Fast atom bombardment mass spectrometry of estrogen glucuronides and sulfates

Fast atom bombardment (FAB) mass spectra of 13 intact, underivatized glucuronides and/or sulfate salts are reported. Spectra are characterized by abundant ions formed by attachment of a proton, [M+H]+, or of an alkali ion, [M+alkali]+, to the glucuronide or sulfate salt. Fragment ions were of low intensity. FAB spectra can be used to obtain the molecular weight of a sample, to assess its purity and to identify the nature of the alkali of the glucuronide or sulfate salt.     

Comparison of 252californium plasma desorption and fast atom bombardment mass spectrometry for analysis of small peptides

The data obtained with 252Cf plasma desorption (PD) and fast atom bombardment mass spectrometry of eight tri-, tetra- and pentapeptides were compared. Good spectra were obtained with 1-10 nmol of peptide. In both techniques molecular weight information was obtained. The PD mass spectra are often dominated by the cationized molecular ions in contrast to the fast atom bombardment (FAB) mass spectra, where cationization is rarely observed. Amino acid content is reflected in the immonium ions equally well in both techniques. The fragmentation patterns observed with the two techniques are almost identical. However, practical sequencing of peptides based on either FAB or PD mass spectrometry of underivatized peptides alone is difficult. This is due to the unpredictable and sometimes absent cleavage yield at certain peptide bonds. Another difficulty is the many simultaneous fragmentation pathways. However, for many peptides enough information is present to allow sequence determination for at least a major part of the molecule.     

Collision-induced dissociation-mass-analysed ion kinetic energy (CID-MIKE) analysis of ions generated by fast atom bombardment of isomeric bile salts

Molecular protonated ions and alkali attachment ions, generated by fast atom bombardment of isomeric bile salts, were analysed by collision-induced dissociation-mass-analysed ion kinetic energy (CID-MIKE) spectroscopy. Within each series of isomers, CID-MIKE spectra were similar, but showed subtle differences in the middle mass region. The most abundant ions were formed by fragmentation of the side chain. The positive charge was found to reside on the sulfonate or carboxylate moiety of the ionized salts and did not seem to migrate to other functional groups. Ions in the middle mass region were probably formed by fragmentation in the steroid ring system and therefore reflected structural differences of the bile conjugates. The usefulness of CID-MIKE spectroscopy in the structural analysis of isomeric materials was demonstrated.     

Study of the structure of anthracycline antibiotics by ERIAD mass spectrometry

Fragmentation of antibiotics daunorubicin, carminomycin, doxorubicin and their semisynthetic analogues under conditions of the new mass spectrometry method ERIAD is discussed. Signals of protonated molecular ion (M + H)+ and ions of fragments are present in all the mass spectra. The results are compared with literary data obtained by means of other (EI and FAB MS) mass spectrometry methods.     

Fast atom bombardment and tandem mass spectrometry for structure determination of steroid and flavonoid glycosides

The combination of fast atom bombardment (FAB) and tandem mass spectrometry (MS-MS) was tested for its applicability to generate useful structural information for steroid and flavonoid glycosides. The following compounds were investigated: quercetin, myricitrin, apigetrin, fraxin, rutin, neohesperidin, hesperidin, naringin, apiin, cymarin, digoxin, digitoxin, xanthorhamnin, and frangulin. Upon FAB, the sample molecules are desorbed as (M + H)+, (M - H)-, or as (M + Na)+ or (M + K)+. Collisional activation of (M + H)+ or (M - H)- ions in the MS-MS experiment leads to sequential losses of glycoside moieties in a manner which permits the sequence of glycosides to be established. Some glycosides occur as mixtures of homologs. Proper interpretation of the MS-MS or collisional activation decomposition spectra often allows the homology to be located. In addition to the simple and highly selective fragmentations observed in this combined experiment, FAB and MS-MS also remove interference caused by the ubiquitous matrix ions which are desorbed by FAB.     

Comparison of electron and chemical ionization mass spectrometry of sialic acids

Sialic acids were analyzed as per-O-trimethylsilylated compounds by gas-liquid chromatography/mass spectrometry either on electron or chemical ionization by isobutane. Electron ionization mass spectra of these derivatives are very similar to those of the corresponding methyl esters described earlier whereas chemical ionization mass spectra are characterized in the high mass range by loss of the C-2 and the C-4 substituents from the M + 1 ion. Together with other fragment ions the seven different sialic acids analyzed could be clearly identified.     

