SCFAs measurements in plasma and cecal samples

Basic questions from students; resources for projects and reports.

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Hello, everyone.

I am a student who has just entered the master’s program. We are setting up a method to measure Short Chain Fatty Acids (SCFA) using GC/MS with Plasma and Cecal contents (fecal) in our laboratory.

So I looked up some papers related to SCFA measurement and I am trying to follow the protocol of the following paper, but I have a few questions.

DOI: 10.1038/s41467-017-02490-4

Methods:

Sample preparation

1. Cecal samples (100mg) or 50 uL of Serum weight into a 2 mL microtube
2. The tubes keep in a cool rack throughout the extraction 33% HCL (50uL of cecal contents or 5uL for serum)
3. Vortexed for 1 min.
4. Add 1mL of diethyl ether, and vortex for 1 min
5. Centrifuge for 3 min at 4°C
6. Transfer organic phase into a 2mL GC vial

GC/MS: GCMS QP2010Plus (Shimadzu)

Column: Famewax, 30 m × 0.25 mm iD, 0.25 μm df capillary column

Question

It may be too basic a question, but I have so many things I don’t know yet that I am asking with a heavy heart.

- Is the reason for using 33% HCL in the sample preparation process to make the pH acidic and cause some action?
- In this process, the derivatization process was omitted, is the derivatization process not essential for measuring SCFA?
- Is it okay not to perform the protein precipitation process separately in the above process? Or is the protein precipitation process accompanied by the above process?
- Should HCL and diethyl be used as GC-specific reagents?

Also, can multistandards be used as standards? (MilliporeSigma: CRM46975)

In addition, if the above protocol is appropriate, can you recommend related reagents from Sigma?

1. 33% HCL
2. Diethyl ether
3. Standards (Acetic acid, Propionic acid, Butyric acid)

Thank you for your reply.
Yes. Your first acidification stage is likely to make sure that your acids are protonated. 33% might be a little extreme BUT 33% is also only about 9 M so it's not super concentrated. You could probably get the same result with 1 M acid - if the purpose is to just keep the free acids in their protonated form. Salts are less likely to transfer from the aqueous phase to the organic phase during the extraction. Neutral species will transfer. At high acid concentration, you may get some hydrolysis of fats (triacylglycerides) if they are present but you usually have to heat them to hydrolyze the ester linkages in the triacylglycerides.

Free carboxylic acids and amines are some of the most difficult materials to get through a chromatographic system. They adsorb to everything. If you can make methyl esters of your acids you'll be way ahead from a reliable "quantitative analysis" standpoint - mostly because the final integration of the peaks will be straightforward and consistent.

I suppose of you don't have to worry about the proteins if they don't cause you a problem in your analysis. I don't see how you're going to get "false positives" on your acids measurement coming from the protein - unless you are using a flame detector and you have coeluters. Most everything responds in a flame detector. If coeluters aren't a problem, then the proteins shouldn't cause you a problem.

Purity of your solvents is all about minimizing the possibility of interferences. I your brand of solvent doesn't show something that coelutes with an analyte, then it is good enough - at least in my opinion, it's good enough.

Sigma is a very reliable supplier of all chemicals. They're my favorite. I go there first when I need something.
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