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I think your comment is missing in your reply message.
Kourosh
Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.
kouroshh1 wrote:
Hi Mattmullaney and Kenn,
Actually, I tried infusing Indole 3 carbinol (I3C) into the MS directly by using syringe and I did not have a problem. The intensity was high and no condensation product could have been seen.
The problem is when the I3C reference standard is running through the LC the condensation will occur. Unfortunately, when the condensation product from I3C is reaching into the ion source it will be fragmented into I3C mass transitions before reaching into the first quadruple. So, in my multiple reaction monitoring (MRM) method as a precursor ion (Q1) I have 130 m/z and as a product ion Q3 I have 103.1 m/z in a positive polarity. Due to the in-source fragmentation, I only get the transition for I3C that is 130 m/z----> 103 m/z. So it is very hard to identify the mass of the second peak because it has a same mass transition as I3C unless as you said we purchase the standard which have not done it yet and compare it against the second peak.
Unfortunately, the ratio of those 2 peaks does not remains constant over the course of your sequence.
Regarding the second part, in my MRM method if I have several compounds and some of them are co-eluting together, it is acceptable to count the area peak of each analyte and do the quantification. Am I understood correctly? I can select each peak based on its MRM transition. So, would it be acceptable to calculate the area peak of each peak independently because it has its own MRM transition?
Thank you for your time and help
kouroshh1 wrote:
Hi Kenn,
I think your comment is missing in your reply message.
Kourosh
kouroshh1 wrote:
Hi Kenn,
Thank you so much for spending time to explain to me in a very clear way. I will try to answer your question below.
* I guess it depends on the objective of your study and what type of study it is.
I am planning to extract I3C from the plant shoot. In general, when the brassica plants are stressed with for instance pathogen ,they expresss the enzyme which break down glucobrassicin (plant secondary metabolite) into I3C. By quantifying I3C, I will ensure that plant are defending themselve against the pathogen. So, quantification of this conpound is important for me.
* Does your high standard return the same area cts after say 20 injections?
Actually, I have tried only 3 injections and the intensity of I3C across 3 injections was not that much different. But i will try it with more injections to enure the result is consistent.
In addition, I haven't made I3C calibration curve yet. I will make it soon and as you sai. If the R square is very high then I will not care as you said about the artifact from source fragmentation.
* Are you running analyst?
Yes, we have Linear iontrap triple quadruple LC-MS/MS 3200 from Sciex in our lab and I am working with an analyst software.
* Set it up in quant method to quantitate each mass transition individually, there is no need for separation.
I didn’t know, it would be acceptable for a publication. So, I will not care that much in the future to get a super perfect resolution of the peaks.
And may I finally ask how can I improve the sensitivity of I3C while i am using basic mobile phase such as methanol with 10 mM ammonium formate at pH of 8.5 with NH4OH? In basic mobile phase I3C will stay mainly as a single peak but the sensitivity is very poor. So, if I can improve the sensitivity my problem will be solved.
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