SOLID-PHASE EXTRACTION STUDY AND PHOTODIODE-ARRAY RP-HPLC ANALYSIS OFXANTHINE DERIVATIVES IN HUMAN BIOLOGICAL-FLUIDS

An automated reverse phase high performance liquid chromatography (HPL C) - 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, theo bromine, paraxanthine, theophylline and caffeine. The separation metho d development was based on mobile-phase optimisation and off-line soli d-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 quantitatio n was performed at 270 nm. Isocaffeine was used as internal standard a t a concentration of 3.06 ng/mu L. High extraction recoveries were ach ieved from Merck RP-18 cartridges using 1% hydrochloric acid as eluent , requiring small volumes, 40 mu L, of blood serum and 100 mu L of uri ne. The separation of xanthines was achieved on octylsilica, using a S ilasorb C-8, 10 mu m, 250x4.6 mm i.d. analytical column thermostated a t 32 degrees C and proved to be highly selective, sensitive, reproduci ble, accurate and rapid regarding the nine compounds. Detection limits ranged from 2 to 3 ng for 20 mu L injected volume while linearity hol ds up to 20 ng/mu L for each compound.