Metabolite Identification Using Multiple Mass Defect Filters and Higher Energy Collisional Dissociation on a Hybrid Mass Spectrometer

May 01, 2008
By Spectroscopy Editors

An integral part of drug discovery and development is the identification of drug metabolites formed through phase I and phase II metabolic reactions. These metabolites may either have intrinsic pharmacological activity or display specific toxicity. Liquid chromatography coupled with mass spectrometry (LC–MS) has become the cornerstone in drug metabolite identification because of its sensitivity and ability to analyze complex mixtures. In particular, LC–MS n employing linear ion-trap MS has become widely used because of its speed, sensitivity, and robustness in generating rich structure information. However, challenges still remain in detecting and identifying metabolites in the presence of highly complex biological matrices.

Coupling a mass analyzer to a linear ion trap greatly facilitates the task of metabolite identification because it not only enables parallel data acquisition with high mass accuracy and resolution, but also provides ion manipulations that occur after the linear ion trap. High resolution and accurate mass help to resolve and identify metabolite peaks from background matrix ions and also allow the use of post-acquisition data processing tools such as the mass defect filter (MDF) to reduce the number of false positives by removing the vast majority of matrix-related background ions. Higher energy collisional dissociation (HCD) was introduced recently on the Thermo Scientific LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, California) as an alternative dissociation method by adding a new collision cell behind the C-trap region (Figure 1). HCD can be used to generate low mass diagnostic ions in MS-MS mode, and can be used in combination with collision induced dissociation (CID) in the linear ion-trap mass spectrometer for MS n experiments.


Figure 1
MMDF is a new feature in MetWorks software (Thermo Fisher Scientific). This postacquisition data processing tool allows the user to combine the results from as many as six MDFs. To explain MMDF, one needs to first understand the terms mass defect and MDF.

The term mass defect originates from the fact that only the monoisotopic element 12C has an integer value for atomic weight, that is, 12.000000. Mass defect refers to the difference between the exact mass of an element (or a compound) and its closest integer value. It can be positive (larger than the nominal mass) or negative (smaller than the nominal mass). MDF refers to a postacquisition data filtering technique based on the mass defect of the parent drug and its metabolites. To use MDF, it is critically important to use high mass accuracy data from high-resolution analyses.

Each parent compound has a mass defect, which will be associated with its metabolites because a large portion of the parent compound structure usually remains unchanged during biotransformation. In other words, the mass defect of metabolites will lie within a relatively narrow range. Based upon the molecular weight of the parent compound, estimation also can be made regarding the range of molecular weight in which these metabolites will occur. MDF can be applied to filter out all ions that fall outside of the expected molecular weight range, as well as those ions that are within the expected molecular weight range but exceed the expected mass defect range. This data reduction technique allows users to focus on the analysis of species that are potential drug metabolite candidates.

Irinotecan (CPT-11; 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy-camptothecin) is a water-soluble carbamate prodrug of camptothecin and is activated in vivo to SN-38, a potent topoisomerase I inhibitor (1,2). Currently, irinotecan, combined with 5-fluorouracil and leucovorin, is approved by the U.S. Food and Drug Administration as first-line therapy in the treatment of metastatic carcinoma of the colon or rectum (2). In this study, MMDF and HCD on a hybrid mass spectrometer were used to study the biotransformations of irinotecan in hepatocyte incubation and identify its metabolites.


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