I tried to measure this with an ion exchange column, Rezex ROA, just to give it a try but we were not successful. We have a concentration of Fructose 2, 6- bisphosphate of 1mM (theoretical given the solution is really old and it's not commercially available anymore).
Any ideas or suggestions?
Ion-pairing reverse phase HPLC methods may be useful. Here are some papers on the subject. There are probably many others.
http://chromsci.oxfordjournals.org/content/24/5/198.abstract
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863137/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989532/
http://www.ncbi.nlm.nih.gov/pubmed/16970336
If you can get the enzyme phosphofructokinase 2, you can make fructose 2,6-bisphosphate yourself using fructose 6-phosphate + ATP as substrates.
In our lab they use HPLC coupled to infrared or a radiolabel detector. This was using reverse phase chromatography. They got very nice results.
Otherwise, I agree with Xia Ge, NMR, especially 31P (as this is the naturally abundant isotope), should work fine for concentrations aroun 1mM. I personally use NMR to detect changes in fructose-6-phosphate and fructose 1,6, bisphosphate.
When using NMR be aware that the three compounds you are interested in quantifying are very close to each other in the ppm range, almost on top of each other.
I would recommend using NMRPy by J.J. Eicher for deconvolution of your peaks. It is an open source python package available on github for the quantification of NMR spectra. I use this for my NMR data. It is wonderful and there is a tutorial included.
When using NMR just remember the intrnal standard. We use TEP as it is metabolically inert.
Check out the paper on determining enzyme kinetics using NMR spectroscopy by J.J. Eicher, it will explain everything I have just said in detail. It is available on researchgate. Co-authors are J.M. Rohwer an J.L. Snoep.
Hope this is of use.
Another option is to use the combination of high pH anion exchange with electrochemical detection. Figure 20 of the technical note (http://www.dionex.com/en-us/webdocs/5023-TN20_LPN032857-04.pdf) shows a separation of all of the fructose phosphates you mention along with a number of other sugar phosphates.
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