Metabolic fingerprinting using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.

Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOF-MS) is applied to the comparative metabolic fingerprinting of physiological fluids. Stable isotope-labeled internal standards plus norvaline serve as extraction standards and are added to the blanks, controls and patient samples prior to protein precipitation with methanol. The extracts are evaporated to complete dryness and derivatized in two steps using methoximation with methoxylamine hydrochloride (MeOx) and silylation with N-methyl-N-trimethylsily-trifluoroacetamide (MSTFA). Between derivatization steps a second internal standard containing odd-numbered, saturated straight chain fatty acids is added for quality control and to normalize retention time shifts. After GC × GC-TOF-MS analysis raw data are processed, aligned, and combined in one data matrix for subsequent statistical evaluation. Both a custom-made and the NIST 05 library are used to preliminarily identify significant metabolites. For verification purposes, commercial standards are run individually. Absolute quantification of selected metabolites is achieved by using a multi-point calibration curve and isotope-labeled internal standards.