of the rearrangement depended on the position of the hydroxyl group in amino acids or peptides, and the N --> O rearrangement mechanism was proposed to involve the participation of the hydroxyl group. From previous solution-phase experiments and theoretical calculations, it was found that the beta-OH group was more active than gamma-OH, and the corresponding difference in negative-ion ESI-MS/MS was consistent with those previous findings.     

Non-enzymatic model glycation reactions--a comprehensive study of the reactivity of a modified arginine with aldehydic and diketonic dicarbonyl compounds by electrospray mass spectrometry

Non-enzymatic glycation (Maillard reaction) of long-lived proteins is a major contributor to the pathology of diabetes, and possibly aging and Alzheimer's disease. Among the amino residues in proteins arginine plays an important role, and its modification by sugar moieties generates the so-called advanced glycation end products (AGEs). Moreover, alpha-dicarbonyl compounds have been found as the main participants in those modifications.Four alpha-dicarbonyl compounds, aldehydic and ketonic, were reacted with the modified amino acid N(alpha)-acetyl-L-arginine (AcArg), in an attempt to establish structure/activity relationships for the reactivity of alpha-dicarbonyls with the amine compound. Electrospray ionization mass spectrometry (ESI-MS), combined with tandem mass spectrometry (MS/MS), was used to identify and characterize reagents, intermediates and reaction products. The fragmentation patterns of precursor ions showed similarities in all reaction systems studied, in which fragmentation of the amino acid residue prevails, especially for the dehydrated and/or multiple dehydrated precursor ions. For the non-hydrated ion species, fragmentation of the arginyl guanidino group was mainly observed. Specific information regarding the nature of the ions formed, in which the dicarbonyl electrophile character played an important role, was obtained. As an example, singly and doubly hydrated acetyl-argpyrimidine ions were detected for the methylglyoxal reaction only. For symmetrical dicarbonyls, glyoxal and diacetyl, the importance of steric contributions with respect to the energetic ones is discussed. Furthermore, the dehydrated acetyl-tetrahydropyrimidine ions for methylglyoxal and phenylglyoxal reactions revealed fragment ion compositions including the protonated molecules of acetyl-argpyrimidine, -hydroimidazolone and -5-methylimidazolone. An explanation for the acetyl-argpyrimidine formation from the acetyl-hydroimidazolone formation reaction is proposed. Aspects such as the amount of acetyl-hydroimidazolone formed, the response of the hydration equilibria of the dicarbonyl forms to the new unhydrated dicarbonyls introduced by the reversal of the acetyl-hydroimidazolone formation reaction and the stability of the dicarbonyl intermediate involved in the acetyl-argpyrimidine formation are proposed, as being responsible to control the formation of acetyl-argpyrimidine.     

Sequence specific stabilization of a linear analog of the antifungal lipopeptide iturin A2 by sodium during low energy electrospray ionization mass spectrometry conditions

The structures and stability of sodiated species of 8-Beta, a linear lipopeptide analog (beta-aminotetradecanoyl-NYNQPNS) of the antifungal peptide iturin A2, were evaluated by electrospray ionization mass spectrometry (ESI-MS). Association of the lipopeptide, 8-Beta, with sodium afforded protection from fragmentation at high cone voltages and increasing collision energy conditions in the ESI-MS. The order of decreasing stability was found as 8-Beta 1Na > 8-Beta 2Na > 8-Beta 3Na > 8-Beta. Substantial differences were found between fragmentation patterns of the free and sodiated molecular species. Breakage of the N-terminal peptide bond of L-Pro generated the major product ions of the free 8-Beta parent ion. Impaired fragmentation of the sodium adducts of 8-Beta, indicated that this bond is protected by sodium complexation. Fragmentation patterns of the sodiated lipopeptide further revealed two specific binding sites for a nonsolvated sodium ion within the two type II beta-turn sequences (beta-aminotetradecanoyl-NYN and QPNS) of the natural iturin A2. It is proposed that specific interaction with sodium takes place with most of the peptide bond oxygens in these turns, and with the Gln sidechain. This interaction leads to stabilized structures in which the peptide backbone, specifically the peptide bonds in which L-Pro participates, is protected against low-energy fragmentation during ESI-MS.