Simultaneous determination of theobromine, paraxanthine, theophylline, and caffeine in urine by reversed-phase high-performance liquid chromatography with diode array UV detection

摘要

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 C_(18) column using 14:86 methanol:buffer (25mM KH_2PO_4 adjusted to pH 4 with ortho-phosphoric acid) solution as mobile phase under isocratic conditions at a flow rate 1mLmin~(-1). An ultraviolet absorption at 274 nm was monitored. In these conditions, the LOD was 0.03 μgmL~(-1) for theobromine, 0.02 μgmL~(-1) for paraxanthine, 0.04 μg mL~(-1) for theophylline, and 0.08 μgmL~(-1) for caffeine. The method has been applied to urine samples.