Abstract: OBJECTIVE To rapidly identify the impurity peak detected in tacrolimus by 2D-LC-IT-TOF-MS. METHODS Firstly, the chromatography was Thermo Hypersil GOLD C₁₈(4.6 mm×150 mm, 3 μm) column and the mobile phase containing a mixture of acetonitrile-1,1-dimethylethyl-phosphoric acid. Then a InertSustain C₁₈(2.1 mm×50 mm, 2 μm) column was used with 0.1% formic acid-acetonitrile(30:70) as mobile phase in a gradient elution mode. Electrospray ionization source was tested in positive ion modes, nebulized gas flow was 1.5 L·min^-1. Dry gas flow was 10 L·min^-1. The desolvation tube temperature was kept at 200℃. Relatedstances were characterized according to multi-level MS behaviors. RESULTS The material structure of chromatographic peak at specific retention time in tacrolimus raw material was deduced, and the detected peak was not ascomycin impurity provided by USP but was tacrolimus isomer Ⅰ with pharmacological activity, content should be involved in calculation. CONCLUSION The established method is suitable for rapid identification of impurities in tacrolimus, which can be applied as a useful analytical tool for quality control and drug process optimization.