Hi Mahesha,

    I am not sure about GC though I have carried out 5'nucleotidase assay using ion-pair reverse phase HPLC. Kindly look at the below mentioned paper and its supplementary information for details on the protocol.

Srinivasan B et al (2014) Allosteric regulation and substrate activation in cytosolic nucleotidase II from Legionella pneumophila.FEBS J.  281(6), 1613-1628.

Hi Dr Bharath,

Thank you very much. I will go through it...

Hello Mahesha,

it is possible to analyse these nucleotides via GC/MS, if only mono-phosphorylated. Di- and triphosphorylated nucleotides won't work.

However, I think the most crucial step is the sample preparation, as nucleotides can be cleaved in presence of water, so that ATP is degraded to AMP, or even to phosphate, adenine and the ribose backbone. So take care, that you don't analyse artifacts.

For analysis of the monophosphorylated nucleotides via GC/MS, the dried, i.e moisture-free extract has to be derivatised, e.g. with methoxyamine hydrochloride (dissolved in pyridine) and MSTFA.

Use a ZB-5MS or similar GC column. With this column you can also analyse nucleosides and nucleobases. If your instrument is equipped with an electron ionisation source, detection and identification of your targets will be easy using a corresponding spectral library for VAR5 columns, TMS-derivatives, alkane retention index.

Dear Dr. Michael, Thanks for your exciting answer. It is really interesting. Is there ant articles regarding this procedure? so that I might get detailed information on the procedure

Dear Mahesha,

I have no particular article in mind; it might be best to search yourself in google, using keywords like mstfa, gc-ms, nucleotides, zb-5ms (or db-5ms) to find an article with a method applicable to your sample type.

You can find a gc-ms method for the analysis of polar metabolites, including monophosphoryIated nucleotides in one of my publications:

DOI number: 10.1002/pmic.201200513

There is an article by Paul Fraser which is very helpful, regarding different sample preparation and analytical approaches. Here, a similar method for the analysis of monophosphorylated nucleotides via gc-ms was used. Have a look in the supplementary material to find retention times for your target compounds.

DOI number: 10.1016/j.mimet.2014.07.037

Dear Dr. Hensler, That's really great! Thank you so much for your information. I will definitely go through it.  Best wishes. MP

Dear Mahesha, Did you tried the method in the end? Does It Work? We are interested in the method expecially for GMP and IMP in dried samples, do have any advice?

Thanks

Dear Borghi, 

Nope! I did not try with GC as I did not find any recommended articles. I working with HPLC now..