Chromatography Forum

Hi all,

I am working on histamine detection in feed sample. I am using 5% TCA extraction procedure. I am doing pre-column online derivatisation using OPA and FMOC. I have observed following things.

1. When I inject the just histamine standard (50 ppm), I am getting only one peak which has the area 26618959.
2. When I inject the standard 17 Amino acid mixture (25 uM) + Histamine (50 ppm), I am getting the area of 44513761.
3. When I inject just amino acid, there is no peak at that Rt.
4. When I inject just sample, I am getting area 38252235.
5. When I inject sample + histamine (50 ppm), I am getting the area of 50616344.

This data doesn't making sense to me. What could be the reason for higher area specifically when I inject with standard Amino acid mixture with histamine the area is higher. The histamine peak isn't merged with other amino acid?

Are these data repeatable? How many injections of each sample did you do?

Are these data repeatable? How many injections of each sample did you do?

I did twice.

Hi Rajan,

Just for quick clarification: are all these area values that you provide from the total area of the chromatogram (besides 1) or for strictly the histamine standard's peak? Could it be an issue with your integration settings that could be including an amino-acid with the histamine peak? You would be able to check this in post-run in your integration settings. It may simply be that at the current resolution (which may look great) the integration could be programmed incorrectly. Is the peak itself visually any different (ie, wider, different height)?
How does the baseline look when you compare the amino-acid mixture with the pure histamine?

Sorry for all of the questions, but the more information you provided will allow others to help!