Determination of the intermediates in glycolysis and tricarboxylic acid cycle with an improved derivatization strategy using gas chromatography-mass spectrometry in complex samples.

Traditional Chinese medicine (TCM) is recognized as a complex matrix, and improved analytical methods are crucial to extract the key indicators and depict the interaction and alteration of the complex matrix. Shenqi Fuzheng Injection (SQ), a water extract of Radix Codonopsis and Radix Astragali, has demonstrated preventative effects on myotube atrophy induced by chemotherapeutic agents. To achieve the improved analytical capability of complex biological samples, we established a highly reproducible, sensitive, specific, and robust gas chromatography-tandem mass spectrometry (GC-MS) method to detect glycolysis and tricarboxylic acid (TCA) cycle intermediates with optimized factors in the extraction and derivatization process. Our method detected fifteen metabolites and covered most intermediate metabolites in glycolysis and TCA cycles, including glucose, glucose-6-phosphate, fructose-6-phosphate, dihydroxyacetone phosphate, 3-diphosphoglycerate, phosphoenolpyruvate, pyruvate, lactate, citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate, and malate. Through methodological verification of the method, it was found that the linear correlation coefficients of each compound in the method were greater than 0.98, all of which had lower limits of quantification, the recovery rate was 84.94-104.45%, and the accuracy was 77.72-104.92%. The intraday precision was 3.72-15.37%, the interday precision was 5.00-18.02%, and the stability was 7.85-15.51%. Therefore, the method has good linearity, accuracy, precision, and stability. The method was further applied to study the attenuating effects of the SQ in a chemotherapeutic agents-induced C2C12 myotube atrophy model to evaluate the changes in the tricarboxylic acid cycle and glycolytic products under the action by the complex systems of TCM and disease model. Our study provided an improved method to explore TCM's pharmacodynamic constituents and action mechanisms.