Abstract: It is important that to analysis of the isotopic abundance and chemical purity of Sudan I-D₅, which is the internal standard of isotope dilution mass spectrometry. The isotopic abundance of Sudan I-D₅ is detected by “mass cluster” classification method and LC-MS. The repeatability and reproducibility experiments were carried out by using different mass spectrometers and different operators. The RSD was less than 0.1%, so the repeatability and reproducibility were satisfactory. The accuracy and precision of the isotopic abundance analysis method was good with the results of F test and t test. The high performance liquid chromatography (HPLC) had been used for detecting the chemical purity of Sudan I-D₅ as external standard method.