baseline increase at the start of the gradient

Hello All,

I am experiencing a huge baseline increase issue during my plasma metabolome analysis exp.

The BPC traces are as follows ( in both list and overlay modes) 

It is supposed to be separated like the K13 sample (second from top) but somehow it starts to be broadened and not separeted in most of the samples.

The sample(s) are methanol extracted plasma (5 ul injection volume).

I initially suspected column overlading but dilution did not help.

The second thought was the that the jump from A% to B% was steep and I have also slightly added a 4 mins isocratic step again not much improvement.

The only thing I have not tried is to increase the gradient time from 23 mins to sth like 40-50 mins. But this also does not make sense to me as the column is HSST3 (1.8 um, 50 mm, 2.1 mm) 

Any possible solution or suggestions?

Thanks in advance,


  • Basri-

    You might try making your sample solvent match the starting mobile phase and see if that helps.

    A 1-2 minute hold at 99% A at the beginning of the run may help a little.

    What major ions are associated with the raised signal and the beginning of the run and do they form chromatographic peaks (check out the EIC of some of the more intense ions).

  • Dear Andy,

    Thanks for the reply. I have tried your suggestions. You will see they indeed helped a bit

    My reconstitution solvent was (90:10 H20/MeOH)  which was actually very close to the starting mobile phase (99:1|)

    The top (black) and bottom (red) traces are 23 and 36 min runs. 

    I have looked into the broad peak at the start and it was EDTA (m/z 293).

    It looks like our clinical collaborator did not do a great good job in extracting the plasma samples as they are contaminated by EDTA. 

    Now the question becomes how to deal with EDTA. We will next try 2 and 4 fold diluted samples to see if the EDTA effect can be minimized.

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