Chen M, Howe D, Leduc B, Kerr S, Williams D A
Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, Boston, MA, USA.
Xenobiotica. 2007 Sep;37(9):954-71. doi: 10.1080/00498250701620734.
This study reports the development of a specific and sensitive liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) assay for the quantification of the in vitro O-glucuronidation of chloramphenicol (CP), the determination of the kinetic parameters for the O-glucuronidation of CP in pooled human liver microsomes (HLM), the biosynthesis of the CP glucuronides (CPGlu), and identification of the structures of CPGlu by (1)H-nuclear magnetic resonance (NMR) and MS. Two glucuronyl derived metabolites of CP were obtained from the incubation of alamethicin-activated HLM with CP and uridine 5'-diphosphoglucuronic acid (UDPGA) in pH 7.4 TRIS buffer. Their identification and structural confirmation were achieved by beta-glucuronidase hydrolysis, in the presence and absence of UDPGA, and by (1)H-NMR and LC-MS/MS. These two metabolites were biosynthesized, isolated, and purified using high-performance liquid chromatography (HPLC). Their structures were further identified as the 1-O-CPGlu (the minor glucuronide formed at the secondary alcohol of CP) and 3-O-CPGlu (the major glucuronide formed at the primary alcohol of CP) by LC-MS/MS and two-dimensional NMR. The enzymatic kinetic parameters K(m) and V(max) in HLM for the 3-O-CPGlu were determined to be 650 microM and 0.26 nmoles min(-1) mg(-1), respectively, and for the 1-O-CPGlu to be 301 microM and 0.014 nmoles min(-1) mg(-1), respectively. This study also provides a sensitive and specific method for the measurement of in vitro CP-UDP-glucuronosyltransferase (UGT) activity.
本研究报告了一种特异性和灵敏的液相色谱-串联质谱检测（LC-MS/MS）分析方法的开发，该方法用于定量氯霉素（CP）的体外O-葡萄糖醛酸化、测定CP在人肝微粒体（HLM）中O-葡萄糖醛酸化的动力学参数、CP葡萄糖醛酸苷（CPGlu）的生物合成以及通过氢核磁共振（NMR）和质谱鉴定CPGlu的结构。在pH 7.4的TRIS缓冲液中，将阿拉霉素激活的HLM与CP和尿苷5'-二磷酸葡萄糖醛酸（UDPGA）孵育，得到了CP的两种葡萄糖醛酸衍生代谢物。通过在有无UDPGA的情况下进行β-葡萄糖醛酸酶水解以及氢核磁共振和LC-MS/MS实现了它们的鉴定和结构确认。使用高效液相色谱（HPLC）对这两种代谢物进行了生物合成、分离和纯化。通过LC-MS/MS和二维核磁共振进一步将它们的结构鉴定为1-O-CPGlu（在CP仲醇处形成的次要葡萄糖醛酸苷）和3-O-CPGlu（在CP伯醇处形成的主要葡萄糖醛酸苷）。HLM中3-O-CPGlu的酶动力学参数K(m)和V(max)分别测定为650 μM和0.26纳摩尔·分钟⁻¹·毫克⁻¹，1-O-CPGlu的分别为301 μM和0.014纳摩尔·分钟⁻¹·毫克⁻¹。本研究还提供了一种灵敏且特异的方法来测量体外CP-尿苷二磷酸葡萄糖醛酸基转移酶（UGT）活性。
