Mass spectrometry of sterols. Electron ionization induced fragmentation of C-4-alkylated cholesterols

The electron impact ionization of C-4-alkylated cholest-5-en-3β-hydroxysterols has been investigated. The mass spectra of the C-4-alkylated cholesterols contain a number of ions in the high mass region for which analogous ions are not found in the spectrum of cholesterol. Detailed studies of the composition and origin of these ions have been made by high resolution mass spectrometry and analysis of metastable ions. In addition, a large number of isotopically (deuterium and ¹⁸O) substituted C-4-alkylated analogues have been prepared to assist in the interpretation of the spectra. The combined results indicate the occurrence of a number of very complex and unusual electron ionization induced fragmentations. Most notable of the findings reported herein concerns the demonstration of the formation of an ion involving loss of the elements of ring A with an intramolecular shift of the oxygen and hydrogen atoms of the hydroxyl function to the charge-retaining species.     

Chemical ionization mass spectrometry of trimethylsilylated carbohydrates and organic acids retained in uremic serum

After appropriate sample pretreatment and derivatization, uremic serum was investigated by combined high resolution gas chromatography and mass spectrometry, using both electron impact and chemical ionization methods. Electron impact and chemical ionization spectra of a number of identified (trimethylsilylated) carbohydrates and organic acids are compared. The utilization of chemical ionization mass spectrometry, with isobutane as the reagent gas, is discussed in detail. The influence of the reagent gas pressure on the total ion current and on the spectral appearance was studied. The identification of compounds, based on electron impact mass spectral data, was confirmed and often aided appreciably by using this technique. The chemical ionization spectra of trimethylsilyated alditols and aldonic acids, as well as of other organic acids showed protonated molecular ions, whereas aldoses did not. Differences with electron impact spectra are found mainly in the high mass region. The loss of one or more trimethylsilanol groups becomes the predominating fragmentation route at higher reagent gas pressures.     

Gas chromatographic/mass spectrometric analysis of specific isomers of polychlorodibenzofurans

The gas chromatographic/mass spectrometric characteristics of 26 congeners of polychlorinated dibenzofurans, previously characterized by specific synthetic routes and by standard spectroscopic techniques, have been evaluated. The electron impact mass spectra are not particularly isomer-specific, though 2,3,7,8-tetrachlorodibenzofuran is distinguishable on this basis from the three other tetrachloro isomers investigated in this work. Positive ion methane chemical ionization mass spectra do show a greater degree of isomer distinction, and are reasonably reproducible. Electron attachment negative ion spectral characteristics are also presented. Preliminary results on negative ion chemical ionization mass spectra, obtained using methane plus small amounts of oxygen as reagent gas, are reported.     

Mass spectra of complex molecules. I. Chemical ionization of sphingolipids

Methane chemical ionization spectra of acetylated and perdeutero-acetylated ceramides, cerebrosides, and ceramide dihexosides have been analyzed and compared with electron impact ionization spectra of the same compounds. Abundant fragment ions for the loss of acetic acid from the protonated molecular ion in C.I. spectra readily enable the determination of the molecular weights of the principal species of sphingolipid mixtures. Complete structures can be elucidated by the combination of E.I. and C.I. mass spectra.     

The N terminus of human myelin basic protein consists of C2, C4, C6, and C8 alkyl carboxylic acids.

Peptide 1-21, generated by cyanogen bromide cleavage of each of two highly purified components of human myelin basic protein, components 1 and 8, gave a series of peaks in the fast atom bombardment mass spectra with m/z 2299, 2327, 2355, 2383, and 2411, indicating additions of 42, 70, 98, 126, and 154 atomic mass units respectively with m/z 2327 and 2355 as the dominant species. The pentafluorobenzyl esters prepared from an acid hydrolysate analyzed by negative ion chemical ionization gas chromatography mass spectrometry confirmed that C6, C8, and C10 fatty acids were present. These data demonstrated (i) that the N terminus of a myelin basic protein is not simply acetylated but contains C2, C4, C6, C8, and C10 fatty acids with C4 and C6 as the dominant species, (ii) the two components studied (C-1 and C-8) showed different relative amounts of C2 and C8 in particular, and (iii) human myelin basic protein is the first protein to be reported with a complex N terminus consisting of several alkyl carboxylic acid species.     

