Abstract: OBJECTIVE To establish a method for determination of related substances in metadoxine. METHODS The HPLC-ELSD method was performed on Agilent Zorbax SB-C₁₈ column(250 mm×4.6 mm, 5 μm) with the mobile phase of 0.2 mol·L^-1 trifluoroacetic acid-methanol(92∶8), the folw rate was 1.0 mL·min^-1 and the drift tube temperature of the detector was 55 ℃(low temperature design), the gas pressure was 420 Pa. RESULTS The main impurities and the metadoxine had a good separation, the established method was effective to detect the non-UV-absorbing impurities. CONCLUSION The method is simple, fast, accurate, and may be used in quality control of metadoxine.