LC-MS/MS 法测定大鼠血浆中阿昔洛韦富马酸酯及其活性代谢物 602076 的浓度：在药代动力学研究中的应用。
Determination of alamifovir disoproxil fumarate and its active metabolite 602076 in rat plasma by LC-MS/MS: application to a pharmacokinetic study.
机构信息
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, PR China.
出版信息
J Pharm Biomed Anal. 2012 Mar 5;61:70-7. doi: 10.1016/j.jpba.2011.11.031. Epub 2011 Dec 6.
Alamifovir disoproxil fumarate (ADF) is a novel ester prodrug of alamifovir, which is currently as a promising antiviral candidate under investigation. This paper is aimed to develop rapid, sensitive and specific LC-MS/MS methods for the quantification of ADF and its active metabolite 602076 in rat plasma. According to the significantly different chemical properties of the compounds, two sets of liquid chromatography and ionization modes were used for determining the concentration of ADF and 602076 in rat plasma, separately. Following liquid-liquid extraction with n-hexane:dichloromethane:isopropanol (100:50:5, v/v/v), the ADF and internal standard (gliclazide) were separated on a Phenomenex Gemini C(18) column (150 mm × 2.0 mm, 5 μm) with a mobile phase consisting of methanol:water:formic acid (70:30:0.1, v/v/v). A tandem mass spectrometer equipped with electrospray ionization (ESI) source was used as the detector and operated in positive ion mode. The metabolite 602076 and the internal standard ZHY81018 were extracted from plasma by protein precipitation with acetonitrile. Chromatographic separation was performed on a Capcell MG C(18) column (150 mm × 2.0 mm, 5 μm) with a mobile phase consisting of acetonitrile and water (20:80, v/v). The MS/MS detection was operated in negative ion mode using an ESI source. The linear concentration ranges of the calibration curves were 2.5-500 ng/mL for ADF and 2.5-1000 ng/mL for 602076. The intra-assay RSD for quality control (QC) samples were from 3.3% to 6.7% for ADF, and 4.0% to 6.1% for 602076. The inter-assay RSD for QC samples were from 4.9% to 14.7% for ADF, and 2.6% to 4.4% for 602076. The relative errors for QC samples were from -10.6% to 1.9% for ADF, and 0.2% to 2.6% for 602076. The methods were successfully applied in the investigation of the pharmacokinetic profile of ADF, alamifovir and 602076 in rats. The results showed that ADF was rapidly metabolized to its active metabolite 602076 after oral absorption, with no detectable unchanged drug. The oral bioavailability of ADF was about 3 times higher than that of alamifovir.
富马酸替诺福韦二异丙酯（ADF）是替诺福韦的新型酯前药，目前作为一种有前途的抗病毒候选药物正在研究中。本文旨在开发快速、灵敏和特异的 LC-MS/MS 方法，用于定量测定大鼠血浆中的 ADF 和其活性代谢物 602076。根据化合物的化学性质显著不同，分别采用两组液相色谱和离子化模式来测定 ADF 和 602076 在大鼠血浆中的浓度。采用正己烷：二氯甲烷：异丙醇（100:50:5，v/v/v）进行液-液萃取后，用 Phenomenex Gemini C(18)柱（150mm×2.0mm，5μm）和甲醇：水：甲酸（70:30:0.1，v/v/v）作为流动相分离 ADF 和内标（格列齐特）。串联质谱仪配备电喷雾电离（ESI）源作为检测器，以正离子模式运行。代谢物 602076 和内标 ZHY81018 通过用乙腈沉淀蛋白从血浆中提取。用 Capcell MG C(18)柱（150mm×2.0mm，5μm）和乙腈-水（20:80，v/v）作为流动相进行色谱分离。MS/MS 检测采用 ESI 源，以负离子模式运行。ADF 的校准曲线线性浓度范围为 2.5-500ng/mL，602076 的校准曲线线性浓度范围为 2.5-1000ng/mL。ADF 的 QC 样品的日内 RSD 为 3.3%-6.7%，602076 的 QC 样品的日内 RSD 为 4.0%-6.1%。ADF 的 QC 样品的日间 RSD 为 4.9%-14.7%，602076 的 QC 样品的日间 RSD 为 2.6%-4.4%。ADF 的 QC 样品的相对误差为-10.6%-1.9%，602076 的 QC 样品的相对误差为 0.2%-2.6%。该方法成功应用于大鼠体内 ADF、替诺福韦和 602076 的药代动力学研究。结果表明，ADF 口服吸收后迅速代谢为其活性代谢物 602076，无检测到未变化的药物。ADF 的口服生物利用度约为替诺福韦的 3 倍。
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