A RP-HPLC method was established to separate B3 and B3iso desamido human insulin in recombinant human insulin injection and HPLC-QTOF mass spectrometry was used to identify the amino acid sequence.On the base of the method of recombinant human insulin injection in Chinese pharmacopeia(edition 2010,second part), the pH of mobile phase was optimized using 0.2 mol/L sulfate buffer solution with pH 3.6 acetonitrile(90∶10) as phase A,and acetonitrile-water(50∶50) as phase B by gradient elution.The peak 1 and peak 2 were collected,desalted and concentrated,then digested with V8 enzyme,and reduced with TCEP,then identified by LC-MS/MS.The peaks were identified as B3 and B3iso desamido human insulin.Using the optimized method to compare the related substance results of recombinant human insulin injection on market,the test results revealed that different brands of insulin injection had different degree of B3 and B3iso desamido human insulin.The optimized method did not influence other impurities determination.It is useful for effectively monitoring the increasing trend of deamido insulin during storage.