Fig 1.
Analytical characterization of derivatized SCFA standards.
(A) Reaction scheme for the aniline derivatization of SCFAs. (B) Overlaid extracted ion chromatograms (EIC) corresponding to a 12C-and 13C-derivatized SCFA mix standard solution detected in selected reaction monitoring mode. Red HPLC traces correspond to the 13C-labeled analytes. (Inset) A single reporter MS/MS fragment was used for analyzing all SCFA: m/z [M+H]+ → 94.1. (C) Relative MS signal intensities of a derivatized mixed solution of 12C- and 13C- SCFA analytes at a concentration of 10 μM and 5 μM, respectively. Data were acquired in full scan mode. EDC, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; CE, collision energy in eV.
Table 1.
LLOD and LLOQ for the selected SCFAs.
Table 2.
Intra-and inter-day precision (%RSD) and accuracy (%Error) of the LC-MS/MS method.
Fig 2.
Correlation between known and observed 12C:13C ratios.
Plot of linear regression for known 12C:13C ratios versus observed 12C:13C ratios for 11 standard mix solutions with 12C- and 13C- SCFAs concentration ratios ranging from 0.5 to 20. Slopes for fitted regression lines and r2 corresponding to plots are shown. Dashed line represents a slope of 1. Error bars represent standard deviation, n = 3 technical replicates.
Table 3.
Summary of tested experimental parameters.
Fig 3.
Relationship between 12C:13C isotope ratio for SCFAs and the observed absolute error.
The dotted line denotes an arbitrary 25% error threshold. Colored square highlights the range of concentration ratios (0.5 < 12C:13C ratio < 20) where the absolute error is below 25%. (Insets show plots for the observed MS signal intensity ratio versus the known concentration ratio for (left) concentration ratios between 0.001 and 0.05 and (right) concentration ratios between 50 and 1000). Error bars indicate standard deviation, n = 3 technical replicates.
Fig 4.
Microbial production of SCFAs in mice and in vitro cultures.
SQUAD was used to analyze (A) caecal samples from germ free (GF, n = 5) and specific-pathogen-free (SPF, n = 4) mice, and (B) growth media of five species of microbes cultured for 4 hours in Mueller Hinton medium (n = 4 isolates per species). Error bars denote standard deviation. Abbreviations: MH, Mueller Hinton medium; E.co, Escherichia coli, E.fa, Enterococcus faecalis; K.pn, Klebsiella pneumoniae; P.au, Pseudomonas aeruginosa; S.au, Staphylococcus aureus.
Fig 5.
Extension of SQUAD to other SCFAs.
Overlaid EICs corresponding to a 12C- (10 μM) and stable isotope labeled-(2 μM) derivatized SCFA standard mix solution detected in selected reaction monitoring mode. Red HPLC traces correspond to the 13C- or deuterated derivatized standards. Ac, Prop and But as previously described. (a) Indicates the HPLC trace for the 2-methylbutyrate standard (an isomer of valerate and isovalerate). Abbreviations: Isobut, isobutyrate; Val, valerate; Isoval, isovalerate.