Mass spectrometry of uronic acid derivatives. Part V. Fragmentation of methyl derivatives of methyl 4-deoxy-beta-L-threo-hex-4-ehopyranosiduronic-acid.

The basic fragmentation mechanism of methyl(methyl 4-deoxy-2,3-di-O-methyl-β-l-threo-hex-4-enopyranosid)uronate has been deduced by deuteromethylation analysis, metastable transition measurements, and by interpreting the spectra of weakly excited foregoing molecules. The differences in fragmentation of partially methylated derivatives of methyl 4-deoxy-β-l-threo-hex-4-enopyranosiduronic acid compared to that of the fully methylated substance are discussed in detail and the criteria are proposed for identification of the compounds concerned by mass spectrometry.     

Mass-spectral fragmentation of methyl 2,3,4-tri-O-methyl-α-D-glucopyranosiduronamide and its analytical application

Fragmentation pathways for methyl 2,3,4-tri-O-methyl-α-D-glucopyranosiduronamide are proposed, based on 70- and 12-eV spectra of the compound specifically labelled with CD₃ and ND₂ groups. The presence of the NH₂ group in the molecule gives rise to new fragmentation series. The number and positions of CD₃ groups can be unequivocally determined from the mass spectra. Partially methylated derivatives of hexuronic acids, obtained by methylation analysis of hexuronic acid-containing substances, can be identified by exhaustive trideuteriomethylation and conversion into readily obtainable crystalline amides.     

Gas-liquid chromatography and mass spectrometry of methylated and acetylated methyl glycosides. Application to the structural analysis of glycoprotein glycans

The mass spectra of 52 partially methylated and acetylated methyl glycosides of galactose, mannose, glucose, and N-acetylglucosamine have been determined. Each derivative was identified on the basis of its gas-liquid chromatography retention time and mass spectra. The analysis of methyl ethers obtained by methanolysis of fully methylated glycans of α1-acid glycoprotein is described as an application of the method.     

O-methyl oximes of sugars. Analysis as O-trimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry

The mass spectra of aldoses, partially methylated aldoses, deoxyaldoses, and ketoses containing 3–7 carbons, were recorded on the ethers of the trimethylsilyl O-methyl oxime derivatives. Each compound gave a distinctive spectrum indicating the carbon-chain length and the location of substituents. The gas-liquid chromatographic properties of most compounds in this study were also examined.     

Micro-determination of plasma diphenylhydantoin by gas—liquid chromatography

A selective, sensitive and precise gas—liquid chromatographic method for the determination of diphenylhydantoin in micro samples of blood plasma is described. After a double extraction with chloroform containing an analogue of diphenylhydantoin as an internal standard, the drug and standard are N,N-dimethylated in alkaline aqueous solution with methyl iodide followed by extraction into acetone. The methylated derivatives are separated gas chromatographically and measured using a flame-ionization detector. The lowest concentration of diphenylhydantoin in plasma which can be measured in a 100-μl sample is 1 μg/ml, which is well below the normal therapeutic concentration of 10–20 μg/ml in plasma. The methylated derivatives of diphenylhydantoin and the internal standard have been identified by their proton magnetic resonance spectra and mass spectra.     

Synthesis and mass fragmentographic analysis of partially O-methylated 2-N-methylglucosamines.

A series of partially O-methylated N-methylglucosamines was synthesized by limited-time methylation of methyl-2-N-methylacetamido-2-deoxyglucopyranoside by Kuhn's procedure, followed by acid hydrolysis. These partially O-methylated N-methylglucosamines were separated satisfactorily by gas chromatography on a column of OV-17 on Gas-chrom Q as amino alditol acetates and identified from their mass spectra. For specific analysis of the methylated aminosugar derivatives, a mass fragmentographic method was established. Methylated aminosugars can be successfully determined in amounts as low as about 1 ng by this method.     

