TITLE: Caltech-SCFA-methods fecal
AUTHORS: rabdelha  

[DESCRIPTION]
This protocol details the Caltech-SCFA-methods for measurement of short-chain fatty acids in mouse fecal samples.

[STEPS]
SECTION: Sample preparation
2. 
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 3 min at Room temperature and sonicate for 15 min. 

SECTION: Sample preparation
3. Centrifuge the suspension at 18000 x g for 15 min at 4 °C. 

SECTION: Sample preparation
4. Derivatize an aliquot of  100 µL  subsequently using a final concentration of 10 millimolar (mM) aniline and 5 millimolar (mM) EDC for 120 min at 4 °C. 

SECTION: Sample preparation
5. Quench derivatization reaction using a final concentration of 18 millimolar (mM) succinic acid and 4.6 micromolar (µM) 2-mercaptoethanol for 120 min at 4 °C. 

SECTION: Sample preparation
6. Store all samples at  4 °C until analysis on the same day.

SECTION: Sample preparation
7. 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. 

SECTION: Liquid Chromatography Mass Spectrometry (LC-MS):
8. 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. 
SECTION: Liquid Chromatography Mass Spectrometry (LC-MS):
9. Subject the samples to reverse phase C18 separation (Phenomenex Scherzo SS-C18) 100 x 2 mm and collect data in positive ion mode. 
SECTION: Sample preparation
1. Extract mouse fecal samples and derivatize as described previously.
 

SECTION: Liquid Chromatography Mass Spectrometry (LC-MS):
10. Acquire the data from 50 to 750 m z-1 at 2 spectra s-1. Set Electrospray ionization (ESI) source conditions as follows: 

 

SECTION: Liquid Chromatography Mass Spectrometry (LC-MS):
11. 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.

SECTION: Liquid Chromatography Mass Spectrometry (LC-MS):
12.  Equilibrate the column at 15% B for 1 min and introduce a sample.

SECTION: Liquid Chromatography Mass Spectrometry (LC-MS):
13. Increase the solvent ratio from 15% B to 100% B over 13 min and reduce back to 15% B over  2 min. Injection volume is 5 µL and column temperature of 45 °C. 

SECTION: Liquid Chromatography Mass Spectrometry (LC-MS):
14. 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.
SECTION: Liquid Chromatography Mass Spectrometry (LC-MS):
15. 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.