Investigation into oximation methods to improve the selective detection of small α-ketoacids in cell extracts by...

Small α-ketoacids are important metabolic intermediates that influence metabolic states associated with disease. Measuring α-ketoacid levels and labeling in stable-isotope tracing studies using chromatography coupled to mass spectrometry is sometimes challenging due to their small mass, low cellular abundance, chemical instability, and enzymatic utilization post-quenching. Chemical derivatization is often employed to increase mass-to-charge, improve chromatographic separation in complex samples, and to stabilize α-ketoacids for detection by GC- or LC-MS. Methoximation is one of the most used derivatization strategies to modify α-keto groups present on metabolic α-ketoacids for GC-EI-MS. However, methoxime groups do not significantly increase analyte mass nor contribute to retention on the mid-polar GC columns widely used for metabolic labeling studies. In this study, we evaluate different oximation reagents to improve the selective detection of small α-ketoacid oxime-TBDMS derivatives by GC-EI-MS. We find that oximation using hydroxylamine, ethoxyamine, or O-benzylhydroxylamine offer comparable performance to methoxyamine with distinct effects on column retention and main fragment ion mass-to-charge. We also demonstrate how these approaches may be applied to cell extracts. Our results highlight hydroxylamine or O-benzylhydroxylamine as preferred derivatization reagents to increase alpha-ketoacid mass-to-charge or increase column retention, respectively.