高效阴离子交换色谱-脉冲安培检测法用于重组人促红素中唾液酸的含量测定  

Content determination of sialic acid in recombinant human erythropoietin by high-performance anion-exchange chromatography-pulsed amperometric detection

作　　者：李响[1] 于雷[1] 范文红[1] 周勇[1] 饶春明[1] LI Xiang;YU Lei;FAN Wenhong;ZHOU Yong;RAO Chunming(Division of Recombinant Biological Products,National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院重组药物室,北京100050

出　　处：《中国医药导报》2018年第27期102-105,共4页China Medical Herald

基　　金：国家科技重大专项项目(2015ZX09501008-001)

摘　　要：目的建立高效阴离子交换色谱-脉冲安培检测法(HPAEC-PAD)测定重组人促红素(rhEPO)中唾液酸含量的方法。方法将rhEPO样品用5 mmol/L的磷酸盐缓冲液(pH 7.0)换液后,调整浓度到0.33 mg/mL,取20μL样品溶液加入10 m U/μL的神经氨酸酶20μL,37℃孵育1 h后,加入0.04 mmol/L的尤罗索尼克酸(KDN)内标物50μL,再加入70μL超纯水,混匀后分析测定。采用CarboPac PA1分析柱,Carbo Pac PA100保护柱,流动相为150 mmol/L氢氧化钠与125 mmol/L乙酸钠混合溶液,柱温30℃,流速1.0 mL/min,等度洗脱,数据采集时间15 min。根据唾液酸峰面积与KDN内标峰面积的比值与含量的量效关系,计算样品中的唾液酸含量。结果唾液酸标准品浓度在0.0015～0.0075μg/mL范围内与唾液酸峰面积与KDN内标峰面积的比值呈良好的线性关系,R2>0.99;重复性RSD<4%;(低、中、高浓度)样品回收率分别为94.9%、94.3%、96.7%。用建立的HPAEC-PAD法测定了6批rhEPO中唾液酸含量,与间苯二酚测定法测定结果比较差异无统计学意义(P=0.7488)。结论本研究建立了rhEPO中唾液酸含量测定的HPAEC-PAD方法,该方法线性、重复性、稳定性良好,适用于rhEPO的唾液酸含量的常规测定。Objective To develop a high-performance anion-exchange chromatography-pulsed amperometric detection（HPAEC-PAD） method to determine the content of sialic acid in recombinant human erythropoietin（rhEPO）. Methods The rhEPO samples were prepared by changing rhEPO bulks to 5 mmol/L phosphate buffer（pH 7.0）, and adjusted to0.33 mg/mL. Taking 20 μL sample solution, and then 20 μL of 10 mU/μL neuraminidase was added, followed by incubation at 37℃ for 1 h. Finally, 50 μL 0.04 mmol/L ketodeoxynonulosonic acid（KDN） and 70 μL milli-Q water were added and mixed thoroughly followed by detection. CarboPac PA1 separation column and CarboPac PA100 guard column were used, with mobile phase being mixture of 150 mmol/L sodium hydroxide solution and 125 mmol/L sodium acetate solution, column temperature being 30℃, flow rate being 1.0 mL/min, and the data acquisition time being 15 min with isocratic elution. The sialic acid content was calculated based on the dose-effect relation of the ratio of peak area of sialic acid to KDN and the content of sialic acid. Results The concentrations of sialic acid standard solutions and the ratios of peak area of sialic acid to KDN showed a good linearity relationship between 0.0015 and 0.0075 μg/mL with R2 being higher than 0.99, the repeatability RSD was below 4%. The recovery rates of samples at low, medium and high concentration were 94.9%, 94.3% and 96.7%, respectively. Six batches of rhEPO were tested by the developed HPAEC-PAD method and the results had no significant differences compared with the results of resorcinol method（P =0.7488）. Conclusion This research has successfully established the HPAEC-PAD method to determine the content of sialic acid in rhEPO, with good linearity, repeatability and robustness, which can be used in routine determination of sialic acid in rhEPO.

关 键 词：高效阴离子交换色谱-脉冲安培检测法 重组人促红素 唾液酸 含量 

分 类 号：R917[医药卫生—药物分析学]