An assay for separating and quantifying four bilirubin fractions in untreated human serum using isocratic high-performance liquid chromatography.
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.
Osawa
Susumu
S
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
Sugo
Shin
S
Yoshida
Toshihiko
T
Yamaoka
Toshihiko
T
Nomura
Fumio
F
D001663:Bilirubin / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D001774:Blood Chemical Analysis / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D012016:Reference Values; D015203:Reproducibility of Results
D001663:Bilirubin