Bile acid preparation and comprehensive analysis by high performance liquid chromatography-high-resolution mass spectrometry

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 0.24 pmol/sample to 1000 pmol/sample, corresponding to 0.024-100 pmol on column) of up to 36 naturally occurring BAs from as little as 10 mu l of plasma or serum, I ml of urine, or 10 mg 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. (C) 2016 Elsevier B.V. All rights reserved.