Objective To establish an HPLC method for determination of related substance in Salmon Calcitonin Nasal Spray.Methods The separation was performed on Macherey-Nagel Nucleosil 100-5 C18AB column(250 mm×4.0 mm,5 μm).The mobile phase A was 0.04 mol/L tetramethyl ammonium hydroxide buffer(adjusted with phosphoric acid to pH 2.5)-acetonitrile(9∶1),and the mobile phase B was 0.04 mol/L tetramethyl ammonium hydroxide buffer(adjusted with phosphoric acid to pH 2.5)-acetonitrile(4∶6),with gradient elution.The detection wavelength was 220 nm,the flow rate was 1.0 mL/min,injection volume was 100 μL,column temperature was 65℃,and sample temperature was 10℃.The contents of calcitonin C and N-acetylcysteine salmon calcitonin(impurity A),9-D-leucine salmon calcitonin(impurity B),and de-22-tyrosine salmon calcitonin(impurity C)were determined by principal component self-control with correction factor.Results The linear ranges of calcitonin C and impurity A,B,and C were all 0.20—7.50 μg/mL.The average recoveries were 98.1%,98.9%,100.5%,and 99.7% with RSD values of 1.6%,1.5%,1.0%,and 1.1%,respectively.The correction factors of impurity A,B,and C were all above 0.90—1.10.Conclusion The method is simple,rapid,and can be used for the determination of related substances in Salmon Calcitonin Nasal Spray.
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