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Analysis of a Verapamil Microsomal Incubation using Metabolite ID and Mass FrontierTM

trong>Overview
Metabolite ID and Mass Frontier have been designed to work in conjunction with each other to efficiently extract information from LC/MS data files obtained from in vivo and in vitro metabolism samples. In order to fully utilize the capabilities of these software packages, MS2 as well as MS data should be acquired. Following a typical work flow, Metabolite ID is used to identify both expected and unexpected metabolites. Once the modifications in the sample have been determined, MS2 spectra can then be exported from the Metabolite ID software package to the Mass Frontier software package for more in-depth characterization. For example, Metabolite ID might determine that a drug molecule has three distinct sites of hydroxylation. Mass Frontier would then be used to locate the specific sites in the molecule where the hydroxylation occurs.

In this application note, Metabolite ID and Mass Frontier are utilized for the analysis of microsomal incubations of Verapamil, a calcium channel blocker used to treat various cardiovascular conditions. The goal of this publication is to describe how Metabolite ID and Mass Frontier can be used to extract the maximum amount of information from the data.

Experimental
Verapamil hydrochloride (Sigma-Aldrich) at a concentration of 100 um was incubated for four hours with liver microsomes (In Vitro Technologies) and Uridine diphosphate glucuronic acid (UDPGA) before termination with an equal volume of acetonitrile. A high drug concentration and a long incubation time were used in order to generate the maximum number of metabolites. The UDPGA was added to generate phase two glucuronide conjugates. Microsomes from male Cynomolgus monkeys, male Wistar rats, and male Beagle dogs were used to probe interspecies variations in metabolism. The incubation mixtures were analyzed without any additional sample preparation or cleanup
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