Optimization of a high-performance liquid chromatography method to quantify bilirubin and separate it from its photoproducts: Effect of column length, pH, mobile phase composition, and flow rate

A rapid reversed-phase (RP) high-performance liquid chromatography method for the isolation of bilirubin from its photoproducts (e.g., biliverdin) is reported. The method is based on isocratic elution using methanol:water as the mobile phase. A 2⁴ full-factorial experimental design approach was adopted. For the optimization, the best separation was obtained using a flow rate of 1.50 mL/min, a mobile phase of 99:1 methanol:water (v/v) at pH 3.60, and a 150 × 4.6 mm id RP (C₁₈) column containing 5-μm particles. These conditions produced the fastest total retention time of 3.38 ± 0.055 min, and other chromatographic parameters were acceptable. Under the optimum conditions, a linear calibration curve for bilirubin was obtained over the 1.0-40.0 μg/L concentration range studied. The limit of quantification was 0.79 g/L and the limit of detection was 0.24 μg/L. Bilirubin in solution was monitored by ultraviolet detection at 450 nm.