Identification of methylated flavonoid regioisomeric metabolites using enzymatic semisynthesis and liquid chromatography-tandem mass spectrometry

Discrimination of isomeric methylated metabolites is an important step toward identifying genes responsible for methylation, but presents substantial challenges because authentic standards are often unavailable and mass spectra of isomers have been considered indistinguishable. In this report, an approach is described for identifying methyl group positions in multiply methylated flavonoid metabolites using combinations of tandem mass spectrometry, liquid chromatography retention, and site-selective methylation by recombinant O-methyltransferases from Solanum habrochaites LA1777. The basis for observed fragment ions in tandem mass spectra of multiply methylated myricetin was further established using enzymatic incorporation of deuterium-labeled methyl groups using S-adenosylmethionine-d ₃ as precursor.     

Methylated flavonoids from cistus ladanifer and cistuspalhinhae and their taxonomic implications

Three methylated kaempferol and two methylated apigenin derivatives were identified in the leaf resins of Cistus ladanifer and C. palhinhae. The two species produced identical secreted flavonoids which supports their close affinity based on morphological similarities.     

Flavonoid aglycones from Eriodictyon californicum resin and their implications for herbivory and UV screening

The flavones luteolin and chrysoeriol and five methylated derivatives of the flavonone eriodictyol were identified in the external leaf resin of Eriodictyon californicum. These flavonoids comprise approx. 80% of the mass of the resin. The flavonones can be classified into 7-hydroxy- or 7-methoxy-compounds, and these two groups of compounds can be separately quantified in a crude resin extract using UV spectroscopy in the presence of sodium methoxide. The different properties of these flavonoids as herbivore deterrents and UV screens may explain the complexity of the leaf resin mixture.     

Identification of keto acids in arctic bramble,Rubus arcticus L. as methyl esters of their 2,4-dinitrophenylhydrazones

The major keto acids in arctic bramble,Rubus arcticus L. were investigated. The acids were isolated with anionic and cationic ion-exchange resins, converted to 2,4-dinitrophenylhydrazones, and purified with an Al₂O₃ column. The derivatives were separated on a silica gel G thin-layer plate and esterified with methanol-HCl and the methyl esters of the keto acid 2,4-dinitrophenylhydrazones formed were analyzed on an OV-1-glass capillary gas-liquid chromatography column and with mass spectrometry. 2-Oxoglutaric, pyruvic, oxaloacetic, and glyoxylic acids were identified. The mass spectra of the derivatives are presented.     

Occurrence,structure and synthesis of 3-(N-Methylamino)glutaric acid,an amino acid from Prochloron didemnii

3-(N-Methylamino)glutaric acid has been identified as a new free amino acid in extracts from Prochloron didemnii (Lewin), a unique prokaryotic algal symbiont associated with certain didemnid ascidians. Its structure was established by elucidation of the mass spectra of its TMSi and other derivatives and confirmed by synthesis.     

The chemistry and control of hereditary lipid diseases

Direct inlet mass spectrometry has been performed on different derivatives of a hematoside (a triglycosylceramide of a tumour) and the major monosialoganglioside of brain (a pentaglycosyl-ceramide). As a confirmation of earlier results it was shown that trimethylsilyl derivatives gave information on ceramide structure (fatty acids and long-chain bases) but no specific information on carbohydrate structure. Fully methylated derivatives on the other hand, not analyzed before, gave in addition to ceramide fragments, specific ions for the sialic acid as well as carbohydrate sequence and branching. Using these derivatives molecular ions were not obtained for the brain ganglioside. However, by reduction of the methylated derivatives with LiA1H₄ (amide groups of ceramide and amino sugars were reduced to the corresponding amines) and trimethylsilylation of the converted sialic acid ester group, molecular weight ions were obtained for both gangliosides. In addition very strong peaks were found for the complete carbohydrate plus the fatty acid, of importance for the determination of the type and exact ratio of sugars, and also the fatty acid composition of the molecules. Ions were also obtained for a conclusive information on carbohydrate sequence and branching. It is concluded that a combined mass spectrometric use of methylated and methylated plus reduced ganglioside derivatives affords structural information on the complete molecules, which will be of considerable help in the characterization of gangliosides on a microscale.     

