Quantification and isotope abundance determination of 13C labeled intracellular sugar metabolites with hydrophilic interaction liquid chromatography

Abstract

Metabolic flux analysis (MFA) using stable isotope labeled tracers is a powerful tool to estimate fluxes through metabolic pathways. It finds applications in studying metabolic changes in diseases, regulation of cellular energetics, and novel strategies for metabolic engineering. Accurate and precise quantification of the concentration of metabolites and their labeling states is critical for correct MFA results. Utilizing an ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) system, an analytical method for simultaneously quantifying the concentration of sugar metabolites and their mass isotopologue distribution (MID) was developed. The method performs with good linearity and coefficient of determination (R²) > 0.99, while the detection limit ranged from 0.1 to 50 mg L⁻¹. Seven sugar metabolites were detected in a labeled Brevibacterium flavum sample using the method. The detected quantities ranged from 6.15 to 3704.21 mg L⁻¹, and ¹³C abundance was between 12.77% and 66.67% in the fermentation fluid and 16.28% and 91.93% in the bacterial body. Overall, the method is efficient, accurate, and suitable for analysis of labeled sugar metabolites in ¹³C MFA studies.