HPLC determination of related substances and identification of main impurity in insulin detemir

Objective: To explore and establish a method for determination of purity of insulin detemir and to identify the main impurity. Methods: The related substances in insulin detemir were separated and analyzed by RP-HPLC. The column was Protronavi C4( 250 mm ×4.6 mm,5 μm,300 ); the mobile phase A was0. 2 mol·L- 1sodium sulphate buffer( pH 2. 3)- acetonitrile( 82∶ 18); the mobile phase B was water- acetonitrile( 40∶ 60) with gradient elution( 0 min,35% B; 45 min,67% B; 65 min,67% B; 66 min,35% B; 80 min,35% B). The flow rate was1. 0 mL·min- 1; the detection wavelength was set at 214 nm; the column temperature was 45 ℃. Chromatographic separation was achieved on an Acquity UPLC BEH300 C18column( 2. 1 mm × 100 mm,1. 7 μm) with a gradient solvent system composed of 0. 1% aqueous formic acid( A) and acetonitrile with 0. 1% formic acid( B),and the gradient profile was 0- 30 min,5% B→30% B. The flow rate was 300 nL·min- 1with 30 min elution. The samples were analyzed with a nanoliter electrospray ionization interface set in positive ionization mode. The main impurity was collected and identified using LC- MS /MS. Results: The analytical procedures were validated and the results showed that the method was qualified and could be applied to accurate determination of the related substances in insulin detemir. The main impurity was identified by LC- MS /MS as the deaminated product of the 21 st asparagine residue in A chain of insulin detemir. Conclusion: The RP- HPLC and LC- MS /MS methods established are accurate and effective for qualitative determination and identification of related substances in insulin detemir,and can be applied to specification research and quality control of insulin detemir.