摘要
目的:建立重组人溶菌酶含量及纯度的反相高效液相色谱(RP-HPLC)检测方法。方法:采用Welch Ultimate XB-C_(18)色谱柱(4.6 mm×250 mm,5μm),含0.1%三氟乙酸和0.9%氯化钠的水溶液为流动相A;含0.1%三氟乙酸和0.9%氯化钠水溶液-乙腈(40∶60)为流动相B;检测波长280 nm,柱温32℃,流速1.0 mL·min^(-1),线性梯度洗脱。结果:本方法专属性良好;供试品48 h内稳定,RSD为0.51%;重复性RSD为0.46%,中间精密度不同工作日间的RSD为0.52%,不同实验员间的RSD为0.53%;定量下限200 ng、检测下限30 ng;在进样量10～60μg范围内,线性关系良好,r=0.999 9;平均回收率(n=9)99.7%,RSD为0.94%。测定3批重组人溶菌酶冻干粉含量分别为36.5%、32.8%和31.1%;纯度均大于98%。结论:方法学验证表明,该方法符合定量检测重组人溶菌酶含量和纯度的要求,适用于重组人溶菌酶的质量控制。
Objective:To establish a method to determine( recombinant human lysozyme rhLYZ)by reversed phase high performance liquid chromatography(RP-HPLC),including quantitative analysis and purity analysis. Methods:Chromatography was performed on Welch Ultimate XB-C18 chromatographic column(4.6 mm×250 mm, 5μm),using 0.1% trifluoroacetic acid and 0.9% sodium chloride solution as mobile phase A;and 0.1% trifluoroacetic acid and 0.9% sodium chloride solution-acetonitrile(40∶60),as mobile phase B,with linear gradient elution at a flow rate of 1.0 mL·min^-1. The detection wavelength was 280 nm,and the column temperature was 32 ℃ . Results:The method has a good specificity. The sample solution is stable within 48,RSD was 0.51%. Repetitive RSD was 0.46%,the intermediate precision(RSD)at different workdays was 0.52% and the RSD was 0.53% from different lab technicians. Quantitative limit was 200 ng and detection limit was 30 ng. In a sample range of 1060 μg,linearity was good,r was 0.999 9. The average recovery was 99.7%(n=9),with RSD of 0.94%. The content of three batches of rhLYZ were 36.5%,32.8% and 31.1%,respectively,with purity greater than 98%. Conclusion:Methodology validation shows that this method conforms to requirement of the quantitative determination of rhLYZ content and purity,and an be applied to quality control.
作者
史瑾
邓晗
王维
郝东
赵金礼
杨小琳
SHI Jin;DENG Han;WANG Wei;HAO Dong;ZHAO Jin-li;YANG Xiao-lin(Shaanxi Huikang Bio-tech Co.,Ltd.,Xian 710054,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2018年第12期2167-2174,共8页
Chinese Journal of Pharmaceutical Analysis
