Chromatography Forum

Developing a method for a compound. Using the LTQ IOn Trap MS. I am also using a Kinetix Column.

When I inject I get two peaks on the Mass Chromatogram. I am injecting the compound of interest into Bovine Serum and extracting.

I am scanning MSMS on mass 161 in this run by the way. There are two peaks, each giving 144 as the major
product ion. This is in CID mode.

When I do this with just pure standard I do not get the double peaks. Unless this is a brand new phenomena and if I try the standard again it will show.
I have run this 5 times in a row with the same results.

Should I try to run the pure standard again to see if I get the same peaks as the Bovine Digest?
Is it possible some of the compound is piggy backing with something and coming out early?


Thanks

what is the compound of interest?

Anatabine

Are you sure the second peak isn't endogenous to bovine serum? 161 is a pretty small mass, and -17 is a fairly non-selective loss.

Well, no I am not sure. The second peak is the correct peak. It is the first that is troubling. The initial loss to 144 is not sepcific. But the secondary peak is also the same. I am not sure at the moment what that loss is.

What are some good ways to determine this?

Run a blank bovine serum extract--to see if mystery peak is present. If not, spike the bovine serum extract with your compound--to see if mere presence of bovine serum (or extraction solvent) components are enough to cause your peak to split. That will at least tell you where to focus your method development efforts.

Oh. Is the mystery 1st peak in the chromatographic void volume? If so, your sample extract solvent is probably too strong. Add the weaker mobile phase to see if it moves backwards or goes away entirely.

It is also possible the unsaturated ring of your compound vacillates between "chair" and "boat" conformation, with a high enough energy barrier between the two that you can get chromatographic separation under the right (i.e., wrong) conditions. I've seen that phenomenon with some aminoglycosides.

I do not believe that the peak is in the void volume as I am running at 300 uL per minute and using a Kinetex 2.6 um column. the peak comes out at I believe 1.3 minutes or so minutes.
I dont know the volume of these columns though so it is possible?

pi*r^2*l, with both r and l in cm, will give you the upper bound of your column volume

Mary has some good advice.
Run a blank bovine serum extract and see if the peak is still there.
In fact, extract a few bovine serum blanks so you can make sure your results are in fact reproduceable and not a fluke.
If you samples have been dried down they should be reconstituted with solution close to the mobilephase starting condition (if you run gradient). If the blanks are clean then you can reconstitue some of these blanks with a solution with your analyte to see if it causes a double peak.

Retention time is no way to judge void volume. You have to take column dimensions and flow rate into consideration. Even the void volume of the autosampler and hplc lines can be significant under some conditions.

In the past I have injected a dilute NaI solution and monitored for I- to get a void volume. (on a reverse phase column or with no column).

Alp

I have run 6 samples and got the double peak.

Now I am extracting 5 bovine blanks and 5 bovine spikes to see what happens there. If I get nothing in the blanks and something in the spikes I will be highly annoyed!
I started these this morning and am almost done with them now. The crappy thing about this is these extracts take time to do and I have better things to be doing!

Thanks for the help guys. Ill post my results. With maybe a chromatogram tomorrow.