HPLC法测定鲑降钙素鼻用喷雾剂中有关物质

Determination of related substance in Salmon Calcitonin Nasal Spray by HPLC

目的建立测定鲑降钙素鼻用喷雾剂中有关物质的HPLC方法。方法使用Macherey-Nagel Nucleosil 100-5 C18AB色谱柱(250 mm×4.0 mm,5μm);以0.04 mol/L四甲基氢氧化铵溶液(用磷酸调pH 2.5)–乙腈(9∶1)为流动相A,以0.04 mol/L四甲基氢氧化铵溶液(用磷酸调pH 2.5)–乙腈(4∶6)为流动相B,梯度洗脱;检测波长为220 nm;体积流量为1.0 m L/min;进样量为100μL;柱温为65℃;样品室温度:10℃。按加校正因子的主成分自身对照法计算降钙素C、N-乙酰半胱氨酰鲑降钙素(杂质A)、9-D-亮氨酸鲑降钙素(杂质B)、去-22-酪氨酸鲑降钙素(杂质C)。结果降钙素C和鲑降钙素杂质A、B、C在0.20~7.50μg/mL线性关系良好,平均回收率分别为98.1%、98.9%、100.5%、99.7%,RSD值分别为1.6%、1.5%、1.0%、1.1%。杂质A、B、C的校正因子均超出0.90~1.10。结论方法简便、快速,可作为鲑降钙素鼻用喷雾剂中有关物质的检测方法。

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,5μm).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 100μL,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—7.50μg/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.