Electrospray tandem mass spectrometric study of alkyl 1-methylpyridinium ether derivatives of alcohols.

The fragmentation of 15 alkyl 1-methylpyridinium ether derivatives (D+) of primary and secondary alcohols, benzylic alcohols and phenyl-substituted alcohols was investigated using energy-resolved tandem mass spectrometry. Fragmentation pathways and mechanisms, including the influence of substituents, on the formation of the major product ions, which appear at m/z 110, [D - 109]+ and [D - 111]+, were postulated. Comparison of the abundances of these ions in the product ion spectra of isomeric alcohol derivatives, obtained at the same center-of-mass collision energy (2.0 eV), was found to provide the ability to differentiate among some isomers.     

Electron-impact promoted fragmentation of alkyl-N-(1-phenylethyl)-carbamates of primary,secondary and tertiary alcohols

The mass spectra of twenty N-alkyl-(1-phenylethyl)-carbamates derived from primary, secondary and tertiary alcohols have been investigated using deuterium labeling and high resolution mass spectrometry. These derivatives are suitable for the determination of the isotopic purity of primary and secondary, but not of tertiary alcohols. Several of the primary and secondary alcohol derivatives yield an ion formally equivalent to the product ion of a McLafferty rearrangement. Deuterium labeling established, however, that the usual site specificity associated with the McLafferty rearrangement process was lacking in these carbamate derivatives. In addition a double hydrogen rearrangement process was observed in the mass spectra of several of the carbamates derived from tertiary alcohols.     

Structural analysis of 2,6-[bis(alkyloxy)methyl]-phenyltin derivatives using electrospray ionization mass spectrometry

Electrospray ionization (ESI) mass spectrometry was applied to the structural analysis of 23 2,6-[bis(alkyloxy)methyl]phenyltin(IV) derivatives. The mass spectra were measured in both polarity modes and multistage tandem mass spectrometric (MS(n)) measurements were performed on the ion trap analyser for positively charged tin-containing ions. The sum of complementary ions observed in the positive-ion mode (i.e. [M-R(3)](+) ion) and in the negative-ion mode (i.e. [R(3)](-) ion) permits molecular mass determination in spite of the fact that the molecular adducts were often missing even in the first-order mass spectra. The subsequent fragmentation of [M-R(3)](+) ions studied by MS(n) and the correlation of observed fragment ions with the expected structures of synthesized organotin(IV) compounds allowed us to understand the fragmentation behaviour and the mechanism of the ion formation for studied compounds. The typical neutral losses are alkenes, alcohols and aldehydes. The fragmentation pattern of one selected compound was supported by MS(n) measurements of an isotopically labelled analogue to confirm unusual ion-molecule reactions of some fragment ions with water in the ion trap.     

Destabilized carbenium ions. Secondary and tertiary α-acetylbenzyl cations and α-benzoylbenzyl cations

The secondary α-acetylbenzyl and α-benzoylbenzyl cations, as well as their tertiary analogues, have been generated in a mass spectrometer by electron impact induced fragmentation of the corresponding α-bromoketones. These ions belong to the interesting family of destabilized α-acylcarbenium ions. While primary α-acylcarbenium ions appear to be unstable, the secondary and tertiaiy ions exhibit the usual behaviour of stable entities in a potential energy well. This can be attributed to a ‘push-pull’ substitution at the carbenium ion centre by an electron-releasing phenyl group and an electron-withdrawing acyl substituent. The characteristic unimolecular reaction of the metastaible secondary and tertiary α-acylbenzyl cations is the elimination of CO by a rearrangement reaction involving a 1,2-shift of a methyl group and a phenyl group, respectively. The loss of CO is accompanied by a very large kinetic energy release, which gives rise to broad and dish-topped peaks for this process in the mass-analysed ion kinetic energy spectra of the corresponding ions. This behaviour is attributed to the rigid critical configuration of a corner-protonatei cyclopropanone derivative and a bridged phenonium ion derivative, respectively, for this reaction. For the tertiary α-acetyl-α-methylbenzyl cations, it has been shown by deuterium labelling and by comparison of collisional activation spectra that these ions equilibrate prior to decomposition with their ‘protomer’ derivatives formed by proton migration from the α-methyl substituent to the carbonyl group and to the benzene ring.     

