Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection.

ABSTRACT

A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from C1 was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from C1 after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 601560 mmol/l in water). Diluted serum and urine could be injected onto C1, and C1 was backflushed after the transfer of uric acid from C1 to C5. Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 mumol/l, respectively) was 98.9 +/- 0.56% and 100.9 +/- 1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 mumol/l, respectively) was 99.4 +/- 0.72% and 98.7 +/- 1.45%, respectively (mean +/- R.S.D., n = 6).