title: 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.
abstract: 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 407nm. This method enables the separation of IX-α biliverdin and IX-α bilirubin in 11min with limit of detection (LOD) and limit of quantitation (LOQ) for biliverdin of 1.2nM and 3nM, and 1nM and 2.8nM 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 0.5nM and 1.5nM, respectively. In parallel, an isocratic HPLC-DAD method was developed for benchmarking against HPLC-TLS methods. The LOD and LOQ for biliverdin were 6nM and 18nM, and 2.5nM and 8nM 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.
authors.affiliation: University of Nova Gorica, The Laboratory for Environmental Research, Vipavska 13, POB 301, SI-5001 Nova Gorica, Slovenia.
authors.forename: Mitja
authors.initials: M
authors.lastname: Martelanc
authors.affiliation: University of Trieste, Department of Life Sciences, via L. Giorgeri 1, 34127 Trieste, Italy.
authors.forename: Lovro
authors.initials: L
authors.lastname: Žiberna
authors.affiliation: University of Trieste, Department of Life Sciences, via L. Giorgeri 1, 34127 Trieste, Italy.
authors.forename: Sabina
authors.initials: S
authors.lastname: Passamonti
authors.affiliation: University of Nova Gorica, The Laboratory for Environmental Research, Vipavska 13, POB 301, SI-5001 Nova Gorica, Slovenia. Electronic address: mladen.franko@ung.si.
authors.forename: Mladen
authors.initials: M
authors.lastname: Franko
mesh_terms: D001663:Bilirubin; D001664:Biliverdine; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male
chemical_list: D001664:Biliverdine; D001663:Bilirubin
keywords: DAD; Free bilirubin; HPLC; Human serum; TLS; Unbound biliverdin