Abstract: 2,3-Pyrizine dicarboxylic anhydride of active pharmaceutical ingredient (API) was easily hydrolyzed to 2,3-pyrazine dicarboxylic acid of related substance, and it could not be directly determined by high performance liquid chromatography (HPLC). Based on this, after derivatizing 2,3-pyrazine dicarboxylic anhydride with methanol, the main component and impurities of 2,3-pyrazine dicarboxylic acid and 2-pyrazine formic acid in API of 2,3-pyrazine dicarboxylic anhydride were determined by HPLC. The sample and methanol were added in the mass to volume ratio (mg:mL) of 10:7, followed by ultrasonic derivatization for 20 min, and then analyzed by HPLC after dilution. YMC Triart C₁₈ column was used as the stationary phase, and a mixed solution of 0.5% (φ) trifluoroacetic acid solution and methanol at different volume ratios was used as the mobile phase for gradient elution, with detection wavelength of 270 nm. As shown by the results, the resolutions among 2,3-pyrazine dicarboxylic acid, 2-pyrazine formic acid and 2,3-pyrazine dicarboxylic anhydride were both greater than 1.5. Linear relationships between values of peak area and mass concentration of them were found in definite ranges, with detection limits of 0.057 5, 0.054 8, 0.010 2 mg·L^-1, respectively. The mixed control solution and test solution were analyzed 6 times, and RSDs of the determined values were less than 2.0%. The spiked recoveries of 2,3-pyrazine dicarboxylic acid and 2-pyrazine formic acid were 95.2% and 98.3%, respectively. This proposed method was applied for the analysis of three batches of actual samples, and the mass fraction of 2,3-pyrazine dicarboxylic anhydride and 2,3-pyrazine dicarboxylic acid were 97.83%-97.93% and 2.07%-2.17%, respectively, while 2-pyrazine formic acid was not detected.