Taking the detection of three fat-soluble vitamins in human serum as an example,an analytical method for human endogenous metabolites was explored.A mixed human serum matrix was prepared by adding standard substances and internal standards followed by background subtraction of endogenous substances,and another simulating matrix substrate was made of bovine serum albumin(BSA).They both were detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS).Results showed that the limits of detection for vitamin A,D3 and E in the mixed human serum matrix were 4.2,0.8 and 67.2 ng/mL,while the limits of quantitation were 13.7,2.6 and 221.9 ng/mL,respectively.There existed good linear relationships for the two methods with good accuracy and reproducibility,and their correlation coefficients were both above 0.996.The recoveries for the actual samples at low and high spiked concentrations were between 90.7% and 112%,with relative standard deviations(RSD) of 1.0%-4.5%.There were no statistical differences between the two methods for 40 unknown samples.Therefore,for the endogenous metabolites from human body,the accurate analysis on the matrix matched between the standard sample and the sample to test could be achieved by adding the standard substance and deducting the background from the human serum matrix,which is beneficial to the convenient diagnosis on clinical related diseases.