Determination of Intracellular Darunavir by Liquid Chromatography Coupled with Fluorescence Detection.
Determination of Intracellular Darunavir by Liquid Chromatography Coupled with Fluorescence Detection.
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液相色谱结合荧光检测测定细胞内达芦那韦。
DOI：
10.1093/chromsci/bmt147
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Yamamoto K.
中科院分区：
文献类型：
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作者：
Nagano D;Araki T;Nakamura T;Yamamoto K.
据报道，外周血单个核细胞（PBMC）中的地瑞那韦（DRV）浓度会影响患者的临床症状和耐药病毒的产生。我们开发了一种简单且高度灵敏的方法，使用高效液相色谱荧光检测来定量人PBMC中DRV的浓度。使用可商购获得的管收集PBMC样品用于密度梯度离心。为了破坏细胞，我们使用了加热破碎方法。为了弥补加热破碎方法的缺点，使用液-液分配通过使用有机溶剂降解蛋白质来破坏未破碎的细胞。作为分析柱，我们使用ODS柱。移动的流动相为20 mmol/L磷酸钾缓冲液（pH4.3）/乙腈（57/43，v/v），流速为1.0 mL/min，定量下限为5 ng/106个细胞。线性范围为5-100 ng/106细胞。批内和批间精密度和准确度<15%。由于我们的方法使得在没有串联质谱系统的医疗机构或实验室中测量PBMC中的DRV浓度成为可能，我们相信它将有助于临床研究，并将改善DRV感染的人类免疫缺陷病毒的医学治疗。
The concentration of darunavir (DRV) in peripheral blood mononuclear cells (PBMCs) was reported to affect the clinical symptoms of patients and the development of drug-resistant viruses. We developed a simple and highly sensitive method to quantify the concentration of DRV in human PBMCs using high-performance liquid chromatography with fluorescence detection. PBMC samples were collected using commercially available tubes for density-gradient centrifugation. To disrupt the cells, we used a heating fragmentation method. To compensate for the disadvantages of the heating fragmentation method, liquid–liquid partitioning was used to destroy the unbroken cells by the degeneration of proteins using an organic solvent. As an analytical column, we used an ODS column. The mobile phase consisted of 20 mmol/L potassium phosphate buffer (pH 4.3)/acetonitrile (57/43, v/v) and was pumped at 1.0 mL/min. The lower limit of quantification was 5 ng/106cells. Good linearity was obtained with 5–100 ng/106cells. The intra- and inter-assay precision and accuracy were <15%. Because our method makes possible the measurement of DRV concentration in PBMCs at medical facilities or laboratories without tandem mass spectrometry systems, we believe that it will contribute to clinical studies and will improve the medical treatment of human immunodeficiency virus infections with DRV.