Chromatography Forum

Dear all,

I am working with LC-MS/MS system (Hybrid Triple Quadrupole – Linear Ion Trap). My mobile phase is consisting of A: H2O 100 % and B: MeOH 100 %. To both solvent i.e A and B, 10 mM ammonium acetate was added and pH was set to 8.5 with ammonium hydroxide. The flow rate is 0.3 ml per min. The information of LC column is as followed: Synergi 4µm Fusion-RP 80A HPLC Column 250 x 2.0 mm from Phenomenex.

I am working with indole 3 carbinol (I3C) reference standard. The standard is dissolved in pure water and injection volume is 10 microliter. After LC-MS analysis the intensity of the I3C peak is quite low in a positive mode with electrospray ionization after using MRM scan mode. So, something is preventing my analyte from ionization. May I ask what do you think is the main reason to loose the intensity of the peak in the condition i explained above?

Sounds more like a MS side issue than HPLC. You didn't give any of your MS parameters, so all I can do is guess. In general, you need to tune your ESI conditions for each compound to yield maximum sensitivity. Your compound should generate positive ions in MS under acidic conditions; I am not sure why you are using basic conditions, which certainly will affect your positive ion formation. Try tuning your MS conditions to your specific compound and try running an acidic buffer.