A comparison of electrospray tandem mass spectra of some sialic acid derivatives: Ion trap and high resolution QqToF mass spectrometers

Using O-acetyl-N-acyl derivatives of O-methyl sialoside methyl esters, it was shown that an ion trap and a hybrid analyzer (linear quadrupole–time-of-flight analyzer, reflectron) give comparable, though not identical secondary mass spectra for the [M + Na]⁺ and [M + K]⁺ ions. A parallel use of an ion trap and a hybrid QqToF instrument gives information about the fragmentation pathways of ions of sialic acid derivatives under collisional activation. In this case, the sequence of fragmentation may be established using an ion trap, whereas a QqToF instrument offers a possibility of revealing the elemental composition of fragment ions quickly and unequivocally.     

Statistical evaluation of electrospray tandem mass spectra for optimized peptide fragmentation

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the analysis of complex peptide mixtures. It combines the separation power of nanoflow LC with highly specific sequence analysis, allowing automated peptide sequencing with high resolution and throughput. For peptide fragmentation, the current experimental setup uses predefined parameters based on the mass-to-charge ratio of the individual precursor. Suitable parameters are typically established by empirical evaluation of fragment spectra of individual peptides used as standards. As a result, nonoptimal fragment spectra are obtained if peptides show fragmentation behavior different from these standards, which often result in the loss of sequence-specific fragment ion information. Here we describe a statistical approach for the systematic evaluation of the quality of individual peptide fragment spectra based on the calculation of their arithmetic mean and standard deviation. The method utilizes the dependence of these parameters on the difference in electric potential across the collision cell to determine the value that results in maximum information content. We show that the method is applicable to fragment spectra generated from a variety of multiply-charged tryptic peptides, over a wide concentration range, and on different types of mass analyzers. We also show how this novel approach can be used to define optimized collision energy settings over a wide mass-to-charge range.     

Effect of electrospray ionization conditions on low-energy tandem mass spectra of peptides

The effect of electrospray ionization (ESI) conditions on low-energy tandem mass spectra of peptides in the relative molecular mass range 400–1200 was examined. For singly charged peptide ions the source skimmer potential (which determines the degree of acceleration of the ions through the intermediate pressure region in the source) can strongly influence the extent of fragmentation observed in tandem mass spectra, especially at low collision energies. For each peptide there is an optimum skimmer potential which represents a balance between generating ions with sufficient internal energy for subsequent tandem mass spectrometric experiments and inducing the onset of other processes such as source fragmentation. The fragmentation which can be achieved in tandem mass spectra with high skimmer potentials differs from ESI source fragmentation for the same peptides. We have found that fragmentation in ESI mass spectra depends both on skimmer potential and on solvent pH, presumably because the latter determines the proportion of doubly charged species generated from a given peptide. Low-energy tandem mass spectra of peptides following ESI are equally as sensitive to peptide structure and the type of adduct studied (e.g. [M + H]⁺ vs. [M + NH₄]⁺) as tandem mass spectra obtained following older ionization methods such as fast atom bombardment.     

Negative ion electrospray high‐resolution tandem mass spectrometry of polyphenols

Representative compounds with a 1,3‐dihydroxybenzene substructure belonging to different important polyphenol classes (stilbenes, flavones, isoflavones, flavonols, flavanones, flavanols, phloroglucinols, anthraquinones and bisanthraquinones) were investigated based on detailed high‐resolution tandem mass spectrometry measurements with an Orbitrap system under negative ion electrospray conditions. The mass spectral behaviour of these compound classes was compared among each other not only with respect to previously described losses of CO, CH₂CO and C₃O₂ but also concerning the loss of CO₂ and successive specific fragmentations. Furthermore, some unusual fragmentations such as the loss of a methyl radical during mass spectral decomposition are discussed. The obtained results demonstrate both similarities and differences in their mass spectral fragmentation under MSⁿ conditions, allowing a characterization of the corresponding compound type. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparison of negative ion electrospray mass spectra measured by seven tandem mass analyzers towards library formation

