An assay for separating and quantifying four bilirubin fractions in untreated human serum using isocratic high-performance liquid chromatography

Abstract

Background The quantification of serum bilirubin fractions has been widely performed with both the diazo-method and an enzymatic method; however, the accuracy of these methods has not been evaluated because quantitative fractional high-performance liquid chromatography (HPLC) reference methods have yet to be established. Methods Samples were analyzed using HPLC and Shodex® Asahipak GS-320HQ columns. Human serum was subjected to HPLC using direct injection, then eluted with acetonitrile: 0.3 mol/l phosphate buffer (pH 6.5) containing 1% Brij 35 and 0.08% sodium ascorbate (30 : 70, v/v). Results Serum bilirubin was separated into 4 fractions; retention times of 9.24, 19.92, 24.07, 35.75 min were identified as δ bilirubin, bilirubin diglucuronide, bilirubin monoglucuronide, and unconjugated bilirubin, respectively. Mean recovery was 93.0%–99.2%. Total precision of peak retention time, height and area exhibited < 4.26% variation. Detection range was 3.1 to 348 mg/l. Hemoglobin (6 g/l) and immunoglobins produced a small positive interference. β-carotene (20 mg/l), vitamin-B 2 (370 μg/l) and B 12 (9.5 μg/l) did not interfere with this analysis. Results ( n = 30) with this method were closely correlated to those by Adachi's HPLC method as r = 0.9941 to 0.9960, slope = 0.88 to 1.27, intercept = − 3.2 to + 4.9, for each fraction. Conclusions Since this method was a precise quantitative HPLC method for serum bilirubin fractionation, it might be used to evaluate the accuracy and the characteristics of various routine methods for bilirubin measurement.