Determination of methylxanthines in urine by liquid chromatography with diode array UV detection

A liquid chromatography-diode array UV detection (LC-UVDAD) method for the simultaneous determination of four methylxanthines (caffeine, theobromine, paraxanthine and theophylline) is described. The chromatographic separation was achieved on a LC-18-DB column using 20:80 methanol:buffer (5mM citric acid adjusted to pH 5 with triethylamine) as mobile phase. The method has been applied to urine samples. The overall procedure had % recoveries ranging from 81.6 +/- 2.6 (theophylline) to 99.3 +/- 6.3 (theobromine). The within-day (n = 5) and between-days (n = 5 over 5 days) coefficients of variation in urine ranged from 2.9% (theophylline) to 3.4% (theobromine) and from 5.2% (theophylline) to 6.2% (theobromine). Estimated LOD and LOQ in urine ranged from 0.15microg/ml (theophylline) to 0.3microg/ml (theobromine) and from 0.8microg/ml (theophylline) to 1.2microg/ml (theobromine), respectively. Urine samples naturally contaminated with the target analytes were found.