A library of negative ion electrospray ionization mass spectra and tandem mass spectra (MS/MS) of sulfonated dyes has been developed for fast identification purposes. The uniform protocol has been elaborated and applied to the measurements of more than 50 anionic dyes. Three collision energies are selected in our protocol which ensures that at least one of them provides a suitable ratio of product ions to the precursor ion. The robustness is investigated with altered values of tuning parameters (e.g. the pressure of the nebulizing gas, the temperature and the flow rate of drying gas, and the mobile phase composition). The results of the inter-laboratory comparison of product ion mass spectra recorded on seven different tandem mass spectrometers (three ion traps, two triple quadrupoles and two hybrid quadrupole time of flight instruments) are presented for four representative anionic dyes--azo dye Acid Red 118, anthraquinone dye Acid Violet 43, triphenylmethane dye Acid Blue 1 and Al(III) metal-complex azo dye. The fragmentation patterns are almost identical for all tandem mass analyzers, only the ratios of product ions differ somewhat which confirms the possibility of spectra transfer among different mass analyzers with the goal of library formation.     

Approaches towards the automated interpretation and prediction of electrospray tandem mass spectra of non-peptidic combinatorial compounds

Combinatorial chemistry is widely used within the pharmaceutical industry as a means of rapid identification of potential drugs. With the growth of combinatorial libraries, mass spectrometry (MS) became the key analytical technique because of its speed of analysis, sensitivity, accuracy and ability to be coupled with other analytical techniques. In the majority of cases, electrospray mass spectrometry (ES-MS) has become the default ionisation technique. However, due to the absence of fragment ions in the resulting spectra, tandem mass spectrometry (MS/MS) is required to provide structural information for the identification of an unknown analyte. This work discusses the first steps of an investigation into the fragmentation pathways taking place in electrospray tandem mass spectrometry. The ultimate goal for this project is to set general fragmentation rules for non-peptidic, pharmaceutical, combinatorial compounds. As an aid, an artificial intelligence (AI) software package is used to facilitate interpretation of the spectra. This initial study has focused on determining the fragmentation rules for some classes of compound types that fit the remit as outlined above. Based on studies carried out on several combinatorial libraries of these compounds, it was established that different classes of drug molecules follow unique fragmentation pathways. In addition to these general observations, the specific ionisation processes and the fragmentation pathways involved in the electrospray mass spectra of these systems were explored. The ultimate goal will be to incorporate our findings into the computer program and allow identification of an unknown, non-peptidic compound following insertion of its ES-MS/MS spectrum into the AI package. The work herein demonstrates the potential benefit of such an approach in addressing the issue of high-throughput, automated MS/MS data interpretation.     

High resolution and tandem fourier-transform mass spectrometry with californium-252 plasma desorption

For ions formed by plasma desorption (PD) in a Fourier-transform mass spectrometer, high resolution measurements are demonstrated, such as 65,000 (FWHH) for the protonated molecular ion of gramicidin S (MW 1140.7). Resolution is substantially improved by delaying measurements until a significant ion concentration has built up in the cell, and by collisionally deactivating the orbital kinetic energy of the ions. This also makes the ions available for subsequent dissociation steps, so that tandem mass spectrometry can be demonstrated for PD ions. With this for larger ions, collisionally activated dissociation (CAD) is affected with> 85% efficiency. The CAD spectra of (M + Na)⁺ and of fragment ions from the PD of gramicidin S provide structurally useful information.     

An electrospray ionisation tandem mass spectrometric investigation of selected psychoactive pharmaceuticals and its application in drug and metabolite profiling by liquid chromatography/electrospray ionisation tandem mass spectrometry

A tandem mass spectrometric investigation of the collision-induced dissociation of five commonly prescribed psychoactive pharmaceuticals, risperidone, sertraline, paroxetine, trimipramine, and mirtazapine, and their metabolites has been carried out. Quadrupole ion trap mass spectrometry was employed to generate tandem mass spectrometric (MS/MS) data of the compounds under investigation and structural assignments of product ions were supported by quadrupole time-of-flight mass spectrometry. These fragmentation studies were then utilised in the development of a liquid chromatographic method to identify the drugs and their metabolites in human hair and saliva samples, thus providing relevant profiling information.     

