Simultaneous Determination of Theobromine,Paraxanthine, Theophylline,and Caffeine in Urine by Reversed-Phase High-Performance Liquid Chromatography with Diode Array UV Detection

A reversed-phase high performance liquid chromatographic method was improved for the simultaneous determination of theobromine, paraxanthine, theophylline, and caffeine in urine. The method includes a liquid-liquid extraction at alkaline pH with ethylacetate. The 7-(2,3-dihidroxypropyl) theophylline was used as an internal standard (ISTD). The separation was achieved on a C₁₈ column using 14:86 methanol:buffer (25 mM KH₂PO₄ adjusted to pH 4 with ortho-phosphoric acid) solution as mobile phase under isocratic conditions at a flow rate 1 mL min^?1. An ultraviolet absorption at 274 nm was monitored. In these conditions, the LOD was 0.03 μg mL^?1 for theobromine, 0.02 μg mL^?1 for paraxanthine, 0.04 μg mL^?1 for theophylline, and 0.08 μg mL^?1 for caffeine. The method has been applied to urine samples.