Chromatography Forum

I'm trying to implement a project to measure intracellular metabolites of bacteria using a Agilent LC Triple Quad MS. The metabolites of question are: ATP, G6P, NAD, F6P and Pyr. However, the main focus of the project is phosphorylated metabolites like DMAPP and IPP.

I'm trying to get the MS work done by going straight to the MS in ESI- however, as of now it's not going well (I think my mobile phase is incorrect). (10 mM Tributylamine solution at roughly pH 4.5):(methanol)

However, my main question is what type of column is preferable in my situation? I initially setup my project using tributylamine in my mobile phase with the idea that a RP column will be used. I found that TBA lingers in ESI+ mode and because the machine I'm on is shared (eg: not owned by my department) this is suboptimal.

I'm currently thinking about HILIC or PGC columns (with HILIC being the possible favorite).

TL;DR - Undergrad student needs help selecting column/mobile phase setup for phosphorylated metabolites on an LC-qqqMS.

PS - I do have a stack of journal articles to get through however, I'm still searching for a good resource for the fragmentation and collision methodology applicable to my situation (voltages and the such).

Thank you in advance.

Not an easy area.

Hilic works, but it won't separate the hexose phosphates. There are several hilic papers out there that claim to measure hexose phosphates, but they don't list F6P and G6P separately, and I certainly haven't separated them (using Phenomenex's Luna Hilic and Luna NH2 column, but with little optimisation). Unfortunately in my hands they don't fragment differently enough to produce some clever MS-based approach either.

I believe others have tried ion exchange and some whizzy box you can buy from Dionex that removes the buffer before the sample hits the MS.

I haven't been trying to keep up in this area, but the lab of Mark Stitt has a good track record. Try Plant Journal 59 824- and Biochemical J. 397 139- as starting points.

I agree that ion pair reagents in shared instruments are likely to get you in serious trouble with other users. They can be very hard to get rid of.