Differentiation of estriol glucuronide isomers by chemical derivatization and electrospray tandem mass spectrometry

This paper describes a way of differentiating between the three isomers of estriol glucuronide by the use of chemical derivatization and liquid chromatography/electrospray tandem mass spectrometry (MS/MS). In their native form, these isomers gave rise to almost identical product ion spectra, involving the neutral loss of 176 Da (i.e. monodehydrated glucuronic acid), which made it impossible to determine the position of conjugation by MS/MS alone. In order to change the fragmentation pathways, positive charges were introduced into the analytes by chemical derivatization. The following reagents were tested: 2-chloro-1-methylpyridinium iodide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2-picolylamine. Interestingly, derivatization using a combination of all three reagents gave a selective fragmentation pattern that could differentiate between the isomers estriol-16-glucuronide and estriol-17-glucuronide. Estriol-3-glucuronide, which lacks a free phenolic group, could be differentiated through a different type of reaction product when exposed to 2-chloro-1-methylpyridinium iodide. Furthermore, in order to assist structural assignment of the fragments, their accurate masses were determined using a hybrid quadrupole time-of-flight mass spectrometer and fragmentation pathways were elucidated by the use of MS3 on an ion trap mass spectrometer.     

Sequence analysis of alginate-derived oligosaccharides by negative-ion electrospray tandem mass spectrometry

Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with (0,2)A- and (2,5)A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Z(int)-ions (Z(int) - 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [(2,5)A(red)]/[(0,4)A(red)] (red: denotes reducing terminal).     

Differentiation of flavonoid glycoside isomers by using metal complexation and electrospray ionization mass spectrometry

The elucidation of flavonoid isomers is accomplished by electrospray ionization tandem mass spectrometry (ESI-MS/MS) via formation and collisional activated dissociation (CAD) of metal/flavonoid complexes containing an auxiliary ligand. Addition of a metal salt and a suitable neutral auxiliary ligand to flavonoids in solution results in the formation of [M(II) (flavonoid-H) ligand]⁺ complexes by ESI which, upon collisional activated dissociation, often result in more distinctive fragmentation patterns than observed for conventional protonated or deprotonated flavonoids. Previously, 2,2′-bipyridine was used as an auxiliary ligand, and now we compare and explore the use of alternative pyridyl ligands, including 4,7-diphenyl-1,10-phenanthroline. Using this technique, three groups of flavonoid glycoside isomers are differentiated, including glycosides of apigenin, quercetin, and luteolin.     

Differentiation of aminomethyl corrole isomers by mass spectrometry

Two new isomeric aminomethyl corrole derivatives of [5,10,15‐tris(pentafluorophenyl)corrolato]gallium(III) were synthesized with pyridine (py) molecules as axial ligands. When investigated by electrospray ionization mass spectrometry, in the positive and the negative ion modes, these compounds showed an unusual gas‐phase behavior that could be used for their differentiation. In the positive ion mode, the differentiation was achieved through the formation of diagnostic fragment ions formed from [M‐py + H]⁺ precursors, by (CH₃)₂NH and HF losses. An unusual addition of water to the main fragment ions provides an alternative route for isomer identification. Semi‐empirical calculations were performed to elucidate the structures and stabilities of the main ionic species formed in the positive ion mode. In the negative ion mode isomer discrimination is accomplished via the fragmentation of the methoxide adduct ions [M‐py + CH₃O]^‐ through (CH₃)₂ N^. and HF losses. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of dissolved naphthenic acids in natural waters by using negative-ion electrospray mass spectrometry

Naphthenic acids (NAs) have been implicated as some of the most toxic substances in oil sands leachates and identified as priority substances impacting on aquatic environments. As a group of compounds, NAs are not well characterized and comprise a large group of saturated aliphatic and alicyclic carboxylic acids found in hydrocarbon deposits (petroleum, oil sands bitumen, and crude oils). Described is an analytical method using negative-ion electrospray ionization mass spectrometry (ES/MS) of extracts. Preconcentration was achieved by using a solid-phase extraction procedure utilizing a crosslinked polystyrene-based polymer with acetonitrile elution. Recovery of the Fluka Chemicals NA mixture was highly pH-dependent, with 100% recovery at pH 3.0, but only 66 and 51% recoveries at pHs 7 and 9, respectively. The dissolved phase of the NA was very dependent on sample pH. It is thus critical to measure the pH and determine the appropriate mass profiles to identify NAs in natural waters. The ES/MS analytical procedure proved to be a fast and sensitive method for the recovery and detection of NAs in natural waters, with a detection limit of 0.01 mg/L.     

