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EA433
Detection of Glucuronides – Human Liver Microsomes
This assay is used to detect glucuronidation of a test compound after incubation with human liver microsomes in the presence of UDPGA
Required from Customer
- Either a minimum of 1.2 mL of test compound at 10 mM in DMSO, or 20 mg of powder
- Exact molecular mass of test compound and its salt form
- MSDS or handling and storage information (e.g., light sensitive, store at -20°C, etc.)
Deliverables
- Presence or absence of glucuronide conjugates formed from test and control compounds
Substrate
- Test compound at a single concentration, typically 50 µM, dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)
Assay System
- Pooled, mixed-gender, human liver microsomes from ≥10 donors, with NADPH, UDPGA and alamethicin added
- LC-MS/MS is used to detect, but not quantify, peaks present in incubation with test or control compound but not in blank, and corresponding to glucuronide conjugates based on mass differential vs. parent compound
Assay Conditions
- Run the assay with a single incubation (N=1)
- Incubate test compound at 37°C in buffer containing
- 0.5 mg/mL microsomal protein, sampling at 0 and 60 minutes
- Incubate reaction mixture without test compound (blank) at 37°C, sampling at 60 minutes
- Incubate positive control (7-hydroxycoumarin) in parallel, sampling at 60 minutes
Assay QC
- The control compound, 7-hydroxycoumarin, is run in parallel to verify the enzymatic activity of the microsomes
Notes
- The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
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