Solid-phase analytical derivatization for gas-chromatography–mass-spectrometry-based metabolomics

A novel derivatization method for gas chromatography/mass spectrometry (GC/MS)-based metabolomics was developed, based on solid-phase analytical derivatization (SPAD) with methoximation followed by trimethylsilylation. This SPAD method realized derivatization on solid phases combining strong anion exchange with strong cation exchange. To omit a sample condensation process, GC/MS injection was performed using a large-volume injection mode. This mode uses a stomach-shaped insert, and enables a large quantity of sample to be vaporized and introduced into the GC/MS system. In the present study, several parameters were investigated for each SPAD step. The optimal derivatization conditions were determined to be 3-min-methoximation with 5 μL of >5% methoxyamine solution, and 10-min-trimethylsilylation with 25 μL of N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA). Derivatized analytes were effectively eluted with 25 μL of n-hexane. The influences of coexisting substances were also investigated. Coexisting saccharides did not significantly affect the derivatization of analytes. Moreover, saccharides were efficiently washed out using 80% (v/v) acetonitrile in water. The influences of coexisting sodium chloride were negated by dilution of the sample solution with water. The developed method enables the derivatization of both anionic and cationic metabolites, and high-throughput sample preparation. The coverage of detectable metabolites for the developed method was similar to that of the conventional method. This is the first report of a SPAD-based human plasma metabolome analysis protocol.