新型二维液相色谱快速测定丙戊酸血药浓度的研究

Measurement of valproic acid in blood by a novel two-dimensional liquid chromatographic system

目的:采用新型二维液相色谱(2D-LC-UV)建立无需衍生即可测定丙戊酸血药浓度的方法。方法:2D-LC-UV由一维和二维液相色谱系统及中间转移柱构成,样品去除蛋白后直接进样,在一维色谱柱Aston SC2(4.6mm×25mm,5μm)上进行在线富集及初级分离,通过转移柱Aston SH(3.0mm×10mm,5μm)捕集保留,转移到二维色谱柱Aston SCB(4.6mm×100mm,5μm)上进一步分离检测。一维流动相为VCV^(-1)D移动相(V_(20.0mmol·L^(-1))磷酸铵:V_(乙腈)∶V_(甲醇)=3∶1∶1,磷酸调pH至5.4),流速1.0mL·min^(-1);转移柱流动相为纯水;二维流动相为OPI^(-1)有机移动相:BPI^(-1)碱性流动相:API^(-1)酸性流动相=24∶40∶36(V∶V∶V),流速1.0mL·min^(-1);柱温40℃;紫外检测波长305nm。结果:丙戊酸在5.99~191.73μg·mL^(-1)范围内与峰面积呈良好的线性关系(r=0.999 9),可在10 min内完整出峰;方法回收率高于96%;日内、日间的精密度RSD均小于3.56%;稳定性试验RSD均小于5%。结论:本方法无需衍生、稳定性良好、简便快速、结果准确、自动化程度高,不依赖于专门的技术人员,适用于临床快速检测。

OBJECTIVE To establish a method based on a novel type of two-dimensional liquid chromatographic system（2 DLC-UV）for detecting plasma concentration of valproic acid without derivation.METHODS 2 D-LC-UV contains the 1 st and 2 nd liquid chromatographic systems and intermediate transfer column.The sample was directly injected for determination after being deproteinized,and valproic acid was online concentrated and primarily separated on the 1 st Aston SC2 column,then trapped on Aston SH column and separated by Aston SCB column.The mobile phase of the 1 st liquid chromatographic system,transfer column and the 2 ndliquid chromatographic system were VCV-1 D（flow rate at 1.0 mL·min-1）,pure water and mixed mobile phase（VOPI-1 organic mobile phase∶V（BPI-1 basic mobile phase）∶V（API-1 acidic mobile phase）=24∶40∶36,1.0 mL·min-1）,respectively.The column temperature was maintained at 40 ℃,and the UV adsorption wave length was set at 305 nm.RESULTS The calibration curves of valproic acid sodium showed good linear regression in the range of 5.99-191.73μg·mL-1（r=0.999 9）.Valproic acid had a complete chromatographic peak within 10 minutes;the method recovery was higher than 96%;intra and inter-day variations were less than5%.CONCLUSION Because of its stability,accuracy,automation,no derivation,and independence of specialized technical personnel,the established chromatographic method is ideal for clinical rapid test.