Caffeine analysis in urine by gas chromatography mass spectrometry: A non-derivatization detection and confirmatory method

Background and Aims: Caffeine is a xanthine alkaloid found naturally in plants. Caffeine has cardiotonic and stimulant effects in humans and animals. For this reason, caffeine is on the monitoring list for human sports and is listed as a feed contaminant in horse racing. The aim of this study was to develop a rapid, practical, and specific method for the determination of caffeine in horse urine.Methods: In the new method, the pH of the sample was adjusted by the addition of phosphate buffer, and after solid phase extraction, it was dissolved in methanol before being analysed by gas chromatography mass spectrometry without derivatization. The method was validated according to the European Commission’s 2002/657/EC criteria.Results: The effects of different cartridge brands, pH, and elution solution were determined. Intraday and interday CV% values are 2.8 and 5.2 for the International Residue Limit (IRL), respectively. Five levels (blank, 0.5xIRL, IRL, 1.5xIRL, and 2xIRL) were used in constructing the curve, and the R2 value was greater than 0.99. The analysis run was 11.8 min. The decision limit (CCα) was determined to be 56.7 ng/mL due to IRL. The detection limit of the method was calculated to be 3.3 ng/mL. The method was determined to be robust according to changes in extraction pH, phosphate buffer concentration, centrifugation time, hexane volume in the wash step, different grades of methanol, inlet temperature, and operator.Conclusion: The applicability of the method was demonstrated by analysing positive and negative horse urine samples. Validation parameters showed the method to be selective, specific, and easy to apply.