Protocol detail

HPLC Quantification of Unconjugated Bilirubin

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For a detailed analysis of UCB (isomers), the method of HPLC was applied (after)50 , as had been used and published by our group4 (link)51 (link) and others52 (link) previously. Briefly, fasting serum samples (stored light-protected in amber vials) were diluted in isocratic mobile phase (methanol, water, n-dioctylamine and acetic acid) and centrifuged. Supernatants were run on a chromatograph (Merck, Hitachi, LaChrom), equipped with a photodiode array detector (PDA, Shimadzu) and a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm), with a Phenomenex C18 HPLC guard column (4 × 3 mm). Sample preparation and analysis followed the previously published protocol4 (link). Unconjugated bilirubin (Frontier Scientific Europe, Carnforth, Lancashire, UK) served as an external standard/quality control. As an internal standard, a reference serum sample was run in each analysis.

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Mölzer C., Wallner M., Kern C., Tosevska A., Schwarz U., Zadnikar R., Doberer D., Marculescu R, & Wagner K.H. (2016). Features of an altered AMPK metabolic pathway in Gilbert’s Syndrome, and its role in metabolic health. Scientific Reports, 6, 30051.

Publication 2016

PDA Fortis C18 HPLC column C18 HPLC guard column

Acetic acid Amber Bilirubin Chromatograph Hplc Isomers Light Methanol Serum

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

Unconjugated bilirubin levels

control variables

Fasting serum samples
Isocratic mobile phase (methanol, water, n-dioctylamine and acetic acid)
Chromatograph (Merck, Hitachi, LaChrom) equipped with a photodiode array detector (PDA, Shimadzu) and a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm), with a Phenomenex C18 HPLC guard column (4 × 3 mm)
Sample preparation and analysis following the previously published protocol4 (link)

controls

Positive control: Unconjugated bilirubin (Frontier Scientific Europe, Carnforth, Lancashire, UK) served as an external standard/quality control
Negative control: A reference serum sample was run in each analysis as an internal standard

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