The use of signal averaging techniques for the quantitation and mass measurement of high molecular weight compounds using fast atom bombardment mass spectrometry

The measurement and recording of peak signals at high mass is often difficult and susceptible to a large degree of error. Signal averaging of repetitive scans has been used to improve detection limits and aid nominal mass assignment by peak matching. This is illustrated using glucagon as the reference material to determine the mass of the [MH]⁺ peak in the molecular ion cluster of Vasoactive Intestinal Peptide, and caesium iodide as reference to mass-measure a 13-unit oligonucleotide.     

Fast atom bombardment mass spectrometry of cationic complexes containing homo- and hetero-atomic cyclic units trihapto-bonded to the metal atom

The fast atom bombardment mass spectra of a series of ionic compounds of formula [(triphos)M(η³-E₂X)]Y (triphos = 1,1,1-tris(diphenylphosphinomethyl)ethane; M = Ni, E = X = P, Y = BF₄ or PF₆; M = Co, E = P, X = S or Se; E = As, X = S, Se or Te; Y = BF₄) are reported. The cations contain a homo- or hetero-atomic trimembered inorganic ring trihapto bonded to a metal-triphos unit. Both in glycerol and thioglycerol the complexes exhibit a characteristic fragmentation which involves the stepwise loss of the three atoms of the inorganic ring and the ejection of organic radicals from the triphos ligand. Adduct ions with sulphur atoms or sulphur organic radicals are, furthermore, observed in thioglycerol. Such adducts infer interactions of the matrix with: (a) the atoms of the inorganic ring of the complex cations, and (b) the metal atom of the (triphos)M moiety.     

Continuous-flow fast atom bombardment mass spectrometry of oligonucleotides

Although frit-fast atom bombardment (frit-FAB) and continuous-flow FAB mass spectrometry have become standard methods for the analysis of peptides and peptide mixtures, these techniques have not been applied previously to the analysis of oligonucleotides. Mobilephase composition, flow rate, and sample size were optimized for the analysis of oligonucleotides by negative ion frit-FAB mass spectrometry (a type of continuous-flow FAB mass spectrometry). With a mobile phase consisting of methanol/water/triethanolamine (80:20:0.5, v/v/w), flow injection frit-FAB analysis of oligonucleotides showed lower limits of detection compared to standard probe FAB mass spectrometry. For example, in order to obtain a signal-to-noise ratio of 3:1, 38 prnol of d(GTIAAC) were required for frit-FAB mass spectrometry and 62 pmol were required for standard probe FAB mass spectrometry. The largest difference between frit-FAB and standard probe FAB was observed for d(pC)₅, for which the limit of detection by frit-FAB was approximately 11-fold lower than by standard FAB mass spectrometry. Adjustment of the mobile phase to pH 7 with trifluoroacetic acid increased the limit of detection (reduced sensitivity) a minimum of sixfold. Equimolar mixtures of two or three oligonucleotides produced deprotonated molecules in identical relative abundances whether analyzed by frit-FAB or standard probe FAB mass spectrometry. Finally, frit-FAB liquid chromatography mass spectrometry was demonstrated by separating mixtures of oligonucleotides on a β -cyclodextrin high-performance liquid chromatography column with a mobile phase containing methanol, water, and triethanolamine.     

Mechanism for dehalogenation reactions in fast atom bombardment mass spectrometry

The mechanism of a dehalogenation reaction that occurs during fast atom bombardment (FAB) mass spectrometry was examined using halogenated nucleosides as model compounds. For aglycone-halogenated nucleosides, an inverse linear relationship exists between the extent of FAB dehalogenation and the calculated electron affinity of an individual nucleoside. The degree of dehalogenation for a given nucleoside also varies inversely with the calculated electron affinity of most FAB matrices. The observed dehalogenation reaction can be completely inhibited when matrices with positive electron affinities, such as 3-nitrobenzyl alcohol and 2-hydroxyethyl disulfide, are used. High-performance liquid chromatographic analysis of the bulk glycerol matrix following exposure to the FAB beam indicates measurable amounts of dehalogenated product, suggesting that this reaction occurs in the condensed phase prior to gas-phase ion formation. A dehalogenation mechanism involving thermal electron capture and subsequent negative charge stabilization is consistent with these observations.     

Negative-ion fast atom bombardment mass spectrometry of N-phosphoamino acids

The fragmentation patterns of N-phosphoamino acids in negative-ion fast atom bombardment mass spectrometry (FABMS) showed different characteristics to those in positive-ion FABMS. Six typical N-diisopropyloxyphorphorylamino acids all had intense [M ? 1]^? peaks, and they underwent similar fragmentation pathways. In general, the elimination of one alkene molecule followed by the loss of one molecule of alcohol occurred. They also favoured an N → O rearrangement reaction, followed by fragmentation to (RO)₂ PO₂^? and (RO) (HO)PO₂^?.     

