Hi all,
I am doing NMR-metabolomics in cancer cells. Now, I am facing problem regarding concentration determination of metabolites. I use TSP as internal standard and using TSP concentration other concentrations are determined. But, for TSP, though I am adding a certain volume from a stock solution, I get TSP peaks are coming different (in terms of intensity) each time . It is very problematic for concentration measurement.
- I want to know what other commercially available standards I can use? There are several amino acids (glycine, serine, glutamine) and other metabolites (ex- Sodium pyruvate, Sodium acetate, lactate, glucose and many more).So, can anyone suggest what will be appropriate for this work?
- Along with that I have seen in a review that Formic acid has been recommended as one of internal standards. As it comes in liquid form, what volume I should use and how can I calculate its concentration because concentration of standards are required to measure other metabolites concentration.Will it not hamper the pH of NMR samples? We maintain sample pH at 7.4 using NaP.
Please guide me with your valuable suggestions.
Thanks in advance.
Thank you Eva Baranovicova .
I will try to be careful enough to remove the proteins from my sample during extraction. I don't have any IS other than TSP. So I am trying to overcome the issue with multiple standards. Let's see.
Do you need an internal standard? Potentially you could use a different approach like ERETIC 2 or QUANTAS. This would give fairly good quantification. I wouldn't use TSP as it has a long T1 and so requires very long relaxation delays. If you must use an internal standard then you could pick any material as long as it doesn't interfere with your signals of interest. I have used potassium hydrogen phthalate in the past - it is cheap, available pure and has a relatively short T1.
Thank you John Hollerton .
Sir, I do use Chenomx for metabolic profiling, so, I need a internal standard to calculate concentration.
I would like to know that, if I use ERETIC method (in that case I can use any external standard to quantify) how can I determine absolute contribution of a specific metabolite under a certain peak area?
[ It seems very hard as more than one metabolite can appear at a certain ppm].
First, we need to distinguish between ERETIC and ERETIC 2. The former uses the injection of a frequency into the probe, a hardware injection, the second is a software approach and is very similar to QUANTAS. In QUANTAS, the instrument is standardised by running a sample of known concentration and this is used to calibrate the signal that is digitally added post acquisition. This signal can be made to appear anywhere in the spectrum. It is then possible to export this spectrum to any 3rd party software. I would recommend that you read our paper about QUANTAS for full details (or look at the Bruker documenation for ERETIC 2)
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