How can I improve ionization in a new analytical method?

[IlariaConcardi]

Good morning

We are developing an analysis method to determine the following analytes: Chloramphenicol, 2,4-di-tert-butylphenol, Palmitic acid, Propylparaben and Stearic acid.

We are developing the method in negative and apart from Chloramphenicol, even injecting a 1 ppm standard, we do not see the peaks in chromatography but only extracting the ions and they are very low.
We are using an Agilent 1260 Infinity II combined with Agilent 6530 LC/Q-TOF.
as a source we have a Dual AJS ESI.
the QTOF parameters are:
Gas temp:250
Sheat Gas Temp: 250
V Cap: 3500
Fragmentor: 150
The column is a Poroshell 120 EC-C18 3x150 mm 2.7µL.
T column : 45 C
Mobile Phase-A ACN + 2 drops of ammonia
Mobile Phase H2O + 2 drops of ammonia
The gradient we use is:
0 min 10% Mobile Phase-A
1 min 10% Mobile Phase-A
5 min 90% Mobile Phase-A
20 min 90% Mobile Phase-A
25 min 10 % Mobile Phase-A
35 min 10% Mobile Phase-A
Flow:0.4 ml/min
do you have any suggestions or ideas to improve the ionization?

[DR]

1) 2 drops of ammonia - is not robust. What's the buffering capacity of 2 drops? My guess is that it is exceeded by your sample. Is your MP's pH consistent? Do the A and B phase pHs match?

2) what is the pKa of your analyte? If it is close to the pH of the MP, you're going to have problems with some of it being ionized and some of it being in free base form (where it won't show up on the MSD).

3) what does your sample prep look like?

4) I'm having doubts that you can separate and ionize your analytes in the same run as your fatty acids and polymers. That's a tall order. Your palmitate, paraben and stearic acids may all be stuck to the column. I would expect normal phase chromatography to be needed to separate those.

[IlariaConcardi]

We will try to make the mobile phases more robust, making a buffer at 0.1% ammonium formate in H2O and brought to pH=8 with ammonia and as organic phase acetonitrile 0.1% ammonium formate.

The method we are developing is for an E&L screening and at the moment we are injecting only a mix of standards in acetonitrile.

For the separation we are trying to apply an old method from another laboratory that had developed it on similar molecules but with the same instrument and they with almost neutral phases were able to see the peaks of the analytes well.