Application of acetate derivatives for gas chromatography–mass spectrometry: Novel approaches on carbohydrates, lipids and amino acids analysis (original) (raw)
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Journal of Chromatography B, 2003
Carbohydrates, either alone or as constituents of glycoproteins, proteoglycans and glycolipids, are mediators of several cellular events and (patho)physiological processes. Progress in the ''glycome'' project is closely related to the analytical tools used to define carbohydrate structure and correlate structure with function. Chromatography, electrophoresis and mass spectrometry are the indispensable analytical tools of the on-going research. Carbohydrate derivatization is required for most of these analytical procedures. This review article gives an overview of derivatization methods of carbohydrates for their liquid chromatographic and electrophoretic separation, as well as the mass spectrometric characterization. Pre-column and on-capillary derivatization methods are presented with special emphasis on the derivatization of large carbohydrates.
Journal of Chromatography A, 2013
Gas chromatographic analysis of complex carbohydrate mixtures requires highly effective and reliable derivatisation strategies for successful separation, identification, and quantitation of all constituents. Different single-step (per-trimethylsilylation, isopropylidenation) and two-step approaches (ethoximation-trimethylsilylation, ethoximation-trifluoroacetylation, benzoximation-trimethylsilylation, benzoximation-trifluoroacetylation) have been comprehensively studied with regard to chromatographic characteristics, informational value of mass spectra, ease of peak assignment, robustness toward matrix effects, and quantitation using a set of reference compounds that comprise eight monosaccharides (C 5 -C 6 ), glycolaldehyde, and dihydroxyacetone. It has been shown that isopropylidenation and the two oximation-trifluoroacetylation approaches are least suitable for complex carbohydrate matrices. Whereas the former is limited to compounds that contain vicinal dihydroxy moieties in cis configuration, the latter two methods are sensitive to traces of trifluoroacetic acid which strongly supports decomposition of ketohexoses. It has been demonstrated for two "real" carbohydrate-rich matrices of biological and synthetic origin, respectively, that two-step ethoximation-trimethylsilylation is superior to other approaches due to the low number of peaks obtained per carbohydrate, good peak separation performance, structural information of mass spectra, low limits of detection and quantitation, minor relative standard deviations, and low sensitivity toward matrix effects.
Biochemistry, 2003
A GC/MS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GC/MS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GC/MS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or diand trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GC/MS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.
European Journal of Mass Spectrometry, 2008
The sugar fraction of α-1 acid glycoprotein (AGP) was studied using porous graphitized carbon (PGC) chromatography coupled to mass spectrometry. Electrospray ionization provides efficient control over fragmentation; at low collision energy only molecular species were observed, allowing accurate oligosaccharide profiling. PGC chromatography was useful separating 18 sugars differing in monosaccharide composition. Most of these were separated into several isomeric forms; altogether 49 different oligosaccharides were found in AGP.
Mass spectrometry of 2-acetamido-2-deoxy-glycose containing disaccharides
Tetrahedron, 1971
Ah&r&-The mass spectra of six trimethylsilyl 2-acetamido-2deoxy-aldohexosyl-aldohexoses with (1 + 2), (1 -+ 3). (1 -+ 4) and (I -+ 6) glycosidic linkages were compared. The spectra could be divided in two main groups on the basis of the ratio of the intensities of the peaks at m/e 217 and m/e 204 (217/2&Q: (1 -, 2), (1 + 3) disaccharides and (1 + 4), (I + 6) disaccharides. (1 + 2) and 41 + 3) disaccharides could be distinguished on the basis of some ratios of peak intensities, (1 + 4) and (1 + 6) disaccharides on the basis of the presence or absence of one intense peak (m/e 552). Further, the mass spectrum of an aldo-hexosy1-Q -+ 6)-2-acetamido-2-deoxy-aldohexose is discussed. In all cases the sequence of the monomers c&d bc determined by using the sum of the intensities of two related peaks.
Characterization ofO-trimethylsilyl oximes of disaccharides by gas chromatography-mass spectrometry
Chromatographia, 2002
Gas chromatography (GC) data (linear retention indices and relative areas) and mass spectra (most representative m/z fragments) of 12 reducing trisaccharides as trimethylsilyl oximes (TMSO) and four non-reducing as trimethylsilyl (TMS) ethers have been described for the first time and related to their structural features. Some trends have been observed: earlier elution of non-reducing compounds and fructotrioses; aldotrioses bearing the reducing end with link in position 6 showing the highest retention. Abundance of several fragment ions and their ratios were useful for trisaccharide characterization; some of these features seem to be useful for the characterization of new trisaccharides.
Oligosaccharide analysis by graphitized carbon liquid chromatography–mass spectrometry
Analytical and Bioanalytical Chemistry, 2009
Structural analysis of complex mixtures of oligosaccharides using tandem mass spectrometry is regularly complicated by the presence of a multitude of structural isomers. Detailed structural analysis is, therefore, often achieved by combining oligosaccharide separation by HPLC with online electrospray ionization and mass spectrometric detection. A very popular and promising method for analysis of oligosaccharides, which is covered by this review, is graphitized carbon HPLC-ESI-MS. The oligosaccharides may be applied in native or reduced form, after labeling with a fluorescent tag, or in the permethylated form. Elution can be accomplished by aqueous organic solvent mixtures containing low concentrations of acids or volatile buffers; this enables online ESI-MS analysis in positive-ion or negative-ion mode. Importantly, graphitized carbon HPLC is often able to resolve many glycan isomers, which may then be analyzed individually by tandem mass spectrometry for structure elucidation. While graphitized carbon HPLC-MS for glycan analysis is still only applied by a limited number of groups, more users are expected to apply this method when databases which support structural assignment become available.
Journal of Chromatography A, 1993
Pneumatically assisted electrospray was demonstrated to be a powerful ionization source for the analysis of oligosaccharides. A mass spectrometer was interfaced to an HPLC system, using this interface, to determine oligosaecharides from the enzymatic digestion of heparin separated on a reversed-phase column. To set up the technique, and particularly to clarify the ionization process, purified disaccharides, from enzymatic digestion of chondroitin sulphates, were measured. The use of a suitable counter ion in the mobile phase, tetrapropylammonium (TPA), to optimize the HPLC separation, gave, with sulphated di-and oligosaccharides, adducts [M + nTPA -(n + m)H] m-, which were unexpectedly stable to fragmentation; molecular ions [M -(n + 1)H] n-, in the presence of the counter ion, were observed only with desulphated or monosulphated disaccharides. The stability of the adducts and the use of a deuterated ion-pair reagent permitted an exact evaluation of the molecular masses of disaccharides and 'oligosaccharides of unknown structure. Spectra obtained in the absence of the counter ion contained singly or multiply charged molecular ions and fragmentation ions mainly from loss of the sulphate groups; under these ionization conditions the exact mass determination and interpretation of the spectra were difficult. After removal of the counter ion, tandem mass spectra could be obtained with some interesting data for the characterization of these molecules. Complete spectral analyses were performed with amounts of samples of 50/~g but, using microbore columns, one twentieth of this amount may give good spectra.