Quantitation of 6-Amino-2-phenylamino-9-[beta]-D-ribofuranosyl-9H-purine (CV-1808) and Its Metabolite, 2-(4-Hydroxyphenyl) aminoadenosine, in Human Serum and Urine by High Performance Liquid Chromatography using a Fluorimetric Detector

A high performance liquid chromatographic method using a fluorimetric detector for determination of the quantities of 6-amino-2-phenylamino-9-β-D-ribofuranosyl-9H-purine (CV-1808, 1) and its metabolite, 2-(4-hydroxyphenyl) aminoadenosine (2), in human serum and urine is presented. Compounds 1 and 2, after chromatographic extraction from urine or serum with a Sep-Pak C18 cartridge, are allowed to react with propionic anhydride in the presence of triethylamine and the quantities of the resulting propionyl derivatives of 1 and 2 (1-P and 2-P) are determined by high performance liquid chromatography on a μPorasil column. The detection limits of 1 and 2 are 5.0 and 10.0 ng/ml in urine and 1.0 and 2.0 ng/ml in serum, respectively. For a more sensitive determination of the amount of 1 in serum, a concentrated eluate of 1-P from the μPorasil column is rechromatographed on a minicolumn (10 cm×2 mm I.D.) packed with Lichrosorb SI-60 (5μm). With this method, a detection limit of 0.1 ng/ml for 1 in serum is obtained.