Zinc-finger motif noncovalent interactions with double-stranded DNA characterized by negative-ion electrospray ionization mass spectrometry

A zinc-finger motif recognizes specific sequences on the double helical structure of DNA. This sequence recognition property offers great promise for various biotechnology applications. Accordingly, it is crucially important to characterize zinc-finger binding characteristics for further developments. Although the gel shift assay or phage display is traditionally used for determining the binding characteristics of zinc-fingers for double stranded DNA, in the present study we utilize electrospray ionization mass spectrometry as an advanced and convenient characterization tool because of the rich information it provides, and its quantitative sensitivity, operational simplicity, and no need for radioactive labeling. Here we demonstrate the use of negative-ion electrospray ionization mass spectrometry for competition-based quantitative comparison of the zinc-finger motif sequence specificity, stoichiometry, and metal ion dependence.     

Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization

Negative-ion electrospray ionization tandem quadrupole mass spectrometry provides a useful method for the structural characterization of ceramides. Fragment ions referring to the identities of the fatty acid substituent and of the long chain base of the molecules are readily available and the structure of ceramides can be easily determined. A unique fragmentation pathway which leads to formation of the fatty acid carboxylate anions (RCO2) was observed. This fragmentation is initiated by cleavage of the C2-C3 bond of the LCB to yield a N-acylaminoethanol anion ([RCONHCH2CH2O]-), followed by rearrangement to a carboxyethylamine ([RCO2CH2CH2NH]-) intermediate, which further dissociates to a RCO2- ion. This pathway is confirmed by the CAD tandem mass spectrum of the synthetic N-acylaminoethanol standard and of the deuterated analogs of ceramides obtained by H-D exchange. The observation of RCO2- ion species permits an unambiguous identification of the fatty acyl moiety of ceramides. Tandem mass spectrometry methods for characterization of structural isomers of ceramides using product-ion scanning and for identification of specific ceramide subclasses in biological mixtures using neutral loss scanning are also demonstrated.     

Differentiation of positional isomers of nitro meso-tetraphenylporphyrins by tandem mass spectrometry

We studied by tandem mass spectrometry two isomers of nitro meso-tetraphenylporphyrin, one with a nitro group in the para position of a phenyl ring and the other with the same group in a beta-pyrrolic position, and their copper complexes. Collisional activation of the molecular ions of both free-base porphyrins and of their copper complexes produces an array of product ions that permit ready differentiation of the two positional isomers. The diagnostic ions, when the nitro group is in a beta-pyrrolic position, may be produced through intramolecular and double cyclization processes, triggered by the interaction of the nitro substituent with the neighboring meso-phenyl ring. These diagnostic ions do not form when the nitro group is in the para position. The gas-phase processes have precedents in solution chemistry.     

Differentiation of three pairs of aconite alkaloid isomers from Aconitum nagarum var. lasiandrum by electrospray ionization tandem mass spectrometry

Three pairs of isomers of aconite alkaloids from Aconitum nagarum var. lasiandrum have been investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) employing ion-trap and quadrupole time-of-flight mass spectrometers in positive mode. Based on the differences of their fragmentation pathways and special fragment ions, three pairs of isomers of aconite alkaloids were differentiated. In addition, fragmentation laws of some veatchines and the discrepancy of fragmentation mechanisms between veatchine-type and aconitine-type alkaloid were also concluded. In the case of veatchines, a radical would be formed by homolysis of C18--C4 or C18--H bonds, followed by elimination of a series of C(2)H(2) and C(2)H(4). Moreover, the retro-Diels-Alder (RDA) reaction occurred in the E-ring and double-electron transfer triggered by the positive charge on C1 led to the formation of diagnostic ions at m/z 216. With regard to aconitine-type alkaloids, the N-substituent is not eliminated easily. Although there is no carbonyl group on some aconitine-type alkaloids, with hydroxyl and methoxyl on C15 and C16 respectively, CO was readily eliminated through tautomerization.     

Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry

Diiodothyronines 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,3'-diiodothyronine (3,3'-T2) are important metabolites of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3). In this paper, a novel and rapid method for identifying and quantifying 3,5-T2, 3',5'-T2 and 3,3'-T2 has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed on the basis of our data obtained by ESI-MS/MS. MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate 3,5-T2, 3',5'-T2 and 3,3'-T2. On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation.     

Differentiation of monoiodothyronines using electrospray ionization tandem mass spectrometry

In this work two monoiodothyronines, 3-T1 and 3'-T1, have been analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS. MS2 spectra in either negative or positive ion mode can be used to differentiate 3-T1 and 3'-T1. Based on the relative abundance of fragment ions in MS2 spectra in the negative ion mode, quantification of the 3-T1 and 3'-T1 isomers in mixtures is achieved without prior separation. Solid-phase extraction in combination with ESI-MS/MS provides a practicable procedure that can be used to determine the molar ratio of 3-T1 and 3'-T1 in human serum with an error less than 3%. The detection limits for 3-T1 and 3'-T1 were 0.5 and 0.7 pg/microL, respectively.     

Stereochemical differentiation of C‐7 hydroxyltaxane isomers by electrospray ionization mass spectrometry

In this study, different electrospray ionization mass spectrometric (ESI‐MS) methods were utilized to analyze several pairs of taxane stereoisomers including paclitaxel and 7‐epi‐paclitaxel. Both ESI‐MS and tandem mass spectrometry (MS/MS) techniques provided stereochemically dependent mass spectra in negative‐ion mode, and all studied stereoisomers could be easily distinguished based on their characteristic ions or distinct fragmentation patterns. MS/MS experiments for several taxane analogues at various collision energies were performed to elucidate potential dissociation pathways. The gas‐phase deprotonation potentials were also calculated to estimate the most thermodynamically favorable deprotonation site using DFT B3LYP/6‐31G(d). The results of the theoretical studies agreed well with the fragmentation patterns of paclitaxel and 7‐epi‐paclitaxel observed from MS/MS experiments. In addition, it was found that liquid chromatography (LC)/ESI‐MS was a useful and sensitive technique for assignment of C‐7 taxane stereoisomers from realistic samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Differentiation and quantification of linear alkyl benzenesulfonate isomers by liquid chromatography-ion-trap mass spectrometry

Discrimination and quantitation of the 20 positional isomers of C10-C13 linear alkyl benzenesulfonates (LASs), based on the use of reversed-phase liquid chromatography-electrospray ionisation in negative ion mode ion-trap mass spectrometry, was undertaken. Discrimination was achieved by LAS MS-MS analysis into the ion trap, by monitoring specific fragment ions resulting from the benzylic cleavage of the carbon alkyl chain on both side of the LAS phenyl group. Parameters affecting the electrospray ionisation source and the ion-trap operation were optimised. Calibration curves for the different isomers were established and this permitted their quantitation by mass spectrometry for the first time. MS-MS responses were dependent on both the position of the phenyl group on the alkyl chain and the length of this alkyl chain, these responses being higher for the external isomers and the longer alkyl chain homologues. The precision, expressed as relative standard deviation ranged between 9 and 13%. Detection limits for LAS isomers were between 0.03 and 0.07 mg/l and therefore the method is sensitive enough to be applied to environmental samples.     

Collision-induced dissociation of a series of coumarins studied by positive- and negative-ion electrospray triple quadrupole tandem mass spectrometry

Aryl-substituted 4-hydroxycoumarins (1-57) were investigated by electrospray ionisation (ESI) mass spectrometry. Their fragmentation in the ion source or in the collision cell of a triple quadrupole mass spectrometer was investigated. The effect of the substituents in the aromatic ring on the fragmentation of the 4-hydroxycoumarin derivatives is shown. The influence of the tautomerism on the formation of quasimolecular ions and mass spectral fragmentation was explained. Mass spectral studies on some deuterated compounds proved some of the proposed fragmentation pathways. Results obtained are very useful in the process of detection and characterisation of 4-hydroxycoumarins, as well as for structural elucidation of their more complex derivatives.     

