NMM肿瘤DNA疫苗乙二胺四乙酸二钠残留量高效液相色谱检测方法的建立及验证

Development and verification of high performance liquidchromatography for determination of ethylenediaminetetraacetic acid disodium salt residue in NMM tumor DNA vaccine

目的 建立NMM肿瘤DNA疫苗原液中乙二胺四乙酸二钠(ethylenediaminetetraacetic acid disodium salt,EDTA-2Na)残留量高效液相色谱(high performance liquid chromatography,HPLC)检测方法,并进行验证,用于DNA疫苗质量控制。方法 将NMM肿瘤DNA疫苗原液与硫酸铜络合后,建立EDTA-2Na残留量HPLC检测方法:色谱柱为Agilent ZORBAX SB-C18(150 mm×4.6 mm,5μm);流动相为水-10%四丁基氢氧化铵-乙腈溶液(74.5∶0.5∶25);检测波长为254 nm;流速为0.8 mL/min;柱温为20℃;进样量为20μL。对方法的专属性、线性范围、检测限及定量限、溶液稳定性、耐用性、准确度、精密度进行验证。用建立的方法对多批次DNA疫苗原液中EDTA-2Na残留量进行检测。结果EDTA-2Na对照品及加入EDTA-2Na的DNA疫苗原液与硫酸铜反应后,在5.3 min附近出现吸收峰,而DNA疫苗原液与硫酸铜反应后未见吸收峰;对照品在4~400μg/mL范围内,与峰面积呈良好的线性关系,R^(2)=0.999 9;该方法检测限为10 ng/mL,定量限为40 ng/mL;对照品及供试品溶液放置12 h,溶液稳定性良好;方法检测条件发生微小变动时(不同流动相比例、不同流速、不同柱温),对检测结果的影响在可接受范围内;低、中、高浓度加标样品EDTA-2Na回收率均值为101.38%,RSD为0.39%;0.1 mg/mL对照品溶液连续进样6次,峰面积RSD为0.04%,6份供试品溶液均未检出EDTA-2Na,含EDTA-2Na的供试品溶液峰面积RSD为0.02%,中间精密度良好。多批次DNA疫苗原液均未检测出EDTA-2Na残留。结论 建立的方法操作简便,准确可靠,专属性强,可用于DNA疫苗原液中EDTA-2Na残留量检测。

Objective To develop and verify a high performance liquid chromatography(HPLC) method for the determination of ethylenediaminetetraacetic acid disodium salt(EDTA-2Na) residues in the bulk of NMM tumor DNA vaccine for the quality control of DNA vaccine.Methods After NMM tumor DNA vaccine bulk was complexed with copper sulfate,a HPLC method for the determination of EDTA-2Na residues was developed with Agilent ZORBA XSB-C18(150 mm × 4.6 mm,5 μm) as the chromatographic column,water,tetrabutylammonium hydroxide 10% and acetonitrile solution(74.5:0.5:25)as the mobile phase.The detection method was as follows:the detection wavelength was 254 nm,the flow rate was 0.8 mL/min,the column temperature was 20 ℃ and the injection volume was 20 μL.The method was verified for the specificity,linear range,limit of detection(LOD),limit of quantification(LOQ),solution stability,durability,accuracy and precision,and used to detect the EDTA-2Na residues in several batches of DNA vaccine bulk.Results When EDTA-2Na control and DNA vaccine bulk with EDTA-2Na reacted with copper sulfate,the absorption peak appeared at around 5.3 min,while no absorption peak was observed when DNA vaccine bulk reacted with copper sulfate;In the range of 4~400 μg/mL,the control solution concentration showed a good linear relationship with the peak area,R~2=0.999 9;The LOD of the method was 10 ng/mL,and the LOQ was 40 ng/mL;The solution of control and sample was stable after placed for 12 h;When the detection conditions changed slightly(different mobile phase ratio,flow rate and column temperature),the influence on the detection results was within acceptable range;The average recovery rate of EDTA-2Na in low,medium and high concentration standard added samples was 101.38% with the RSD of 0.39%;0.1 mg/mL control solution was injected continuously for 6 times,and the peak area RSD was 0.04%.EDTA-2Na was not detected in 6 sample solution,and the peak area RSD of DNA vaccine bulk with EDTA-2Na solution was 0.02%,indicating a good intermediate precision.EDTA-2Na residue was not detected in these batches of DNA vaccine bulk.Conclusion The developed method is simple,accurate,reliable with good specificity,which can be used for the determi-nation of EDTA-2Na residues in DNA vaccine bulk.