Novel very long-chain methyl-branched alcohols and their esters,and methyl-branched alkanes in pupae of the cabbage looper,Trichoplusia ni (Hubner)

Four homologous series of very long-chain methyl-branched alcohols were found in the internal lipids of cabbage looper, Trichoplusia ni, pupae both as free alcohols and as esters. These alcohols were identified based on mass spectra of their TMS and acetate derivatives, and on the Kovats Indices and mass spectra of the alkanes obatained by reduction of their bromide derivatives with LiAlH₄. The four homologous series (from C36 to C46) consisted of monomethyl-, two dimethyl- and a trimethyl-branched alcohol series. The major alcohols (with the corresponding alkanes in parentheses) were identified as 24-methyltetracontan-1-ol (17-methyltetracontane), 24,28-dimethyltetracontan-1-ol (13,17-dimethyltetracontane), 24,36-dimethyltetracontan-1-ol (5,17-dimethyltetracontane) and 24,28,36-trimethyltetracontan-1-ol (5,13,17-trimethyltetracontane). The minor components had an odd number of carbon atoms (C37, C39, C41 and C43) in the carbon chain and the methylalkanes formed from their reduction had methyl branches on the 18- and 14,18-positions. The methylalkanes found internally in pupae were similar to those previously reported in larval cuticular lipids (de Renobales and Blomquist, Insect Biochem. 13, 493–502, 1983). Additional novel methylalkanes identified were 2,X-dimethylalkanes, 5,15-dimethylhentriacontane, 5,23-dimethyltritriacontane, 5,17,23-trimethyltritriacontane and 5,15,23-trimethylpentatriacontane. The methyl branching positions of the major methyl-branched alcohols were different from those of the major methylalkanes.     

Amino acid sequencing by gas chromatography-mass spectrometry using perfluoro-dideuteroalkylated peptide derivatives: B. Interpretation of the mass spectra

The mass spectra of the O-trimethylsilylated trifluoro-dideuteroethyl polyamino alcohols, produced by LiAlD₄-reduction and O-trimethylsilylation of N-trifluororacetyl oligopeptide methyl esters, are evaluated. Characteristic mass spectra of derivatives are shown which are derived from peptides containing all protein amino acids including Arg, His, Trp, Gln, Asn and carboxyl terminal amides as well as modified Cys-residues. The mass spectra of these derivatives can be easily interpreted in terms of the amino acid sequence of the original peptides since they contain abundant and intensity-balanced sequence-determining ions.     

Determination of the linkages in some methylated,sialic acid-containing,meningococcal polysaccharides by mass spectrometry

The application of gas-liquid chromatography-mass spectrometric (g.l.c.-m.s.) analysis to a number of sialic acid-containing polysaccharides of meningococcal origin has been studied. Methylation of these polysaccharides by the Hakomori conditions resulted in both O- and N-methylation. Methanolysis of the methylated polysaccharides from serogroup C [(2→9)-linked], colominic acid [(2→8)-linked], and serogroups Y and W-135 [both (1→4)-linked], yielded the respective 4,7,8,4,7,9-, and 7,8,9-tri-O-methyl derivatives of methyl N-acetyl-N-methyl-β-D-neuraminate methyl glycoside. As model compounds, methyl N-acetyl-4,7,8,9-tetra-O-methyl-α-D-neuraminate methyl glycoside and its N-methyl derivative were also synthesized. All of the methylated derivatives could be identified on the basis of their typical fragmentation-patterns, indicating that this method is applicable to the determination of the position of linkages to sialic acid residues in biopolymers.     

Separation and detection of uv-absorbing derivatives of fecal bile acid metabolites by high-performance liquid chromatography

Major fecal bile acid metabolites related to lithocholic acid were resolved by high-performance liquid chromatography (hplc). The uv-absorbing p-nitrobenzyl ester derivatives of lithocholic, isolithocholic, 3-keto-5β-cholanic, and 5β-cholanic acids were prepared using the reagent o,p-nitrobenzyl-N,N′-diisopropylisourea. Separation was achieved in less than 20 min on a microparticulate silica column using isocratic elution with 2% isopropanol in isooctane as the mobile phase. The p-chlorobenzoyl esters of methylated lithocholic and isolithocholic acids were also prepared but required purification by thin-layer chromatography before separation by hplc. These derivatives were eluted from a Porasil T column using 5% diisopropyl ether in isooctane as the mobile phase. Lithocholic and isolithocholic acids produced by microbial metabolism of [¹⁴COOH]taurolithocholic acid were separated and identified by preparing p-nitrobenzyl derivatives and monitoring the column effluent for both uv and radioactivity. This technique is a rapid and sensitive method for isolating bile acid metabolites.     

