Bilirubin is an endogenous antioxidant in the human blood. While measurement of a total serum
bilirubin, comprising of protein-bound (indirect bilirubin) and bilirubin glucuronide (direct bilirubin), is a
routine clinical test, direct determination of free bilirubin represents an analytical challenge. Free bilirubin,
which is the bioactive form, accounts for a minor fraction (<100 nM). Here we show the direct analysis of
free bilirubin in human and animal blood serum samples using a state-of-the-art system comprised of newly
developed high-performance liquid chromatography (HPLC) on reversed-phase (RP) C18 support coupled
with thermal lens spectrometric detection (TLS), based on excitation wavelength of 457.9 nm using argon
laser. The method excels in ultra-high sensitivity with limit of detection (LOD) and limit of quantitation (LOQ) of
90 pM and 250 pM, respectively. As a result, we present here the direct assay of free bilirubin in serum (around
10 nM), which can be employed as an assessment of antioxidative blood status.
