title: Direct determination of free bilirubin in serum at sub-nanomolar levels.
abstract: Direct analysis of free bilirubin in human and animal blood serum samples is reported for the first time. A state-of-the-art system comprised of newly developed high-performance liquid chromatography (HPLC) on reverse-phase (RP) C18 support coupled with thermal lens spectrometric detection (TLS), based on excitation at λ=457.9 nm by an argon laser was used for this purpose. This HPLC-TLS method enabled a baseline separation of all three structural isomers of bilirubin (XIII-α, IX-α and III-α) and the respective degradation products in isocratic mode in fewer than 7 min. The method excels in ultra-high sensitivity with limit of detection (LOD) and limit of quantitation (LOQ) of 90 pM and 250 pM, respectively. Moreover, this method also affords high precision and accuracy, with correlation coefficients R(2)>0.997 over a broad linear range (0.250-150 nM) and R(2)=0.9998 in a concentration range of clinical interest (0.500-25 nM). The method's boosted sensitivity enabled to streamline sample preparation to just one serum ultrafiltration step, which made qualitative evaluation of sample preparation possible for the first time. The performance of the HPLC-TLS method was assessed to have 20-fold enhanced sensitivity when compared to a comparable method incorporating HPLC coupled with diode array detector (DAD), which is also a novel method by itself, and could be applied for free bilirubin determination in patients with elevated bilirubin levels.
authors.affiliation: University of Nova Gorica, 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 Science, via L. Giorgeri 1, 34127 Trieste, Italy.
authors.forename: Lovro
authors.initials: L
authors.lastname: Žiberna
authors.affiliation: University of Trieste, Department of Life Science, via L. Giorgeri 1, 34127 Trieste, Italy.
authors.forename: Sabina
authors.initials: S
authors.lastname: Passamonti
authors.affiliation: University of Nova Gorica, 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: D000328:Adult; D000818:Animals; D001663:Bilirubin / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D064368:Healthy Volunteers; D006801:Humans; D008297:Male
chemical_list: D001663:Bilirubin
keywords: Diode array detection; High-performance liquid chromatography; Human and animal blood serum; Thermal lens spectrometry; Unbound and unconjugated bilirubin