Determination of bovine beta2-microglobulin and albumin in urine by a reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (HPLC) was used to analyze beta2-microglobulin and albumin in bovine urine. The urine samples were chromatographed on TSK-gel ODS-120T column with an acetonitrile gradient. Urinary beta2-m and albumin were detected at 220 nm. For the pre-treatment, there were two steps proceeding injection: dialysis of urine with distilled water overnight, followed by concentration by solid-phase extraction method using a Sep-Pak cartridge. The retention times of beta2-microglobulin and albumin were 25.35 +/- 0.85 and 32.20 +/- 0.20 minutes (n=5), respectively. The mean analytical recoveries of beta2-microglobulin and albumin added to 0.1 ml of urine samples were 94.5 and 100.5%, respectively. The within-run coefficients of variation ranged from 1.5 to 5.3% for beta2-microglobulin and from 2.3 to 7.0% for albumin. The sensitivity for quantification of each protein was 0.5 microg in 100 microl injected urine samples. Urine samples from healthy cows and from cows with different types of proteinuria were analyzed by this reversed-phase HPLC. Results revealed albumin was remarkable in the urine from a cow with glomerulonephritis, and beta2-microglobulin was, in the urine from a cow with tubular dysfunction.