Institut des Biomolécules Max Mousseron (IBMM), UMR 5247-CNRS-UM-ENSCM, Montpellier, France.
Institut régional du Cancer de Montpellier (ICM), Département de Pharmacie et Pharmacologie, Montpellier, France.
J Chromatogr A. 2018 Dec 7;1579:121-128. doi: 10.1016/j.chroma.2018.10.017. Epub 2018 Oct 15.
Developing an easy to use, cheap and fast analytical methodology is highly demanded for clinical practices, such as therapeutic drug monitoring (TDM). The present work deals with the development of an analytical methodology for the analysis of four basic anticancer drugs, namely tyrosine kinase inhibitors (TKIs), in human plasma by combining salting-out assisted liquid-liquid extraction (SALLE) with capillary electrophoresis (CE). This SALLE-CE methodology makes a full use of the advantages of both techniques by combining extraction, on-line concentration and separation in a simple way. First, plasma samples containing TKIs are mixed with acetonitrile (ACN) in appropriate volumes to precipitate proteins. After vortexing and centrifugation, sodium chloride (NaCl) is added to the plasma-ACN mixture to induce a two phases separation. TKIs are efficiently extracted (60-100% extraction efficiency) in the upper (mostly organic) phase which is directly analyzed by capillary electrophoresis (CE) coupled to UV detection. The high content of ACN in the upper phase allows the stacking of the analytes in the capillary (on-line stacking) during analysis. For the first time thanks to this electrophoretic process, the injected sample volume can be as large as 80% of the capillary volume (till the detector window). Good linearity was obtained for each TKI in the concentration range 60-2000 ng/ml with correlation coefficient (r²) between 0.997 and 0.999. LOD and LOQ in human plasma with such large injected volume were determined from 16 to 280 ng/ml and from 62 to 900 ng/ml respectively depending on the TKI. Recoveries for the four TKIs ranged from 60 to 100%. The repeatability of the SALLE-CE methodology for the analysis of TKIs in human plasma was evaluated with injected sample volume equal to 80% of the capillary volume till detector window. Relative standard deviations (RSDs) of less than 1.24 and 2.84% on migration times and corrected peak areas respectively were obtained at the LOQ. The sensitivity was enhanced by 61 to 265 folds confirming the applicability of the proposed methodology for the assay of TKIs in patients' plasma.
开发一种易于使用、廉价且快速的分析方法对于临床实践（如治疗药物监测 (TDM)）非常重要。本工作涉及开发一种分析方法，用于通过结合盐析辅助液 - 液萃取 (SALLE) 和毛细管电泳 (CE) 分析人血浆中的四种基本抗癌药物，即酪氨酸激酶抑制剂 (TKI)。这种 SALLE-CE 方法通过以简单的方式结合提取、在线浓缩和分离，充分利用了两种技术的优势。首先，将含有 TKI 的血浆样品与适量的乙腈 (ACN) 混合以沉淀蛋白质。涡旋和离心后，向血浆-ACN 混合物中加入氯化钠 (NaCl) 以诱导两相分离。TKI 在上相（主要为有机相）中被有效地提取（提取效率为 60-100%），然后直接通过与紫外检测耦合的毛细管电泳 (CE) 进行分析。在上相中高浓度的 ACN 允许在分析过程中（在线浓缩）将分析物堆积在毛细管中。由于这种电泳过程，首次可以将注入的样品体积增加到毛细管体积的 80%（直到检测器窗口）。对于每种 TKI，在 60-2000ng/ml 的浓度范围内均获得了良好的线性，相关系数 (r²) 在 0.997 到 0.999 之间。在如此大的注入体积下，LOD 和 LOQ 在人血浆中分别为 16-280ng/ml 和 62-900ng/ml，取决于 TKI。四种 TKI 的回收率在 60-100%之间。当注入的样品体积等于毛细管体积直至检测器窗口的 80%时，SALLE-CE 方法分析人血浆中 TKI 的重复性通过相对标准偏差 (RSD) 进行评估。在 LOQ 时，迁移时间和校正峰面积的 RSD 分别小于 1.24%和 2.84%。灵敏度提高了 61 至 265 倍，证实了所提出的方法在测定患者血浆中的 TKI 方面的适用性。
