ANALYTICAL METHOD FOR QUANTITATION OF BREFELDIN A IN MOUSE PLASMA

Brefeldin A（NSC 89671），一种从青霉菌中分离的大环内酯 Brefeldianum作为一种抗肿瘤药物正在进行临床前评价 在国家癌症研究所。 布雷菲德菌素A的特异性测定是 开发涉及其从血浆中提取到乙醚， 通过在气体前用七氟丁酰咪唑衍生化 色谱法与电子捕获检测。 最低浓度 在50微升的 血浆浓度为0.1 μ g/ml（0.36 μ M）。 该试验已用于 研究布雷菲德菌素A在小鼠血浆中药代动力学。 但由于 小鼠（102 ml/min/kg）和犬（53 ml/min/kg），血浆水平仅可监测约60 min 在静脉推注给予最大耐受剂量（小鼠 约26 mg/kg;犬约8 mg/kg）。 因此，交替 寻求各种办法，努力开发一种更敏感的方法。 使用反相HPLC和UV检测，定量测定 布雷菲德菌素A在生物体液中的含量。 采用的方法 通过用叔丁基醚进行液/液萃取分离分析物 甲基醚 在蒸发有机溶剂和 复溶后，通过反相HPLC分离分析物， 在215 ng下通过UV吸收检测。 的定量下限 100微升血浆样品中的布雷菲德菌素A为0.1微克/毫升。 而 这并不代表灵敏度比GC方法有任何改进， HPLC测定允许对药物进行特异性测定， 技术上比GC分析更方便。 采用LC/热喷雾MS，布雷菲德菌素A的最低浓度 在50 μ l血浆中可定量的浓度为0.005 μ g/ml（0.014 μ M）。 方法的线性范围为0.005 ~ 0.500 μ g/ml g/ml，相关系数通常为0.99或更大。 这 代表灵敏度的提高， 以前的方法。 方法的验证和应用 目前正在进行中。

Brefeldin A (NSC 89671), a macrolide isolated from Penicillium brefeldianum, is undergoing preclinical evaluation as an antitumor agent at the National Cancer Institute. A specific assay for brefeldin A was developed involving its extraction from plasma into diethyl ether followed by derivatization with heptafluorobutyrylimidazole prior to gas chromatography with electron capture detection. The lowest concentration of brefeldin A determined with acceptable reproducibility in 50 micro l of plasma was 0.1 micro g/ml (0.36 micro M). This assay has been utilized to investigate brefeldin A plasma pharmacokinetics in mice. However, due to the rapid clearance of the drug by mice (102 ml/min/kg) and dogs (53 ml/min/kg), plasma levels could only be monitored for approximately 60 min following bolus i.v. administration of maximum tolerated doses (mouse approximately 26 mg/kg; dog approximately 8 mg/kg). Thus, alternate approaches were sought in an effort to develop a more sensitive method. Using reversed-phase HPLC with UV detection, an assay for the quantitation of brefeldin A in biological fluids was developed. The method employed isolation of the analyte by liquid/liquid extraction with tert-butyl methyl ether. Following evaporation of the organic solvent and reconstitution, the analyte was separated by reversed-phase HPLC and detection by UV absorption at 215 ng. The lower limit of quantitation for brefeldin A in a 100 micro l sample of plasma was 0.1 micro g/ml. While this did not represent any improvement in sensitivity over the GC method, the HPLC assay permitted specific determination of the drug and was technically more convenient than the GC assay. Employing LC/thermospray MS, the lowest concentration of brefeldin A quantifiable in 50 micro l of plasma was 0.005 micro g/ml (0.014 micro M). The dynamic linear range of the assay extended from 0.005 to 0.500 micro g/ml, with a correlation coefficient typically 0.99 or greater. This represents an improvement in sensitivity by about a factor of 20 compared to previous methods. Validation and application of the method are currently in progress.