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 x 4.6 mm id RP (C18) column containing 5-microm 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 microg/L concentration range studied. The limit of quantification was 0.79 g/L and the limit of detection was 0.24 microg/L. Bilirubin in solution was monitored by ultraviolet detection at 450 nm.
Al-Hamdi
A M H
AM
Department of Chemistry, College of Science, Sultan Qaboos University, Al-Khod 123, Sultanate of Oman, United Arab Emirates.
Williams
J R
JR
Al-Kindy
S M Z
SM
Pillay
A E
AE
D001663:Bilirubin / Q000302:isolation & purification* / Q000528:radiation effects; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008027:Light; D044085:Microfluidics / Q000379:methods; D044367:Phase Transition; D010777:Photochemistry / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001663:Bilirubin