Structure and thermal interconversion of cyclobilirubin IX alpha.

One of the two main photoproducts in bilirubin metabolism during phototherapy in neonatal hyperbilirubinaemia is (EZ)-cyclobilirubin. However, it has not yet been possible to come to a final conclusion as to its chemical structure, despite the fact that much effort has been expended on the problem. The present paper demonstrates that (EZ)-cyclobilirubin is formed by the intramolecular cyclization of the C-3-vinyl group with the position at C-7 rather than at C-6, without delta-lactone-ring formation. The evidence comes from 13C-n.m.r. spectra, which indicate that an oxygen-bound quaternary carbon atom is not present, and from 1H-n.m.r. spectra, which indicate that the orientation of the methyl group at C-2 is equatorial; these findings are supported by mass spectra. The existence of both an epimeric relationship at C-7 between (EE)- and (EZ)-cyclobilirubins A and B and of steric isomers of the hydrogen atom and methyl group at C-2 is supported by the fact that the methyl-group protons at C-2 and C-7 are observed as a paired signal in 1H-n.m.r. spectra, and that new signals at C-7, C-2 and C-3 beta appear in 13C-n.m.r. spectra, that mass spectra of (EZ)-cyclobilirubins A and B are extremely similar and that, furthermore, thermal interconversion between (EE)- and (EZ)-cyclobilirubins A and B is observed.     

Mass spectrometry of high-mannose oligosaccharides after trifluoroacetolysis and periodate oxidation.

High-mannose oligosaccharides were treated with a mixture of trifluoroacetic acid and trifluoroacetic anhydride under conditions where reducing terminal N-acetylglucosamine residues are specifically degraded. The resulting mannose-containing products were, after further chemical modifications, analyzed by mass spectrometry in fast atom bombardment and electron ionization modes. Binding positions between monosaccharide residues were deduced from mass spectra of peracetylated compounds, which, prior to the derivatization, had been subjected to periodate oxidation and borodeuteride reduction.     

Principles of electrospray ionization

Electrospray ionization is today the most widely used ionization technique in chemical and biochemical analysis. Interfaced with a mass spectrometer it allows the investigation of the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which thus enables even the investigation of large noncovalent protein complexes. Its high ionization efficiency profoundly changed biomolecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion. This review article focuses mainly on the exploration of the underlying ionization mechanism. Some ionization characteristics are discussed that are related to this mechanism. Typical spectra of peptides, proteins, and noncovalent complexes are shown and the quantitative character of spectra is highlighted. Finally the possibilities and limitations in measuring the association constant of bivalent noncovalent complexes are described.     

New approach to the mass spectroscopy of non-volatile compounds

A new method has been developed to obtain mass spectra of non-volatile compounds from the solid state at room temperature. Volatilization and chemical ionization of the sample are effected using a ²⁵²Cf fission fragment source. Mass spectra are measured using a new time-of-flight mass spectrometer featuring single ion counting and fast electronics. We present positive and negative ion mass spectra for the amino acids arginine and cystine which are characterized by intense quasi-molecular ions.     