Sites of protonation in phenothiazines: Methane and ammonia chemical ionization mass spectra

Positive ion methane and ammonia chemical ionization mass spectra for ten phenothiazine derivatives are reported. The fragmentations observed in the chemical ionization mass spectra are rationalized in terms of the location of the added proton. High-resolution measurements are used to confirm empirical formulae of the ions in the mass spectra. Changes in the mass spectra with a change in the chemical ionization reagent gas from methane to ammonia are described. A comparison with positive ion secondary ion mass spectra of the same compounds show that the amount of fragmentation is higher in the secondary ion mass spectra, but the same types of ions are observed in spectra produced by both ionization methods.     

The STEP method (statistical test of equivalent pathways): application to pharmaceuticals

The STEP method (Statistical Test of Equivalent Pathways), recently developed to determine primary and secondary fragmentation in the MS/MS of peptides and carbohydrates, is applied in the current study to common pharmaceutical antibiotics. The classification of product ions as primary or secondary is then utilized to construct genealogy diagrams that aid in the structural characterization of the product ions. Four compounds were subjected to the MS/MS conditions used for the STEP method, and the method was used to correctly identify primary and secondary ions in three of the four pharmaceuticals. Calculated STEP values for erythromycin did not match previously characterized fragmentation assignments. This provided an opportunity to explore potential limitations of STEP analysis. It was determined that inaccurate STEP assignments could result, if the starting compound is classified as "fragile", because fragile ions, such as erythromycin can produce abnormally low STEP ratios. While this finding represents a limitation of using the STEP method to determine whether product ions are due to primary or secondary fragmentation for fragile ions, it suggests the possibility of identifying the presence of "fragile ions" by STEP analysis.     

Structures and fragmentation of [Cu(uracil-H)(uracil)]+ in the gas phase

Complexes of copper (II) ions and uracil were studied using tandem mass spectrometry (Fourier transform ion cyclotron resonance, FTICR, mass spectrometry) including extensive isotopic labeling as well as theoretical calculations. Positive ion electrospray mass spectra of aqueous solutions of CuCl(2) and uracil show that the [Cu(Ura-H)(Ura)](+) ion is the most abundant ion even at low concentrations of uracil. Sustained off-resonance irradiation collision-induced dissociation (SORI-CID) experiments show that the lowest energy decomposition pathway for [Cu(Ura-H)(Ura)](+) , surprisingly, is not the loss of uracil, but the loss of HNCO followed by HCN as the most abundant secondary fragmentation product. MS(n) studies identified primary, secondary and tertiary fragmentation products. Extensive isotopic labeling studies, as well as computational studies allowed for a detailed fragmentation scheme for the [Cu(Ura-H)(Ura)](+) ion, beginning with the lowest energy structure.     

Fragmentation of negative ions from carbohydrates: Part 2. Fragmentation of high-mannose N-linked glycans

[M + NO3]- And [M + (NO3)2]2- ions were produced by electrospray from neutral high-mannose ([Man](5-9)[GlcNAc]2, [Glc](1-3)[Man](4-9)[GlcNAc]2) N-linked glycans and their 2-aminobenzamide derivatives sprayed from methanol:water containing ammonium nitrate. Low energy collision-induced decomposition (CID) spectra of both types of ions were almost identical and dominated by cross-ring and C-type fragments, unlike the corresponding spectra of the positive ions that contained mainly B- and Y-type glycosidic fragments. This behavior could be rationalized by an initial proton abstraction from various hydroxy groups by the initially-formed anionic adduct. These negative ion spectra were more informative than the corresponding positive ion spectra and contained prominent ions that were diagnostic of structural features such as the composition of individual antennas that were not easily obtainable by other means. C-ions defined the sequence of the constituent monosaccharide residues. Detailed fragmentation mechanisms are proposed to account for many of the diagnostic ions.     

