Accurate Determination of 13C Isotopic Abundance of Free Intracellular Amino acids with Low Concentration by GC-MS-Selective Ion Monitoring Method

13C isotopic abundance of intracellular free amino acid with a characteristic of fast-turnover can quickly reflect the changes in intracellular metabolic state. However, the concentration of intracellular free amino acid was low and the existed 13C isotope detection method based on GC-MS could not satisfy the requirement with full scan mode. In this study, selected ion monitoring method with theoretically higher detection accuracy was used for the analysis of 13C isotopic abundance of free intracellular amino acid. The fracture law of different amino acids was investigated by full scan mode, and also a characteristic fragment library for the 16 kinds of free intracellular amino acids was established. According to this characteristic fragment library, only the specific m/z signal was detected in the sample analysis, which realized the selected ion monitoring and improved the quality of signal. The results of amino acid standards indicated that, in comparison with the full scan mode, the signal-to-noise ratio (S/N), measurement precision and accuracy were improved by 17, 2 and 3.8 times, respectively. In the analysis of coenzyme Q10 producing strains of samples, 13C isotopic abundance of 8 kinds of free intracellular amino acids were successfully detected. This method was quite feasible for the detection of 13C isotopic abundance of the intracellular free amino acids in cell metabolism research.

Graphical abstract13C-isotopic abundance of intracellular free amino acids were successfully accomplished by gas chromatography-mass spectroscopy with full scan and SIM mode for 13C metabolic flux analysis. The optimization of instrument parameters and derivative conditions were investigated, and 16 kinds of amino acids were effectively separated under the optimum derivative and GC/MS parameters conditions.Figure optionsDownload full-size imageDownload as PowerPoint slide