Department of Clinical Chemistry/Preclinical Pharmacology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Jul 15;901:9-17. doi: 10.1016/j.jchromb.2012.05.033. Epub 2012 Jun 8.
NVP-BEZ235 is a novel dual inhibitor of PI3K/mTOR and currently undergoing phase I/II clinical trials for advanced solid tumors. We developed a sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) assay with fluorometric detection for quantification of NVP-BEZ235 in biological matrices. Liquid-liquid extraction with tert-butyl methyl ether was used for sample pre-treatment, yielding a recovery of >84%. Chromatographic separation of NVP-BEZ235 and the internal standard (IS) NVP-BBD130 was achieved on a GraceSmart C-18 column by isocratic elution with a mobile phase which consisted of acetonitrile, methanol, and milliQ water adjusted with acetic acid to pH 3.7 (20:36:44, v/v/v). Fluorescence detection using excitation and emission wavelengths of 270 and 425 nm, respectively, provided a selectivity and sensitivity allowing quantification down to 1 ng/ml in human plasma and linear calibration curves within a range of 1-1000 ng/ml. The assay was validated for human plasma, mouse plasma and a range of tissues. The accuracy, within-day and between-day precision for all matrices, was within the generally accepted 15% range. NVP-BEZ235 was stable for 72 h in pretreated samples in reconstitution mixture (acetonitrile-water (30:70, v/v)), but unstable in mouse tissue homogenates upon repeated freeze-thaw cycles or long term storage (≥24 h) at room temperature. A pilot pharmacokinetic study in mice demonstrated the applicability of this method for pharmacokinetic purposes. Overall, this assay is suitable for the pharmacokinetic studies of NVP-BEZ235 in mice and in human plasma.
NVP-BEZ235 是一种新型的 PI3K/mTOR 双重抑制剂，目前正在进行用于晚期实体瘤的 I/II 期临床试验。我们开发了一种灵敏且选择性的反相高效液相色谱（HPLC）荧光检测法，用于定量生物基质中的 NVP-BEZ235。采用叔丁基甲醚进行液液萃取作为样品预处理，回收率>84%。NVP-BEZ235 和内标（IS）NVP-BBD130 的色谱分离在 GraceSmart C-18 柱上通过等度洗脱实现，流动相由乙腈、甲醇和用醋酸调节至 pH 3.7 的 MilliQ 水（20:36:44，v/v/v）组成。荧光检测采用激发和发射波长分别为 270nm 和 425nm，具有选择性和灵敏度，可在人血浆中定量至 1ng/ml，线性校准曲线范围为 1-1000ng/ml。该方法已在人血浆、小鼠血浆和一系列组织中得到验证。所有基质的准确度、日内和日间精密度均在可接受的 15%范围内。在复溶混合物（乙腈-水（30:70，v/v））中，预处理样品中 NVP-BEZ235 在 72 小时内稳定，但在小鼠组织匀浆中，在反复冻融循环或室温下长期储存（≥24 小时）时不稳定。在小鼠中进行的初步药代动力学研究表明，该方法适用于 NVP-BEZ235 的药代动力学研究。总的来说，该方法适用于 NVP-BEZ235 在小鼠和人血浆中的药代动力学研究。