Automated deconvolution and deisotoping of electrospray mass spectra

Electrospray ionization (ESI) of peptides and proteins produces a series of multiply charged ions with a mass/charge (m/z) ratio between 500 and 2000. The resulting mass spectra are crowded by these multiple charge values for each molecular mass and an isotopic cluster for each nominal m/z value. Here, we report a new algorithm simultaneously to deconvolute and deisotope ESI mass spectra from complex peptide samples based on their mass-dependent isotopic mean pattern. All signals corresponding to one peptide in the sample were reduced to one singly charged monoisotopic peak, thereby significantly reducing the number of signals, increasing the signal intensity and improving the signal-to-noise ratio. The mass list produced could be used directly for database searching. The developed algorithm also simplified interpretation of fragment ion spectra of multiply charged parent ions.     

Analysis of oxysterols by electrospray tandem mass spectrometry

Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier, and oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. Oxysterols are difficult to analyze by atmospheric pressure ionization mass spectrometry on account of the absence of basic or acidic functional groups in their structures. In this communication, we report a method for the derivatization and analysis of oxysterols by electrospray mass spectrometry. Oxysterols with a 3beta-hydroxy-Delta5 structure were converted by cholesterol oxidase to 3-oxo-Delta4 steroids and then derivatized with the Girard P reagent to give Girard P hydrazones, which were subsequently analyzed by tandem mass spectrometry. The improvement in sensitivity for the analysis of 25-hydroxycholesterol upon oxidation and derivatization was over 1000.     

Pyridine N-oxide and pyridine-d5 N-oxide: an electrospray/tandem mass spectrometric study carried out at high mass resolution

A mass spectrometric study of pyridine N-oxide and pyridine-d5 N-oxide was carried out with a hybrid quadrupole/time-of-flight (TOF) mass spectrometer coupled with an electrospray (ES) source. In addition to the observation of protonated, sodiated, and proton-bound dimers of pyridine N-oxide and pyridine-d5 N-oxide, mass scans revealed the presence of several doubly-charged ion species. Doubly-charged ions of m/z 191 were identified as diprotonated tetramers of pyridine N-oxide; a structure has been proposed for the diprotonated tetramer and its energy relative to that of protonated pyridine N-oxide has been obtained from geometry optimizations. The principal ion species observed were subjected to collision-induced dissociation; accurate mass measurements were made of each fragment ion so as to determine its elemental composition. On the basis of mass spectrometric evidence, it is suggested that dissociation of pyridine N-oxide may occur during the ES process and the resulting fragments become embedded in doubly-charged ions. The proton affinity for both pyridine N-oxide and pyridine-d5 N-oxide was calculated; the difference between these proton affinities was compared with an experimentally determined difference between the proton affinities of pyridine N-oxide and pyridine-d5 N-oxide.     

Reliability of veterinary drug residue confirmation: High resolution mass spectrometry versus tandem mass spectrometry

Confirmation of suspected residues has been a long time domain of tandem triple quadrupole mass spectrometry (QqQ). The currently most widely used confirmation strategy relies on the use of two selected reaction monitoring signals (SRM). The details of this confirmation procedure are described in detail in the Commission Decision 93/256/EC (CD). On the other hand, high resolution mass spectrometry (HRMS) is nowadays increasingly used for trace analysis. Yet its utility for confirmatory purposes has not been well explored and utilized, since established confirmation strategies like the CD do not yet include rules for modern HRMS technologies.     

