Chromatography Forum

Dear all,
We are facing an analytical nightmare with N-nitroso diclofenac analysis by UHPLC/MS (SIR). We can not get a valid method due to issues with recovery/stability issues.
Due to nitrosamines unstability, we set the injector temperature to 8 ºC, but even with standards in water, the signal decay over time was significant. We also tried adding ascorbic acid (1%) to avoid oxidation, getting no improvement. Surprisingly, the following day we always recover (or even exceed) the signal intensity by injecting the same samples stored in the injector. This blowed our mind, it could not be an estability issue, wright?
We did the same test at 18 ºC and we also find a higher signal and a slight to moderate signal reduction with time, but at that time, if we set the injector temperature to 8ºC again, then we see a significant reduction in the peak area and height. ¿A solubility issue? At concentrations around 1-10 ng/mL this is unlikely...
We tried different aspiration speeds with no chanhe in signal.

We are lost... any idea?? HELP!!

Hello Aganza,

N-nitroso compounds are indeed a pain! I did some digging and some papers on environmental analytical chemistry suggest these compounds can readily oxidize or react with water. They actually suggest readers to avoid any contact between sample and N-nitroso-diclofenac... which is definetly not ideal.

There are examples in literature where the degradation of N-nitroso compounds in general have their kinectics slowed down a bit by acidification. You have only mentioned water and 1% ascorbic acid. some of the options could be to use a lower pH dilution mixture for your sample (be sure to have a column that withstands low pHs!) or use 100% of a very dry organic solvent such as ACN as your dilution solvent - please note that injections of a sample that contains a higher elution strength in comparison to your mobile phase will most probably cause some peak deterioration, you could partially solve this by using a high loadability column or lowering your injection volume. Unfortunately we are working on an optimization and best case scenario has already been excluded.

you could also try to investigate the stability of this analyte in alkaline media. but for this i suggest you to develop a method in alkaline mobile phase just so you do not inject an alkaline sample into an acid mobile phase... this never turns out well! also, be sure to have a hybrid silica column that withstands this condition, like a Triart!