Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research, Sector 67, SAS Nagar 160 062, Punjab, India.
Eur J Pharm Sci. 2010 Sep 11;41(1):173-88. doi: 10.1016/j.ejps.2010.06.006. Epub 2010 Jun 15.
Rifabutin, an anti-mycobacterial agent, is reported to be extensively metabolized in vivo into more than 20 biotransformation products, with similar profile both in humans and rats. Among the metabolites formed, only seven have been characterized, the remaining are unknown. Hence, the purpose of the present study was to fill this gap by using modern in silico tools combined with advanced liquid chromatography-mass spectrometry (LC-MS) techniques. Initially a comprehensive mass fragmentation pattern for rifabutin was established using Frontier 5.1 software coupled with the data collected from multiple-stage MS (MS(n)), high resolution MS (HR-MS) and hydrogen/deuterium exchange MS (HDE-MS) experiments. The metabolites were then predicted in silico by using different software like MetaSite, Metabolite Predict and MetWorks. The in silico results were verified through in vivo studies by administration of 20mg/kg drug to rats followed by LC-MS analyses. The urine was collected post-dose at different time intervals, and subjected to sample preparation involving sequentially protein-precipitation, liquid-freeze separation, and solid-phase extraction. The drug and metabolites were separated on an HPLC column followed by LC-MS studies. The difference of accurate masses of the drug and metabolites, and differences in their mass fragmentation pattern helped to assign structures to the metabolites and define the site of change. Here also, in silico detection tools were used, which provided complementary information. Using this strategy, 23 metabolites were detected and identified in rat urine without their isolation. The new sixteen metabolites were monohydroxy (05), dihydroxy (04), N-dealkyl (01), 25-O-desacetyl-27-O-demethyl (01), 25-O-desacetyl-23-O-acetyl (01), 25-O-desacetyl-monohydroxy (01), 27-O-demethyl-monohydroxy (01) and dehydrogenated (02) rifabutin.
利福布汀是一种抗分枝杆菌药物，据报道，它在体内广泛代谢为 20 多种生物转化产物，在人和大鼠中的代谢产物谱相似。在形成的代谢产物中，只有七种已被鉴定，其余的则未知。因此，本研究的目的是使用现代计算工具结合先进的液相色谱-质谱（LC-MS）技术来填补这一空白。首先，使用 Frontier 5.1 软件结合多阶段 MS（MS(n)）、高分辨率 MS（HR-MS）和氢/氘交换 MS（HDE-MS）实验收集的数据，建立了利福布汀的综合质谱裂解模式。然后，使用不同的软件，如 MetaSite、Metabolite Predict 和 MetWorks，在计算机上预测代谢产物。通过给大鼠静脉注射 20mg/kg 的药物，然后进行 LC-MS 分析，对计算机预测的结果进行体内研究验证。在不同的时间间隔收集给药后的尿液，进行样品制备，包括依次进行蛋白沉淀、液体冷冻分离和固相萃取。药物和代谢产物在 HPLC 柱上分离，然后进行 LC-MS 研究。药物和代谢产物的精确质量差异及其质量裂解模式的差异有助于对代谢产物进行结构分配，并确定变化的部位。在这里，也使用了计算机检测工具，提供了补充信息。使用这种策略，在大鼠尿液中无需分离就检测和鉴定了 23 种代谢产物。新的 16 种代谢产物为单羟基（05）、二羟基（04）、N-去烷基（01）、25-O-脱乙酰-27-O-去甲基（01）、25-O-脱乙酰-23-O-乙酰基（01）、25-O-脱乙酰-单羟基（01）、27-O-去甲基-单羟基（01）和脱氢（02）利福布汀。