Mass spectrometry in structural and stereochemical problems—CLVIII electron impact promoted fragmentation of triphenylmethyl ethers

The triphenylmethyl (trityl) moiety is frequently used for the protection of alcohols but the mass spectra of such trityl ethers have hitherto escaped scrutiny. It has now been found that triphenylmethyl derivatives of primary alcohols yield abundant molecular ions which permit the determination of the isotopic purity of the parent alcohol. Upon electron impact the triphenyl-methyl entity directs the fragmentation of trityl ethers as demonstrated by a detailed study of n-pentyl trityl ether and its deuterated analogs. Ions formed by migration of phenyl groups were observed in the mass spectra of the trityl ethers investigated as well as in the spectrum of triphenyl-methane itself.     

Vacuum-ultraviolet photoionization and mass spectrometric characterization of lignin monomers coniferyl and sinapyl alcohols

The fragmentation mechanisms of monolignols under various energetic processes are studied with jet-cooled thermal desorption molecular beam (TDMB) mass spectrometry (MS), 25 keV Bi(3)(+) secondary ion MS (SIMS), synchrotron vacuum-ultraviolet secondary neutral MS (VUV-SNMS) and theoretical methods. Experimental and calculated appearance energies of fragments observed in TDMB MS indicate that the coniferyl alcohol photoionization mass spectra contain the molecular parent and several dissociative photoionization products. Similar results obtained for sinapyl alcohol are also discussed briefly. Ionization energies of 7.60 eV ± 0.05 eV for coniferyl alcohol and <7.4 eV for both sinapyl and dihydrosinapyl alcohols are determined. The positive ion SIMS spectrum of coniferyl alcohol shares few characteristic peaks (m/z = 137 and 151) with the TDMB mass spectra, shows extensive fragmentation, and does not exhibit clear molecular parent signals. VUV-SNMS spectra, on the other hand, are dominated by the parent ion and main fragments also present in the TDMB spectra. Molecular fragmentation in VUV-SNMS spectra can be reduced by increasing the extraction delay time. Some features resembling the SIMS spectra are also observed in the desorbed neutral products. The monolignol VUV-SNMS peaks shared with the TDMB mass spectra suggest that dissociative photoionization of ion-sputtered neutral molecules predominate in the VUV-SNMS mass spectra, despite the extra internal energy imparted in the initial ion impact. The potential applications of these results to imaging MS of biomolecules are discussed.     

Selective reagents in chemical ionization mass spectrometry: Tetramethylsilane with aliphatic alcohols

Mixtures of tetramethylsilane and helium have been found to form [M + 73]⁺ adducts, hydrated trimethylsilyl ions and alkyl ions with aliphatic alcohols. The adduct ions were found to be formed by displacement of water from the hydrated trimethylsilyl ion. Ratios of the abundances of the adduct ions to the hydrated trimethylsilyl ion can be used to differentiate among primary, secondary and tertiary alcohols. Sensitivities for a number of alcohols with the tetramethylsilane/helium chemical ionization reagent system are approximately equal.     

Electron Transfer Dissociation of Milk Oligosaccharides

For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS(3) by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS(3) techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra, and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.     

Electron impact and isobutane and ammonia chemical ionization mass spectrometry of some tetrahydropyranyl acetals

The electron impact induced fragmentations of 24 tetrahydropyrane ethers of different types of alcohols were studied by exact mass measurement and metastable ion analysis. The most characteristic features of the spectra were associated with the tetrahydropyrane ring, As the size of the molecule increased, the fragmentations with respect to exocyclic oxygen became more important allowing differentiation among the isomeric aliphatic alcohols. Under isobutane chemical ionization, loss of neutral alcohol from [M + H]⁺ leading to the formation of a tetrahydropyranium ion was dominant for aliphatic alcohol derivatives, whereas loss of 2-hydroxy tetrahydropyrane giving rise to an alkyl ion was dominant for bicyclic alcohol derivatives. Under ammonia chemical ionization, nucleophilic substitution reactions were always most important.     