Positive and negative electrospray ionisation tandem mass spectrometry as a tool for structural characterisation of acid released oligosaccharides from olive pulp glucuronoxylans

Xylo-oligosaccharides with degrees of polymerisation 5-13, formed by partial acid hydrolysis from an extract representative of olive pulp glucuronoxylans (GX), were analysed by electrospray ionisation mass spectrometry (ESI-MS), both in positive and negative modes. The positive spectrum showed the presence of xylo-oligosaccharides in the mass range between m/z 500 and 1500 corresponding to singly [M+Na](+) charged ions of neutral (Xyl(7-9)) and acidic xylo-oligosaccharides (Xyl(5-9)MeGlcA), and doubly [M+2Na](2+) charged ions of Xyl(9-13) and Xyl(7-11)MeGlcA. Ammonium adducts [M+NH(4)](+) were also observed for Xyl(5-9)MeGlcA. The negative spectra showed the contribution of ions in the mass range between m/z 600 and 1400, ascribed to the deprotonated molecules [M-H](-) of Xyl(3-9)MeGlcA. Tandem mass spectrometry (MS/MS) of the major ions observed in the MS spectra was performed. The MS/MS spectra of the [M+Na](+) adducts showed the loss of MeGlcA residues as the major fragmentation pathway and glycosidic fragment ions of Xyl(n) and Xyl(n)MeGlcA structures. The MS/MS spectra of the [M+NH(4)](+) adducts suggests the occurrence of isomers of Xyl(5-9)MeGlcA oligosaccharides with the MeGlcA residue at the reducing end and at the non-reducing end of the molecules, although other structural isomers can also occur. Both glycosidic bond and cross-ring cleavages in the MS/MS spectra of the [M-H](-) ion suggest the occurrence of Xyl(3-9)MeGlcA with the substituting group at the reducing end position of the xylose backbone, as the main fragmentation ions. The results obtained by ESI-MS/MS, both in positive and negative modes, of Xyl(7-13)- and Xyl(5-11)MeGlcA, allow to identify fragmentation patterns of the structural isomers with MeGlcA linked to the terminal xylosyl residues of the oligosaccharides. The occurrence of these higher molecular weight oligosaccharides with a low substitution pattern allows to infer a scatter and random distribution of MeGlcA along the xylan backbone of olive pulp.     

Fast atom bombardment and collisional activation mass spectrometry of retinyl phosphate mannose synthesized by liver membranes

Fast atom bombardment (FAB) and collisional activation dissociation (CAD) mass-analysed ion kinetic energy (MIKE) spectra have confirmed the structures of retinyl phosphate (Ret-P), retinyl phosphate mannose (Ret-P-Man) and guanosine 5'-diphospho-D-mannose (GDP-Man). Ret-P-Man was made in vitro while Ret-P and GDP-Man were chemically synthesized. Positive ion FAB mass spectrometry of Ret-P showed an observable short-lived spectrum with a mass ion at m/z 367 [M + H]+, and a major fragment ion at m/z 269 [M + H - H3PO4]+. Negative ion FAB mass spectrometry of Ret-P showed a strong stable spectrum with a parent ion at m/z 365 [M - H]-, a glycerol (G) adduct ion at m/z 457 [M - H + G]- and a dimer ion at m/z 731 [2M - H]-. GDP-Man showed an intense spectrum with parent ion at m/z 604 [M - H]- and cationized species at m/z 626 [M + Na - 2H]- and 648 [M + 2Na - 3H]-. Negative ion FAB mass spectrometry of Ret-P-Man showed a parent ion at m/z 527 [M - H]- and a fragment ion at m/z 259 [C6H12PO9]-. The CAD-MIKE spectra showed structurally significant fragment ions at m/z 442 and 361 for the [M - H]- ion of GDP-Man, and at m/z 509, 406, 364 and 241 for the [M - H]- ion of Ret-P-Man. FAB and CAD-MIKE spectra have been applied successfully to confirm the structure of Ret-P-Man made in vitro from Ret-P and GDP-Man.     

