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.

  • Hi, thanks for the response.

    I tried increasing the gain, and it did increase the instrument response for all the compounds in the matrix (for instance, see the comparison below for Tribromoacetamide). However, when I run the Optimizer and run the Unknown analysis in the optimizer, it is not picking up any of the compounds at all with the scan data acquired with increased gains. 

  • EPA 552 corresponds to the Haloacetic acids (HAAs), I had already developed an MRM method for the HAAs. Now I'm working on the HAMs. 

  • Please check your data files and make sure the solvent peak is completely excluded. I have found that some data files I cannot detect ANY analytes of interest in Optimizer, even though I can identify them in standalone Unknowns Analysis. Without fail I find that the very tail end of the solvent peak is recorded in the data file. If I reinject the same sample with a longer solvent delay that excludes the solvent peak tail, then the peak identification process in Optimizer works as intended.

    The TIC shown in your original message looks fine to me, but just to rule this out as a possibility try setting solvent delay to 6 minutes, collect a data file, and check whether it resolve the issue. If it does back the solvent delay to shorter and shorter times to include any early eluters you need.

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