王忠元 杨华丽 崔杏 朱高峰 汤磊 廖伟科

中圖分类号 969.1;R917 文献标志码 A 文章编号 1001-0408（2021）11-1325-05

DOI 10.6039/j.issn.1001-0408.2021.11.08

摘 要 目的：建立测定肝微粒体中Foretinib衍生物LWK-126质量浓度的方法，研究其在大鼠、比格犬、人肝微粒体中的代谢稳定性。方法：采用体外肝微粒体孵育体系，将LWK-126分别溶于由还原型烟酰胺腺嘌呤二核苷酸磷酸溶液启动的大鼠、比格犬、人肝微粒体孵育体系中，于37 ℃水浴下分别孵育0、5、10、20、30、60 min时加入含内标（1 μg/mL甲苯磺丁脲）的乙腈终止反应，并采用超高效液相色谱-串联质谱法检测各孵育体系中LWK-126的质量浓度。以Waters BEH C18为色谱柱，以水（含0.01%甲酸）-乙腈（含0.01%甲酸）为流动相进行梯度洗脱，流速为0.4 mL/min，柱温为30 ℃，进样量为2 μL;离子源为电喷雾电离源，采用正离子模式采集，扫描范围为m/z 50→1 200。以孵育0 min时LWK-126的质量浓度为参照，计算其在不同孵育体系中的剩余百分率与酶动力学参数。结果：LWK-126检测质量浓度的线性范围为0.05～15 μg/mL（R 2=0.999 2），定量下限为0.05 μg/mL，最低检测限为0.01 μg/mL，精密度、准确度、提取回收率和基质效应均符合生物样品定量分析的要求。LWK-126在人、大鼠、比格犬肝微粒体中孵育60 min后的剩余百分率分别为（33.17±4.52）%、（3.14±6.73）%、（1.38±5.85）%，半衰期分别为33.15、11.76、5.62 min，清除率分别为38.45、118.81、245.76 μL/（min·mg）。结论：成功建立了肝微粒体中LWK-126质量浓度的测定方法。LWK-126在不同种属肝微粒体中的代谢稳定性排序依次为人>大鼠>比格犬。

关键词 LWK-126;超高效液相色谱-串联质谱法;肝微粒体;代谢稳定性

ABSTRACT   OBJECTIVE： To establish the method for the concentration determination of foretinib derivative LWK-126 in liver microsomes， and to study its metabolism stability in liver microsomes of rats， Beagle dogs and human. METHODS： In the in vitro incubation system of liver microsomes， LWK-126 was dissolved in liver microsomal incubation systems of rats， Beagle dog and human initiated by reduced nicotinamide adenine dinucleotide phosphate solution. After incubation in water at 37 ℃ for 0，5，10，20，30 and 60 min， the reaction was terminated with acetonitrile containing internal standard （1 μg/mL tolbutamide）. UPLC-MS/MS method was applied to determine the concentration of LWK-126 in the incubation systems. The determination was performed on Waters BEH C18 column with mobile phase consisted of water （containing 0.1% formic acid）-acetonitrile （containing 0.1% formic acid） by gradient elution at the flow rate of 0.4 mL/min. The column temperature was 30 ℃， and the sample size was 2 μL. The mass spectral analysis was performed in a positive electrospray ionization mode，and the full MS experiment was run with the selective reaction monitoring mode with a scanning range of m/z 50→1 200. Taking the concentration of LWK-126 at 0 min as reference，the remaining percentage and the enzyme kinetic parameters were calculated. RESULTS： The linear range of LWK-126 was 0.05-15 μg/mL （R 2=0.999 2）; the lower limit of quantification was 0.05 μg/mL， and the lowest detection limit was 0.01       μg/mL. The precision， accuracy， extraction recovery and matrix effect all met the analysis requirements of biological samples. The remaining percentage of LWK-126 in liver microsomes of human， rats and Beagle dogs for 60 min were （33.17±4.52）%，（3.14±6.73）%，（1.38±5.85）%; t1/2 of them were 33.15， 11.76， 5.62 min; the clearance rates were 38.45， 118.81， 245.76 μL/（min·mg）， respectively. CONCLUSIONS： The method for the content determination of LWK-126 in liver microsomes is established successfully. The order of metabolic stability of LWK-126 in liver microsomes of different species is human>rats>Beagle dogs.

KEYWORDS   LWK-126; UPLC-MS/MS; Liver microsomes; Metabolic stability

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