Identification and characterization of two chloramphenicol glucuronides from the in vitro glucuronidation of chloramphenicol in human liver microsomes.
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.
Chen
M
M
Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, Boston, MA, USA. mchen@combinatorx.com
Howe
D
D
Leduc
B
B
Kerr
S
S
Williams
D A
DA
D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics; D001711:Biotransformation; D002701:Chloramphenicol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics*; D002853:Chromatography, Liquid / Q000379:methods; D004285:Dogs; D020719:Glucuronides / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008252:Macaca fascicularis; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013045:Species Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods
D000900:Anti-Bacterial Agents; D020719:Glucuronides; D002701:Chloramphenicol