Unconjugated bilirubin were measured from serum samples, as described previously12 (link), using a high-performance liquid chromatograph (Merck, Hitachi, LaChrom, Vienna, Austria) equipped with a photodiode array detector (PDA, Shimadzu,) and a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm) and a phenomenex C18 HPLC guard column (4.0 × 3.0 mm). An isocratic mobile phase consisting of 0.1 M n-dioctylamine in methanol/water (95:5; v/v) and glacial acetic acid was used. Unconjugated bilirubin IX alpha (Frontier Scientific Europe, Carnforth, Lancashire, UK) served as an external standard. Sample and standard preparation and analysis were performed as previously published12 (link).
C18 hplc guard column
Manufactured by Phenomenex
Sourced in Austria
The Phenomenex C18 HPLC guard column is a pre-column component designed to protect the primary analytical column in high-performance liquid chromatography (HPLC) systems. It is made with a C18 stationary phase and serves to trap particulates, salts, and other contaminants before they reach the analytical column, thus extending the column's lifetime and maintaining optimal performance.
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Lab products found in correlation
4 protocols using c18 hplc guard column
1
Quantification of Unconjugated Bilirubin
2
Serum Bilirubin and Heme Analysis
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Serum samples for UCB measurement were stored in dark tubes, immediately after separation, as described previously37 (link). Unconjugated bilirubin and heme were measured in serum samples, using a high-performance liquid chromatograph (Merck, Hitachi, LaChrom, Vienna, Austria) 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 (4·0 × 3·0 mm). Sample preparation and analysis were performed as previously published37 (link). Unconjugated bilirubin and haemin (both Frontier Scientific Europe, Carnforth, Lancashire, UK) served as external standards.
Free bilirubin was calculated from serum UCB and albumin levels, using a formula kindly provided by Dr. Silvia Gazzin and Dr. Claudio Tiribelli.
Free bilirubin was calculated from serum UCB and albumin levels, using a formula kindly provided by Dr. Silvia Gazzin and Dr. Claudio Tiribelli.
3
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.
4
HPLC Analysis of Unconjugated Bilirubin Isomers
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For a detailed analysis of UCB (isomers), HPLC was conducted (after Brower et al.66 (link)), as had been used and published by our group9 (link), 10 (link), 12 (link) and others67 (link) previously. Samples were protected from light exposure and kept at 4 °C throughout all processing steps. Briefly, fasting serum samples (stored 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 protocol12 (link). For the purpose of quality control unconjugated bilirubin (Frontier Scientific Europe, Carnforth, Lancashire, UK) with an isomeric purity of >99% served as an external standard. Additionally, to assure consistency in analyte recovery a reference serum sample was run in each analysis.
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