Stability in aqueous solutions is a fundamental requirement to a successful drug candidate. Degradation may be caused by a variety of mechanisms: hydrolysis, oxidation, light-catalysed degradation and others. At early stages of drug discovery, screening for stability in buffer solutions at acidic, neutral and basic pH is desirable for eliminating potentially troublesome candidates.
Chemical stability analyses are often performed by HPLC with UV-Vis detection at 3 wavelengths. Yet, in some cases degradation products may be poorly separated from parent compounds, causing inaccuracy in analysis. For this reason in our lab this analysis is typically performed by LC/MS (although UV-Vis detection can also be performed upon request).
The assay is performed in a reusable 96-well Teflon plate (Millipore) to avoid possible artifacts caused by adsorption of certain compounds to polypropylene surfaces. Upon request, polypropylene plates can be used in the same assay setup for the non-specific binding assessment, which can be conveniently combined with the chemical stability assay, as PTFE (Teflon®) and polypropylene have different binding characteristics.
Glycine buffer (pH 8 – 11), PBS (pH 7 – 8) and acetate buffer (acidic – pH 4-6) are used in this assay to cover main pH ranges used in ADME. Stock solutions with concentration of 10 mM of the test compounds are prepared in DMSO. These solutions are stored at -20°C and are used for preparing working solutions in DMSO and buffers (1-5 μM). The compound solutions are incubated in experimental buffers at 37°C for a specified time interval. The sample aliquotes are taken at 6 time points: 0, 60, 120, 180, 240 and 300 min. To stabilize time point samples prior to HPLC/MS analysis, they are stored at 4°C in 50% acetonitrile/buffer, covered by adhesive sealing film. The HPLC/MS measurements are performed using Shimadzu VP HPLC system coupled with tandem mass spectrometer API3000 (PE Sciex). Data acquisition and analysis are performed using Analyst 1.5.2 software (PE Sciex).
In this assay, propantheline is used as a control. This compound is stable at acidic pH, slightly unstable at pH 7.4 and unstable at pH 9.4. The newly obtained data for propantheline is always compared to the previous measurements for quality control.
Sample requirement is at least 100 µL of 20 mM stock compound solutions in DMSO for kinetic solubility measurements, and 2 x 2 µmoles of dry compound for thermodynamic solubility measurements.