Liquid chromatographic determination of four purine bases using porous graphitic carbon column

A high performance liquid chromatographic method has been developed for the simultaneous determination of hypoxanthine (Hx), xanthine (Xa), guanine (Gu) and adenine (Ad) in shellfish. The separation of these compounds was performed on a porous graphitic carbon column (100 x 4.6 mm I.D.) using acetonitrile-phosphate buffer (23/77 v/v) containing 0.5% trichloroacetic acid at pH 2.0 as the mobile phase. The flow rate was fixed to 1 mLmin(-1) and the detection was monitored at 254 nm. Calibration curves were found to be linear in the concentration range from 0.3 to 100 mugmL(-1) with correlation coefficients (r(2)) > 0.999. The lowest detectable concentrations of Hx, Xa, Gu and Ad were 0.04, 0.06, 0.08, 0.07 mug mL(-1) respectively. Recovery of all purine standards added to sea urchin gonad was above 97%. The proposed method was successfully applied for the determination of Hx, Xa, Gu and Ad in sea urchin gonad extract.