purpose
Metabolites of 1 μM verapamil obtained by in vitro incubation of human liver microsomes were identified using the ACQUITY UPLC ® /XevoTM G2 QTof Mass Spectrometry System and MetaboLynxTM XS Application Management Software.
background
In recent years, as more and more first-line drugs have withdrawn from the market due to safety concerns, more attention has been paid to drug metabolism and toxicity studies in drug development. Nowadays, the trend of early drug metabolism research in the drug discovery and development phase has become more apparent. It is common practice to conduct in vitro metabolite studies of parent drugs to quickly identify weaknesses in the early stages of drug development.
One challenge in metabolite identification during the drug discovery phase is the need to provide a rapid and versatile method, and the method should be sensitive enough that in vitro incubation studies can be performed at low μM levels, bringing them closer to the compound The role of the body.
A typical in vitro metabolic study also includes analysis of the metabolic rate and pathway of the parent drug. The ideal analytical protocol for such studies needs to provide an analysis speed and sensitivity for the detection of metabolites at substrate concentrations that mimic in vivo conditions.
Using the Xevo G2 QTof mass spectrometry system coupled with UPLC/MS E , in vitro metabolite studies can be performed at low μM levels with good speed, sensitivity and selectivity.
Figure 1. Incubation results of human liver microsomes verapamil (1 μM) are shown in the MetaboLynx browser.
solution
Verapamil at a concentration of 1 μM was incubated with human liver microsomes at 37 ° C and the reaction was stopped by adding an equal volume of cold acetonitrile at 0, 15, 30, 60, 120 and 240 minutes, respectively. The sample was centrifuged and the supernatant was injected directly.
Using Waters ACQUITY UPLC ® system, ACQUITY UPLC HSS T3 column (1.7 μm, 2.1 x 100 mm ), chromatographed. The mobile phase consisted of 0.1% aqueous formic acid (A) and acetonitrile (B) in an injection volume of 5.0 μL. In the ESI positive ion mode, the Uevo/MS E technology is used for data acquisition using the Xevo G2 QTof mass spectrometer, so that both the parent ion and the product ion data can be obtained simultaneously in one injection.
MetaboLynx XS application management software is used for data mining, and the results are displayed in the MetaboLynx browser (as shown in Figure 1). The product ion information is processed simultaneously and displayed in the Fragmentation Analysis window in the MetaboLynx browser (Figure 2). By analyzing the sample injection at multiple incubation time points, the parent drug clearance curve and the metabolite formation curve can be acquired simultaneously in the same experiment (as shown in Figure 3).
Figure 2. MS/MS information displayed in the Fragment Analysis window.
Figure 3. Clearance curve of verapamil (3A) and its metabolite formation curve (3B).
By using the UPLC/MS E data acquisition strategy, coupled with the chemical intelligence of the MetaboLynx XS data processing workflow, all metabolite identification can be done quickly with a single LC injection. By injecting at multiple time points, the metabolic rate and pathway of the target drug at low concentration (μM) incubation levels can be relatively easily obtained. Therefore, the goal of maximizing production capacity can be easily achieved.
to sum up
This application demonstrates that by using the Xevo G2 QTof mass spectrometry system with UPLC/MS E and MetaboLynx XS workflows, in vitro metabolite studies can be performed at low concentrations (μM) with good speed, sensitivity and selectivity.
The Xevo G2 QTof system and its innovative QuanTofTM and Engineered SimplicityTM technology are the perfect combination with the ACQUITY UPLC system. It is currently the most sensitive benchtop oaTOF instrument with 1 ppm accurate mass measurement capability and data acquisition speeds of up to 20 spectra per second.
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