二维液相色谱测定伏立康唑血药浓度的研究

Determination of voriconazole blood concentration by two-dimensional liquid chromatography

目的:采用全自动二维液相色谱(2D-LC-UV)建立快速测定伏立康唑血药浓度的方法,并应用于临床。方法:样品去除蛋白后直接进样,待测物在一维柱Aston SC2(4.6 mm×25 mm,5μm)上初步分离,通过中间柱Aston SH (3.0 mm×10 mm,5μm)截取保留,转移到二维色谱柱Aston SCB(4.6 mm×100 mm,5μm)上进一步分离。一维流动相为VCV-1D移动相,流速0.7 mL·min^-1;二维流动相为OPI-1有机移动相-BPI-1碱性移动相-MPI-1移动相(30∶48∶22),流速1.0 mL·min^-1;柱温40℃;紫外检测波长262 nm。结果:伏立康唑与各杂质分离良好,在0.38~12μg·mL^-1范围内呈良好的线性关系(r=0.999 9),可在10 min内完整出峰,方法回收率高于95%;日内、日间的精密度RSD均小于5%;稳定性试验RSD小于5%。所建立方法对79名肾移植住院患者进行了伏立康血药浓度检测共280例次,常规剂量未达到治疗浓度的占55%,超过安全浓度的占10.4%,在治疗浓度内的占34.6%。结论:本方法简便快速,结果准确,稳定性良好,自动化程度高,不依赖于专门的技术人员,适用于临床伏立康唑快速检测。

Objective:To establish a method for rapid determination of voriconazole blood concentration by automated two-dimensional liquid chromatography(2D-LC-UV) and apply it to clinical practice. Methods:The sample was directly injected for determination after being deproteinized. The sample was preliminarily separated on one-dimensional Aston SC2 column,then was trapped on intermediate Aston SH column(3.0 mm×10 mm,5μm) and separated by two-dimensional Aston SCB column(4.6 mm×100 mm,5 μm). The mobile phase of the onedimensional and two-dimensional liquid chromatography were VCV- D(flow rate was 0.7 mL·min^-1) and mixed mobile phase [VOPI-1 organic-VBPI-1 basic-VMPI-1 acidic(30∶48∶22)](flow rate of 1.0 mL·min^-1),respectively. The column temperature was maintained at 40 ℃ and the UV adsorption wavelength was set at 262 nm. The established method was applied to the patients with kidney transplantation to monitor the voriconazole blood concentration and the preliminary results were evaluated. Results:Under the established chromatographic conditions,voriconazole was separated well from other impurities and linear range was 0.38-12 μg·mL^-1(r=0.999 9). Voriconazole had a complete chromatographic peak within 10 minutes. The recovery of the method was higher than 95%. Intraday and interday precisions were less than 5% and the RSD of stability test was less than 5%. A tolal of 280 voriconazole blood concentrations were measured in 79 patients addmitted to the kidney transplant department. In the routine dose of voriconazole,55% of the patients did not reach the therapeutic comcentratin,and 10.4% exceeded the safe concentration,accountcd for 34.6%. Conclusion:The eatablished method is simple,rapid,accurate,stable,highly automated,independent of specialized technicians,and is suitable for rapid detection of voriconazole in hospitals.