Determination of Bovine β₂-Microglobulin and Albumin in Urine by a Reversed-Phase High-Performance Liquid Chromatography

Abstract

Reversed-phase high-performance liquid chromatography (HPLC) was used to analyze β₂-microglobulin and albumin in bovine urine. The urine samples were chromatographed on TSK-gel ODS-120T column with an acetonitrile gradient. Urinary β₂-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 β₂-microglobulin and albumin were 25.35 ± 0.85 and 32.20 ± 0.20 minutes (n=5), respectively. The mean analytical recoveries of β₂-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 β₂-microglobulin and from 2.3 to 7.0% for albumin. The sensitivity for quantification of each protein was 0.5 μg in 100 μl 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 β₂-microglobulin was, in the urine from a cow with tubular dysfunction.