Structural characterisation by both positive- and negative-ion electrospray mass spectrometry of partially methyl-esterified oligogalacturonides purified by semi-preparative high-performance anion-exchange chromatography

The off-line coupling of high-performance anion-exchange chromatography (HPAEC) to electrospray ionisation/ion trap mass spectrometry (ESI-ITMS) is described. The Dionex carbohydrate membrane desalter (CMD) has been assessed as an on-line chromatographic desalting system to remove the high sodium concentration necessary for the HPAEC separation of partially methyl-esterified oligogalacturonides. The developed HPAEC configuration proved to be suitable for indirect coupling with ESI-ITMS. This paper provides some interesting features of positive- and negative-ion multistage tandem mass spectrometry (MS(n)) analysis of these acidic oligosaccharides. The spectra acquired in both negative- and positive-ion modes show characteristic fragment ions resulting from glycosidic bond and cross-ring cleavages. Some new mass spectrometric fragmentation routes are also described. The positive-ion mode gave more complex spectra but was as informative as the negative-ion mode. ESI-ITMS was revealed to be, as previously reported from direct use on an unseparated enzymatic digest, a powerful sequencing technique for the determination of linkage type and the methyl ester distribution of partially methyl-esterified oligogalacturonides. Moreover, unlike matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS), it gives valuable information on the elution behaviour of these oligomers in relation to their structure, namely the HPAEC co-elution of isomeric structures.     

Glycoprotein profiling by electrospray mass spectrometry

This work compares several different methods of site-specific analysis of glycoproteins using electrospray mass spectrometry. The glycoprotein, oLHalpha (ovine luteinizing hormone, alpha-subunit) was chosen as an appropriate example protein for these studies because of its biological relevance and extreme microheterogeneity. More than 20 unique glycoforms were detected for this glycoprotein at the Asn(56) site of oLHalpha. The carbohydrates present at this site affect receptor binding affinity, so understanding the great variety in the composition of these carbohydrates is important in studying ligand binding interactions. MS data was acquired on a quadrupole ion trap, a triple quadrupole, and a quadrupole time of flight mass spectrometer, and carbohydrate composition at the Asn(56) site of oLHalpha was determined using these instruments. Additionally, neutral loss and precursor ion scanning modes were also used to identify the glycoforms present, and these techniques were compared to the standard MS data. Of the three instruments compared in the study, the qTOF mass spectrometer achieved the lowest sample consumption, but all three instruments were useful in profiling the glycopeptide composition.     

Differentiation of isomeric polyaromatic hydrocarbons by electrospray Ag(I) cationization mass spectrometry

Abundant Ag(I)-cationized complexes of 13 polyaromatic hydrocarbons (PAHs), [Ag+PAH](+) and [Ag+2(PAH)](+), were readily generated by electrospray ionization (ESI). In-source collision-induced dissociation (CID) of the [Ag+2(PAH)](+) complex yielded the monomer complex [Ag+PAH](+), which fragmented further to yield the radical molecular ion [PAH](+.). Based on significant differences in relative intensities of [Ag+2(PAH)](+), [Ag+PAH](+) and [PAH](+.), isomeric PAHs can be differentiated. The [PAH](+.)/[Ag+PAH](+) ion intensity ratio was found to increase with decreasing ionization potentials (IPs) of PAHs. The ratio was significantly different for the isomeric PAHs studied over a wide range of PAH concentrations (1.6-100 nmol/mL), and showed good measurement reproducibility; the coefficient of variation of inter-day measurements was in the range 3-12% for four representative PAHs (n = 5). Detection limits for phenanthrene, pyrene, chrysene and benzo[a]pyrene, in the form of the monomer complexes [(107)Ag+PAH](+) and measured in the selected-ion monitoring (SIM) mode, were 0.31, 0.63, 0.16 and 1.25 pmol/5 microl injection, respectively (S/N ratio approximately 2-3).     

Analysis of xanthates by negative-ion fast atom bombardment mass spectrometry

A new technique using negative-ion fast atom bombardment mass spectrometry for the analysis of xanthates and related compounds is described. Electron impact and positive-ion fast atom bombardment mass spectrometry produced no structurally related fragment ions or observable molecular ions at the expected m/z values. It was demonstrated that negative-ion fast atom bombardment ionization was the most suitable method of ionization for structure elucidation studies for the compounds described.