Caltech-SCFA-methods fecal
rabdelha
Abstract
This protocol details the Caltech-SCFA-methods for measurement of short-chain fatty acids in mouse fecal samples.
Attachments
Steps
Sample preparation
Extract mouse fecal samples and derivatize as described previously.
Briefly add, ice-cold extraction solvent (1:1 v/v ACN/water) to fecal sample at a ratio of 2µL: 1mg sample and internal standard mix to a final concentration of 100micromolar (µM) and subject to vortex mixing for 0h 3m 0s at Room temperature and sonicate for 0h 15m 0s.
Centrifuge the suspension at 18000x g,0h 0m 0s for 0h 15m 0s at 4°C.
Derivatize an aliquot of 100µL subsequently using a final concentration of 10millimolar (mM) aniline and 5millimolar (mM) EDC for 2h 0m 0s at 4°C.
Quench derivatization reaction using a final concentration of 18millimolar (mM) succinic acid and 4.6micromolar (µM) 2-mercaptoethanol for 2h 0m 0s at 4°C.
Store all samples at 4°C until analysis on the same day.
Mix calibrators of acetic acid, propanoic acid, butyric acid and isobutyric acid (10 nM - 10×103nM) together with single- and double- blanks, spiked with internal standard mix (Acetic acid-d3, propanoic acid-d2, butyric acid-d2) to a final concentration of 100micromolar (µM) , prepare and subject to the same sample preparation procedure as fecal samples.
Liquid Chromatography Mass Spectrometry (LC-MS):
Analyze derivatized samples using an ultra-high performance liquid chromatography (UHPLC) system 1290 connect to a quadrupole time of flight (Q-TOF 6545) mass spectrometer from Agilent Technologies (Santa Clara, CA, USA) equipped with an orthogonal DUAL AJS-ESI interface.
Subject the samples to reverse phase C18 separation (Phenomenex Scherzo SS-C18) 100 x 2 mm and collect data in positive ion mode.
Acquire the data from 50 to 750 m z-1 at 2 spectra s-1. Set Electrospray ionization (ESI) source conditions as follows:
| A | B |
|---|---|
| Gas temperature | 325 °C |
| Drying gas | 9 L min-1 |
| Nebulizer | 35 psi |
| Fragmentor | 125 V |
| Sheath gas temperature | 350 °C |
| Sheath gas flow | 8 L min-1 |
| Nozzle voltage | 1000V |
Use, a two-solvent gradient running at 0.3mL min-1 (Mobile Phase: A: 100:0.1 Water: Formic Acid, B: 100:0.1 Isopropanol: Formic Acid) for reverse phase C18 chromatographic separation.
Equilibrate the column at 15% B for 0h 1m 0s and introduce a sample.
Increase the solvent ratio from 15% B to 100% B over 0h 13m 0s and reduce back to 15% B over 0h 2m 0s. Injection volume is 5µL and column temperature of 45°C.
Acquire the LC-MS/MS data using Agilent Mass Hunter Workstation (.d files) and process in quantitative analysis software (Agilent Technologies) for quantitative analysis of samples.
Construct the linear calibration plots for acetic acid, propanoic acid, butyric acid and isobutyric acid using peak area ratios of each analyte to the IS versus the concentrations of calibrators (x) with 1/x weighting, and obtain the least squares linear regression equations as the calibration equations for individual analytes.
