Fecal butyrate and deoxycholic acid quantitation for rapid assessment of the gut microbiome

The intestinal microbiome is composed of myriad microbial species with impacts on host health that are mediated by the production of metabolites. While loss of bacterial species and beneficial metabolites from the fecal microbiome is associated with development of a range of diseases and medical complications, there are currently no clinical diagnostic tests that rapidly identify individuals with microbiome deficiencies. This method aims to rapidly quantify fecal concentrations of butyrate and deoxycholic acid, as depletion of these two metabolites are associated with adverse clinical outcomes and result from the loss of a subset of health-associated bacterial species. We present a rapid diagnostic screen based on 3-nitrophenylhydrazine derivatization and ultrahigh-performance liquid chromatography-mass spectrometry that measures fecal butyrate and deoxycholic acid concentrations as markers of microbiome function. A matrix-matched calibration curve was developed using a simulated fecal mixture to optimize accuracy and facilitate adherence to clinical laboratory regulations. The assay resulted in an analytical measurement range from 4.30-3030 µM (LLOQ = 3.71 µM) for butyrate and from 0.9-64.9 µM (LLOQ = 0.7 µM) for deoxycholic acid. Precision evaluation demonstrated a coefficient of variation <15% at all quality control levels tested. The rapid liquid chromatography-mass spectrometry screen can be performed in under an hour from extraction to provision of quantitative results, enabling the rapid identification of patients with defective microbiome function.