Analysis of unconjugated bilirubin in serum by reversed-phase high performance liquid chromatography

Abstract

A high performance liquid chromatographic method for the analysis of unconjugated bilirubin in neonatal serum is presented. Bilirubin was dissociated from protein with caffeine reagent and extracted with chloroform. An isocratic, reversed-phase HPLC system based on a C₁₈ column was used. Bilirubin was detected at 450 nm. Bilirubin SRM 916a from National Insititute of Standards and Technology, USA was the primary calibrator. An average recovery factor of 0.996 (SD = 0.018; N = 16) was obtained for bilirubin added to neonatal cord sera. The measurement range extended from 25 to 500 μmol 1⁻¹ L⁻¹. The method is proposed as a reference method for unconjugated bilirubin in neonatal sera.

Key Words:

• HPLC analysis
• neonatal serum
• reference method
• unconjugated bilirubin