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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 chromatographic method for the determination of AP25 and related substances. A COSMOSIL C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase A was acetonitrile-mobile phase B with 0.05 mol / L sodium dihydrogen phosphate (adjusted to pH 2.5 with phosphoric acid) The wavelength was 220 nm. The volume flow rate was 1.0 mL / min. The column temperature was 25 ℃. The injection volume was 20 μL. Under the chromatographic conditions, the peaks of all related substances could be well separated from the main peak of AP25, the theoretical plate number was no less than 5 000, the AP25 concentration was 0.8-1.2 mg / mL (r = 0.999 6, n = 5) The linear range was 42μg / mL (r = 0.999 5, n = 5). The detection limits and the limits of quantification were 0.5 ng and 1 ng respectively. The repeatability test RSD values for the content and related substances were 0.60% and 0.48% respectively (n = 6). The RSD values of intermediate precision test were 0.35% and 0.80% respectively (n = 12). The contents of the three batches of APIs and the test results of related substances were 99.56%, 99.84%, 99.74% and 2.17%, 0.98% and 2.19% respectively. The method is simple and convenient, has high sensitivity, good durability, good specificity, accurate and stable, and can be used for determining the content of polypeptide AP25 and related substances.