HPLC测定重组Exendin-4-FC融合蛋白注射液中抗氧剂及其降解物

Determination of Antioxidants and Their Degradation Products in Recombinant Exendin-4-FC Fusion Protein Injection by HPLC

关键词:  重组Exendin-4-FC融合蛋白注射液  抗氧剂  高效液相色谱法

Key words:  recombinant Exendin-4-FC fusion protein injection  antioxidants  HPLC

摘要:

目的 建立HPLC对重组Exendin-4-FC融合蛋白注射液中11种抗氧剂及其降解物含量测定方法。方法 采用饱和硫酸铵沉淀蛋白，离心后上清液转移至经甲醇活化后的C₁₈固相萃取小柱中，分别用4 mL甲醇、5 mL乙酸乙酯依次洗脱小柱，洗脱液用甲醇-乙酸乙酯(2∶3)混合溶剂定容后过0.22 μm PTFE疏水滤膜，经HPLC分析，外标法定量。色谱条件：Kinetex^® XB-C₁₈ 100Å(100 mm×4.6 mm，2.6 μm)色谱柱，检测波长230 nm，柱温箱30 ℃，进样量5 μL，流速0.4 mL·min^–1，流动相0.1%甲酸-甲醇(A)-0.1%甲酸水溶液(B)，运行时间45 min。结果 11种目标物在2.5~35 μg·mL^–1内线性关系良好(R²≥0.99)。在25，10，5 μg·mL^–1 3个浓度水平下，平均加样回收率为88.1%~106.5%，RSD为0.10%~9.05%。6份样品重复性RSD为2.01%~4.77%，12份样品中间精密度RSD为2.58%~9.75%，对3批次重组Exendin-4-FC融合蛋白注射液的0月，25 ℃加速3月及6月正/倒置样品进行检测，结果显示所有批次样品在不同检测点均未有抗氧剂及其降解物浸出。结论 该方法专属性好、准确度与精密度高、溶液稳定性好、方法耐用性高，能够用于对药品中抗氧剂类物质的含量检测。

Abstract:

OBJECTIVE To establish a method for determining the content of 11 antioxidants and their degradation products in recombinant Exendin-4-FC fusion protein injection by HPLC. METHODS The protein was precipitated with saturated ammonium sulfate. After centrifugation, the supernatant was transferred to a C₁₈ solid phase extraction cartridge activated by methanol. Then the cartridge was eluted with 4 mL of methanol and 5 mL of ethyl acetate respectively, and the eluent was diluted with methanol-ethyl acetate(2∶3) mixed solvent and passed through a 0.22 μm PTFE hydrophobic filter. It was analyzed by HPLC and quantified by external standard method. Chromatographic conditions: Kinetex^® XB-C₁₈ 100Å (100 mm×4.6 mm, 2.6 μm)column, the detection wavelength was 230 nm, the column oven was 30 ℃, the injection volume was 5 μL and the flow rate was 0.4 mL·min^–1, mobile phase was 0.1% formic acid-methanol(A)-0.1% formic acid aqueous solution(B), the running time was 45 min. RESULTS The 11 target substances showed a good linear relationship in the range of 2.5-35 μg·mL^–1 with R² ≥0.99. At three different concentration(25, 10, 5 μg·mL^–1) of spiked samples, the average recovery rates of 11 antioxidants ranged from 88.1% to 106.5%, with RSDs in the range of 0.10%–9.05%. The RSDs of 6 repeatable samples was 2.01%–4.77%, which of 12 intermediate precision samples was 2.58%–9.75%. The positive/inverted samples of three batches of recombinant Exendin-4-FC fusion protein injection were detected at 0 month, 3 months and 6 months(25 ℃), and the results showed that there was no antioxidant and its degradation leaching in all batches of samples at different detection points. CONCLUSION The method has good specificity, high accuracy and precision, good solution stability, high durability and can be used for the content detection of antioxidants in drugs.