Currently we are trying to develop methods of HPLC analysis for the kynurenine pathway metabolites, however we are having difficulties in the case of quinolinic acid. So far we have tried detection at 274 nm and 220 nm in mobile phase consisting of water and MeOH (80-50:20-50) but we end up with big peak tailing, poor method sensitivity and short Rt. We obtained similar results in mobile phase composed of acetate buffer pH 2,8 or 4,5 + MeOH (97-50:3-50). Furthermore, addition of tetrabutylammonium bromide (as an ion-paring agent) into the above-mentioned mobile phases did not bring any improvement.
We then tried reaction with Fe2+ as it is supposed to form coordination complexes, however mixture of 25-100 ng/ml of quinolinic acid with Mohr's salt (ammonium ferrous sulfate) in conc. 10-1 mM showed no maximum in visible light.
So if anybody has worked with it or would have some recommendations I would highly appreciate it.
Thank you very much in advance,
Rona
I assume you are using reversed phase chromatography with a C18 column. I have never analyzed quinolic acid using RP but looking at the pKa values for the molecule you likely have a speciation situation, thus you need to lower the pH further if possible (by the way acetate is not buffering anything at pH 2.8). You need to protonate both carboxylic groups to lower hydrophilicity of the molecule. Also you need to start with no organic solvent in the system and run an organic solvent gradient. Finally if you are using an ion-pair agent you need to ensure having one type of quinolinic acid species (one where a negative charge dominates obviously).
Hi Rona,
You tried to do right things using metal additives to the mobile phase to bind quinolinic acid and to improve peak shape of chromatographic peaks. The only problem is that Fe2+ is not a good choice. Firstly, it can be easily oxidised by the residual air in the eluent and form Fe3+ with possible complications due to hydrolysis. Secondly, iron has an elevated affinity to O,O' chelating ligands, so it will be presumably coordinated by two carboxyls from the molecule of quinolinic acid rather than on pyridinic nitrogen and carboxyl group. It means that this aromatic nitrogen is still available for secondary interactions causing peak tailing. Finally, Fe2+ forms rather weak complexes, which are not too stable at acidic pH. The possible solution is to use addition of Cu2+ instead Fe2+. Cu2+ forms stable N,O type complexes even under acidic conditions, stable to red-ox reations and can significantly improve sensitivity of UV detection due to formation of complexes.
It should improve peak shape.
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