摘要
建立多肽AP25含量测定及有关物质检查的高效液相色谱方法。采用COSMOSIL C18色谱柱(250 mm×4.6 mm,5μm);以0.05 mol/L磷酸二氢钠(用磷酸调pH值至2.5)为流动相A,乙腈为流动相B,进行梯度洗脱;检测波长为220 nm;体积流量为1.0 mL/min;柱温为25℃;进样体积为20μL。在该色谱条件下,各有关物质峰均可与AP25主峰良好分离,理论塔板数不低于5 000,AP25浓度在0.8～1.2 mg/mL(r=0.999 6,n=5)和18～42μg/mL(r=0.999 5,n=5)范围内与峰面积呈良好线性关系;检测限和定量限分别为0.5 ng和1 ng;含量和有关物质检测方法的重复性试验RSD值分别为0.60%和0.48%(n=6),中间精密度试验RSD值分别为0.35%和0.80%(n=12)。3批原料药的含量和有关物质检测结果分别为99.56%,99.84%,99.74%和2.17%,0.98%,2.19%。本方法简便易行,灵敏度高,耐用性、专属性良好,准确稳定,可用于测定多肽AP25的含量及有关物质。
To establish a high performance liquid chromatography （ HPLC ） method for content determination of the peptide AP25 and its related substances ,the separation was performed on a COSMOSIL Cls column （250 mm ×4.6mm,5μm） . The mobile phase A consisted of 0.05 mol/L sodium dihydrogen phosphate （ adjusting the pH value to 2.5 with the phosphonic acid）, the mobile phase B was acetonitrile, the separation process was gradient elution：the ratio of mobile phase B rised from 10% to 40% within 30 minutes. The retention time of AP25 main peak was kept at about 13 minutes. The detection wavelength was at 220 nm and the flow rate was 1.0 mL/min, the column temperature was 25℃, and the volume of injection was 20 μL. Under this chromatographic condition, an excellent separation was achieved for AP25 and its related substances, the degree of separability was more than 1.5, and the number of theoretical plates with AP25 was more than 5 000. There was a good linear relationship between the peak area and the concentration of AP25 in the range of 0. 8 mg/mL - 1.2 mg/mL （r = 0. 999 6,n =5） and 18 μg/mL -42μg/mL （r = 0. 999 5, n = 5 ） , respectively. The minimal detectable level and the minimal quantifiable level were 0. 5 ng and 1 ng. In method of analysis of AP25 and its related substances ,the relative standard deviation （RSD） of the reproducibility assay was 0. 60% and 0. 48% （n = 6）, the relative standard deviation （RSD） of the intermediate precision was 0. 35% and 0. 80% （ n = 12） ,respectively. The contents of AP25 （ n = 3 ） in substance were 99.56%, 99.84%, 99.74%, and the values of its related substances （ n = 3 ） were 2. 17%, O. 98%, and 2. 19%, respectively. The method was simple, easy, sensitive, accurate and stable, having a good durability and specificity. The results indicated that the method can be successfully applied in the analysis of AP25 and its related substances, and also can be used in the quality control of AP25 and its preparations.
出处
《药物生物技术》
CAS
2013年第3期250-255,共6页
Pharmaceutical Biotechnology
基金
十二五国家科技重大专项候选药物项目(No.2012ZX09103301-004)
江苏省"产学研联合创新资金-前瞻性联合研究项目"(No.SBY2010-20063)
国家863计划"多肽分子药靶发现与药物设计技术"(No.2012AA020304)
作者简介
尼格尔热依·亚迪卡尔（1987-），女，新疆维吾尔自治区乌鲁木齐人，硕士研究生，研究方向：药物质量分析，Tel：025—86185436，E—mail：nigary0115@sina．com。
通讯作者：徐寒梅，教授，中国药科大学博士生导师，主要从事抗肿瘤新药研究开发，Tel：025-83271007，025-86185437,Tax：025-83271007，E-mail：xuhanmei@yahoo．com．cn；xuhanmei@cpu.edu.cn。
