Negative peak response

[Robotjock]

Virtual colleagues,
I have encountered one of those "beat-your-head-against-a-wall" problems.

About 3 weeks ago I successfully obtained UV/TIC data for a Decitabine, 5-aza-2'-deoxycytidine, intermediate. The separation is accomplished using a Synergi Hydro-RP 250 x 4.6 column w/ water/THF (90/10 v/v) as MP A and ACN/THF (90/10, v/v) as MP B on an Agilent 1200 LC coupled to an Agilent MSD SL G1956B run ES positve mode.

Of course, now I can't reproduce the analysis. I was having a relatively high background, ~ >500,000 response units, the second time around. I contacted Agilent and was directed to flush the system w/ a cleaning "cocktail". This appears to have been succesful as various configurations of the system - w/, w/o column, direct to nebulizer from pump, different channels, etc., provides very similar background traces ~ 50,000 response unit. However, when I injection the sample solution (in ACN) I obtain good UV trace, but I also obtain negative peak responses in the TIC.

I have no idea what's going on and would appreciate comments/suggestions.


Regards,

[Kostas Petritis]

You need to add some acid in your mobile phase (0.1% of formic acid should do the job) in order to help with the ionization of your compound in MS...

[Robotjock]

Mr. Petritis,

Note - the LC conditions are per a client's SOP used to obtain Assay and Impurity data for lot release. I would like to use exact conditions so the elutions times are similar for impurity peak tracking.

Having said that, I did use formic acid (0.1 %) in some of my initial separation attempts to ensure ionization in solution. However, while this did lead to the appearance of some peaks, it significantly changed the peak elution profile.

I then prepared fresh mobile phases w/o acid modifer and tried APCI mode. The data I obtained led me to return to ES mode - using the same mobile phases. I was able obtain two successful runs over different mass ranges. Subsequently, I can not reproduce the separation as mentioned in my initial post

[Kostas Petritis]

Absence of any acid when you are working in positive ion mode ESI-MS is not a very good practice. Your previous experiments might have worked because of traces of acid which ionized your compounds and now that your acid has completely washed out, no more ionization is achieved.

I understand that you need to use the client's SOP so my recommendation would be to introduce acid either through a T after your UV detector or through the use of sheath liquid. In this way your chromatographic retention time won't change and you should be able to now ionize and detect your compounds in MS...