Mass spectrometry of uronic acid derivatives. XI--Structure elucidation by mass spectrometry of aldobiouronic and pseudoaldobiouronic acids and their beta-elimination products as per-o-methyl derivatives

Electron impact mass spectra of a series of aldobiouronic and pseudoaldobiouronic acid per-O-methyl derivatives and of the corresponding 4,5-unsaturated analogues, found normally among the products of methylation of uronic acid containing disaccharides as a result of methylation accompanying beta-elimination, have been studied. Using labelling experiments, metastable transition measurements and high resolution mass spectrometry, the fragmentation mechanisms of substances of this class have been deduced. Application of the information to the structure elucidation of this type of compound is discussed. It is concluded that from the mass spectra alone it is possible to determine the molecular weight, the cycle masses as well as the mode of linkage between the monomeric units. The appearance potentials of ions formed by cleavage of the glycosidic linkages have also been determined and the energetic differences encountered in the fission of the glycosidic linkages of various types of uronic acid containing oligosaccharides are discussed.     

A tandem mass spectrometric study of bile acids: interpretation of fragmentation pathways and differentiation of steroid isomers

Bile acids are steroids with a pentanoic acid substituent at C-17. They are the terminal products of cholesterol excretion, and play critical physiological roles in human and animals. Bile acids are easy to detect but difficult to identify by using mass spectrometry due to their poly-ring structure and various hydroxylation patterns. In this study, fragmentation pathways of 18 free and conjugated bile acids were interpreted by using tandem mass spectrometry. The analyses were conducted on ion trap and triple quadrupole mass spectrometers. Upon collision-induced dissociation, the conjugated bile acids could cleave into glycine or taurine related fragments, together with the steroid skeleton. Fragmentations of free bile acids were further elucidated, especially by atmospheric pressure chemical ionization mass spectrometry in positive ion mode. Aside from universally observed neutral losses, eliminations occurred on bile acid carbon rings were proposed for the first time. Moreover, four isomeric 5β-cholanic acid hydroxyl derivatives (3α,6α-, 3α,7β-, 3α,7α-, and 3α,12α-) were differentiated using electrospray ionization in negative ion mode: 3α,7β-OH substituent inclined to eliminate H(2)O and CH(2)O(2) groups; 3α,6α-OH substituent preferred neutral loss of two H(2)O molecules; 3α,12α-OH substituent apt to lose the carboxyl in the form of CO(2) molecule; and 3α,7α-OH substituent exhibited no further fragmentation after dehydration. This study provided specific interpretation for mass spectra of bile acids. The results could contribute to bile acid analyses, especially in clinical assays and metabonomic studies.     

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.     

Oligosaccharide sequence determination using B/E linked field scanning or tandem mass spectrometry of phosphatidylethanolamine derivatives

Product ion mass spectral data of [M + H]+ ions of oligosaccharides, mainly tetra- and pentasaccharides, as their dipalmitoyl phosphatidylethanolamine derivatives were obtained using both liquid secondary ion mass spectrometry with B/E linked scanning and fast atom bombardment ionization with collision-induced dissociation/tandem mass spectrometry. Both methods give similar positive product ion spectra of equivalent high sensitivity (detection limits of approximately 50 pmol) that principally contain glycosidic cleavage ions retaining the reducing end of the molecule from which monosaccharide sequence can be deduced. A series of ions from fission of the phosphate ester bond together with glycosidic cleavage are present in the tandem mass spectra and B/E linked scan spectra when helium collision gas is used. Monosaccharide linkage position of isomeric molecules is reflected in the intensity of glycosidic fragmentation, without retention of the oxygen atom, with decreasing cleavage in the order 1-3 greater than 1-4 greater than 1-6 linkage. Fucose and N-acetylhexosamines show an increased degree of fragmentation over hexose sugars. The application of product ion spectra of derivatized oligosaccharides is demonstrated for characterizing mixed samples and also the acquisition of spectra directly from the silica surface of high-performance thin-layer chromatography plates.     

