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.     

Identification of guanosine diphosphate derivatives of d-xylose, d-glucose and d-galactose in mature strawberry leaves

The nucleotides from a trichloroacetic acid extract of mature strawberry leaves were separated into ten main fractions by chromatography on a Dowex 1 (formate form) column with ammonium formate as the eluting agent. One of these fractions, which was suspected to contain not only ADP but also GDP-sugars, was separated into a number of subfractions by further chromatography on a Dowex 1 (formate form) column with the formic acid system as the eluting agent. One of these subfractions was identified from its ultraviolet spectra, from its position on the two ion-exchange columns and by thin-layer chromatography as a GDP-sugar. Mild acid hydrolysis gave GDP and a mixture of sugars. The sugars, after a preliminary separation on a paper chromatogram, were identified by an isotope-dilution method. The sugars were condensed with sodium [(14)C]cyanide, the [(14)C]nitriles were hydrolysed and one of the epimeric acids was isolated, either as lactone or amide, by co-crystallization with a non-radioactive carrier. This method distinguishes between enantiomorphic sugars. d-Mannose, d-xylose, d-glucose and d-galactose were present in the proportions 40:10:1:1 respectively. The total amount of the GDP-sugars was approx. 0.1mumole/100g. of fresh leaves.     

Studies on trimethylsilyl derivatives of 1,2-dialkylglycerols by gas-liquid chromatography mass spectrometry

Trimethylsilyl derivatives of 1,2-dihexadecyl- and 1,2-dioctadecyl-glycerols were subjected to analysis by a gas chromatograph mass spectrometer system. The mass chromatographic identification of four kinds of glycerophospholipids, 1,2-dihexadecyl, 1-hexadec-1-enyl-2-hexadecanoyl, 1-hexadecyl-2-hexadecanoyl- and 1,2-dihexadecanoyl-glycerol is also described.     

The analysis of 2-acetamido-2-deoxyaldose derivatives by gas-liquid chromatography and mass spectrometry

Syntheses are reported of 4-substituted, 4-deoxy analogs of methyl β-D-galactopyranoside (the 4-amino-4-deoxy, 4-azido-4-deoxy, 4-bromo-4-deoxy, 4-chloro-4-deoxy, 4-deoxy-4-fluoro, 4-deoxy-4-iodo, and 4-thio derivatives) as potential substrates of D-galactose oxidase. These syntheses involved nucleophilic displacement of the 4-(p-bromophenylsulfonyl)oxy group of appropriate D-glucose derivatives, although the more reactive (trifluoromethylsulfonyl)oxy group was also utilized as a novel leaving-group. Formation of the bromo and iodo derivatives was accompanied by appreciable halogen exchange and a resulting overall retention of configuration, and formation of the thio compound was attended by competing reactions. Optical rotatory characteristics of the halogeno compounds, their triacetates, and tribenzoates are described, and “anomalous” behavior of the last group is noted.     

Analysis of acylcarnitines as their N-demethylated ester derivatives by gas chromatography-chemical ionization mass spectrometry.

A novel approach to the analysis of acylcarnitines has been developed. It involves a direct esterification using propyl chloroformate in aqueous propanol followed by ion-pair extraction with potassium iodide into chloroform and subsequent on-column N-demethylation of the resulting acylcarnitine propyl ester iodides. The products, acyl N-demethylcarnitine propyl esters, are volatile and are easily analyzed by gas chromatography-chemical ionization mass spectrometry. For medium-chain-length (C4-C12) acylcarnitine standards, detection limits are demonstrated to be well below 1 ng starting material using selected ion monitoring. Well-separated gas chromatographic peaks and structure-specific mass spectra are obtained with samples of synthetic and biological origin. Seven acylcarnitines have been characterized in the urine of a patient suffering from medium-chain acyl-CoA dehydrogenase deficiency.     

Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry.

We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.     

Use of t-butyldimethylchlorosilane/imidazole reagent for identification of molecular species of phospholipids by gas-liquid chromatography mass spectrometry

The t-butyldimethylsilyl derivatives of 1,2-diakyl, 1-alk-1'-enyl-2-acyl, 1-alkyl-2-acyl and 1,2-diacyl glycerols were analysed with a gas chromatograph mass spectrometer system. The characteristic fragment ions were as follows. The molecular weight determining ion was [M-57]+, which was formed by cleavage of the t-butyl radical from the molecular ion. The nature of the alk-1'-enyl residue could be determined by the presence of ions at [RCH-CH 56]+ and [RCH = CH + 130]+ (RCH = CH = alk-1'-enyl), and the alkyl residue by the ion at [R + 130]+(R = alkyl group). Ions giving information about the acyl group, [RCO]+, [RCO + 74]+ and [M-RCH = CHO, -RO or -RCOO]+ were also observed. The mass spectra of pairs of trimethylsilyl and t-butyldimethylsilyl derivatives showed differences in several respects. The t-butyldimethylsilyl derivatives gave more effective information for elucidating the structure of phosphoglycerides.     

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.     

