Microsomal Clearance/Stability Assay

Metabolism is a major clearance mechanism that predominantly occurs in the liver. Liver microsomes are a subcellular fraction of the liver that can be derived from a variety of animals and used to determine the half-life (t1/2) or in vitro intrinsic clearance (CLint, app) of test compounds. Microsomal stability is often used in early lead-optimisation, due to the relatively low costs and high-throughput capability. Measuring microsomal clearance not only assists in ranking/triaging compounds for in vivo studies, but also plays a role in predicting in vivo hepatic clearance.

Domainex’s Standard Experimental Procedure:

Test compounds are incubated with liver microsomes in the presence of NADPH at 37 °C. The reaction mixture is sampled at allocated timepoints into a cold stop plate containing acetonitrile and an internal standard. The samples are subsequently assessed by Ultra-High Performance Liquid Chromatography (UHPLC)-Mass Spectrometry (MS) to quantify depletion of the test compound. Two control compounds are also assessed in each run to ensure intrinsic clearance values fall within the acceptance criteria. Example microsomal turnover data is shown in Figures 1-2. 

Figure 1: Depletion of test compounds is monitored following incubation with human liver microsomes (HLM). 

microsomal feature icons
microsomal icons
microsome feature icons
* Other options available upon request

Data Analysis:

Microsomal extracts are analysed using Waters Acquity UHPLC TQ-S, TQS-micro or TQ-XS instruments. Triple quadrupole mass spectrometers, operated in multiple reaction monitoring (MRM) mode, provide accurate quantification with excellent sensitivity, selectivity and reproducibility.   

Plotting ln compound concentration against time allows determination of half-life and intrinsic clearance using the equations below:

microsomal equations


The results are reported in Excel file format as CLint, app (µl/min/mg protein), t1/2 (mins) and parent remaining (%) including standard error of mean (SEM). Any relevant comments about compound stability/solubility/binding are also included in the report.

Turnaround time from receipt of the test compounds to release of data is typically less than 2 weeks.

rate of HLM turnover graphs

Figure 2: Natural Log linear plots of compound turnover allow Clint,app and t1/2 values to be calculated.