Metabolite Profiling and Identification using High Resolution Accurate Mass Spectrometry

Understand the drug metabolism of your compound by identifying which metabolites are formed during in vitro or in vivo studies.

Metabolite profiling and identification is included in Cyprotex's portfolio of ADME services. Cyprotex deliver consistent, high quality data in line with regulatory guidelines, and can adapt protocols based on specific customer requirements.

Profiling and identification of metabolites

Understanding which metabolites are likely to be formed in vivo is essential for interpreting pharmacology, pharmacokinetic and toxicology data.
Cyprotex’s metabolite profiling and identification service uses either the AB Sciex TripleTOF^® 6600 or the Waters Xevo^® G2-S QTof to determine high resolution accurate mass of any metabolites – leading to increased sensitivity and enhanced structural characterization.
Service can be combined with the LabLogic Beta-Ram^® for radiochemical detection and quantification of metabolites or with gas chromatography (APGC) for analysis of different types of chemistry.
Critical information on the formation of metabolites including, where appropriate, both Phase I and Phase II metabolism, and comparison of drug metabolism routes in different species is provided.
Metabolites can be investigated in a number of different matrices including in vitro microsomal incubations, hepatocyte incubations, expressed enzyme incubations as well as in vivo samples.
Cyprotex offers a range of metabolite profiling services depending upon the level of detail and interpretation required.

We encourage the identification of differences in drug metabolism between animals used in nonclinical safety assessments and humans as early as possible during the drug development process. The discovery of disproportionate drug metabolites late in drug development can potentially cause development and marketing delays.

¹FDA Guidance for Industry: Safety Testing of Drug Metabolites (February 2008)

Protocol

Metabolite profiling and identification protocol

Matrices Analyzed	Typically, microsomal incubations, hepatocyte incubations, expressed enzyme incubations, and plasma (others available on request)
Instruments	AB Sciex TripleTOF^® 6600 Waters Xevo^® G2-S QTof LabLogic Beta-Ram^® (for radiolabelled studies)
Data Delivery	Various options are available depending on the depth and breadth of metabolite profiling or identification that is required. Typically data is delivered as either: Summary document with chromatograms and ion spectra Customized report without structural elucidation Comprehensive report with structural elucidation Data delivery format is discussed with the client prior to study initiation. This way the appropriate information is returned in the most timely and cost effective manner.

Data

Data from the metabolite profiling and identification service

A summary of all potential metabolites is provided.

Name	Formula	Mass Difference	m/z Found	Mass Error (ppm)	Identifier	Rt (min)
Parent	C₁₈H₂₅NO	-	272.02010	-1.5	-	8.33
Dehydration	-H₂O	-18.0118	254.1896	-4.9	M5	9.09
Demethylation	-CH₂	-14.0159	258.1855	-1.0	M3	8.28
Demethylation	-CH₂	-14.0154	258.1860	0.9	M1	5.72
Oxidation	+O	+15.9935	288.1949	-1.4	M4	8.85
Oxidation	+O	+15.9946	288.1930	-0.3	M2	6.67

Figure 1
Metabolite profiling of dextromethorphan metabolism in human liver microsomes.

Q&A

Questions and answers on metabolite profiling and identification

Why is understanding the routes of metabolism of a compound important?

There are several reasons why understanding the metabolic profile of a compound is important. Firstly, the FDA Guidance for Safety Testing of Drug Metabolites (2008) recommends in vitro evaluation of interspecies differences in drug metabolism between humans and those animals which are expected to be used in preclinical safety assessments. If a metabolite is formed only in humans and is absent in the animal test species, or if the metabolite is present at disproportionately higher levels in humans than in the animal species, then it may be necessary to assess the preclinical safety of the metabolite in question. Secondly, understanding the metabolic liability of a compound is important in directing chemistry. If a compound is very rapidly cleared, then identifying which functional groups are undergoing metabolism will be valuable in understanding how the chemistry may be altered to reduce compound degradation.

What do you recommend as an appropriate strategy for evaluation of the routes of metabolism of a compound?

Before embarking on an in vitro metabolite profiling study, it is recommended that the compound has previously been investigated in the in vitro drug metabolizing system of choice. This will give an indication of the extent of metabolism and whether sensitivity by mass spectrometry is likely to be a problem.

The decision to progress to metabolite profiling and/or identification, and the level of interpretation which is required is driven by the goal of the study. For example, screening multiple compounds through microsomal clearance may only require the identification of the major metabolites, in order to localize any metabolic hot-spots that can then be designed out. A single compound that is screened through multiple hepatocyte species is likely to be require a more complete interpretation if it is to aid in the decision point for pre-clinical toxicology models (minimize the chances of human specific metabolites). Cyprotex are able to offer services ranging from data acquisition through to full structural elucidation.

What are the advantages of high resolution accurate mass spectrometry instruments for metabolite profiling studies?

By utilizing high resolution, accurate mass spectrometry, specificity is improved without compromising on sensitivity. Greater confidence in structural elucidation can occur as both MS precursor and 'MS/MS' products ions can only be rationalized with a very small number of elemental combinations – therefore increasing the confidence in structural assignment.