Solid-Phase Extraction Study and Photodiode Array RP-HPLC Analysis of Xanthine Derivatives in Human Biological Fluids

Abstract

An automated reverse phase high performance liquid chromatography (HPLC)—photodiode array method using a multi linear gradient elution is described for the simultaneous analysis of nine xanthines: xanthine, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, isocaffeine, theobromine, paraxanthine, theophylline and caffeine. The separation method development was based on mobile-phase optimisation and off-line solid-phase extraction (SPE) from human biological fluids: blood serum and urine. Eluent consisted of 0.05 M CH₃COONH₄ and methanol (90:10 v/v) changing to (70:30 v/v) over a period of 20 min. Identification of xanthines was achieved by photodiode—array detector and quantitation was performed at 270 nm. Isocaffeine was used as internal standard at a concentration of 3.06 ng/μL. High extraction recoveries were achieved from Merck RP-18 cartridges using 1% hydrochloric acid as eluent, requiring small volumes, 40 μL of blood serum and 100 μL of urine. The separation of xanthines was achieved on octylsilica, using a Silasorb C₈, 10μm, 250 × 4.6 mm i.d. analytical column thermostated at 32 °C and proved to be highly selective, sensitive, reproducible, accurate and rapid regarding the nine compounds. Detection limits ranged from 2 to 3 ng tor 20 μL injected volume while linearity holds up to 20 ng/μL for each compound.