Improved micro-method for the HPLC analysis of caffeine and its demethylated metabolites in human biological fluids after SPE

An improved high performance liquid chromatographic-photodiode array method, using a multi-linear gradient elution, is described for the simultaneous determination of caffeine and its eight primary metabolites: xanthine (XA), 7-methylxanthine (7-MX), 3-methylxanthine (3-MX), 1-methylxanthine (1-MX), isocaffeine (IC), theobromine (TB), paraxanthine (PA) and theophylline (TP). The separation method Is based on mobile phase optimization and off-line solid-phase extraction (SPE) from human biological fluids: blood serum and urine. The eluting system consisted of ammonium acetate 0.05 M (pH=7) and methanol (90:10 v/v) changing to (40:60 v/v) over a period of 30 min. Identification of metabolites was achieved by photodiode array detector at 270 nm resulting in 2 ng limit of detection, while linearity held up to 20 ng/mu L for each compound. Lamotrigine was used as internal standard at a concentration of 10.0 ng/mu L. The statistical evaluation of the method was examined performing intra-day (n=8) and inter-day (n=8) and was found to be satisfactory with high accuracy and precision results. High extraction recoveries were achieved from blood serum and Furine using Merck C-8 400 mg and Oasis HLB cartridges respectively, requiring small volumes, 40 mu L of blood and 100 mu L of urine. The separation was achieved on octylsilica, using a Silasorb C-8, 10 mu m, 250 x 4.6 mm analytical column at ambient temperature and proved to be highly selective, sensitive, reproducible and rapid regarding the nine compounds.