Application of high-performance liquid chromatography combined with ultra-sensitive thermal lens spectrometric detection for simultaneous biliverdin and bilirubin assessment at trace levels in human serum

We present the applicability of a new ultra-sensitive analytical method for the simultaneous determination of biliverdin and bilirubin in human serum. The method comprises isocratic reversed-phase (RP) C18 high-performance liquid chromatography (HPLC) and thermal lens spectrometric detection (TLS) based on excitation by a krypton laser emission line at 407 nm. This method enables the separation of IX-α biliverdin and IX-α bilirubin in 11 min with limit of detection (LOD) and limit of quantitation (LOQ) for biliverdin of 1.2 nM and 3 nM, and 1 nM and 2.8 nM for bilirubin, respectively. In addition, a step-gradient elution was set up, by changing the mobile phase composition, in order to further enhance the sensitivity for bilirubin determination with LOD and LOQ of 0.5 nM and 1.5 nM, respectively. In parallel, an isocratic HPLC-DAD method was developed for benchmarking against HPLC-TLS methods. The LOD and LOQ for biliverdin were 6 nM and 18 nM, and 2.5 nM and 8 nM for bilirubin, respectively. Additionally, both isocratic methods were applied for measuring biliverdin and free bilirubin in human serum samples (from 2 male and 2 female healthy donors). Combining isocratic HPLC method with TLS detector was crucial for first ever biliverdin determination in serum together with simultaneous free bilirubin determination. We showed for the first time the concentration ratio of free bilirubin versus unbound biliverdin in human serum samples.