May 23, 2022
Caltech-SCFA-methods fecal
- 1California Institute of Technology
External link: https://doi.org/10.7554/eLife.81453
Protocol Citation: rabdelha 2022. Caltech-SCFA-methods fecal. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61rrkvqe/v1
Manuscript citation:
Abdel-Haq R, Schlachetzki JC, Boktor JC, Cantu-Jungles TM, Thron T, Zhang M, Bostick JW, Khazaei T, Chilakala S, Morais LH, Humphrey G, Keshavarzian A, Katz JE, Thomson M, Knight R, Gradinaru V, Hamaker BR, Glass CK, Mazmanian SK, A prebiotic diet modulates microglial states and motor deficits in α-synuclein overexpressing mice. eLife doi: 10.7554/eLife.81453
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 12, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 62496
Keywords: Caltech-SCFA-methods, fecal, Liquid Chromatography Mass Spectrometry, ASAPCRN
Abstract
This protocol details the Caltech-SCFA-methods for measurement of short-chain fatty acids in mouse fecal samples.
Attachments
434-922.docx
105KB
Sample preparation
Sample preparation
Extract mouse fecal samples and derivatize as described previously.
Note
(J.C. Chan, DH.Y. Kioh, G.C. Yap, B.W. Lee, E.C. Chan A Novel LCMSMS Method for Quantitative Measurement of Short-Chain Fatty Acids in Human Stool Derivatized With 12 C- And 13 C-labelled Aniline.J. Pharm. Biomed Anal., 138 (2017), pp. 43-53).
Briefly add, ice-cold extraction solvent (1:1 v/v ACN/water) to fecal sample at a ratio of 2 µL : 1 mg sample and internal standard mix to a final concentration of 100 micromolar (µM) and subject to vortex mixing for 00:03:00 at Room temperature and sonicate for 00:15:00 .
18m
Centrifuge the suspension at 18000 x g for 00:15:00 at 4 °C .
15m
Derivatize an aliquot of 100 µL subsequently using a final concentration of 10 millimolar (mM) aniline and 5 millimolar (mM) EDC for 02:00:00 at 4 °C .
2h
Quench derivatization reaction using a final concentration of 18 millimolar (mM) succinic acid and 4.6 micromolar (µM) 2-mercaptoethanol for 02:00:00 at 4 °C .
2h
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 - 10×103 nM) 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 100 micromolar (µM) , prepare and subject to the same sample preparation procedure as fecal samples.
Liquid Chromatography Mass Spectrometry (LC-MS):
Liquid Chromatography Mass Spectrometry (LC-MS):
16m
16m
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 0.3 mL 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 00:01:00 and introduce a sample.
1m
Increase the solvent ratio from 15% B to 100% B over 00:13:00 and reduce back to 15% B over 00:02:00 . Injection volume is 5 µL and column temperature of 45 °C .
15m
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.