Haloacetamides - MRM Method Development - Optimizer - Precursor Ion Selection


I am developing an MRM (multiple reaction monitoring) method for Haloacetamides (HAMs). I've completed the following steps:

(1) Ran the full scan and identified the peaks using Unknown Analysis
(2) The HAMs I am working with are not present in the NIST library (except for one), so I made a custom library after completing the analysis.
(3) In total, I have 6 HAMs - the peaks for them are pretty small, as can be seen in the chromatogram here:
(DCAM - Dichloroacetamide) (BCAM - Bromocholoracetamide) (DBAM - Dibromoacetmaide) (BDCAM - Bromodichloroacetamide) (DBCAM - Dibromochloroacetamide) (TBAM - Tribromoacetamide)

(4) Now, when I have made the library, and I am proceeding to the step of adding identifying the precursor ions in the Optimizer software, it is only picking up the 4 compounds (DCAM, BCAM, BDCAM, DBCAM) and not the TBAM and DBAM

Here is a screenshot from the Optimizer menu where I am using these settings to analyze peaks from a full-scan data file (the RT window that I am using is from 3 to 20)


And here is the library that I have made:

I am preparing the HAMs standards in 1mg/L concentration in methanol (1mL) and then diluting it with MTBE because my primary elution solvent is MTBE.

What settings can I change, try, or take steps to get the optimizer to pick these two compounds (TBAM and DBAM)?

Any help would be highly appreciated

  • It sounds like you are doing everything correctly. I am curious if reducing the library match score to 10 has any difference (long shot, and it shouldn't be doing that with a custom library made from that same sample.. but it is worth trying.)

    What scan range and scan time is being used? You could also try increasing the gain a bit--try bringing the max abundance up to 2e7 in the base peak chromatogram. I wonder if the low response factor of these two compounds is causing the trouble. If you still can't get it please let me know, I'd be curious to look closer at the problem if you can't fix it.

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