Effect of stereochemistry on the electrospray ionization tandem mass spectra of transition metal chloride complexes of monosaccharides

The effect of stereochemistry on the complexation of aldohexoses (glucose, mannose, galactose, allose and talose) and ketohexoses (fructose, tagitose and sorbose) with transition metal chlorides (CoCl(2), NiCl(2), MnCl(2) and ZnCl(2)) has been investigated by electrospray ionization tandem mass spectrometry. Electrospray ionization of methanolic solutions of hexoses containing metal chlorides gave abundant ions corresponding to [M + MetCl](+) and [2M + MetCl](+) which on collision-induced dissociation gave characteristic fragment ions. The fragmentation pathways have been confirmed by examining methyl glucoside and several isotopically labeled glucoses. Eliminations of H(2)O and HCl, C-C cleavages and elimination of metalhydroxychloride are the competing fragmentation pathways observed. All these pathways seem to be influenced by the stereochemistry of the molecule. The fragmentation of the dimeric complexes, [2M + MetCl](+), is also controlled by the stereochemistry of the molecule. The abundance of the product ions corresponding to elimination of HCl is found to increase with increasing number of axial hydroxyl groups in aldohexoses. [2M + MetCl](+) dissociates by elimination of HCl followed by C(2)H(4)O(2) in aldohexose complexes and by elimination of HCl followed by C(3)H(6)O(3) in ketohexose complexes.     

Formation of cyclic acylphosphoramidates in mass spectra of N‐monoalkyloxyphosphoryl amino acids using electrospray ionization tandem mass spectrometry

The fragmentation reactions of N‐monoalkyloxyphosphoryl amino acids (N‐MAP‐AAs) were studied by electrospray ionization tandem mass spectrometry (ESI‐MS). The sodiated cyclic acylphosphoramidates (CAPAs) were formed through a characteristic pentacoordinate phosphate participated rearrangement reaction in the positive‐ion ESI‐MS/MS and HR‐MS/MS of N‐MAP‐AAs, in which the fragmentation patterns were clearly different from those observed in the corresponding ESI‐MS/MS of N‐dialkyloxyphosphoryl amino acids/peptides and N‐phosphono amino acids. The formation of CAPAs depended on the chemical structures of N‐terminal phosphoryl groups, such as alkyloxy group, negative charge and alkali metal ion. A possible integrated rearrangement mechanism for both P‐N to P‐O phosphoryl group migration and formation of CAPAs was proposed. The fragmentation patterns of CAPAs as novel intermediates in gas phase were also investigated. In addition, it was found that the formation of α‐amino acid CAPAs was more favorable than β‐ or γ‐CAPAs in gas phase, which was consistent with previous solution‐phase experiments. Copyright © 2010 John Wiley & Sons, Ltd.     

High mass resolution breath analysis using secondary electrospray ionization mass spectrometry assisted by an ion funnel

In this study, we used secondary electrospray ionization mass spectrometry assisted by an ion funnel (IF) operating at ambient pressure to find compounds in the mass range of 100–500 m/z in online breath fingerprinting experiments. In low‐resolution experiments conducted on an ion trap instrument, we found that pyridine is present in breath of individuals long after drinking coffee. In high‐resolution experiments conducted on a Fourier transform ion cyclotron resonance, we found more than 30 compounds in the mass range of 100–500 m/z in analogous online breath experiments. More than a third of these compounds have molecular weights above 200 Daltons and have not been mentioned in previous studies. In low‐resolution experiments as well as experiments without the IF, these compounds could not be detected. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

A qualitative study of amlodipine and its related compounds by electrospray ionization tandem mass spectrometry

A comprehensive structural analysis of amlodipine and certain related compounds was performed by electrospray ionization tandem mass spectrometry. Triple quadrupole and quadrupole time-of-flight instruments were used to provide collision-induced dissociation and accurate mass measurement for selected product and second-generation product ions. A unique ion rearrangement was observed, which was found to be characteristic of certain dihydropyridines. This study provides a fundamental understanding of the fragmentation of these compounds. The structural elucidation of an unknown impurity is presented as an example.     

Structural characterization of glycoporphyrins by electrospray tandem mass spectrometry

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.