Evidence for the contribution of humic substances to conditioning films from natural waters

The presence of humic substances in conditioning films deposited on solid surfaces from natural waters was investigated using electron impact (EI), chemical ionization (CI) and secondary ion time-of-flight mass spectrometry (TOFSIMS). EI and CI spectra of a freshwater sample from a pond in Centennial Park, Sydney, Australia, showed a high degree of similarity with spectra of humic acids purchased from Fluka and Sigma as well as with reference humic acid and fulvic acid from the International Humic Substances Society, suggesting that most of the organic matter in the pond water was of humic origin. All the complex electron impact mass spectra feature series of high-intensity ions separated by 14 Da or 18 Da, which can be attributed to CH(2) and OH(2) respectively. Thermal desorption profiles of all samples generated by EI and CI were qualitatively similar. The secondary desorption peaks were less well-defined in CI compared to EI. Positive ion thermal desorption profiles displayed a two-step ionisation, with a sharp and well-defined initial desorption peak at t～50s, followed by a broader desorption peak with a maximum intensity at t ～ 100 s post-heating. The Centennial Park natural organic matter (NOM) differed from the other humic fractions in having two additional broad desorption peaks between the two described previously, and a less-defined initial peak. Infrared spectroscopy showed that proteinaceous matter in the lake water was insignificant in comparison with functional groups indicative of humic substances. TOFSIMS characterization showed almost identical spectra for Aldrich humic acid and Centennial Park NOM in the high mass region of 2000 Da to 3000 Da. Each spectrum contains approximately 25 groups of ion peaks, separated by 74 Da from group to group. Each group is composed of 6 or 7 individual peaks. The spectral features are consistent with a macromolecular structure of humic acid where aromatic rings are joined to the macrostructure via aliphatic spacer molecules.     

Chemical ionization and electron impact mass spectra of oligosaccharides derived from sphingoglycolipids

Chemical ionization (CI) mass spectra with isobutane and ammonia for the oligosaccharides obtained from sphingoglycolipids were compared with their electron impact (EI) mass spectra. The oligosaccahride moieties were liberated from the parent glycolipids and were further reduced with sodium borohydride. They were analyzed as their permethyl peracetyl and pertrimethylsilyl derivatives. In the CI spectra, peaks corresponding to QM+ and/or [M-59]+ were observed in all of the peracetylated oligosaccharides examined. In CI with ammonia as the reagent, H+ was transferred to nitrogen-containing saccharides to produce [MH]+ and NH4 was transferred to nitrogen-free saccharides to yield [M+NH4]+ as QM+. Non-reducing ends yielded very intense peaks in CI spectra. On the other hand, the reduced end, glucitol, produced rather prominent peaks in EI spectra. Fragment ions due to cleavage of glycosidic bonds were major ones under the CI conditions, and they could be used for elucidating the sugar sequence in the oligosaccharides. An additional characteristic feature in the CI spectra was that ions due to scission of hexosaminyl glycosidic linkages were observed with very high intensities.     

Fast ATOM Bombardment Mass Spectra of Nucleosides. Comparison with Electron Impact and Chemical Ionization Mass Spectra

Abstract

The fast atom bombardment (FAB) mass spectra of the eight major nucleosides found in RNA and DNA, pseudouridine and 2′,3′-O-isopropylidene adenosine are described and compared to El, CI, and desorption chemical ionization (DCI) spectra reported in the literature or obtained in this laboratory. Bcty, cocltl nun FAB spectra are reported. The FAB spectra are simple and provide unambiguous molecular weight information along with structurally significant fragment ions. Mechanisms of ion formation are thought to closely parallel those suggested earlier to be operating in the CI mode. Advantages and disadvantages of FAB relative to the standard ionization modes are discussed.     

Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides

The positive and negative ion fast atom bombardment (FAB) mass spectra and fast atom bombardment collisionally activated decomposition (CAD) spectra of a series of nucleosides and two dinucleotides are reported. The nucleosides studied are substituted forms of guanosine, adenosine, nebularine, tubercidin, uridine, and related pyrimidines. The FAB and CAD data both contain similar information. The CAD spectra are found to provide some structural information not found in the FAB mass spectra. Tandem mass spectrometry also allows emphasis to be put on weak fragments which are either not observed in the FAB mass spectrum or are lost in the matrix ion signals.     

