Background: The in vitro stability screening of drugs solutions utilized as membrane markers, internal cellular standardization and permeability class markers in typical biopharmaceutical applications is essential for accurate drug exposure and cellular response. The stability of the stock drug solutions were tested by high-performance liquid chromatography (HPLC) under various storage, preparation and standard experimental temperature conditions and timepoints used for biopharmaceutical studies.

Methods: Atenolol, FITC-dextran, furosemide, piroxicam, theophylline were evaluated in pH 6.8 and 7.4 buffers at 22°C, 37°C, 4°C, -20°C or -80°C over 7 days.  Drug stock solutions samples were tested was on an Agilent 1100 HPLC with UV and fluorescence detection.  Separation was obtained on Phenomenex Luna C18 (Torrance, CA) column with phosphate buffers (pH =3.0, 6.9, 7.0) and acetonitrile (10-30%).

Results: Furosemide and theophylline were stable at all temperature conditions.  FITC-dextran decreased 7% after 2 weeks at -80°C. Piroxicam at  -20°C showed 3% decrease after 3 weeks.  At -20°C, atenolol had a 2% decrease after 2 weeks. All drugs at 37°C were stable for 6 hours. There was less than 10% variation for each drug at all conditions. There was no difference between conventional biopharmaceutical cellular test pH’s of 6.8 and 7.4 at all temperature. 

Conclusion: A standard screening protocol was developed and tested for commonly used in vitro drug stock solutions. Screening data indicated that avoidance of drug loss or degradation, usage must occur in 1 week. Efficient screening protocols are essential for the application of regulatory risk management tools such as the Biopharmaceutical Classification Guidance.