High‐resolution isotope‐dilution mass spectrometry using metabolism of isotope‐labeled compounds: Application to drug metabolites

Abstract

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) 0 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 mM b-hydroxy-T and 4 0 -hydroxy-D at 60 min starting with 10 mM parent drug as determined by LC/RAM. It was assumed that the amounts of [ The study of the movement (disposition) of molecules and atoms in vivo is constrained by the analytical techniques applicable to a particular structure; however, the approach of labeling with radioisotopes and stable isotopes to study endogenous compounds (endobiotics) and xenobiotics (drugs) offers obvious advantages and has been widely applied in biochemical research. In pharmacology, quantitative (kinetic) and qualitative analysis of the radioactive constituents in tissues and fluids in vivo and in excreta following administration of a 14 C-labeled drug is a standard protocol required for drug development and registration (radiolabeled distribution, or 'mass balance', and metabolism study; the quantitative and qualitative analysis of drug-related material). Far earlier in the drug discovery and development process, questions related to the biotransformation of drugs occur and these include: (1) What are the rates of metabolism across species in vitro and in vivo and what is the role of metabolism in drug clearance (CL), and (2) What is the qualitative nature of the drug metabolite profile including formation of reactive metabolites? It is important to determine not only the qualitative nature of the drug metabolic profile, but to also make a reasonable estimate of the amounts of drug metabolites produced in preclinical species and in man. This estimate of exposure to drug metabolites should be viewed in the context of both (1) exposure relative to other drug-related material (parent alone or sum of drug-related material), a percentage, and (2) exposure in an absolute sense, such as mM concentrationtime in plasma data. In specific situations, the accurate quantitative analysis of drug metabolites is important.For situations where the quantitative analysis of drug metabolites is warranted, there are two general approaches that are commonly employed. The first approach is to obtain an authentic standard for the drug metabolite (DM) and use the standard in the conventional manner. In this process, the structures of DMs are partially characterized and authentic standards synthesized (full structural characterization), and a quantitative analytical method developed using an authentic standard. This is an expensive and time-consuming process, although the use of cloned-expressed enzymes followed by purification of product has streamlined the process somewhat and certainly affords considerable cost containmen...

Bio-generation of stable isotope labeled internal standards for absolute and relative quantitation of drug metabolites in plasma samples by LC–MS/MS

“…This process can also be applied to STIL parent compounds to generate STIL metabolites, which has been previously used in elucidation of metabolite structures and biotransformation mechanisms [20][21][22]. The advantage of using such approach for quantitative analysis has also been recently discussed in literature [14]. In this study, we adopted this practice to generate STIL metabolites for the use as IS for quantitation of metabolites in biological samples.…”

“…Accordingly, methods of relative quantitation without reference standards were developed to mitigate this issue. A metabolite-to-parent mass spectrometry (MS) response factor (RF), which can be obtained by MS-to-UV calibration [11,12] MS-to-radioactivity calibration [13,14] or MS-to-NMR calibration [11,15] has been used to normalize analyte-specific response variations, thereby providing a tool to estimate relative exposures across species. Nevertheless, methods using RF usually do not have high sensitivity and can be labor intensive.…”