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目的 采用新型二维液相色谱系统(2D-LC-UV)建立中毒患者尿液中百草枯浓度的测定方法,并对二维色谱特征及方法学进行评估。方法 2D-LC-UV由第一维和第二维液相色谱系统及FLC转运机构构成,尿液100μL直接进样,在第一维Aston SX1色谱柱(4.6 mm×25 mm,5μm)上进行在线富集及初级分离,通过Aston SX捕集柱(4.6 mm×10 mm,5μm)捕集保留,最后转移到第二维色谱柱Aston SX1(4.6 mm×75 mm,5μm)上进一步分离检测。第一维流动相为2 mol·L~(-1)醋酸铵-乙腈=10∶1(V∶V),流速1.0 mL·min~(-1),捕集柱流动相为纯水,第二维流动相为2.5 mol·L~(-1)醋酸铵-甲醇-乙腈=5∶3∶1(V∶V∶V),流速1.0 mL·min~(-1),柱温40℃,紫外检测波长285 nm。结果 所建立的二维色谱方法可以去除样品中大部分杂质,最大可以在线处理500μL样品,目标物在二维系统中转移完整;在20~20 000ng·mL~(-1)内线性关系良好(r=0.999 9),不同来源尿液中未发现干扰;工作曲线可长时间稳定,色谱平衡时间小于15min。结论 本方法稳定性良好、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,适用于临床百草枯中毒患者的快速检测,为临床制定解救方案提供科学依据。
Objective To establish a method for the determination of paraquat in urine of patients with poisoning by a novel two-dimensional liquid chromatography (2D-LC-UV) system and to evaluate the two-dimensional chromatographic characteristics and methodology. Methods 2D-LC-UV consisted of first-dimension and second-dimension LC systems and FLC transporter. 100 μL of urine was injected directly onto the first dimension Aston SX1 column (4.6 mm × 25 mm, 5 μm) Enrichment and primary separation were carried out on an Aston SX trapping column (4.6 mm × 10 mm, 5 μm), and finally transferred to the second-dimension Aston SX1 column (4.6 mm × 75 mm, 5 μm) for further separation and detection. The first mobile phase was 2 mol·L -1 ammonium acetate-acetonitrile = 10:1 (V:V), the flow rate was 1.0 mL · min -1. The mobile phase was pure water. The mobile phase was 2.5 mol·L -1 ammonium acetate-methanol-acetonitrile = 5: 3:1 (V:V:V), the flow rate was 1.0 mL · min -1, the column temperature was 40 ℃, UV detection wavelength of 285 nm. Results The established two-dimensional chromatographic method can remove most of the impurities in the sample and can process up to 500μL of sample online. The target is completely transferred in the two-dimensional system. The linearity is good in 20 ~ 20 000 ng · mL -1 r = 0.999 9). No interference was found in urine from different sources. The working curve could be stable for a long time and the chromatographic equilibrium time was less than 15 min. Conclusion The method is stable, simple, rapid, accurate, highly automated and does not depend on specialized technicians. It is suitable for the rapid detection of clinical paraquat poisoning patients and provides a scientific basis for clinical rescue plan.