Laser-assisted field-desorption mass spectrometry of cyclomalto-hexaose and -heptaose and some 6-alkylthio derivatives

The use of laser-assisted field-desorption mass spectrometry for determination of molecular weight, elucidation of structure, and control of purity is demonstrated for cyclomalto-hexaose and -heptaose and their derivatives. Each compound gave an abundant [M + Na]⁺ ion. The [Na]⁺ ions originate from traces (～0.1%) of salts in the authentic samples. The fragmentation obtained is structurally highly significant, as the sequential loss of 1–5 sugar subunits is observed. Under these conditions, the elimination of water is negligible, but can be induced by applying higher thermal stress, e.g., using higher laser power. When fragmentation was induced, the cyclic oligosaccharides substituted at positions 6 lost substituted sugar units, thus confirming the synthesis pathway.     

Isolation and identification of bile salts conjugated with cysteinolic acid from bile of the red seabream, Pagrosomus major.

Bile salts present in gallbladder of wild and cultured red seabream, Pagrosomus major, a marine teleost were analyzed. The bile from wild red seabream was found to contain two previously unknown bile salts along with two known bile salts, taurocholate and taurochenodeoxycholate. Isolation of each bile salt was performed by column chromatography. Fast atom bombardment mass spectra of the unknown bile salts showed the molecular ions (M-H)- of m/z 544 and 528 which are shifted 30 mass units upfield compared to those (m/z 514 and 498) of taurocholate and taurochendeoxycholate, respectively; this is consistent with the presence of cysteinolic acid (mol wt 155) instead of taurine (mol wt 125). Enzymatic hydrolysis of the bile salts released cholic acid and chenodeoxycholic acid, respectively, and an amino acid that was identified as D-cysteinolic acid by direct comparison with an authentic sample. From these results, the bile salts in the bile of wild red seabream were identified as the conjugates of cholic acid and chenodeoxycholic acid with cysteinolic acid. 1H- and 13C-magnetic resonance spectra of the bile salts were also consistent with the proposed structure. The cysteinolic acid conjugates were found only in wild and not in cultured red seabream; this distinction seems to result from differences in dietary cysteinolic acid.     

Characterization of phosphatidylcholines in rabbit lung lavage fluid by positive and negative ion fast-atom bombardment mass spectrometry

The relative distribution of intact diacylphosphatidylcholine species isolated from the lung lavage fluid of rabbits has been investigated by positive ion fast-atom bombardment (FAB) mass spectrometry. Two different isolation/purification methods were applied and evaluated prior to mass spectrometric analysis. The first method consisted of a Bligh and Dyer extraction of the lung lavage fluid followed by isocratic high-performance liquid chromatographic (HPLC) separation. In the second method a thin-layer chromatographic purification step was introduced between the extraction procedure and the HPLC separation. Further, the FAB matrices glycerol and 3-nitrobenzyl alcohol were used, and their influence on the diacylphosphatidylcholine molecular ion species was studied. The Bligh and Dyer extraction followed by the simple HPLC separation was the method of choice to obtain stable, long-lasting protonated molecular ions and diagnostic fragment ions, which permitted the identification of the polar head-group. In combination with 3-nitrobenzyl alcohol as liquid matrix we established a procedure that yielded a fast sample preparation method, a good signal-to-noise ratio for detecting minor species, and reduced formation of [M + H − 2H]⁺ ion species. The relative fatty acid composition of the diacylphosphatidylcholine fractions isolated from rabbit lung lavage fluid was determined by negative ion FAB mass spectrometry using the carboxylate anions. The mass spectrometric results were compared with those acquired by gas chromatographic determination of the fatty acid methyl esters. Close agreement was found between the data obtained by the two independent methods.     

High-performance liquid chromatography-mass spectrometry of glycosphingolipids: II. Application to neutral glycolipids and monosialogangliosides

