A UHPLC–MS/MS method for the quantification of 7α-hydroxy-4-cholesten-3-one to assist in diagnosis of bile acid malabsorption

Abstract

Bile acids malabsorption (BAM) is encountered in numerous gastrointestinal pathologies and is a good example of a treatable cause of watery diarrhea after ileal resection. The gold standard for diagnosing BAM is the selenium homocholic acid taurine test (SeHCAT), an expensive and complex analysis. An alternative method is the quantification of 7α-hydroxy-4-cholesten-3-one (C4). Here, we present a simple, ultra high-performance liquid chromatography–tandem mass spectrometry method to measure C4 in human serum. To avoid time consuming sample preparation (e.g., derivatization, solid phase extraction), we used absorption chemistry-based extraction plates. This method demonstrates a lower limit of quantification of 5 ng/mL and is linear over a concentration range from 5 to 300 ng/mL (R2 = 0.9977). Inaccuracy and imprecision were less than 15%. The validated method is currently used for routine measurement of C4 from serum in patients to confirm BAM diagnosis.