Measurement of Purine Derivatives and Creatinine in urine by HPLC. In Estimation of Microbial Protein Supply in Ruminants Using Urinary Purine Derivatives

Two HPLC methods to measure the purine derivativcs (PD), including allantoin, uric acid, hypoxanthine and xanthine) and creatinine content in urine are described. PD separation and quantification were achieved using two Spherisorb ODS 2 reversed phase columns connected in series (4.6 x 250 mm) and a Spherisorb ODS 2 Waters pre-column and a Perkin Elmer pump with an autosampler. The mobile phase was NH4H2PO4; NH4H2PO4: acetonitrile (80:20), which was used at a flow rate of 0.8 ml/min and the detection wavelength was at 190 nm. The average recoveries of standard compounds added to urine samples were satisfactory (92-106%) and the low detection limits (0.7-3.4 µM) permitted the precise determination of these compounds in urine. Separation and quantification of crcatinine was achieved using one Spherisorb ODS 2 reversed phase column (4.6 X 250 mm) and one Spherisorb ODS 2 Waters pre-column and a Perkin Elmer pump with an auto sampler. The mobile phase was NH4H2PO4; NH4H2PO4;-acetonitrile (80:20), used at a how rate of 1.00 m1/min and the detection was at 190 nm. The mean recovery (3 measurements) of standard solution added to urine samples was 101%; detection limit was 7.9 µM.