Gas chromatography with electron ionization and mass spectrometry (GC–EI-MS) detection is a workhorse among analytical techniques
in metabolomics. A major challenge in the utilization of GC–EI-MS in metabolomics is the identification of unknowns. The recent
availability of high resolution, accurate mass, time-of-flight mass spectrometry (TOF-MS) systems provides great potential
for identifying unknowns by reducing uncertainty in possible formulas and enhancing detection continuity between sample sets.
A workflow using GC–EI-MS spectra for library identification, with molecular formula information for unknowns provided by
chemical ionization-mass spectrometry (CI-MS) and accurate mass analysis was used for the analysis of blood plasma.
Metabolomics provides a foundation for quantitative, comparative biology and is indispensable for the comprehensive characterization
of molecules and differential analysis of populations. It entails instrumental detection, characterization, and quantification
of small molecules (molecular weight < 1500 Da) produced, or transformed in the cells of living organisms (1,2). Although
no single instrument is capable of fully profiling the metabolome, the unique capabilities of time-of-flight mass spectrometry
(TOF-MS) make it an ideal tool for metabolomic studies. Gas chromatography–time-of-fight-mass spectrometry (GC–TOF-MS) is
a powerful approach for unbiased metabolic profiling and quantitation of a diverse array of metabolites (3,4). High-resolution
TOF-MS provides additional benefits such as mass accuracies below 1 ppm for robust formula determinations and resolving power
in excess of 50,000 for resolution of isobars and minimization of background interferences. A workflow that combines accurate
mass electron ionization (EI) and chemical ionization (CI) high-resolution TOF-MS for confident characterization of different
compound classes was used for the analysis of human blood plasma. It includes recommendations for sample preparation, analytical
conditions, and data analysis.
The majority of metabolites in blood plasma are labile and exhibit low volatility making their analysis by GC difficult. This
is addressed by using a two step derivatization procedure involving the treatment of samples with O-methylhydroxylamine hydrochloride
in pyridine (MEOX) followed by silylation with N-methyl-N-trifluoroacetamide/1% trimethylchlorosilane (MSTFA) (5). This two-step procedure minimizes the number of possible forms of
reducing sugars as shown for glucose in Figure 1. Treatment of glucose with MSTFA (Figure 1b) results in four different forms
(α- and β-furanose and pyranose trimethylsilyl derivatives), while the two-step procedure typically produces two geometric
isomers of the open chain structure of the monosaccharide (Figure 1a). Note that one isomer is produced preferentially in
this two-step derivatization of glucose. This not only simplifies the analysis of blood samples, but also increases the likelihood
of compound detection.
Figure 1: (a) Extracted ion chromatogram showing one major product for the two-step derivatization of glucose. (b) Products
for the one-step MSTFA derivatization of glucose.
GC–MS compound characterization in metabolomics relies primarily on retention time and spectral similarity. A higher confidence
for feature identification can be achieved through the addition of accurate mass formula determinations for molecular ions,
quasimolecular ions, and fragments. Key to the success of analyte identification was a workflow that included both EI and
CI high-resolution TOF-MS data acquisition (Figure 2) (6). CI high-resolution TOF-MS data were critical in characterizing
compounds that produced poor spectral matches in relation to commercially available libraries or did not exhibit molecular
ions in their EI high-resolution TOF-MS mass spectra.
Figure 2: General workflow.