Benno Amplatz1, Evelyn Zöhrer2, Christina Haas1, Maria Schäffer1, Tatjana Stojakovic1, Jörg Jahnel2, Günter Fauler3. 1. Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria. 2. Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Austria. 3. Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria. Electronic address: guenter.fauler@medunigraz.at.
Abstract
BACKGROUND: Accurate analytical methods for bile acid (BA) analysis are important for clinical diagnosis in newborns, adolescents, and adults. Improvements in speed, sensitivity and simplicity enable BA profiling using high performance liquid chromatography (HPLC) together with electrospray ionization (ESI) and high-resolution mass spectrometry (HR-MS). RESULTS: We present a method, validated in different species and tissues, enabling a highly sensitive quantitative determination (in a range of 0.24pmol/sample to 1000pmol/sample, corresponding to 0.024-100pmol on column) of up to 36 naturally occurring BAs from as little as 10μl of plasma or serum, 1ml of urine, or 10mg of dried stool. Chromatographic separation is achieved by HPLC using a C18 reversed phase column and a water/methanol gradient. After ESI, BAs are analyzed through HR-MS using orbitrap technology in full scan mode. 30 different BAs and the corresponding internal standards are separated and analyzed in a single run. Six additional BAs are evaluated in a second run using a pentafluorophenyl (PFP) stationary phase. CONCLUSIONS: This method generates detailed human and rodent BA profiles in full scan mode and accurate mass with the advantage of remarkably low required sample volume.
BACKGROUND: Accurate analytical methods for bile acid (BA) analysis are important for clinical diagnosis in newborns, adolescents, and adults. Improvements in speed, sensitivity and simplicity enable BA profiling using high performance liquid chromatography (HPLC) together with electrospray ionization (ESI) and high-resolution mass spectrometry (HR-MS). RESULTS: We present a method, validated in different species and tissues, enabling a highly sensitive quantitative determination (in a range of 0.24pmol/sample to 1000pmol/sample, corresponding to 0.024-100pmol on column) of up to 36 naturally occurring BAs from as little as 10μl of plasma or serum, 1ml of urine, or 10mg of dried stool. Chromatographic separation is achieved by HPLC using a C18 reversed phase column and a water/methanol gradient. After ESI, BAs are analyzed through HR-MS using orbitrap technology in full scan mode. 30 different BAs and the corresponding internal standards are separated and analyzed in a single run. Six additional BAs are evaluated in a second run using a pentafluorophenyl (PFP) stationary phase. CONCLUSIONS: This method generates detailed human and rodent BA profiles in full scan mode and accurate mass with the advantage of remarkably low required sample volume.
Authors: Katharina Meinel; Doloresz Szabo; Antal Dezsofi; Sina Pohl; Tanja Strini; Theresa Greimel; Victor Aguiriano-Moser; Harald Haidl; Martin Wagner; Axel Schlagenhauf; Jörg Jahnel Journal: Front Pediatr Date: 2022-05-11 Impact factor: 3.569
Authors: Rudolf E Stauber; Günter Fauler; Florian Rainer; Bettina Leber; Andreas Posch; Andrea Streit; Walter Spindelboeck; Vanessa Stadlbauer; Harald H Kessler; Harald Mangge Journal: Wien Klin Wochenschr Date: 2017-10-04 Impact factor: 1.704
Authors: Christopher D Chouinard; Gabe Nagy; Ian K Webb; Sandilya V B Garimella; Erin S Baker; Yehia M Ibrahim; Richard D Smith Journal: Anal Chem Date: 2018-08-24 Impact factor: 6.986
Authors: Stefan Traussnigg; Emina Halilbasic; Harald Hofer; Petra Munda; Tatjana Stojakovic; Günter Fauler; Karl Kashofer; Martin Krssak; Michael Wolzt; Michael Trauner Journal: Wien Klin Wochenschr Date: 2020-09-15 Impact factor: 1.704