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- Location: South Korea
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.