Tu Xiaowei, Lu Youming, Zhong Dafang, Zhang Yifan, Chen Xiaoyan
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, PR China.
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Aug 1;964:202-7. doi: 10.1016/j.jchromb.2014.01.021. Epub 2014 Jan 19.
Clofarabine triphosphate is an intracellular active metabolite of clofarabine. In the present study, we developed and validated a rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood using the Ficoll gradient centrifugation method. Chromatographic separation was performed on a CN column using an isocratic mobile phase comprising acetonitrile/5mM ammonium acetate with 0.001% ammonium hydroxide (20/80, v/v) at a flow rate of 0.60 mL/min. Detection was carried out by MS/MS in the multiple reaction monitoring mode using a negative electrospray ionization interface. The method was validated in concentration ranges of 1.25-100 ng/10(7) cells with acceptable accuracy and precision using 50 μL of cell extract. Clofarabine triphosphate was stable in a series of stability studies with bench-top, auto-sampler, and repeated freeze-thaw cycles. The validated method was successfully used to measure the concentrations of clofarabine triphosphate in PBMCs from cancer patients treated with clofarabine.
三磷酸氯法拉滨是氯法拉滨的一种细胞内活性代谢物。在本研究中，我们开发并验证了一种快速、灵敏且具选择性的液相色谱-串联质谱法（LC-MS/MS），用于定量测定人外周血单核细胞（PBMCs）中三磷酸氯法拉滨的浓度。采用Ficoll梯度离心法从血液中分离出PBMCs。在CN柱上进行色谱分离，使用由乙腈/5mM醋酸铵与0.001%氢氧化铵（20/80，v/v）组成的等度流动相，流速为0.60 mL/min。采用负电喷雾电离接口，在多反应监测模式下通过MS/MS进行检测。该方法在1.25 - 100 ng/10(7)个细胞的浓度范围内进行了验证，使用50 μL细胞提取物时具有可接受的准确度和精密度。在一系列涉及台式、自动进样器以及反复冻融循环的稳定性研究中，三磷酸氯法拉滨表现稳定。该经过验证的方法成功用于测定接受氯法拉滨治疗的癌症患者PBMCs中三磷酸氯法拉滨的浓度。
