Determination of amino acids in colon cancer cells by using UHPLC-MS/MS and [U-¹³C₅]-glutamine as the isotope tracer

Rapid and simple quantitative analysis of intracellular metabolites is a critical tool for monitoring the alteration of biologically significant metabolites in cell lines or in vivo. We established an ultra-high performance liquid chromatography (UHPLC) method, equipped with hydrophilic interaction liquid chromatography (HILIC) column coupled to tandem mass spectrometry (MS/MS) for the simultaneous determination of 19 amino acids and 2 related derivatives in human cell lines. Chromatographic separation was achieved within 20 min using a BEH amide column, with aqueous mobile phase containing 20 mM ammonium acetate and 20 mM ammonium hydroxide, and acetonitrile as the organic mobile phase. Amino acids were analyzed in positive ion multiple reaction monitoring (MRM) mode without the need of derivatization. Intra- and inter-day precisions were less than 13.7%. The method was successfully applied to simultaneously detect the 21 compounds in a human colon cancer cell line DLD1. Moreover, metabolite fate of glutamine-derived carbons into amino acids in DLD1 cells was successfully traced by using [U-¹³C₅] glutamine as the isotope tracer. Metabolic consequences of glutaminolysis inhibition on amino acid metabolism were evaluated. Analysis of ¹²C- and U-¹³C-labeled amino acids revealed the significantly decreased incorporation of [U-¹³C₅]-glutamine derived carbons into aspartate, alanine and ornithine, indicating impaired metabolic flux via the tricarboxylic acid cycle and the urea cycle.