method development - very polar analytes

[trilobite]

Dear all,

I am developing a UPLC method for the simultaneous analysis of 6mercaptopurine (drug) and its metabolites (thioguanine, 6methylmercaptopurine). I am using an Acquity (Waters) system
I have employed a great variety of conditions. The best being the following:

Acquity BEH C18 [1.7 um, 2.1 x 100 mm]
Tcolumn= 25C
A = 96:1:3 0.02M KH2PO4(pH 2.25): MeOH : AcN
B = AcN, 0.1% formic acid
flow rate of 0.5ml/min
gradient conditions of 0.0-10 min, 100%A to 80%A
10-10.5 min, back to 100%A
10.5-12 min, 100%A

I get very nice peaks for all analytes. However, the two metabolites being rather polar elute at 1.322 and 1.165 min, respectively (the drug elutes at 4.551 min). How can I improve elution times (later within run) and peak separation??
I really need your feedback on this..

Kind Regards,
T

[annaand]

What about starting with an isocratic step at the beginning? What is the retention time of these 2 metabolites when you use 100% of mobile phase A?

[sepscientologist]

I agree with the last poster. Can you just add a two minute initial hold at 100% A and try it?

[Rooke123]

If you have to use the reverse phase column for those polar metabolites, ion pairing agents could help you to retain those analytes.

Also, HILIC columns are good alternatives to retain polar compounds, to avoid ion pairing agents,as it causes ion superession with MS

[unmgvar]

remember to check capacity of your peaks
if they are around 2 and up so things are quite good

like said above to improve resolution, see if an isocratic step in the begining of 1-2 minutes helps

[DSP007]

Hi
No Rokie, HILIC is not alternative for the novice operator
- its vary good, but "non robastic" ( poorly reproducible) method.
2Tribolit - its may be mistake . "Jump" substances through column in vary acidic phase.
See
http://www.thefreelibrary.com/Simultane ... 0209622069