Assay: Plasma Stability - Mouse
The stability of test compounds in plasma is an important parameter, which not only affects in vivo results, but also the bioanalytical assay strategy and design. Investigation of plasma stability should be performed early in the discovery process in order to assess potential degradation and/or protein binding issues. ..more
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, Lake Nona, FL)
Network: NIH Molecular Libraries Probe Production Network (MLPCN)
Grant Proposal Number:
Assay Provider: Dr. Layton Smith, Sanford-Burnham Medical Research Institute
The stability of test compounds in plasma is an important parameter, which not only affects in vivo results, but also the bioanalytical assay strategy and design. Investigation of plasma stability should be performed early in the discovery process in order to assess potential degradation and/or protein binding issues.
A solution of test compound in plasma is prepared and incubated for a predetermined time period. Aliquots are removed at pre-defined time points and analyzed by LC/MS/MS. The peak area for the parent compound is compared to the time zero sample in order to assess the amount of compound still available.
1) Di L, Kerns EH, Hong Y, Chen H., Development and application of high throughput plasma stability assay for drug discovery., Int J Pharm. 2005 Jun 13;297(1-2):110-9.
2) Edward H. Kerns, Li Di Plasma Stability Methods Drug-like Properties: Concepts, Structure Design and Methods, 2008, Pages 348-352
3) Li Di, Edward H Kerns Profiling drug-like properties in discovery research Current Opinion in Chemical Biology, Volume 7, Issue 3, June 2003, Pages 402-408
4)E.H. Kerns, L. Di Chemical Stability Comprehensive Medicinal Chemistry II, 2007, Chapter 5.20, Pages 489-507
1) Test compound(s) 40uM in DMSO
2) Control compounds at 40uM in DMSO
Procaine - highly unstable (< 95% remaining)
Procainamide - very stable (> 95% remaining)
Isotonic 1X PBS pH 7.4
4) Plasma (mouse)
5) 37 oC shaking incubator (~300 rpm)
6) Tabletop centrifuge with a plate holder-capable rotor
7) 96-well plates, 350 uL (polystyrene)
8) 96-deep well plates, 2mL (polystyrene)
9) Multi-channel pipette and reagent reservoirs
10) 96-well autosampler for LC/MS or HPLC
1. Thaw plasma to RT
2. Centrifuge at 3000 RPM for 10min to pellet insoluble material
3. Make a 1:1 solution in 1X PBS pH 7.4 (50% plasma v/v)
4. Warm plasma:PBS to 37 oC for 30min while preparing samples
1. Prepare test and control compounds by dissolving or diluting compounds to 40uM in 100% DMSO.
2. Dispense 195 ul of Plasma:PBS into the deep well plate.
3. Add 5 ul of sample stock (2.5% DMSO final).
4. Mix 4x by aspirating and dispensing with multichannel pipette
5. Aspirate 50 uL from sample plate and dispense into t=0 crash plate containing 400 uL of ice cold precipitation solution containing internal standard (e.g. Phenacetin @ 2.00 ug/mL).
6. Cover the plate with breathable sealing tape and incubate at 37 oC on an orbital shaker at 300 RPM for 3.0hr (180 min).
7. Remove 50 ul and transfer into t=180 crash plate containing 400 uL of ice cold precipitation solution containing internal standard. If more than one incubation time desired, remove aliquots when needed and place reaction plate back in the 37 oC shaker.
8. Vortex plate for 5 mins and centrifuge at 3000 RPM for 10 minutes.
9. Transfer supernatant to a new plate.
10. Dilute as needed and perform quantitative measurements by LC/MS/MS.
Compounds with <90% remaining after 180 minutes are considered to be active in this assay.
% Remaining = (100 x Peak Area of compound at t=180)/Peak Area of compound at t=0
Scoring for single-concentration screening:
Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics employs a 3-tiered system:
1) The first tier (0-40 range) is reserved for primary or single-concentration screening data and is not applicable to this assay.
2) The second tier (41-80 range) is reserved for dose-response confirmation data of the primary hits that are cherry picked from the HTS mother plates and is not applicable to this assay.
3) The third tier (81-100 range) is reserved for dry-powder compounds that represent purchased and resynthesized positives and their analogues.
Inactive compounds are assigned a score of 81.
Active compounds have a score between 82 and 100.
100 - (18 * (%compound remaining/90))
** Test Concentration.
Data Table (Concise)