Chromatography Forum

Hello,
I am trying to quantify neopterin and biopterin in urine, serum and CSF with Agilent Triple Quad UHPLC/MS/MS. The problem is that I cannot receive signal of this compounds in any of this samples, while in standards methods works fine – as I suppose. But now I am thinking - maybe all the method is wrong.

Do you think that it is possible to quantify neopterin and biopterin using:
Column: ACE Excel 2 C18-PFP (which I have in my lab, apart from zorbax C-18).
Mobile phase: acetonitrile : water (different combination with gradient and addition of for example formic acid 0,1% in each solvent).

what is your sample prep and expected concentration of those pterins in each matrix?

Are you working with naturally-derived samples in which you expect to find the compounds, or spiked samples? If you can't find anything in a sample, try spiking the sample with a concentration that you think you ought to be able to see, and look to see if it gives you a signal. This will give you a chance to check whether you have a problem with sample prep, a problem with the LC-MS method, or just a general issue that the assay isn't sufficiently sensitive for real life samples. It's also a good way to check whether you have a lot of suppression going on, where components of the real sample matrix reduce the efficiency of ionization of the analytes.