Abstract: Objective To establish a stable, reliable and suitable preparation method of serum metabolomics for the analysis of large samples. Methods The ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS) method was utilized to comparatively analyze the preparation methods of human serum for metabolomic analysis, including direct three-fold acetonitrile precipitation(Method 1), concentration and reconstitution after three-fold acetonitrile precipitation(Method 2), and one-fold acetonitrile precipitation(Method 3), and then the biological quality control sample was used to investigate the precision, stability and repeatability of instruments. Results In ESI+ and ESI- scanning, Method 2 yielded the largest characteristic number of metabolites, Method 1 yielded the least number, and Method 3 was in between. No statistically significant difference in the mean peak intensity of the ion peaks was found between Method 1 and Method 3 in the ESI+ and ESI- scanning (all P>0.05), but the results of both methods were greater than that of Method 2 (all P<0.05). The peak intensity coefficient of variation(CV) values of the most ion peaks of Method 2 and Method 3 were less than 25.00%, whereas that of Method 1 was more than 25.00%. The results indicated that the metabolite extraction of Method 3 had better repeatability, and this method could guarantee the proper detection of both the total characteristic number and the peak intensity of metabolites. The precision, stability and repeatability of the instrument during the experiment were all good. Conclusion The serum preparation method that protein molecules are first one-fold acetonitrile precipitated and then intercepted through the ultrafiltration tube can comprehensively retain and detect small molecule compounds in serum samples with a simple and nice reproducible way, and is suitable for metabolomics research.

Key words: Serum, UPLC, QTOF-MS, Metabolomics, Preparation method