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High-resolution isotope-dilution mass spectrometry using metabolism of isotope-labeled compounds: Application to drug metabolites.
MedLine Citation:
PMID:  23059872     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
RATIONALE: Herein we describe a generic quantitative method using high-resolution, isotope-dilution (HRID) metabolism of isotope-labeled compounds and apply it to the analysis of drug metabolites (DMs) in human plasma. Metabolites (drug) in Safety Testing (MIST) application was one goal.
METHODS: Testosterone (T) and diclofenac (D) were chosen for mass defect characteristics. T, [(14) C]T, [(13) C(3) ]T, D, [(14) C]D, and [(13) C(6) ]D were metabolized separately in vitro to produce test metabolites. Liquid chromatography/radioactivity monitoring (LC/RAM) analysis was used to determine the concentration of the test metabolites in the incubates. The incubates containing 6β-hydroxy-T (6βHT), [(13) C(3) ]6βHT, 4'-hydroxy-D (4'HD) and [(13) C(6) ]4'HD were used to make standard curves. Plasma samples were prepared by 'dilute-and-shoot' and analyzed by LC/MS using SCIEX 5000 and Thermo Orbitrap instrumentation.
RESULTS: Human hepatic microsomes and the S9 fraction produced between 2-6 μM β-hydroxy-T and 4'-hydroxy-D at 60 min starting with 10 μM parent drug as determined by LC/RAM. It was assumed that the amounts of [(13) C(3) ]6βHT and [(13) C(6) ]4'HD produced were similar. Dilutions and standard curves were prepared in human plasma. Analysis of the DMs by LC/MS/MS and LC/HRMS exhibited linear responses over a useable range.
CONCLUSIONS: HRID with metabolism of an isotope-labeled compound reduces the number of analytical variables considerably. Metabolism of the parent drug to DMs represents a simpler alternative quantitative method compared with traditional approaches. The method will have useful applications for evaluating MIST situations. Copyright © 2012 John Wiley & Sons, Ltd.
Authors:
J James Vrbanac; Allen Hilgers; Tyson Dubnicka; F Barclay Shilliday; David Humphries; Roger N Hayes
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Rapid communications in mass spectrometry : RCM     Volume:  26     ISSN:  1097-0231     ISO Abbreviation:  Rapid Commun. Mass Spectrom.     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-10-12     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8802365     Medline TA:  Rapid Commun Mass Spectrom     Country:  England    
Other Details:
Languages:  eng     Pagination:  2569-76     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 John Wiley & Sons, Ltd.
Affiliation:
Discovery Sciences, MPI Research, Mattawan, MI, 49071, USA. jim.vrbanac@mpiresearch.com.
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