Collisional-activated decomposition of benzyloxycarbonyl-protected tripeptide derivatives containing proline in fast atom bombardment mass spectrometry

The effect on the fragmentations of N-benzyloxycarbonyl-protected tripeptide ethyl esters due to the existence of L -proline in the gas phase was examined by the collisional-activated decomposition of the deprotonated molecule and the fragment ions produced by the cleavage of the tripeptide derivatives containing the neutral amino acids (L -alanine, L -leucine and L -phenylalanine) and L -proline, in which changes in both the numbers and positions of the prolyl residues were observed, in negative-ion fast atom bombardment mass spectrometry. The cleavage patterns of these ions in the collisional-activated decomposition mass spectra were observed to depend on the numbers and positions of prolyl residues in the peptide derivatives. These results indicate that the conformational differences in the peptide derivatives due to the existence of L -proline affect the decomposition of the ions containing the neutral amino acids in the gas phase.     

Characteristic fragmentations of peptide derivatives containing proline in negative-ion fast atom bombardment mass spectrometry

Fragmentations of N-benzyloxycarbonyl-protected tripeptide ethyl esters containing proline were compared with those of the corresponding peptide derivatives not containing proline in negative-ion fast atom bombardment mass spectrometry. The fragment ion [M – 109]^? due to loss of the benzyloxy group followed by dehydrogenation from the peptide molecule was the base peak in the negative-ion mass spectra for the peptides not containing proline, whilst it was a very weak fragment ion or not observed at all in those for the peptides containing proline. These results suggest that the fragmentations of the peptide derivatives in negative-ion fast atom bombardment mass spectrometry depend on the conformational difference of the peptide derivatives owing to the existence of proline in the derivative.     

Partial methanolysis and fast atom bombardment mass spectrometry of peptides containing sulphurated ammo acids

Sequence determination by partial methanolysis and fast atom bombardment (FAB) mass spectrometry of peptides containing cysteine and methionine was investigated. Cysteine-containing peptides require methylation of the sulphydryl group by methyl iodide to give a stable S-methylcysteinyl residue prior to partial methanolysis and mass spectrometry. Methionine-containing peptides undergo partially a methylation on sulphur during methanolysis, with formation of an S-methylsulphonium ion which under FAB conditions is extracted from the matrix and eliminates methyl sulphide in the gas phase. The presence of additional peaks due to chemical modifications or gas-phase fragmentations, however, does not interfere with the sequence information of the spectra.     

The analysis of salen complexes by fast atom bombardment mass spectrometry and atmospheric pressure chemical ionization mass spectrometry

Fast atom bombardment (FAB) mass spectrometry provides useful structural information about salen complexes and salen-based oxo transfer catalysts that are not appreciably soluble in organic solvents. It was discovered that initial dissolution of these complexes in trifluoroacetic acid was crucial for producing good FAB mass spectra. Trifluoroacetic acid helps dissolve the salen-based catalysts, concentrates the analyte molecules at the matrix surface, and most importantly, suppresses the reduction process, which is a well-known phenomenon when protic matrices are used. The best FAB matrices for these catalysts were found to be thioglycerol and “magic bullet.” However, dechlorination occurred under the acid conditions for complexes containing iron chloride and manganese chloride. Demetalation also occurred for nickel-containing oxo transfer salen-based complexes. When the salen-based complexes are soluble in LC solvents, they can be analyzed easily by atmospheric pressure chemical ionization (APCI) mass spectrometry without the employment of relatively nonvolatile matrices. In addition, APCI/MS provides much more sensitive detection for manganese-salen complexes when compared with FAB results. No dechlorination or demetalation were observed when a negative ion mode APCI was employed. To our knowledge, this is the first time that an intact molecule of this type of complex has been observed by mass spectrometry.     

In situ N-phosphorylation of oligopeptides for fast atom bombardment mass spectrometry

Positive ion fast atom bombardment mass spectrometry (FABMS) of in situ N-phosphorylated oligopeptides showed intense quasi-molecular ions together with the successive alkene loss fragment ions, which afford multiple checks of the unequivocal reality of the relative molecular mass of the tested samples. More interesting, in a novel cleavage pattern only the N-phosphoryl fragment ions gave intense peaks, the C-terminal series ions being suppressed. For each of the N-terminal ions, losses of alkenes also occur to provide multiple checks for the existence of these ions. The FABMS of the in situ N- phosphorylated oligopeptides might provide an easily accessible routine method for peptide sequencing.     

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.     

Determination of the stability constants of macrocyclic ligand-alkali cation complexes by fast atom bombardment mass spectrometry

Quantitative studies of the complexation of the macrocylic ligands, 18 crown 6 (18C6,1) and cyclogentiotetraose peracetate (CGD4Ac,2), with alkali cations, have been investigated by fast atom bombardment mass spectrometry (f.a.b.m.s.). Complexation curves of 18C6 with Na⁺, K⁺, Rb⁺ and Cs⁺ in glycerol, and of CGD4Ac with Cs⁺ in PEG 200, were obtained by plotting the complex peak intensity against the alkali cation concentration. From these curves we describe a method to calculate the stability constant for an alkali cation-macroyclic ligand complex. There is a good agreement between stability constants obtained either by f.a.b.m.s. or calorimetric techniques for 18C6-alkali cation complexes. These results suggest that the f.a.b. technique can be used to study complex formation and determine the stability constant.     

