Quantitative analysis of betulinic acid in mouse, rat and dog plasma using electrospray liquid chromatography/mass spectrometry
Quantitative analysis of betulinic acid in mouse, rat and dog plasma using electrospray liquid chromatography/mass spectrometry
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DOI：
10.1002/rcm.1155
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发表时间：
2003-01-01
影响因子：
2
通讯作者：
van Breemen, RB
中科院分区：
文献类型：
--
作者：
Cheng, X;Shin, YG;van Breemen, RB
桦木酸作为治疗转移性恶性黑色素瘤的治疗剂正在开发中。为支持药代动力学和毒理学评价，开发了一种基于液相色谱/质谱（LC/MS）的稳健测定方法，用于桦木酸的定量分析。样品制备包括通过加入3体积乙腈和1体积甲醇进行血浆脱蛋白，然后离心。使用偶联负离子电喷雾质谱仪的等度反相高效液相色谱（HPLC）系统分析上清液的等分试样。使用选择离子监测在m/z 455处检测桦木酸和异构体内标油酸的去质子化分子。桦木酸的检测限为0.5 μ g（1.1 fmol），注射到柱上（50 μ g/mL，10穆尔注射体积），定量限为2 μ g（4.4 fmol，200 μ g/mL，10穆尔注射体积）。桦木酸在血浆样品中在-20 ℃下稳定至少3周。日内变异系数≤ 6.4%，日间变异系数≤ 9.0%。通过分析加标到小鼠、大鼠和犬血浆中的桦木酸，通过测定桦木酸与血浆蛋白的结合程度，以及通过测量体内腹膜内或静脉内给药后小鼠和大鼠血浆中的桦木酸，证明了该测定法的实用性。在小鼠、大鼠或狗血浆中，当浓度为15和25 μ L/mL时，桦木酸与血清蛋白的结合率为99.99%，当浓度为5 μ L/mL时，桦木酸与血清蛋白的结合率大于或等于99.97%。版权所有（C）2003约翰威利父子有限公司。
Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fmol) injected on-column (50 pg/mL, 10 muL injection volume), and the limit of quantitation was 2 pg (4.4 fmol, 200 pg/mL, 10 muL injection volume). Betulinic acid was stable in plasma samples at -20"C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were less than or equal to6.4 and less than or equal to9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 muLg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 mug/mL, betulinic acid was greater than or equal to99.97% bound. Copyright (C) 2003 John Wiley Sons, Ltd.