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.     

Soft ionization mass spectral methods for lipid analysis

Chemical ionization (CI), field ionization (FI) and field desorption (FD) are sometimes preferable to electron impact (EI) mass spectrometry as methods for obtaining abundant high-mass ions from lipids. FD often provides mass spectral information which is unobtainable by other methods, and is the best method for obtaining molecular weight information. Fragment ions are observed in the spectra from all the ionization methods, which provide structural information complementing that obtainable from an EI spectrum. Using CI, high-mass ions carrying a large proportion of the total ionization current can be monitored by selected ion monitoring, resulting in enhanced sensitivity for quantitative studies in some cases.     

Chemical properties and stereoisomerism of heterogeneous long chain bases in lysosphingolipids by positive ion fast atom bombardment mass spectrometry and carbon-13 NMR spectroscopy

Positive ion fast atom bombardment (FAB) mass spectrometry of galactopsychosine and glucopsychosine was capable of showing not only the pseudo molecular ion peaks, but also various fragment ion peaks such as protonated sphingosine and its fragment ions. The percent distribution of sphingosine and dihydrosphingosine in each lysosphingolipid was determined by GLC of the trimethyl-silylated derivatives of long chain bases after methanolysis and was comparable to the relative intensities of ion peaks derived from the sphingosine and dihydrosphingosine groups. The FAB mass spectra showed that during the fast atom bombardment the sphingosine more preferentially gave rise to one and/or two fragment ions by loss of one and/or two molecules of water than the dihydrosphingosine did. The stereoisomerism of sphingosylphosphorylcholine containing mainly L-threo-sphingosine could be reconfirmed by carbon-13 NMR spectroscopy. Furthermore, although the carbon-13 NMR signals of sphingosine C-1, C-2, C-3, C-4, and C-5 showed significant chemical shift differences between D-erythro and L-threo-sphingosines of lysosphingolipids, it was concluded that the signal position of sphingosine C-3 was the most important for the determination of D-erythro and L-threo configuration in the long chain base moieties of lysosphingolipids.     

Isolation and identification of 1(3),2-diacylglyceryl-(3)-O-4'-(N,N,N-trimethyl)homoserine from the soil amoeba, Acanthamoeba castellanii

A polar lipid accounting for 12.5% of the total lipid nitrogen has been isolated from the protozoan Acanthamoeba castellanii. On the basis of thin-layer chromatography and mass spectral analysis, the lipid has been identified as diacylglyceryltrimethylhomoserine (DGTS). Fast atom bombardment (FAB) mass spectra of DGTS are reported for the first time and are compared to the FAB mass spectra of phosphatidylcholines and the electron ionization (EI) and field desorption (FD) mass spectra of DGTS. Gas-liquid chromatographic-mass spectrometric (GLC-MS) analysis of the acyl chain composition of this lipid has shown that 87.5% consists of cis-9-octadecenoic acid. Plasma membrane isolated from this organism has shown that labeled DGTS appears in the plasma membrane but is not enriched in this fraction. DGTS has been isolated previously only from a limited number of green plants and one species of fungus. Identification of this lipid in Acanthamoeba indicates that this lipid is distributed among a diverse group of lower eucaryotes.     

The identification of trimethylsilylated dipeptides with chemical ionization mass spectrometry.

The chemical ionization (CI) and electron impact (EI) mass spectra were compared for over 40 trimethylsilylated (Me₃Si) dipeptides. The dipeptides chosen had all 20 common amino acids represented at amino and carboxyl positions. The CI mass spectra of Me₃Si dipeptides typically contain three ions of high abundance used for dipeptide identification: a sequence-determining ion and two molecular weight-determining ions. The intensity of the molecular weight-determining ions relative to that of the ion that characterizes the N-terminal residue (β-cleavage ion) is greater in the CI mode than in the EI mode. Because the available intensity of the β-cleavage ion is similar in both modes, use of the CI mode will extend the lower limit for Me₃Si dipeptide identification.     

Synthesis and spectroscopic analysis of modified bile salts

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.     

