Chromatography Forum

Non stability assay method for Compund X & Y.

Compound X detected using fluorescence,
Compound X counter ion and Compound Y detected using UV.

In a series of 120 sample injections it was noticed there were a number of odd profiles, 5 vials gave no peak at all for compound X with fluorescence where there should be one. In these samples the peak areas for Compound Y and counter ion X were both detected as normal.

On re-vialing and re-injecting the same solutions, the compound Y and counter ion X peak areas were the same but this time there was also a peak for compound X.

Re-injecting the same vial with no compound X peak, vial 1, and the re-vialled one, where a compound X peak was detected, vial 2 (i.e. same solution in 2 different vials);

Original LC system - detection fluorescence- same as original results - vial 1 no response, vial 2 normal response
Different LC system - detection fluorescence- same as original results - vial 1 no response, vial 2 normal response
Original system - UV detection for both compounds X & Y
- vial 1 - peak present but smaller response than expected (approx 1/3rd result of that expected), vial 2 normal response.

Note: The same batch of branded vials was used throughout the analysis.

Is this a classic case of some sort of quenching fluorescence problem? We are very confused. Has anyonme come across instances of this before?

All thoughts/ideas most welcome.

I would speculate that you are losing some of compound X by ion exchange onto the glass surface. That would leave the counter-ion alone. It's actually not an unusual phenomenon with proteins; less common with small molecules.

It suggests poor QC on the part of the vial supplier.