Stability studies of selected doping agents in urine:: caffeine

The stability of caffeine in urine samples has been studied. A high-performance liquid chromatography (HPLC) method for the quantification of caffeine in urine samples was validated for that purpose. The method consists of a liquid-liquid extraction at alkaline pH with chloroform-2-propanol] (9: 1, v/v) with a salting out effect. 7-Ethltheophylline was used as internal standard y (ISTD). Analyses were performed with an Ultrasphere ODS C 18 column using water/acetonitrile (90: 10, v/v) as a mobile phase at a flow rate of I ml/min. Ultraviolet absorption at 280 nm was monitored. Extraction recoveries for caffeine and 7-ethyltheophylline were 81.4 +/- 6.0 and 87.3 +/- 5.7%, respectively. The calibration curves were demonstrated to be linear in the working range of 6-30 mug/ml (r(2) > 0.990). The limit of detection and the limit of quantitation were estimated as 0.7 and 2.0 mug/ml, respectively. Precisions in the range of 1.5-9.2 and 4.1-5.8% were obtained in intra- and inter-assay studies, respectively, using control samples containing 10, 14 and 26 mug/ml of caffeine. Accuracies ranging from 2.9 to 7.4% for intra-assay experiments, and from 3.9 to 5.4% in inter-assay studies were obtained. Stability of caffeine in urine samples was evaluated after long- and short-term storage at different temperature conditions. The batches of spiked urine were submitted to sterilization by filtration. No adsorption of the analyte on filters was observed. Before starting stability studies, batches of reference materials were tested for homogeneity. For long-term stability testing, caffeine concentration in freeze-dried urine stored at 4degreesC and in liquid urine samples stored at 4, -20, -40 and -80degreesC was determined at several time intervals for 18 months. For short-term stability testing, caffeine concentration was evaluated in liquid urine stored at 37 degreesC for 7 days. The effect of repeated freezing (at -20degreesC) and thawing was also studied for up to three cycles. The stability of caffeine was also evaluated in non-sterile samples stored at -20 degreesC for 18 months. No significant loss of the compound was observed at any of the investigated conditions. (C) 2003 Elsevier B.V. All rights reserved.