Liquid ionization mass spectrometry of phospholipids

Several phosphatidylcholines (PC) and a phosphatidylethanolamine (PE) were subjected to liquid ionization (LI) mass spectrometry, in which a sample is ionized through energy transfer from metastable argon atoms under atmospheric pressure. Commercially available and synthesized, saturated or unsaturated fatty acid containing phospholipids and their mixtures were studied. A sample either as a concentrated chloroform-methanol solution or with glycerol (matrix) gave characteristic peaks such as MH+ and four fragment ions. One of the fragment ions (e.g., m/z 551 of PC 16:0, 16:0) containing both fatty acid residues has been commonly observed with other ionization methods such as CI, FD, and FAB, but the other fragment ions have not been observed in other mass spectra with one exception on desorption CI. Ions b and d (e.g., m/z 464 and 328, respectively, for PC 16:0, 16:0) contain one fatty acyl residue and the other ion containing the phosphorylcholine moiety appears at m/z 196 for PC. Thus the masses of the MH+ ion and these fragment ions provide useful structural information even in the case of a mixture. The ion b (e.g., m/z 488 of PC 18:0, 18:2) observed during an early period of heating was formed mainly by the loss of one acyl group at sn-1 of the glycerol backbone and thus may be used to differentiate the positional specificities of the constituent fatty acids. The temperature of the sample, however, should be controlled precisely, because it has a significant effect on the mass spectrum. The present method (LI) also provided useful information for a mixture of PC and PE.     

Enhancement of structural information in FAB ionized carbohydrate samples by neutral gas collision

Fast atom bombardment (FAB) ionization and two coupled analyzers (BE-EB) have been combined with neutral gas collision (C) to enhance structural information in the mass spectra of oligosaccharides. (B and E are abbreviations for magnetic and electric sectors respectively.) FAB ionization and the first analysers (BE) have provided parent ions free from biological and liquid matrix contaminants. Structural detail of these products were observed after collision and daughter ion analysis in a second coupled analyser (EB). Starting from complex mixtures, this instrumental approach, BE-C-EB, has provided specific oligomeric sequence information which was not observed in the normal FAB mass spectra. Collision spectra obtained from isomeric linear and branched oligosaccharides show unique fragments that can be directly related to structure.     

Fast atom bombardment mass spectrometry of bouvardin and selected analogs

The fast atom bombardment (FAB) mass spectra of bouvardin (1) 6-O-methylbouvardin (2), deoxybouvardin (3) and a synthetic analog (4) have been examined. The spectra of the bicyclic compounds 1-3 display site-directed fragmentations resulting from the presence of a phenolic bridged tyrosine moiety unique to this class of compounds. Comparison of the spectrum of 4, which lacks the rigid 14-membered ring, with the spectra of 1-3 shows some similarities, but clearly does not cleave in a site-directed manner as the other bouvardin analogs. Fragmentation pathways are postulated which account for most of the major ions observed in the FAB mass spectra of these potentially useful antitumor agents. Metastable ion analysis confirms the operation of the proposed pathways.     

Incorporation of tandem mass spectrometric detection to the analysis of peptide mixtures by continuous flow fast atom bombardment mass spectrometry

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.     

Application of fast atom bombardment mass spectrometry to biological samples: analysis of urinary metabolites of acetaminophen

Fast atom bombardment (FAB) is useful for the characterization of all major metabolites of the analgesic acetaminophen (APAP). It is particularly useful for providing mass spectra of the polar glucuronide and sulfate conjugates which eluded identification by field desorption and other more conventional methods of ionization. A protocol is described for the use of FAB in the identification of urinary APAP metabolites isolated by reversed phase high-performance liquid chromatography (HPLC) following therapeutic dosages of the drug. A tentative set of recommendations for the off-line use of HPLC and FAB is directed towards solving problems encountered when using these two analytical techniques in concert. In addition, a method for calculating the signal to background ratio (S/B) for analyte peaks in FAB spectra from selected relative ion intensities is proposed. Examples are presented that show the potential of S/B as an empirical parameter for judging the quality of FAB spectra.     

