ABSTRACT
An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C4, 250 x 4 mm, 5 microm, was operated at ambient temperature with back pressure values of 80-110 kg/cm2. The mobile phase consisted of an acetate buffer (pH 3.5)-methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/microl. Detection was performed with a variable wavelength UV-visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-microl injection, while linearity held up to 8 ng/microl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/microl and for caffeine for which it was 20 ng/microl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol-acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 microl of blood serum and 100 microl of urine.
