索马鲁肽原料中N-羟基琥珀酰亚胺残留量反相高效液相色谱检测方法的建立及验证

Development and validation of reverse phase-high performance liquid chromatography for determination of residual N-hydroxy succinimide content in semaglutide

目的建立检测索马鲁肽原料中N-羟基琥珀酰亚胺(N-hydroxy succinimide,NHS)残留量的反相高效液相色谱(reverse phase-high performance liquid chromatography,RP-HPLC)法,并进行验证。方法筛选色谱柱并优化流动相条件(磷酸盐浓度与有机相比例),建立检测索马鲁肽原料中NHS残留量的RP-HPLC法,并验证方法的专属性、适用性、准确度、重复性和稳定性,确定线性范围及定量限、检测限。用建立的方法检测3批索马鲁肽中NHS含量。结果确定的色谱条件如下:色谱柱为CAPCELL PAK ADME(4.6 mm×150 mm,3μm);流动相A为0.05 mol/L磷酸二氢钾溶液-乙腈(98∶2),流动相B为70%乙腈溶液;洗脱梯度为0 min 0%B,10 min 0%B,19 min 90%B,19.1 min 0%B,25 min 0%B;流速为0.8 mL/min;检测波长为260 nm;柱温为30℃。建立的方法在0.2~3.0μg/mL范围内与峰面积呈良好的线性关系,R^(2)=1.0000,检测限和定量限分别为4.8和9.6 ng;专属性、系统适用性良好;3种不同浓度NHS加样样品回收率的相对标准偏差(RSD)为0.58%,准确度良好;6份供试品溶液中NHS含量的RSD为0.16%,重复性良好;室温放置0、4、8、12、16、20和24 h NHS峰面积的RSD为0.34%,稳定性良好。3批索马鲁肽中均未检出NHS。结论建立的RP-HPLC法简单、灵敏度高,可用于索马鲁肽原料中NHS含量的测定。

Objective To develop and validate a reverse phase-high performance liquid chromatography(RP-HPLC)method for determination of residual N-hydroxy succinimide(NHS)content in semaglutide.Methods A RP-HPLC method was developed based on the screening of chromatographic column and optimization of mobile phase(phosphate concentration and the ratio of acetonitrile),validated for specificity,suitability,accuracy,reproducibility and stability,and determined for linear range,limit of quantitation(LOQ)and limit of detection(LOD).The NHS contents in three batches of semaglutide were determined by the developed method.Results The optimal condition for RP-HPLC was as follows:CAPCELL PAK ADME column(4.6 mm×150 mm,3μm)was adopted,serving 0.05 mol/L potassium dihydrogen phosphate solution-acetonitrile(98∶2)as mobile phase A,and 70%acetonitrile as mobile phase B with gradient elution(0 min,0%B;10 min,0%B;19 min,90%B;19.1 min,0%B;25 min,0%B)at a flow rate of 0.8 mL/min.The detection wave length was set at 260 nm,while the column temperature was 30℃.The developed method showed good specificity and systemic suitability,of which the linear range was 0.2~3.0μg/mL(R^(2)=1.0000),while the LOD and LOQ were 4.8 and 9.6 ng respectively.The RSD of recovery rates of NHS samples at three concentrations was 0.58%,indicating a high accuracy.The RSD of NHS contents in six test samples was 0.16%,indicating a high reproducibility.The RSD of peak areas of NHS after storage at room temperature for 0,4,8,12,16,20 and 24 h was 0.34%,indicating a high stability.No NHS was detected in three batches of semaglutide by the developed method.Conclusion The developed RP-HPLC method is simple and sensitive,which may be used for the determination of NHS content in semaglutide.