Keto acids in tissues and biological fluids: O-t-butyldimethylsilyl quinoxalinols as derivatives for sensitive gas chromatographic/mass spectrometric determination

Quinoxalinol t-butyldimethylsilyl ethers were prepared from three branched-chain and from two aliphatic unbranched 2-keto acids. The electron impact (EI) mass spectra display pronounced [M-57]+ ions. With 39-51% of total ion current contained within them, sensitivity is greater than on chemical ionization (CI) mass spectrometry of O-trimethylsilyl derivatives. Mass spectra and chromatographic behaviour of these novel keto acid derivatives are discussed and preliminary quantitative data from rat muscle are given.     

Fast atom bombardment,electron impact and chemical ionization mass spectrometry of the neutral 18-electron species M(CO)2(CNR)2(PR′3)2 (MMo or W)

The molybdenum(0) and tungsten(O) complexes of the type M(CO)₂(CNR)₂(PR′₃)₂ have been studied using a variety of mass spectral techniques, viz. fast atom bombardment (FAB), electron impact (EI), and both positive- and negative-ion chemical ionization (PICI and NICI) mass spectrometry. The FABMS technique gave the most structurally informative spectra with the observation of the molecular ions M⁺ (100% relative abundance in the case of MW) and in some instances the pseudomolecular ion (M + H)⁺. Fragmentation ions arising from competitive ligand loss (CO versus RNC versus PR′₃) were observed, as well as those formed by loss of H from fragment ions and dealkylation of RNC ligands. The EI and PICI spectra were not especially useful due to the relatively low thermal stability of these complexes, while the NICI spectra gave an abundance of ions that resulted from ligand redistribution reactions. Of special note were anions that contained M(CO)₄ and M(CO)₃ fragments. Dealkylation of the RNC ligands to give cyanometallate anions was also prevalent.     

Detection and structural characterization of amino polyaromatic hydrocarbon-deoxynucleoside adducts using fast atom bombardment and tandem mass spectrometry

Fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS) are shown to be useful methods for the detection and structural characterization of nanogram amounts of amino polyaromatic hydrocarbon-nucleoside DNA adducts. The positive ion spectra of four aromatic amine guanosine adducts were studied in detail. The FAB spectra of these adducts exhibit an [MH]+ ion and a more abundant aglycon fragment ion, [AH2]+, which results from the loss of the deoxyribose sugar. The sensitivity of the adducts to FAB was enhanced by preparing trimethylsilyl (TMS) ether derivatives. High-quality full-scan spectra could be obtained on less than 70 ng of the derivatized adducts without signal averaging. With a B/E-linked scan of the [MH]+ ion for the TMS2 species, these same adducts could be detected by examination of their metastable ion spectra at levels as low as 4-5 ng (S/N greater than 10). Collision-induced dissociation (CID) of the [MH]+ ion yields the aglycon fragment and an ion, S1, which results from cleavage through the sugar. The CID spectrum of the aglycon [AH2]+ ion is much more useful, providing structural information relating to the base, the polyaromatic hydrocarbon, and, possibly, the site of covalent attachment. Differentiation of isomeric aminophenanthrene-guanine adducts was demonstrated on the basis of the CID spectra of their respective [AH2]+ ions. The use of TMS derivatives also improves the sensitivity of these methods.     

Interaction of metal ions with N-glycosylamines: isolation and characterization of the products of 4,6-O-benzylidene-N-(o-carboxyphenyl)-beta-D-glucopyranosylamine with different metal ions

Metal-ion complexes of Li(+), Na(+), K(+), Mg(2+), Ca(2+), Ba(2+), Pb(2+), Cd(2+), Hg(2+) with 4,6-O-benzylidene-N-(o-carboxyphenyl)-beta-D-glucopyranosylamine were synthesized and isolated as solid products and characterized by analytical means as well as by spectral techniques, such as, 1H and 13C NMR, FTIR, absorption, FAB mass spectrometry, optical rotation and CD. While the alkali metal ions formed ML type of complexes, the other metal ions formed ML(2) type complexes. Molecular weights of the complexes of Li(+), Na(+) and K(+) were established based on the molecular-ion peaks in the FAB mass spectra. The saccharide portion remains in the beta-anomeric form even after the complexation. The spectral data, as well as the trends observed in the chemical shifts, indicate the interaction preferences between this glycosyl amine and different metal ions, and further reveal certain structural features of the complexes.     

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.