Characterization of gibberellins from dark-grown Phaseolus coccineus seedlings by gas-liquid chromatography and combined gas chromatography-mass spectrometry

Summary An extract from 6000 dark-grown Phaseolus coccineus seedlings was purified by countercurrent distribution and G-10 Sephadex followed by gradient elution from a silicic acid partition column with increasing amounts of ethyl actetate in n-hexane. 25 fractions were collected and tested with the barley-aleurone, Tan-ginbozu dwarf-rice, lettuce, cucumber, dwarf-pea, d-1, d-2, d-3 and d-5 maize, oat first-internode, and sugarcane-spindle bioassays. Major gibberellin (GA)-like activity was detected in fractions 4 (500g GA₃-equivalents) and 12–13 (270 g GA₃-equivalents) with smaller amounts in fractions 6, 8–9, 15–16, 18, 20, 23 and 25. The extracts were also applied to AMO-1618=dwarfed Ph.-coccineus seedlings. Fractions 4, 8 and 12 promoted the growth of both light- and dark-grown seedlings. GA₁, GA₃, GA₄ and GA₈ were active in the Phaseolus bioassay but GA₈-glucoside was inactive.The biological and chromatographic properties of fractions 4, 8–9 and 12–13 correspond with those of GA₄, GA₁₉ and GA₁. The identity of GA₄ in fraction 4 was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of the methyl ester and the trimethylsilyl ether of the methyl ester. Gasliquid-chromatography peaks corresponding to these derivatives of GA₁₉ and GA₁ were detected on QF-1 and SE-33 columns but their intensities were too weak to permit conclusive identification by GC-MS.Supported by an S.R.C. StudentshipSupported by a NATO Grant.Supported by NRC Grant A-5727.     

Characterization of prednisone, prednisolone and their metabolites by gas chromatography—mass spectrometry

Human urinary metabolites of the synthetic corticosteroids prednisone and prednisolone were detected in the course of gas chromatographic steroid profiling as methoxime-trimethylsilyl derivatives. Metabolites were provisionaly identified by combined gas chromatography—mass spectrometry. The major metabolites were 11-keto/11-hydroxy conversion products, 20-hydroxy and 4,5-dihydro analogues of the parent drugs. Cortisone, 6-hydroxy and fully saturated A-ring compounds were minor metabolites. Retention indices and mass spectral data are presented.     

Measurement of plasma testosterone by gas chromatography-negative-ion mass spectrometry using pentafluoropropionic derivatives

Plasma testosterone was measured by gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-MS). The testosterone was extracted from plasma using home-made Extrelut columns and diethyl ether elution. It was quantified as the pentafluoropropionate (PFP) derivative by selected-ion monitoring at m/z 560 (testosterone) and 563 (d₃-testosterone), accounting for about 34% of the total ion. The characteristics of the method were: extraction recovery about 95%; linearity over the range 1.7–71.5 nmol l⁻¹ with linear regression equation y = 1.41x + 0.0217, r = 0.999; detection limit 3.5 fmol injected with a signal-to-noise ratio of 7.4; within-day variation, 3% for GC-MS, and 5.8% for the whole process; day-to-day coefficient of variation, 6.6–11%, depending on the concentrations. There was a good correlation between the results obtained by GC-MS and RIA (r = 0.994), but the GC-MS values were significantly lower (p < 0.05) than those obtained by RIA.     

Characterization of trimethylsilyl derivatives of cerebrosides by direct inlet-chemical ionization mass spectrometry

Submicrogram quantities of trimethylsilyl derivatives of cerebrosides obtained from the spleen of a patient with Gaucher's disease and from bovine brain were analyzed by direct probe inlet-chemical ionization mass spectrometry, using isobutane as the reagent gas. Quasimolecular ions (QM+, M + 73) and other recognizable fragment ions produced by the successive elimination of trimethylsilanol and sugar residue gave useful information about fatty acid compositions. These ions could also be utilized for qualitative analyses of the molecular species of cerebrosides. Cerebrosides with non-hydroxy and hydroxy fatty acids could be discriminated from each other by comparing the intensities of their quasimolecular ions. Cerebrosides with saturated and monounsaturated fatty acids could also be discrimnated from each other, because the mass number decreased by two mass units in cerebrosides with monounsaturated fatty acids. It was concluded that structural information and molecular species determination could be obtained from small amounts of purified cerebrosides.     

Characterization of oligosaccharides from industrial fermentation residues by matrix-assisted laser desorption/ionization,electro spray mass spectrometry,and gas chromatography mass spectrometry

We report here the preliminary characterization of oligosaccharides present in an enzyme-treated industrial fermentation residue using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ion trap mass spectrometry (ESI-ITMS), and gas chromatography mass spectrometry (GC-MS). After sample cleaning with carbon graphite columns, analysis of oligosaccharides present in the sample using MALDI-TOF-MS resulted in identification of molecular ions representing sodiated hexose and pentose oligo/polysaccharides. The GC-MS analyses revealed that the signals observed in the mass spectrum for hexose oligomers represent linear structures, whereas the pentose oligomers were identified as arabinoxylans with a (1-->4) linked Xylp backbone where the Xylp residues were either not substituted or singly substituted with Araf branching residues at positions C-2 or C-3 of the Xylp ring. Analyses by ESI-ITMS of the signals corresponding to arabinoxylan oligosaccharides with four and five monosaccharide residues showed the presence of isomeric structures differing in degree of branching and localization of the branched residue along the Xylp backbone.     

Absence of arabinose in bovine brain hyaluronic acid as analysed by gas-liquid chromatography and mass spectrometry

No arabinose could be detected in a simple of bovine brain hyaluroni acid when examined by gas—liquid chromatography and mass spectrometry. A control, containing only one part arabinose per 1000, gave three readily discernible arabinose components.