Characterisation of reactive metabolites


Our expertise in reactive metabolites analysis

Our reactive metabolite characterisation analytics is combining the benefits of the various approaches. We use a test system with three different stable isotope labeled trapping agents ((13)C2, (15)N-glutathione, K(13)C(15)N and (13)C,(15)N2-semicarbazide, for trapping of the different types of reactive metabolites from microsomal incubations. For detection we use UPLC-TOF/MS. The high mass resolution and accurate mass measurement capability of TOFMS, with the possibility to simultaneously acquire in-source fragment ion data, enables also structural identification of the detected trapped reactive metabolites.

Case study demonstrating superiority of TOF-MS vs triple quadrupole MS approach in reactive metabolite detection for ethinyl estradiol GSH-conjugates. Traces a-b) MS/MS parent ion scanning of m/z 272 / 275 (labeling causes 3 u isotope pattern) 2 detected conjugates; Trace c) MS/MS neutral loss scanning 129 u 1 poorly detected conjugate; Traces d-j) TOF-MS ion chromatograms 10 detected conjugates. See ref: Rapid Communications in Mass Spectrometry, 23, 843–855, 2009.

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Background of reactive metabolites

The small molecule metabolites may be biologically active and confer to different toxicity and/or pharmacology effects, so called reactive metabolites. This phenomenon is well explained in pro-drugs, in which the administered parent compound is inactive and and is metabolized to become the pharmacologically active molecule in a process called metabolic activation.

The reactive drug metabolites are usually electrophilic compounds that easily react with nucleophiles, such as glutathione (GSH) that works naturally in the human body as a scavenger for reactive metabolites. In addition, these compounds can also form covalent adducts with biomolecules, like proteins and nucleic acids. Also their half-life is typically short due to the reactivity. Reactive drug metabolites are believed to be one of the main factors behind unexpected idiosyncratic drug-induced toxicity and therefore their identification early in the drug discovery process is of big importance.

Due to their high reactivity and short half-life, reactive metabolites are not directly detected by traditional analytical methods, but are analyzed by chemical trapping with nucleophilic agents such as glutathione tri-peptide (GSH; most often used), potassium cyanide (KCN; used for hard electrophiles) or semicarbazide, leading to formation of stable conjugates, which are then detected by LC-MS methods.

Common ways for analytics of reactive metabolites

The most common approach for detection of reactive metabolites has been the GSH trapping in microsomal incubations in a manner similar as for metabolite profiling with the inlcusion of GSH as a trapping agent. This is followed by LC-MS/MS analysis by neutral loss scanning of 129 Da due to cleavage of the pyroglutamic moiety from the glutathione conjugate. However, the detection sensitivity of scanning mode data acquisition with triple quadrupole MS is quite poor, in addition all classes of GSH conjugates do not form the neutral loss of 129 Da, leading to false negative detection. Another way of reactive metabolite detection has been to use negative ion mode precursor ion scanning for the fragment ion at m/z 272. However, the detection sensitivity of the precursor ion scanning mode is also relatively poor, and the approach gives only little structural information regarding the reactive metabolite.

Due to these drawbacks of the common ways to detect reactive metabolites, we us a method that combines the benefits form these methods. We use a test system with three different stable isotope labeled trapping agents ((13)C2, (15)N-glutathione, K(13)C(15)N and (13)C,(15)N2-semicarbazide, for trapping of the different types of reactive metabolites from microsomal incubations. See the top of this page for more information.