Identification of iso-TC-1 as a new T-2 toxin metabolite in cow urine

The structure of a new metabolite T-2 toxin (iso-TC-1) has been established as 3,15-diacetoxy-4-hydroxy-8(3-methyl-3'-hydroxy-butyryloxy)-12, 13-epoxytrichothec-9-ene. The compound is an isomer of TC-1 (a recently isolated T-2 derivative) in which the hydroxy and acetoxy groups at the C-3 and C-4 positions, respectively, are reversed. Direct probe analysis by electron impact (EI) of the underivatized iso-TC-1, as well as EI, positive chemical ionization (CI) in methane, and positive CI in ammonia of its trimethylsilylether or trifluoroacetate provided evidence to support the structure assignment of the new metabolite. The mass spectra of iso-TC-1 were compared with those of TC-1, T-2 toxin and iso-T-2 toxin (the isomer of T-2 toxin having reversed substituents at C-3 and C-4) with regard to molecular weight and fragments involving the substituents at C-3, C-4, C-8 and C-15. Although the two isomers, TC-1 and iso-TC-1, were not easily resolved by thin layer chromatography (TLC), a very good separation of their trimethylsilyl and trifluoroacetate derivatives was obtained by capillary gas chromatography. Acetylation of TC-1 or iso-TC-1 gave the same product. Iso-TC-1 is one of the main products of T-2 metabolism in the cow (more abundant than TC-1) and is found in the urine.     

Synthesis and characterization of some aminoarsonium chlorides

R₃As reacts with NR′R″Cl to give good yields of a new homologous series of aminoarsonium chlorides, [R₃AsNR′R″]Cl, in which R = Me, Et, n-Pr, and Ph; R′ and/or R″ = H, Me. IR, NMR, mass, and X-ray spectral data suggest that the arsenic is tetra- coordinate. Electfical conductivity and temperature and concentration dependent NMR studies suggest that hydrogen-bonding interactions are important in solution. Quaternization of the arsenic produces a downfield ¹H NMR chemical shift for the protons in the alkyl chains and a change from non-equivalence to equivalence of the C-1 protons. The NMR data are compared with those for the analogous phosphorus compounds. The electron impact, chemical ionization, and negative ion mass spectral data and fragment ion identities are given for the compounds. Ions corresponding to a variety of AsCl containing species, in addition to those associated .with fragmentation of the R₃As moieties, are observed in the EI mass spectra. AsN, AsNAs, and AsNAsN containing fragments are observed in the Cl mass spectra and AsCl bonding species in the NI mass spectra. A preliminary X-ray diffraction study of [n-Pr₃AsNH₂]Cl indicates near tetrahedral geometry about the arsenic atom.     

A gas chromatographic mass spectrometric chemical ionization assay for haloperidol with selected ion monitoring

A new gas chromatographic mass spectrometric chemical ionization assay for haloperidol with selectedion monitoring is presented which provides for better combined selectivity and sensitivity than previous assays. Levels of haloperidol in 2 ml of human plasma were reproducibly measured down to subnanogram levels. Both methane and methane--ammonia chemical ionization spectra are presented for haloperidol and the internal standard trifluperidol.     

Identification of methotrexate and folic acid analogs by mass spectrometry

Methotrexate and folic acid analogs are polar molecules and attempts to obtain electron impact and chemical ionization mass spectra of these compounds by several laboratories have failed. We have found that methylation of this important class of compounds with diazomethane produces derivatives which are sufficiently volatile to allow their mass spectra to be recorded. The mass spectra of the methyl derivatives of five compounds, 4-amino-4-deoxy-N10-methylpteroylglutamic acid, 4-amimo-4-deoxypteroylglutamic acid, pteroylglutamic acid, 4-amino-4-deoxypteroic acid and N10-methylpteroylglutamic acid are presented, and the fragmentation pathways of these compounds discussed.     

Synthesis and characterization of long-chain 1,2-dioxo compounds

A series of long-chain methyl esters with vicinal oxo groups (1,2-diones; 1,2-diketones) were synthesized by potassium permanganate-based oxidation of methyl esters of mono-unsaturated fatty acids. The presence of two additional carbonyl groups may facilitate the synthesis of other derivatives. The starting materials were selected in such a fashion to give the 1,2-dioxo moiety in consecutive positions from the methyl ester group. The compounds were characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. In mass spectrometry, both electron and chemical ionization (methane as reagent gas) were investigated. The position of the dioxo moiety can be determined in both ionization modes, however, in electron ionization mode the corresponding fragment ions are considerably stronger. In electron ionization mode, a fragmentation mechanism depending on the position of the 1,2-dioxo moiety occurs while the spectra derived from chemical ionization mode are mainly characterized by peaks around the molecular ion with both ionization modes appearing suitable.     

