Direct determination of free bilirubin in serum at sub-nanomolar levels

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• 作者：Mitja Martelanc ; Lovro 沤iberna ; Sabina Passamonti ; Mladen Franko
• 关键词：Unbound and unconjugated bilirubin ; Human and animal blood serum ; High-performance liquid chromatography ; Diode array detection ; Thermal lens spectrometry
• 刊名：Analytica Chimica Acta
• 出版年：27 January, 2014
• 年：2014
• 卷：809
• 期：Complete
• 页码：174-182
• 全文大小：1413 K

文摘

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 位 = 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-伪, IX-伪 and III-伪) 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 R² > 0.997 over a broad linear range (0.250-150 nM) and R² = 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.