My lab is new to metabolomics analysis and for the past few months, we have been searching for a protocol to prepare our human fecal samples for LC-MS analysis, done by a metabolomics lab on campus. We ran a few validation samples using the attached protocol from the Sumner lab. However, I've seen other literature using 1) different ratios of methanol to water (50:50, 20:80, 1:1) and 2) cold methanol (anywhere from -20 to -80 °C).
I'm looking for information on how to decide what ratio of methanol:water is best and what temperature of the methanol you'd recommend? Also, if you have suggestions for other/better extraction solvents, that would be great! If anyone has done something similar, I'd love to hear your rationale behind your choices.
Hi, Honestly speaking - the methods are still developing and methods are yet to be perfected. Having said that recently an excellent review came out that would help you nucleate some methods : Karu et al. A review on human fecal metabolomics: Methods, applications and the human fecal metabolome database : https://www.sciencedirect.com/science/article/pii/S0003267018306354?via%3Dihub and beyond just methanol and water or solvents there is solvent: material ratio, pH, drying methods etc have HUGE bearing on results. Results as outputs of method optimization can only be evaluated in terms of the number of metabolites/ metabolic features a method captures as well as their summed intensity/ abundances and confidence of quantification, I.e., RSD or CV. Without these just visually gauging at peaks one cannot conclude. And this. An result in a decent optimization methods paper too if done properly. Donot forget to include suitable labeled or unlabeled internal standards at the start of extraction. Run multiple replicates for each methods-at least 3-5 samples. Then see. Also matters if targets are non-polar the not to go for water and methanol at all. Thanks, Biswa
Biswapriya Biswavas Misra, thank you so much for your input! The review you shared is extremely helpful and I can't wait to look into it more. I appreciate your time and guidance!
Mrs/miss Shinn,
You can try 1:1 nevertheless both solvents are polar protic ones.
Bojidarka B. Ivanova, thank you! Any reason why you'd recommend 1:1 compared to another ratio?
Scott Petersen, I'm not sure, I'd say mostly different macronutrient derivatives. We have a separate process for bile acids, but we're still reviewing the literature to decide what metabolite groups we should be focusing on, but the more we can cover, the better. I'm not sure if that would require multiple sample preparation techniques or if there's one we could use to cover everything. I guess I'm still not 100% clear on how to interpret our results. For example, we gave our metabolomics center some validation samples and they sent us back the results that listed over 2000 possible compounds that were detected and I'm not sure how we narrow down the results from that point.
Dear Leila Shinn,
You sample may consist of many compound from polar to non-polar nature. Therefore, it is important to use gradient elution method for better separation of your compounds. So. I recommend to you to use gradient elution. You can start methanol to water (5:95), then you can increased it to(95:5) in your program. To elute most non polar compound you can maintain 100% methanol for several minute at the end of the program.
Piyal
A. G. Piyal Aravinna That's an interesting suggestion! Thank you for sharing your expertise with us!
- Badges
- Science method