Clin Chim Acta
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.
Published: April 2006
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.
Download full-text PDF |
Source |
---|---|
http://dx.doi.org/10.1016/j.cca.2005.09.031 | DOI Listing |
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!
© LitMetric 2025. All rights reserved.