Secondary ion formation of low molecular weight organic dyes in time-of-flight static secondary ion mass spectrometry

Time-of-flight static secondary ion mass spectrometry (TOF-S-SIMS) was used to characterize thin layers of oxy- and thiocarbocyanine dyes on Ag and Si. Apart from adduct ions a variety of structural fragment ions were detected for which a fragmentation pattern is proposed. Peak assignments were confirmed by comparing spectra of dyes with very similar structures. All secondary ions were assigned with a mass accuracy better than 50 ppm. The intensity of molecular ions as well as fragment ions has been studied as a function of the type of organic dye, the substrate, the layer thickness and the type of primary ion. A large yield difference of two orders of magnitude was observed between the precursor ions of cationic carbocyanine dyes and the protonated molecules of the anionic dyes. Fragment ions, on the other hand, yielded similar intensities for both types of dye. As the dye layers deposited on an Ag substrate yielded higher secondary ion intensities than those deposited on a Si substrate, the Ag metal clearly acts as a promoting agent for secondary ion formation. The effect was more pronounced for precursor signals than for fragment ions. The promoting effect decreased as the deposited layer thickness of the organic dye layer was increased.     

ToF-SIMS Analysis of Adsorbed Proteins: Principal Component Analysis of the Primary Ion Species Effect on the Protein Fragmentation Patterns

In time-of-flight secondary ion mass spectrometry (ToF-SIMS), the choice of primary ion used for analysis can influence the resulting mass spectrum. This is because different primary ion types can produce different fragmentation pathways. In this study, analysis of single-component protein monolayers were performed using monatomic, tri-atomic, and polyatomic primary ion sources. Eight primary ions (Cs(+), Au(+), Au(3) (+), Bi(+), Bi(3) (+), Bi(3) (++), C(60) (+)) were used to examine to the low mass (m/z < 200) fragmentation patterns from five different proteins (bovine serum albumin, bovine serum fibrinogen, bovine immunoglobulin G and chicken egg white lysozyme) adsorbed onto mica surfaces. Principal component analysis (PCA) processing of the ToF-SIMS data showed that variation in peak intensity caused by the primary ions was greater than differences in protein composition. The spectra generated by Cs(+), Au(+) and Bi(+) primary ions were similar, but the spectra generated by monatomic, tri-atomic and polyatomic primary ion ions varied significantly. C(60) primary ions increased fragmentation of the adsorbed proteins in the m/z < 200 region, resulting in more intense low m/z peaks. Thus, comparison of data obtained by one primary ion species with that obtained by another primary ion species should be done with caution. However, for the spectra generated using a given primary ion beam, discrimination between the spectra of different proteins followed similar trends. Therefore, a PCA model of proteins created with a given ion source should only be applied to datasets obtained using the same ion source. The type of information obtained from PCA depended on the peak set used. When only amino acid peaks were used, PCA was able to identify the relationship between proteins by their amino acid composition. When all peaks from m/z 12-200 were used, PCA separated proteins based on a ratio of C(4)H(8)N(+) to K(+) peak intensities. This ratio correlated with the thickness of the protein films and Bi(1) (+) primary ions produced the most surface sensitive spectra.     

A regioselective etherification of pyridoxine via an ortho-pyridinone methide intermediate

The catalyst-free, regioselective synthesis of 4′-O-substituted pyridoxine derivatives under solventless conditions is described. The methodology relies on the highly regioselective formation of the ortho-pyridinone methide from pyridoxine and subsequent oxa-Michael addition of alcohol nucleophiles. This methodology provides good to excellent yields for primary and secondary alcohols and moderate yields for tertiary alcohols.     

Fragmentation of even electron ions: Protonated amines and esters

The fragmentation of protonated amines and esters is examined by collision induced dissociation of mass selected ions generated by chemical ionization. Four fragmentation types are observed: (i) loss of an alkane moiety, explicable in terms of a four-centered reaction, (ii) loss of alkene, also explicable as a four-centered process, (iii) loss of an alkyl radical, requiring electron unpairing, (iv) losses of other neutral molecules, viz. acids and alcohols from protonated esters, or amines and ammonia from protonated amines. Proposed mechanisms were checked by characterizing selected product ions through their collision induced dissociation spectra. The usefulness of these generalizations is substantiated by the successful prediction of the fragmentation patterns and the order of relative abundances of daughter ions for eight unknowns including some containing functional groups not studied previously.     

Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites in urine by LC‐MS after derivatization and its application for differentiation between positional isomers of prilocaine

The demand for clinical toxicology analytical methods for identifying drugs of abuse and medicinal drugs is steadily increasing. Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites by GC‐EI‐MS and LC‐ESI‐MS/MS is of great analytical challenge. These compounds exhibit only/mostly fragments/product ions representing the amine‐containing residue, while the aromatic amide moiety remains unidentified. This task becomes even more complicated when discrimination between positional isomers of such compounds is required. Here, we report the development of a derivatization procedure for the differentiation and structural elucidation of a mixture of local anesthetic drugs and their metabolites that possess tertiary and secondary amines in water and urine. A method based on two sequential “in‐vial” instantaneous derivatization processes at ambient temperature followed by LC‐ESI‐MS/MS analysis was developed. 2,2,2‐Trichloro‐1,1‐dimethylethyl chloroformate (TCDMECF) was utilized to selectively convert the secondary amines into their carbamate derivatives, followed by hydrogen peroxide addition to produce the corresponding tertiary amine oxides. The resulting derivatives exhibited rich fragmentation patterns, enabling improved structural elucidation of the original compounds. The developed method was successfully applied to the differentiation and structural elucidation of prilocaine and its four positional isomers, which all possess similar GC and LC retention times and four of them exhibit almost identical EI‐MS and ESI‐MS/MS spectra, enabling their structural elucidation in a single LC‐ESI‐MS/MS analysis. The developed technique is fast and simple and enables discrimination between isomers based on different diagnostic ions/fragmentation patterns.     

Collision-induced dissociation mass spectra of positive ions derived from tetrahydropyranyl (THP) ethers of primary alcohols

Peaks for [M + H](+) are not observed when electrospray ionization mass spectra of tetrahydropyranyl (THP) ethers are recorded under acidic conditions. However, gaseous [M + H](+) ions can be generated from ammonium adducts of THP ethers of primary alcohols by in-source fragmentation. The product ion spectra of these proton adducts show two significant peaks at m/z 85 and 103. Tandem mass spectrometric data obtained from appropriately deuteriated derivatives and ab initio calculations indicate that the m/z 85 ion originates from more than one mechanism and represents two structurally different species. A charge-directed E1-elimination mechanism or an inductive cleavage mechanism can produce the 3,4,5,6-tetrahydropyrylium ion as one of the structures for the m/z 85 ion, whereas a charge-remote process with ring contraction can generate the 5-methyl-3,4-dihydro-2H-furylium ion as the other structure. A comparison of the relative abundances of product ions from different isotopologues showed that the charge-remote process is the preferred mechanism. This is congruent with the ab initio calculations, which showed that the dihydrofurylium ion bears the lowest energy structure. The less abundant m/z 103 ion, which represents a protonated tetrahydropyran-2-ol, is formed by a charge-remote process via a proton transfer from the alkyl substituent. This process involves the formation and rearrangement of a carbenium ion in close association with a hydroxypentanal molecule. A proton transfer from the carbenium ion to the aldehyde is followed by elimination of an alkene.     

Mass spectrometry of heterocyclic compounds—III. Electron-impact-induced fragmentation of 1,2-benzisothiazole and some 3-substituted derivatives

The mass spectra of fifteen 1,2-benzisothiazoles are reported; their fragmentation patterns have been investigated by labelling experiments, high resolution mass measurements and defocused metastable ion detection. The parent compound of the series, 1,2-benzisothiazole, eliminates HCN after partial inter-ring hydrogen scrambling. The extent of scrambling has been determined for normal daughter ions produced at different electron beam energies and also for metastable daughter ions, and is compared with the data reported for benzothiazole. Some 3-substituted derivatives show unusual fragmentation patterns. The mechanisms of these processes are under further investigation.