Negative ion chemical ionization mass spectrometry of 2,4-dinitrophenyl amino acid esters

We report the negative ion chemical ionization mass spectra of 2,4-dinitrophenyl (DNP) amino acid methyl esters. For the common amino acids, these derivatives exhibit very simple mass spectra; the molecular anion is the base peak in all cases. The electrophilicity of the DNP group allows selective and sensitive ionization. Amino acids can be identified singly or in mixtures by their molecular weights, and picomole detection is possible. Chromatography is only needed for Leu/Ile differentiation. Amino-terminal analysis of proteins is demonstrated.     

Rapid analysis of naphthalene and its metabolites in biological systems: Determination by high-performance liquid chromatography/fluorescence detection and by plasma desorption/chemical ionizations mass spectometry

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.     

Metabolic studies of explosives. 1--EI and CI mass spectrometry of metabolites of 2,4,6-trinitrotoluene

A series of metabolites of trinitrotoluene (TNT) have been synthesized and analysed by electron impact (EI) and chemical ionization (CI) mass spectrometry. Identification characteristics of these metabolites by their mass spectra have been determined. Differentiation of isomers is made possible by EI ions which are characteristic of the position of the methyl with regard to the nitro groups.     

Systematic analysis of desorption chemical ionization (DCI) mass spectra of nucleic acids--7

The positive ion and negative ion pyrolysis mass spectra of the herring sperm DNA have been studied using desorption chemical ionization. The positive ion desorption chemical ionization spectra have been produced with CH4, i-C4H10, NH3, HCl and Cl2; the negative ones with N2O/CH4, N2O/i-C4H10, Cl2, CCl4, HCl and via electron capture. These spectra have been compared with the electron impact ionization spectra. We have observed an important increase of sensitivity when negative ionization has replaced the positive ionization mode. The series of diagnostic ions resulting from direct chemical ionization belong to the family of base + reagent ion X [BH + X] and base + X - HX ion [B]. Their abundance has increased considerably compared to the electron impact spectra. The application of these new diagnostic ions in nucleic acid studies is interesting especially for the much higher abundance of the usually weak dG fragment ion obtained in the negative ionization mode. The dG-base segment of the DNA is the most nucleophilic centre of the whole nucleic acid and is implicated in numerous important biochemical reactions involving, for example, proteins.     

Characterization of mixtures of dipeptides by gas chromatography/mass spectrometry

The gas chromatographic and mass spectrometric behavior of over 120 different dipeptides has been investigated. The dipeptides were analyzed as their N,O-perfluoropropionyl methyl ester derivatives by combined gas chromatography/mass spectrometry. Mass spectra of the dipeptides were obtained using both electron impact and chemical ionization. Gas chromatographic retention times were obtained for each of the dipeptides studied and utilized for the prediction of the retention times for most of the 400 common dipeptides. These techniques enable the unambiguous identification of dipeptides in mixtures.     

Compositional and molecular species analysis of phospholipids by high performance liquid chromatography coupled with chemical ionization mass spectrometry

High performance liquid chromatography (HPLC) was combined with chemical ionization mass spectrometry (CIMS) by the use of a moving-belt interface. The technique was employed for the analysis of naturally occurring phospholipids. Positive and negative ion mass spectra of various phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin were obtained in the chemical ionization mode with ammonia or methane as the reagent gas. Specific ions for individual phospholipid "bases" were identified. These ions were used in specific ion monitoring of the phospholipids during HPLC-CIMS. CIMS of each phospholipid also provided extensive information on the molecular species of the individual class of phospholipids. Relative abundance of different molecular species of each phospholipid as determined by CIMS agreed well with the results obtained by gas-liquid chromatography. Rat brain phospholipids were analyzed by HPLC-CIMS in about 15 minutes. Routinely, about 5 micrograms of individual phospholipid was analyzed by HPLC-CIMS, however, with specific ion monitoring the method provides a detection capability at the subnanogram level.