How to improve peak shape of tetracyclin by LC/QQQ

[thohry]

Hi all,

We are analyzing tetracyclin by LC/QQQ. The peak is not so good, it's rather tailing while the application shows very nice peak.
Our conditions are:
Column: RP C18 : 50mm, 2.1mm, 1.8 um
Mobile phase A: H2O + 0.1% formic acid
Mobile phase B: Acetonitrile + 0.1% formic acid
Gradient: B% = 5% at 0 min. and increases to 95% at 4min.
Injection volume : 5uL

MS/MS:
ESI positive mode
dry gas: 7L/min, temp = 350 oC, Vcap = 4000, nebulizer = 40 psi
MRM: 445 -> 410 and 445 --> 154.

We have tried many times and it improves a little bit but not as good as the peak in application. Have we done something wrong and what can be done to improve the peak shape.
Thanks for any helps.
Here I post the best peak that we could get:



http://farm7.static.flickr.com/6071/611 ... b725_z.jpg

[Alp]

I took a look at your image.
I have put baselines to far worse looking peaks!
One thing I notice is that you seem to have some "fronting". The reverse of tailing.
There is also a small peak in this fronting, it is either due to tetracycline eluting a bit before the main peak, or it is due to an isomer or other compound with the same mass transition.
I suspect that your sample may not be quite compatible with the starting conditions of your gradient. This can cause imperfect peak shape. Perhaps you could make your samples in 100% 0.1% formic acid or 5% B, 95% A, if you are not already doing so.

Make sure your tubing connections do not have any extra void volume.
You might also try a "virgin" column and see if that helps.

If the peaks in your literature are tall and narrow I would first confirm that the time scale is comparable. Peaks will look wider when 0 to 5 minutes fits across the page for your data but the literature has the time from 1 to 20 minutes.

Good luck

Alp

[thohry]

Thank you very much Alp. Your advice is helpful indeed.

[kmchale]

Tetracyclines are notorious for LC peak tailing. In my experience with tetracyclines, the two key factors for improving LC peak shape are (1) LC column and (2) mobile phase pH.

The LC column must have packing material that is well endcapped. I use a 2.1x100 mm, 3 um Betasil C18. Second, the pH plays a significant role. For the aqueous mobile phase (A), I originally used 0.02% TFA, but found that 0.2% formic acid yielded similar results without the ionization suppression observed with TFA. I also added 10 mM ammonium formate to the aqueous mobile phase, which yielded a pH ~ 2.5. ACN is used as the organic mobile phase (no modifier added).

Finally, make sure the LC gradient is not too steep, as there are epimers of tetracycline that form in solution with time, and will need to be separated, since they have the same SRM transitions.

Good Luck!