Rapid and quantitative analysis of bilirubin in equines by high-performance liquid chromatography

Rapid and quantitative analytical methods for bilirubin using high-performance liquid chromatography (HPLC) with UV detection were developed for samples from equines at a meat inspection site. Sharp HPLC peaks for bilirubins, unconjugated bilirubin (UCBL) and conjugated bilirubin (CBL), were obtained using a simple mobile phase of methanol:0.5 M Tris-HCl buffer (65:35, v/v, pH 7.4). A variable wavelength detector set at 450 nm, 0.01 AUFS and a recorder set at 4 cm/min were used for detection. Peaks for UCBL and CBL occurred at 7.1 min and 4.9 min, the lower limits of detection ranged between 0.16 and 0.78 microgram/ml, respectively. Orange II, for which the retention time was 3.6 min, was selected as an internal standard. When the samples were analysed from healthy equines and those suspected of being jaundiced due to a yellow colour change in the carcasses, only UCBL peaks were recognized, and CBL peaks were never obtained. Aqueous humour is a very common sample at meat inspections but UCBL or CBL peaks were all absent in both healthy and clinical samples from equines. There were reliable correlations between UCBL peaks (microV) using HPLC and total bilirubin value (mg/dl) measured by the total bilirubin assay kit in serum. A correlation between peak height (microV) on HPLC from serum samples and that of pericardial fluid samples was obtained.