Improved HPLC Method For The Simultaneous Determination Of Purine Derivatives And Creactinine In Ruminant Urine

The purine derivatives (PD; allantoin, uric acid, xanthine and hypoxanthine) in the urine of ruminants have been validated to estimate microbial protein. The molar ratio of Urinary purine derivatives to creatinine in the spot urine can be also used as an index of microbial protein supply in ruminants. PD concentration can be estimated by using both colorimetric and HPLC.This paper describes a method of simultaneous determination of PD and creatinine.in the urine of ruminant by reversed-phase HPLC.The HPLC system (Varian, Inc., USA) equipped with a ProStar 335 Diode-array detector set 220nm was used. The analytical column used Varian C18 reversed-phase (250mm×4.6mm I.D., 5μm) with guard column (Varian). The stock standard solutions of uric acid (100μM), xanthine (50μM) and hypoxanthine (50μM) were prepared by dissolving in alkalinized (pH>10) hot water, and allantoin (300μM) and creatinine (100μM) were prepared in water. The stock standard solutions and urine samples diluted 1:10 were adjusted pH 6 with 0.01M H3PO4 and 0.01M KOH and kept at -20oC. The mobile phase was 25mM KH2PO4 solution (pH 4.7).The flow rate was 1ml/min.The column was maintained at 25 oC., 20μl injection was adopted.Allantoin and creatinine are constituents of urine excretion of ruminants with similar polarity. It is very difficult to separate these compounds in biological fluids. This method can make it useful for the simultaneous estimation of PD and creatinine. The retention time for allantoin, creatinine, uric acid, hypoxanthine and xanthine in standards solution were 2.78, 3.43, 4.97, 6.00, 6.73min, respectively. A linear relationship between the peak area and the concentration of standards in water was showed well. The recovery was determined by triplicate analysis of urine samples spiked with standards, which was satisfactorily recovered (96-102%). The within-day and day-to-day coefficients of variations (2.8-4.3%) were calculated by processing aliquots of spiked urine. The detection limits (LOD) of allantoin, creatinine, uric acid, hypoxanthine and xanthine in urine at S/N of 3 were 0.06, 0.01, 0.02, 0.01, 0.01μM, respectively.The present HPLC method is effective in the simultaneous determination of PD and creatinine in ruminant urine, allowing a good separation between the peaks of allantoin and creatinine.