IMPROVED MICRO-METHOD FOR THE HPLC ANALYSIS OF CAFFEINE AND ITS DEMETHYLATED METABOLITES IN HUMAN BIOLOGICAL FLUIDS AFTER SPE

Abstract

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/μL for each compound. Lamotrigine was used as internal standard at a concentration of 10.0 ng/μL. 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 C8 400 mg and Oasis HLB cartridges respectively, requiring small volumes, 40 μL of blood and 100 μL of urine. The separation was achieved on octylsilica, using a Silasorb C8, 10 μm, 250 × 4.6 mm analytical column at ambient temperature and proved to be highly selective, sensitive, reproducible and rapid regarding the nine compounds.