Fortis c18 hplc column

Manufactured by Phenomenex

Sourced in Austria

The Fortis C18 HPLC column is a high-performance liquid chromatography (HPLC) column designed for separation and analysis of a wide range of organic compounds. The column features a silica-based stationary phase with chemically-bonded C18 alkyl chains, providing a hydrophobic surface for reversed-phase chromatography.

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Lab products found in correlation

Potato dextrose agar (pda), by Shimadzu (8 mentions) C18 hplc guard column, by Phenomenex (4 mentions) High performance liquid chromatography (hplc), by Merck Group (3 mentions) Bilirubin alpha, by Merck Group (2 mentions) Securityguard cartridges for c18 hplc columns, by Shimadzu (2 mentions)

8 protocols using fortis c18 hplc column

Quantification of Unconjugated Bilirubin

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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).

Tosevska A., Franzke B., Hofmann M., Vierheilig I., Schober-Halper B., Oesen S., Neubauer O., Wessner B, & Wagner K.H. (2016). Circulating cell-free DNA, telomere length and bilirubin in the Vienna Active Ageing Study: exploratory analysis of a randomized, controlled trial. Scientific Reports, 6, 38084.

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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.

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.

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HPLC-Based Quantification of Circulating UCB

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Circulating UCB levels were measured in serum/plasma samples following a well-established protocol [12 (link),27 (link)] using high-performance liquid chromatography (HPLC, Merck, Hitachi, LaChrom, Vienna, Austria), with a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm), Phenomenex SecurityGuard™ cartridges for the C18 HPLC columns (4 × 3 mm), and a photodiode array detector (PDA, Shimadzu). An isocratic mobile phase contained glacial acetic acid (6.01 g/L) and 0.1M n-dioctylamine in HPLC-grade methanol/water (96.5/3.5%). Before starting the procedure, all aliquots were centrifuged and a 50 μL sample was mixed with 200 μL of mobile phase. After a second centrifugation, 120 μL of the supernatant was injected into the HPLC at a flow rate of 1 mL/min.
Case-control pairs were analyzed in the same plate to minimize batch-to-batch fluctuation. Bilirubin (alpha) (purity≥ 98%, Sigma Aldrich) acted as an external standard (3.3% IIIα, 92.8% Ixα, and 3.9% XIIIα isomers, 450nm). One reference serum sample was assessed per analysis as an internal standard. The coefficient of variation (CV) between each plate was 3.76%.

Seyed Khoei N., Anton G., Peters A., Freisling H, & Wagner K.H. (2020). The Association between Serum Bilirubin Levels and Colorectal Cancer Risk: Results from the Prospective Cooperative Health Research in the Region of Augsburg (KORA) Study in Germany. Antioxidants, 9(10), 908.

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Indirect Bilirubin Quantification by HPLC

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Indirect bilirubin levels (UCB) were measured in plasma samples by performing high-performance liquid chromatography analyses (HPLC; Merck, Hitachi, LaChrom) according to a widely-used protocol [28 (link),29 (link),35 (link)]. For the analyses, a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm), cartridges (Phenomenex SecurityGuard™), and a photodiode array detector (PDA, Shimadzu) were used. All plasma samples were centrifuged and 50 μl sample was mixed with the isocratic mobile phase consisting of glacial acid (6.01 g/L) as well as 0.1 M n-dioctylamine solution in HPLC-grade methanol/water (96.5/3.5%). The samples were centrifuged repeatedly in order to inject 120 μl of the supernatant into the HPLC (flow rate: 1 mL/min). To reduce batch-to-batch fluctuation, all case-control pairs were analyzed using the same plate run and bilirubin α (purity≥98%, Sigma Aldrich) was used as the external standard.

Draxler A., Blaschke A., Binar J., Weber M., Haslacher M., Bartak V., Bragagna L., Mare G., Maqboul L., Klapp R., Herzog T., Széll M., Petrera A., Laky B., Wagner K.H, & Thell R. (2023). Age-related influence on DNA damage, proteomic inflammatory markers and oxidative stress in hospitalized COVID-19 patients compared to healthy controls. Redox Biology, 67, 102914.

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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.

Tosevska A., Moelzer C., Wallner M., Janosec M., Schwarz U., Kern C., Marculescu R., Doberer D., Weckwerth W, & Wagner K.H. (2016). Longer telomeres in chronic, moderate, unconjugated hyperbilirubinaemia: insights from a human study on Gilbert’s Syndrome. Scientific Reports, 6, 22300.

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HPLC Analysis of Unconjugated Bilirubin Isomers

Cited 3 times

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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 group^{9 (link), 10 (link), 12 (link)} and others^{67 (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 protocol^{12 (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.

