title: Simplified absolute metabolite quantification by gas chromatography-isotope dilution mass spectrometry on the basis of commercially available source material.
abstract: In the field of metabolomics, GC-MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or extracellular metabolites is nowadays mostly accomplished by application of diverse LC-MS techniques. Only few groups have so far adopted GC-MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive. Here we present an improved and simplified gas chromatography-isotope dilution mass spectrometry (GC-IDMS) protocol for the absolute determination of intra- and extracellular metabolite levels. Commercially available (13)C-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed.
authors.affiliation: Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany. vielhauer@ibvt.uni-stuttgart.de
authors.forename: Oliver
authors.initials: O
authors.lastname: Vielhauer
authors.forename: Maksim
authors.initials: M
authors.lastname: Zakhartsev
authors.forename: Thomas
authors.initials: T
authors.lastname: Horn
authors.forename: Ralf
authors.initials: R
authors.lastname: Takors
authors.forename: Matthias
authors.initials: M
authors.lastname: Reuss
mesh_terms: D000693:Anaerobiosis; D002247:Carbon Isotopes / Q000032:analysis; D002457:Cell Extracts / Q000737:chemistry; D000460:Chlorophyta; D004867:Equipment Design; D005110:Extracellular Space / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D042541:Intracellular Space / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D055432:Metabolomics / Q000379:methods* / Q000592:standards; D010091:Oximes / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D014297:Trimethylsilyl Compounds
chemical_list: D002247:Carbon Isotopes; D002457:Cell Extracts; D010091:Oximes; D014297:Trimethylsilyl Compounds; C005514:N,N'-monomethylenebis(pyridiniumaldoxime)