高效液相色谱法测定血红蛋白氧载体中残留的戊二醛

Detection of Residual Glutaraldehyde in Hemoglobin-Based Oxygen Carrier with High Performance Liquid Chromatography

建立了一种测定血红蛋白氧载体中戊二醛残余含量的高效液相色谱方法。用10kDa超滤膜通过离心3000r/min×20min将游离戊二醛从待测样品中分离;在pH1.0时,在含有70%乙腈的体系中用2,4-二硝基苯肼在30min内将戊二醛衍生成2,4-二硝基苯腙(n(2,4-二硝基苯肼):n(戊二醛)=60:1),以70%乙腈为流动相,避免了衍生产物生成沉淀。用高效液相色谱法分离测定衍生物,可在20min内完成分离,同时对衍生步骤进行了优化。本方法具有较高的灵敏度和良好的线性关系,检出限为1.0ng,在0.1～10mg/L范围内其线性相关系数为0.9999,重复测定6次的相对标准偏差小于3.0%,回收率为95.26%。

A method for the determination of glutaraldehyde in hemoglobin-based oxygen carrier（HBOC） by high performance liquid chromatography is described.Free glutaraldehyde was separated from HBOC by 10 kDa ultrafiltration membrane under centrifuged 3000 r/min for 20 min and derivatized under the conditions of pH=1.0,2,4-dinitrophenylhydrazine（DNPH）：glutaraldehyde=60：1,reaction time 30 min in 70% acetonitrile medium.Precipitation of derivatives of glutaraldehyde with DNPH was avoided under this condition and it was possible to direct injection of the sample into the LC system.Subsequent reversed phase separation and determination of the derivatives within 20 min with a mobile phase of 70% acetonitile can be achieved.The linear range of this method was from 0.1 to 10.0 mg/L（R2=0.9999） with relative standard deviation of 3.0%（n=6）,the standard addition recovery was 95.3%,and the limit of detection was 1.0 ng.