We previously reported a method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of molecular species of GlcCer and IV3 beta Gal-Gb4Cer [M. Suzuki et al. (1989) J. Biochem. 105, 829-833]. In this paper, we report a modification of this HPLC/FAB/MS method, which was used for the separation and characterization of neutral glycosphingolipids (GlcCer, LacCer, Gb3Cer, Gb4Cer, and IV3 alpha GalNAc-Gb4Cer) and monosialogangliosides [GM3(NeuAc or NeuGc), GM2 (NeuAc or NeuGc), and GM1 (NeuAc or NeuGc)]. Mixtures of the purified neutral glycolipids and monosialogangliosides were subjected to HPLC on a silica gel column, with programmed elution with isopropanol-n-hexane-water, with or without ammonium hydroxide. In order to obtain mass spectra and mass chromatograms of individual components, effluent from the HPLC column was mixed with a methanol solution of triethanolamine, which was used as the matrix for the FAB ionization, and one-thirtieth of the effluent mixture was introduced into a mass spectrometer through a frit interface. A mixture of the five neutral glycolipids, 5 micrograms of each, gave five peaks on a mass chromatogram obtained by monitoring of the corresponding major pseudo-molecular ions. A mixture of the six monosialogangliosides, 5 micrograms of each, gave six peaks on a mass chromatogram obtained by monitoring of the major pseudo-molecular ions, indicating that GM3, GM2, and GM1 were clearly separated, and that separation due to differences in sialic acid species was also achieved. In the mass spectra of the neutral glycolipids and monosialogangliosides, pseudo-molecular ions and fragment ions due to the elimination of sugar moieties were clearly detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide studies using a fast atom bombardment high field mass spectrometer and data system. 4. Disulfide-containing peptides

Ten peptides containing one, two or three disulfides were examined to determine their behavior under fast atom bombardment (FAB) mass spectrometric conditions. The mass spectra for the disulfide and the reduced disulfide forms of each peptide were compared. Several factors were examined that contribute to the fast atom bombardment mass spectra of these peptides: components of the FAB matrix such as alkali cations, acids, bases and reducing agents, the intrinsic molecular properties of the intact peptide, and the effect of reducing conditions on sensitivity. The FAB mass spectra of the disulfide-containing peptides examined in this study displayed accurate molecular weight information and fragmentation which indicated the position of the disulfide in the amino acid sequence.     

Thermospray and particle beam liquid chromatographic—mass spectrometric analysis of coumarin anticoagulants

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography—thermospray mass spectrometry (LC—TSP-MS) and liquid chromatography—electron impact mass spectrometry (LC—EI-MS) to assess the use of LC—MS methods for the determination of these compounds in biological materials. LC—TSP mass spectra showed a single [M + 1]⁺ ion with no fragmentation; LC—EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC—MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC—TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.     

Combinatorial evaluation of the chiral discrimination of permethylated carbohydrates using fast-atom bombardment mass spectrometry

The chiral discrimination abilities of several variously permethylated carbohydrates toward various amino acid 2-propyl esters were combinatorially evaluated from the relative peak intensity of the 1:1 diastereomeric complex ions with the deuterium-labeled L-amino acid 2-propyl ester protonated ion and with the unlabeled D-amino acid 2-propyl ester protonated ions in FAB mass spectrometry. The chiral discrimination abilities evaluated using FAB mass spectrometry approximately corresponded to the ratio of the association constants (K(R)/K(S)) toward each enantiomer in the solution. Therefore, this evaluation method is very useful for the screening of the chiral discrimination abilities of carbohydrates and their derivatives.     

Characterization of glutathione conjugates of pyrrolylated amino acids and peptides by liquid chromatography-mass spectrometry and tandem mass spectrometry with electrospray ionization

High-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was used to identify the products formed upon reaction of lysine-containing peptides with the neurotoxicant 2,5-hexanedione (2,5-HD). In addition, secondary autoxidative reaction products of the resultant alkylpyrroles with the biological thiol, glutathione, were characterized. ES mass spectra of the HPLC-separated conjugates showed intense [M+H]⁺ ions as well as several ions formed by amide and C-S bond cleavage. The glutathione conjugates of pyrrolylated amino acids and peptides were analyzed by ES ionization and MS-MS, and product-ion spectra showed fragmentation pathways typical of glutathione conjugates. ES-MS-MS analysis of a synthetic nonapeptide modeling a sequence found in neurofilament proteins showed pyrrole formation after incubation with 2,5-HD, and sequence ions were used to assign the position of the pyrrole adduct. Subsequent reaction of the pyrrolylated peptide with reduced glutathione was evidenced by a shift in m/z of the sequence ions of the reaction products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobiotic-modified peptides and confirm that stable pyrrole-thiol conjugates are formed by the reaction of biological thils with pyrrolylated peptides.