Indirect Bilirubin Quantification by HPLC

Indirect bilirubin levels (UCB) were measured in plasma samples by performing high-performance liquid chromatography analyses (HPLC; Merck, Hitachi, LaChrom) according to a widely-used protocol [28 (link),29 (link),35 (link)]. For the analyses, a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm), cartridges (Phenomenex SecurityGuard™), and a photodiode array detector (PDA, Shimadzu) were used. All plasma samples were centrifuged and 50 μl sample was mixed with the isocratic mobile phase consisting of glacial acid (6.01 g/L) as well as 0.1 M n-dioctylamine solution in HPLC-grade methanol/water (96.5/3.5%). The samples were centrifuged repeatedly in order to inject 120 μl of the supernatant into the HPLC (flow rate: 1 mL/min). To reduce batch-to-batch fluctuation, all case-control pairs were analyzed using the same plate run and bilirubin α (purity≥98%, Sigma Aldrich) was used as the external standard.