Mölzer C., Wallner M., Kern C., Tosevska A., Zadnikar R., Doberer D., Marculescu R, & Wagner K.H. (2017). Characteristics of the heme catabolic pathway in mild unconjugated hyperbilirubinemia and their associations with inflammation and disease prevention. Scientific Reports, 7, 755.

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HPLC Quantification of Circulating Bilirubin Levels

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Circulating UCB levels were measured in plasma samples following a well-established protocol [30 (link), 31 (link)] using high-performance liquid chromatography (HPLC, Merck, Hitachi, LaChrom, Vienna, Austria), equipped with a Fortis C18 HPLC-column (4.6 × 150 mm, 3 μm), a Phenomenex SecurityGuard™ cartridges for C18 HPLC-columns (4 × 3 mm), and a photodiode array detector (PDA, Shimadzu). An isocratic mobile phase contained glacial acetic acid (6.01 g/L) and 0.1 M n-dioctylamine in HPLC grade methanol/water (96.5/3.5%). Before starting the procedure, all aliquots were centrifuged and 50 μL plasma/serum was mixed with 200 μL mobile phase. After a second centrifugation, 120 μL of the supernatant was injected to the HPLC at a flow of 1 ml/min.
Case-control pairs were analyzed in the same plate to minimize batch-to-batch fluctuation. Bilirubin (alpha) (purity ≥ 98%, Sigma Aldrich) acted as an external standard (3.3% IIIα, 92.8% IXα, and 3.9% XIIIα isomers, 450 nm). One reference plasma sample was assessed per analysis as internal standard. The coefficient of variation (CV) between each plate was 6%.

Seyed Khoei N., Jenab M., Murphy N., Banbury B.L., Carreras-Torres R., Viallon V., Kühn T., Bueno-de-Mesquita B., Aleksandrova K., Cross A.J., Weiderpass E., Stepien M., Bulmer A., Tjønneland A., Boutron-Ruault M.C., Severi G., Carbonnel F., Katzke V., Boeing H., Bergmann M.M., Trichopoulou A., Karakatsani A., Martimianaki G., Palli D., Tagliabue G., Panico S., Tumino R., Sacerdote C., Skeie G., Merino S., Bonet C., Rodríguez-Barranco M., Gil L., Chirlaque M.D., Ardanaz E., Myte R., Hultdin J., Perez-Cornago A., Aune D., Tsilidis K.K., Albanes D., Baron J.A., Berndt S.I., Bézieau S., Brenner H., Campbell P.T., Casey G., Chan A.T., Chang-Claude J., Chanock S.J., Cotterchio M., Gallinger S., Gruber S.B., Haile R.W., Hampe J., Hoffmeister M., Hopper J.L., Hsu L., Huyghe J.R., Jenkins M.A., Joshi A.D., Kampman E., Larsson S.C., Le Marchand L., Li C.I., Li L., Lindblom A., Lindor N.M., Martín V., Moreno V., Newcomb P.A., Offit K., Ogino S., Parfrey P.S., Pharoah P.D., Rennert G., Sakoda L.C., Schafmayer C., Schmit S.L., Schoen R.E., Slattery M.L., Thibodeau S.N., Ulrich C.M., van Duijnhoven F.J., Weigl K., Weinstein S.J., White E., Wolk A., Woods M.O., Wu A.H., Zhang X., Ferrari P., Anton G., Peters A., Peters U., Gunter M.J., Wagner K.H, & Freisling H. (2020). Circulating bilirubin levels and risk of colorectal cancer: serological and Mendelian randomization analyses. BMC Medicine, 18, 229.

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HPLC Quantification of Circulating UCB

Cited 1 time

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Circulating UCB levels were measured in serum by HPLC following a well-established protocol (Wallner et al., 2013b (link); Seyed Khoei et al., 2020 (link)) using HPLC (HPLC, Merck, Hitachi, LaChrom, Vienna, Austria), equipped with a Fortis C18 HPLC-column (4.6 × 150 mm, 3 μm), a Phenomenex SecurityGuard™ cartridges for C18 HPLC-columns (4 × 3 mm), and a photodiode array detector (PDA, Shimadzu). An isocratic mobile phase contained glacial acetic acid (6.01 g/L) and 0.1 M n-dioctylamine in HPLC grade methanol/water (96.5/3.5%) was used. UCB was extracted from serum by mixing 40 μL serum with 160 μL mobile phase. After centrifugation, 50 μL of the supernatant was injected at a flow rate of 1 mL/min.

Zöhrer P.A., Hana C.A., Seyed Khoei N., Mölzer C., Hörmann-Wallner M., Tosevska A., Doberer D., Marculescu R., Bulmer A.C., Herbold C.W., Berry D, & Wagner K.H. (2021). Gilbert’s Syndrome and the Gut Microbiota – Insights From the Case-Control BILIHEALTH Study. Frontiers in Cellular and Infection Microbiology, 11, 701109.

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