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

Osawa S, Sugo S, Yoshida T, Yamaoka T, Nomura F

Division of Biological Sciences and Technology, Department of Health Sciences, School of Medicine, Kyushu University, 3-1-1, Maidashi, Higasshi-Ku, Fukuoka City, 812-8582, Japan. osawas@shs.kyushu-u.ac.jp

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 delta 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. beta-carotene (20 mg/l), vitamin-B2 (370 microg/l) and B(12) (9.5 microg/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.

Published 6 March 2006 in Clin Chim Acta, 366(1): 146-55.
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