SIMULTANEOUS MEASUREMENT OF ALLANTOIN, URIC-ACID, XANTHINE AND HYPOXANTHINE IN BLOOD BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A high-performance liquid chromatographic method for determining catab olism products of nucleic acids and purines, such as oxypurines (i.e. uric acid, xanthine and hypoxanthine) and allantoin in the blood plasm a of ruminants was developed. The plasma was deproteinized with 10% tr ichloroacetic acid. The method enabled determination of oxypurines wit hout derivatization. Allantoin was determined after conversion with 2, 4-dinitrophenylhydrazine to a hydrazone (GLX-DNPH). Separation of conv erted allantoin, uric acid, xanthine and hypoxanthine derivatives was carried out using two reversed-phase C-18 columns. The combination of pre-column derivatization and gradient elution with monitoring of the effluent at 205, 254 and 360 nm provides a simple and selective analyt ical tool for studying oxypurines and allantoin in plasma. The total r un time of the HPLC analysis was 60 min. The recovery of the purine de rivatives (i.e. oxypurines and allantoin) added to the plasma was betw een 95 and 106%. Purine derivatives were stable when the processed sam ples were stored for 7 days at -10 degrees C. The low values of the in tra-assay coefficient of variations (2.5-4.6%) and the low values of t he detection Limits (0.187-0.004 nmol) point to the satisfactory preci sion and sensitivity of the method. (C) 1997 Elsevier Science B.V.