Gas chromatography mass spectrometry based profiling of alkyl coumarates and ferulates in two species of cattail (Typha domingensis P., and Typha latifolia L.)

Several long-chain n-alkyl coumarates and ferulates were identified in cattails (Typha domingensis and Typha latifolia) from the Florida Everglades. Characterization of these compounds was achieved based on the interpretation of mass spectra obtained by GCMS as their trimethylsilyl ether derivatives, comparison with published mass spectra and available standards. Both n-alkyl p-coumarates and n-alkyl ferulates were identified in roots and leaves of both Typha species, featuring unique distribution patterns and differences between leaf and root biomass. For both Typha species, roots have higher concentrations and a much greater diversity of n-alkyl p-coumarates and ferulates but with different side chain carbon numbers ranging from C₁₄ to C₂₈. Typha domingensis leaves only contained n-alkyl ferulates with traces of n-alkyl p-coumarates, while both types of compounds were present in Typha latifolia leaf material. These chemicals were not found in the other dominant wetland vegetation, which suggests their potential for application as phytochemical tracers of fresh cattail-derived organic matter in the Everglades ecosystem.     

Characterization of polyphenols in cell walls of cultured Populus trichocarpa tissues

The amount and composition of cell wall-bound polyphenol (lignin) in cultured Populus trichocarpa tissues which formed numerous xylem elements (xylogenic) or no xylem (non-xylogenic) were compared. Polyphenol accounted for ca 15% of the dry wt of the cell wall and did not differ significantly in amount in xylogenic and non-xylogenic tissues. The syringic acid derivatives, 3,4.5-trimethoxybenzoic acid, was identified as one of the oxidation products of methylated cell walls and was recovered in similar amounts irrespective of xylem formation. In contrast, lignin from xylogenic cultures contained more p-coumaryl alcohol derivatives and less coniferyl alcohol derivatives than lignin from non-xylogenic cultures. In this respect the lignin composition of xylogenic tissues closely resembled that from stems.     

Minor aristolochic acids from Aristolochia argentina and mass spectral analysis of aristolochic acids

The minor aristolochic acids isolated from Aristolochia argentina were identified as 6,7-dimethoxy, 6-hydroxy-7-methoxy, 2-hydroxy-8-methoxy and 7-hydroxy-8-methoxy disubstituted derivatives of the 3,4-methylenedioxy-10-nitro-1-phenanthroic acid, respectively. A. argentina also contains the previously reported aristoloside. The mass spectra of the aristolochic acids, their esters and decarboxylation products have been examined. A number of successive fragmentation processes leading to the formation of aromatic hydrocarbons were observed. Cleavage of the nitro group is a prominent process in the mass spectra of the aristolochic acids and their esters. Evidence is presented that the formation of the [MNO₂]⁺ ion occurs by an intramolecular aromatic substitution reaction with participation of the CO₂R group. The different behaviour of the decarboxylated aristolochic acids is also discussed. A mechanism is proposed for the favourable loss of CH₂O in the 8-methoxy isomer.     

Improved identification of monomethyl paraffin chain branching (close to the methyl end) of long-chain compounds by gas chromatography and mass spectrometry

An improved method for the identification of monomethyl substituted paraffin chains (n?2, n?3 and n?4) by gas chromatography and mass spectrometry is presented. Fatty acids and sphingolipid long-chain bases were converted to their corresponding alcohols, which were analyzed as methyl ethers. These compounds were better separated on packed columns (XE-60) than other derivatives, and the low temperature of analysis minimized the bleeding of stationary phase into the mass spectrometer. Mass spectra of methyl ethers allowed a more conclusive identification of branches than of other derivatives. The method may be generally applied to fatty acids, aldehydes and alcohols.