Structural study of Mn(III)-tetraarylporphyrin complexes by fast atom bombardment mass spectrometry

A series of 27 Mn(III)-tetraarylporphyrins bearing heterogeneous substituents on the phenyl rings at the meso positions were subjected to positive ion fast atom bombardment mass spectrometry The source spectra yielded the molecular ion and a few peaks confirmative of the chemical structure. Major processes were hydrogenolytic loss of the aryl rings or of their substituents. Molecules carrying a side-chain underwent loss of the chain either as a solution process (amide linkage) or as a gas-phase process (phenolic ether linkage). Collisionally activated dissociation mass-analysed ion kinetic energy spectroscopy allowed discrimination between the two types of fragmentation processes and some other previously unreported gas-phase reaction channels to be recognized.     

Field desorption mass spectrometry,fast atom bombardment mass spectrometry and fast atom bombardment tandem mass spectrometry of echinacoside,the main caffeoyl-glycoside from Echinacea angustifolia roots (Asteraceae)

The mass spectral fragmentation of echinacoside, a pharmacologically active caffeoyl-glycoside isolated from Echinacea angustifolia roots, has been investigated using different soft-ionization techniques, field desorption and fast atom bombardment (positive and negative ions) mass Spectrometry. Both ionization modes are successful in molecular mass determination and furnish approximately equivalent structural information. A fast atom bombardment tandem mass spectrometry approach (negative ions) was developed for the study of the fragmentation pathways and for the detection of echinacoside in crude plant extracts. The results demonstrate the usefulness of this technique for the rapid search of this important caffeoyl-glucoside directly in natural complex matrices.     

Use of fast atom bombardment mass spectrometry for the characterization of nitroaromatic isomers

Positive and negative ion fast atom bombardment (FAB) mass spectra of some monosubstituted nitroaromatic isomers are reported. Generally ions carresponding to [M + H]⁺ and M⁺ are observed in the positive ion FAB spectra; ions such as [M ? H] ^? and M^?˙ are observed in the negative ion FAB spectra. The use of FAB mass spectra to distinguish the isomers is discussed. Comparisons of FAB, chemical ionization and electron impact mass spectra of the same isomers (wherever possible) are reported. The structural information obtained in the negative ion FAB spectra complement those obtained in the positive ion FAB spectra.     

Analysis of some eicosanoids by continuous-flow fast atom bombardment mass spectrometry

Continuous-flow fast atom bombardment has been used to analyze eicosanoids by selected-ion monitoring on a sector-field mass spectrometer operating in the negative-ion mode. The method has been optimized with respect to solvent composition and flow-rates. Detection limits were below 50 pg, and under optimal conditions a linear relationship between response and amount of substance was achieved. The method was successfully applied to the analysis of two spiked urine samples.     

Structural characterization of isoxazolidinyl nucleosides by fast atom bombardment tandem mass spectrometry.

4'-Aza-2',3'-dideoxyerythrofuranosyl derivatives of thymine (AdT, 1) and uracil (AdU, 2) are analogues of 2',3'-dideoxyribofuranosyl thymine (ddT, 3) and uracyl (ddU, 4). Compounds 1 and 2 are representative of a new class of antiviral agents where the sugar moiety is replaced by an isoxazolidine ring. The increasing importance of isoxazolidinyl nucleosides has encouraged the exploitation of simple mass spectrometric rules for unambiguously assigning their structure. The species 1, 2, 5 and 6 were therefore synthesized in order to evaluate the role of the basic centre of the modified sugar moiety in their gas-phase chemistry. The tandem mass spectra of these compounds are similar to those of the wild-type nucleosides and display fragment ions corresponding to [B + 2H](+),[M - BH](+) and [B + 27](+) species, where B is the nucleobase. The last species derives from a retrocycloaddition process which is less evident in 2'-deoxyribosides. This behaviour is consistent with protonation of the analytes at the pyrimidine rings. Model isoxazolidines, in which the nucleobase was replaced by a phenyl or a naphthyl moiety, displayed the expected behaviour of species with a localized charge on the N-O moiety of the isoxazolidine ring.     

Differentiation of diastereomeric chlorin derivatives by fast atom bombardment mass spectrometry

The stereochemistry of six pairs of diastereomeric chlorin derivatives was investigated by electron impact (EI), fast atom bombardment (FAB) and direct chemical ionization DCI mass spectrometry. It was demonstrated that FAB and EI mass spectrometry are convenient and rapid methods for distinguishing between diastereomeric chlorin derivatives due to their different fragmentation patterns.