Protocols for stability and homogeneity studies of drugs for its application to doping control

Practical procedures for testing the homogeneity and the stability of drugs in biological fluids, with especial attention to antidoping control, and criteria for data evaluation are proposed. The stability of the analytes was evaluated at -20 and 4 °C in liquid urine and at 4 °C in freeze-dried urine for up to 24 months. A stock of samples was stored at low temperatures (-80 °C) for comparison purposes. Higher temperatures (e.g. -40 or -20 °C) can be used as reference provided that the analyte has proved to be stable under these conditions. For short-term stability testing, aliquots of the urine sample were analyzed after 3 and 7 days of storage at 37 °C. Stability of the analytes after going through three freeze and thaw cycles was also determined. According to the results obtained, long-term stability studies are recommended to be performed by using a sample stored at a reference condition that has to be analyzed at the same time as the test conditions samples. The use of a reference aliquot analyzed in parallel has the potential of allowing the performance of stability studies by using qualitative methods. In these cases, the signal of the analyte is used in the calculations instead of concentration values. This approach has demonstrated to be useful to assess analyte stability independently of the analytical technique applied to analyze urine samples (high-performance liquid chromatography, gas chromatography with nitrogen– phosphorus detection or mass spectrometry detection). The use of qualitative methods can be also considered for homogeneity, and short-term and freeze–thaw stability studies, where the reference sample is also analyzed at the same time as the test samples. The criteria proposed for evaluating the stability and homogeneity are based on the precision of the analytical method. The suitability of the protocols and the criteria for data evaluation has been demonstrated by studying the stability of samples containing caffeine, ephedrine derivatives, amphetamine derivatives, and testosterone and epitestosterone glucuronides.