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Subsequently, I applied the 100 ppb Indole-3-carbinol into LCMS where the MRM scan mode was used. After doing LCMS using 100 % H2O as a aquas solvent and 100 % methanol as an organic solvent, I noticed that I could not see 1 single peak. It was 2 peaks with 2 minutes retention time difference belong to the same analyte Indole-3-carbinol. I did MS and MS/MS analysis and I realized both peaks are indeed belong to Indole-3-carbinol.
I checked at the structure of Indole-3-carbinol and I couldn't find a chiral center so no diastereomer could cause the appearance of 2 peaks.
I checked the Pka value of Indole-3-carbinol and it is around 15. Unfortunately, I couldnt make high pH mobile phase since our column is not resistance to the high pH. I know when we have a basic analyte we should go 2 units above the pH.
Apart from Pka, my question is that do you have any solution for my problem? Why I see 2 peaks instead of one peak because I only applied Indole-3-carbinol into the system and nothing more.
Thank you
Kourosh