PT  - JOURNAL ARTICLE
AU  - Gregory S. Walker
AU  - Jonathan N. Bauman
AU  - Tim F. Ryder
AU  - Evan B. Smith
AU  - Douglas K. Spracklin
AU  - R. Scott Obach
TI  - Biosynthesis of Drug Metabolites and Quantitation Using NMR Spectroscopy for Use in Pharmacologic and Drug Metabolism Studies
AID  - 10.1124/dmd.114.059204
DP  - 2014 Oct 01
TA  - Drug Metabolism and Disposition
PG  - 1627--1639
VI  - 42
IP  - 10
4099  - http://dmd.aspetjournals.org/content/42/10/1627.short
4100  - http://dmd.aspetjournals.org/content/42/10/1627.full
SO  - Drug Metab Dispos2014 Oct 01; 42
AB  - The contribution of drug metabolites to the pharmacologic and toxicologic activity of a drug can be important; however, for a variety of reasons metabolites can frequently be difficult to synthesize. To meet the need of having samples of drug metabolites for further study, we have developed biosynthetic methods coupled with quantitative NMR spectroscopy (qNMR) to generate solutions of metabolites of known structure and concentration. These quantitative samples can be used in a variety of ways when a synthetic sample is unavailable, including pharmacologic assays, standards for in vitro work to help establish clearance pathways, and/or as analytical standards for bioanalytical work to ascertain exposure, among others. We illustrate five examples of metabolite biosynthesis and qNMR. The types of metabolites include one glucuronide and four oxidative products. Concentrations of the isolated metabolite stock solutions ranged from 0.048 to 8.3 mM, with volumes from approximately 0.04 to 0.150 ml in hexadeutarated dimethylsulfoxide. These specific quantified isolates were used as standards in the drug discovery setting as substrates in pharmacology assays, for bioanalytical assays to establish exposure, and in variety of routine absorption, distribution, metabolism, and excretion assays, such as protein binding and determining blood-to-plasma ratios. The methods used to generate these materials are described in detail with the objective that these methods can be generally used for metabolite biosynthesis and isolation.