Optimization of a quantitative protocol for the intermediate metabolites of the glycolysis pathway in human serum using gas chromatography-mass spectrometry

Glycolysis takes place in all cells of the body and plays an important role in the metabolism of the organism. The detection of intermediate metabolites in the glycolysis pathway is critical in understanding metabolic alterations that occur in many metabolic disorders. The intermediate metabolites of glycolysis such as glycerate 3-phosphate (3PG), beta-fructose 6-phosphate (F6P) and alpha-glucose 6-phosphate (G6P) in biological samples have poor stability and low abundance, which makes their separation and detection more challenging. In this work, an optimal protocol for detecting 10 glycolysis metabolites in serum samples was developed using gas chromatography triple quadrupole mass spectrometry. Single factor experiment and response surface methodology were used to optimize the pretreatment of serum samples. The optimal conditions were as follows: the volume of the derivatization reagent, 100 mu L; extraction solvent, 80% methanol; derivatization temperature, 80 degrees C; and derivatization time, 60 min. The protocol showed an acceptable linearity (R-2 >= 0.9872), the lowest detection limit (0.0002-0.2382 mu g mL(-1)), the limits of quantitation (0.0007-0.7940 mu g mL(-1)), and the satisfactory intra-day (RSD% <= 13.67%) and inter-day precision (RSD% <= 12.01%). The results of the stability test showed that it was a better choice to determine metabolites during 2 months when the serum was stored at -80 degrees C in order to avoid the change in 3PG and G6P. Furthermore, the metabolites in serum were relatively stable for detection (RSD% < 18.77%) after 4 freeze-thaw cycles. Finally, this protocol was applied to the quantitative analysis of glycolysis metabolites from gastric cancer patients, and was beneficial to find out the metabolic changes of the glycolysis pathway and explain the pathogenesis of the disease.