McMahon G P, Kelly M T
Department of Chemistry, Royal College of Surgeons in Ireland, St. Stephen's Green, Dublin, Ireland.
Anal Chem. 1998 Jan 15;70(2):409-14. doi: 10.1021/ac9707040.
A column-switching liquid chromatographic method is described for the simultaneous determination of aspirin and salicylic acid in human plasma. Blood samples are taken into chilled tubes containing a fluoride anticoagulant, and the plasma is isolated by centrifugation. Following a simple acidification step, a 200 microL aliquot of the sample is injected directly onto the HPLC system. The C-18 extraction column is washed with acidified water for 2 min, after which time the compounds are removed by back-flushing directly onto the analytical column (C-8 Nucleosil, 5 microns, 250 mm x 4.6 mm). The flow rate through both columns is 1 mL/min, and the analytes are quantified by measurement of their UV absorbance at 225 nm. The mobile phase is a mixture of water-methanol-acetonitrile-orthophosphoric acid (650:200:150:1 v/v/v/v). The method is linear in the concentration ranges 0.10-5.00 micrograms/mL for aspirin and 0.25-15.00 micrograms/mL for salicylic acid. Both compounds have a limit of quantitation of 0.10 microgram/mL and a limit of detection of 0.04 microgram/mL. Extensive stability tests have been carried out, and validation studies reveal the method to be reproducible and repeatable. Excellent recoveries from plasma obviate the need for an internal standard. The procedure is easier to execute and requires less sample handling than methods currently described in the literature. It has been successfully applied to the investigation of the levels of aspirin and salicylic acid in a healthy, nonfasting volunteer following a 600 mg oral dose of aspirin.
本文描述了一种柱切换液相色谱法，用于同时测定人血浆中的阿司匹林和水杨酸。将血样采集到含有氟化物抗凝剂的冷却试管中，通过离心分离出血浆。经过简单的酸化步骤后，将200微升样品等分试样直接注入高效液相色谱系统。用酸化水冲洗C-18萃取柱2分钟，之后通过反冲将化合物直接转移到分析柱（C-8核硅，5微米，250毫米×4.6毫米）上。通过两根柱子的流速均为1毫升/分钟，通过测量其在225纳米处的紫外吸光度对分析物进行定量。流动相是水 - 甲醇 - 乙腈 - 正磷酸（650:200:150:1 v/v/v/v）的混合物。该方法在阿司匹林浓度范围为0.10 - 5.00微克/毫升和水杨酸浓度范围为0.25 - 15.00微克/毫升时呈线性。两种化合物的定量限均为0.10微克/毫升，检测限均为0.04微克/毫升。已经进行了广泛的稳定性测试，验证研究表明该方法具有可重复性。从血浆中获得的优异回收率使得无需内标。该程序比文献中目前描述的方法更易于执行，并且需要的样品处理更少。它已成功应用于一名健康、非空腹志愿者口服600毫克阿司匹林后阿司匹林和水杨酸水平的研究。
