J.-C. PROST¹, C. GROB³, C. BERCHTOLD¹, G. SCHOLTTERBECK³, C. BOVET¹, F. BRUNNER², A. MACPHERSON², M. G. FIEDLER¹, P. JUILLERAT<sup>2

1</sup>University Institute of Clinical Chemistry, Inselspital, Bern University Hospital, Bern, Switzerland, ²Gastroenterology, Clinic for Visceral Surgery and Medicine, Bern University Hospital, Bern, Switzerland, ³Institute for Chemistry and Bioanalytics, University of Applied Sciences and Arts Northwestern Switzerland FHNW, Muttenz,, Switzerland

Bile acids malabsorption is encountered in numerous gastrointestinal pathologies and is a good example of treatable cause of watery diarrhea after ileal resection. Robust validated diagnostic methods to detect malabsorption are not routinely used in practice due to cost, logistics or laboratory equipment limitations. The current available test for the measurement of bile acids malabsorption is the selenium homocholic acid taurine (SeHCAT) test, an expensive and complex nuclear medicine method. An alternative approach could be the quantitative analysis of 7α-hydroxy-4-cholesten-3-one (7αOHC), an intermediate metabolite in the synthesis of bile acids from cholesterol. Methods using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) had demonstrated good sensitivities in the low ng/mL concentrations for 7αOHC. However, these methods usually used time-consuming sample preparation (liquid-liquid (LLE) or solid phase (SPE) extraction) and long analysis time (typically 14-50 minutes).

For the routine measurements of 7αOHC in large patient’s sets, a simple and faster protocol would be beneficial. The aim of this work was to develop an ultra-high performance LC-MS/MS (UHPLC-MS/MS) assay in order to efficiently quantify 7αOHC in human serum. The analyte was extracted using the Tecan Immobilized Coating Extraction (TICE) technology, a special coating in well plates that act as an absorptive extraction phase for small and principally non-polar molecules. The quantification of 7αOHC was then performed in a 6 min run time by reversed-phase chromatography coupled to a triple quadrupole mass spectrometer (Acquity I-Class UPLC coupled to Xevo TQ-S, Waters). The method was then validated over the concentration range of 5-300 ng/mL. Our laboratory is now offering this assay for the verification of the bile acids malabsorption diagnose.