Fig 1.
Reduced fecal butyrate and deoxycholic acid (DCA) in hospitalized patients are associated with adverse medical outcomes.
A same-day fecal screen would enable identification of patients with the low butyrate and DCA levels indicative of higher risk for adverse events in a clinically relevant timeframe.
Fig 2.
Conventional compared to rapid measurement of butyrate and DCA.
Comparison of conventional, targeted short-chain fatty acid and bile acid metabolomics methods, such as those in academic labs (A, B), to the unified measurement of these classes on one mass spectrometry platform (C). Conventional methods require multiple mass spectrometry platforms to measure metabolites with dissimilar chemical characteristics. (A) Short-chain fatty acids (SCFAs) such as butyrate typically require analysis by GC-MS, often with derivatization, while (B) bile acids are routinely analyzed using LC-MS technology. (C) The rapid mass spectrometry method presented here unifies the measurement of SCFAs and bile acids while resolving multiple isomers on a single system that minimizes sample preparation, cost (instrumentation, personnel time) and data acquisition time. (D) Sample preparation, data acquisition, and analysis time to acquire quantitative butyrate and deoxycholic acid concentration data for one biological sample using conventional metabolomics methods and the rapid metabolomics screen (See also S2 Table in S1 File).
Fig 3.
3-NPH derivatization mechanism.
(A) Reaction mechanism of carboxylic acid and aldehyde derivatization with 3-nitrophenylhydrazine (3-NPH) catalyzed by N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC). (B) A five-step procedure to derivatize and prepare patient fecal samples for LC-MS/MS analysis. (C) A 10-minute chromatographic method enables the detection and resolution of butyrate and deoxycholic acid (DCA) from their isomers in the same sample injection.
Fig 4.
MRM butyrate and DCA chromatograms.
Multiple reaction monitoring chromatogram overlays from the lowest rapid metabolomic screen calibrator for butyrate (A; quantifier, m/z 222.1–137.1; qualifier, m/z 222.1–152.0) with D7-butyrate internal standard quantifier transition (B; m/z 229.1–137.1) and deoxycholic acid (C; quantifier, m/z 526.3–152.0; qualifier, m/z 526.3–137.1) with D4-deoxycholic acid internal standard quantifier transition (D; m/z 530.3–137.1).
Fig 5.
Validation metrics for quantifying butyrate and DCA from fecal sample.
The analytical measurement ranges (AMR) and linearity of butyrate (A) and DCA (B) are shown across eleven concentration points for the rapid metabolomic screen. The lower limit of quantification (LLOQ) for butyrate at 3.71 µM (C) and DCA at 0.7 µM (D) are defined by the concentration at which the percent coefficient of variation (% CV) reaches 20%.
Table 1.
Inter-day precision and accuracy for five concentration levels (L1–L5) spanning the analytical measurement range of the rapid metabolomic screen. Concentrations are shown in μM; % CV = percent coefficient of variation.
Fig 6.
Comparison of the rapid metabolomic screen versus measurement by conventional mass spectrometry methods in patient fecal samples.
Linear regression (A) and Bland-Altman (B) comparisons of butyrate concentrations as measured by the rapid metabolomic screen versus measurement by conventional GC-MS metabolite analysis with PFB-Br derivatization (GCMS); Linear regression (C) and Bland-Altman (D) comparisons of DCA concentrations as measured by the rapid metabolomic screen versus measurement by conventional bile acid analysis using QToF LC-MS. See S4 Table in S1 File for patient butyrate and DCA concentrations with clinical metadata.