基于高效液相色谱-三重四极杆质谱联用方法评价透析器对蛋白结合类尿毒症毒素的清除效果

Evaluation on Removal Effect of Dialyzer to Protein-bound Uremic Toxins Based on High Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry

建立了一种评价透析器对硫酸吲哚酚、硫酸对甲酸和马尿酸3种蛋白结合类尿毒症毒素(PBUTs)清除效果的高效液相色谱-三重四极杆质谱联用方法。配制与透析患者体内PBUTs浓度相当的模拟液代替血液,进行透析模拟治疗,取透析前后的模拟液作为待测样品。样品经乙腈沉淀蛋白和超滤离心处理后,采用Hypersil GOLD VANQUISH C18(100 mm×2.1 mm,1.9μm)色谱柱分离,以5 mmol/L乙酸铵溶液和甲醇为流动相进行梯度洗脱,负离子选择反应监测(SRM)模式扫描,内标法进行定量分析。3种PBUTs在25~1 000 ng/mL范围内线性关系良好,相关系数(r)均大于0.99,定量下限为25 ng/mL;在75、250、750 ng/mL加标水平下,回收率为92.5%~114%,日内、日间相对标准偏差(RSD)均小于5.0%,基质效应为80.6%~105%。该方法成本低、前处理简单、分析速度快、回收率高,适用于透析器对PBUTs清除效果的评价。

A method for evaluation on the removal effect of dialyzer to protein-bound uremic toxins(PBUTs) was established based on high performance liquid chromatography-triple quadrupole mass spectrometry.The dialysis simulated fluid with the same concentration of PBUTs as the dialysis patients’ blood was prepared instead of blood to conduct dialysis treatment in vitro due to the difficulty to obtain clinical blood samples and the complexity of patient blood composition,and the simulated fluid before and after dialysis was taken as the sample to be tested.After acetonitrile precipitation of protein and ultrafiltration centrifugation, the samples were separated on a Hypersil GOLD VANQUISH C18(100 mm × 2. 1 mm,1. 9 μm) column by gradient elution,using 5 mmol/L ammonium acetate solution and methanol as mobile phases.The mass spectrometric analysis was carried out under negative ion selective reaction monitoring mode(SRM),and the internal standard method was used for quantitative analysis of 3 PBUTs.The calibration curves of 3 PBUTs showed good linear relationships in the concentration range of 25-1 000 ng/mL,and the correlation coefficients(r) were more than 0. 99.The limits of quantitation(LOQs) were 25 ng/mL.The average recoveries for 3 analytes at three added concentrations of 75,250 and 750 ng/mL ranged from 92. 5%-114%,with the intra-day and inter-day RSDs both less than 5. 0% and the matrix effects of 80. 6%-105%.This method had the characteristics of low cost,simple pretreatment,short run-time and high accuracy,which provided a reliable technical support for evaluation on the removal effect of dialyzer to PBUTs.