High-resolution isotope-dilution mass spectrometry using metabolism of isotope-labeled compounds: application to drug metabolites.
BACKGROUND
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.
Discovery Sciences, MPI Research, Mattawan, MI 49071, USA. jim.vrbanac@mpiresearch.com
J James
JJ
Vrbanac
Allen
A
Hilgers
Tyson
T
Dubnicka
F Barclay
FB
Shilliday
David
D
Humphries
Roger N
RN
Hayes
D004008:Diclofenac / Q000032:analysis / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007554:Isotopes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013739:Testosterone / Q000032:analysis / Q000097:blood / Q000737:chemistry / Q000378:metabolism
D007554:Isotopes; D004364:Pharmaceutical Preparations; D004008:Diclofenac; D013739:Testosterone