I am working on a class of phosphate-containing compounds that is widely present in plants called "Phytates" - This class contains a range of compounds starting from IP6 to IP1 - the numbers here refers to the number of phosphate group that is present in the molecule.
I am developing a protocol using an LC-ESI-MS/MS ion trap. The challenge that I face is that IP6 or phytic acid which is the predominant compound failed to be fragmented in the MS2 (It gives zero signal). However, all other inositol phosphate compounds (IP5, IP4, and IP3) were well fragmented in the MS2. I do not get why the IP6 molecule would not have the same case.
I did direct infusion to the MS and the HPLC-MS/MS. It still not working. I also doubted that the compound could have a problem, but the new IP6 reference standard showed no fragmentation in the MS2. I injected an extract for a sample, it worked well in the MS2 with IP5, but not with IP6. The only difference is one phosphate group between IP5 and IP6.
Currently, I am searching the literature for recommendations on phosphate-containing compounds because there is no data on the fragmentation of phytates by ION TRAP detector to my knowledge so far.
Do you have any extra recommendations for what I can search for or try? Or has anyone experienced such a thing before, and what did you do to solve it?
Thanks in advance.
I guess hexakis structure of the IP6 gives more rigidity to the molecules in terms of electron distribution and this returns as fragmentation resistance. Without holding a particular experience on IP6, I suggest you increase the collision energy to disrupt the intramolecular bonding energies to get some daughter ion information. In this case, alternative fragmentation techniques such as ETD/ECD would be helpful (ETD is known to be very effective in elucidating phosphorylation). But if we discuss only the based on HC/CID fragmentation increasing the collision energy may be a choice...Have you ever tried the stepped collision energies?
Secondly, since this is not related to mass filter configuration (ion trap for your case) but the ionization technique (possibly), maybe the precursor ionization is poor, and therefore further mass fragmentation of the selected precursor is not observed at the ms2 stage. I mean poor ionization of the precursor at the ms1 level may be the core problem of not seeing fragments of the desired IP6... Are you able to compare the signals of IP variants at the MS1 level to confirm this hypothesis?
If this could be true, APCI ionization would be more appropriate to filter out IP6-like structures at the quadrupole stage in high numbers to efficiently provide ion flux through the collision cells which ends as improved MS2 signals...
In addition to these, the target compound is a natural chelating agent. Phytic acid can strongly chelate multivalent metal ions, especially the positively charged ions zinc, calcium, and iron. You are likely trying to acquire the phytate spectrum. Such chemical bonds with multivalent cations can be formed intra- and intermolecularly by one or several phosphate groups of the phytic acid molecule. Therefore it is important to either passivated or use an inert LC system to prevent non-specific loss.
Multivalent adducts of the molecules are generally hard to fragment such as sodium adduct. Try to modify your mobile phase by adding volatile salts, acids, or bases to suppress predominant adduct formation (do not forget to switch the ESI polarity during your survey)...If all indicated suggestions are not possible for your experiment, maybe it is better to keep the IP6 in SIM mode rather than forcing it to the SRM...
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