Chromatography Forum

I'm developing a method for Tylosin(A), pKa = 7.1, in serum or plasma. Most described methods use acidic conditions with a C18 column however retention has been described as due to residual silanol activity rather than a reversed phase process. Also Tylosin is supposed to loose mycarose converting to Tylosin (B) aka Desmycosin in acidic medium. In neutral to basic medium Tylosin (A) adol is formed together with several polar decomposition products.
So the question is given limited resources and time should I work on a high pH or a low method. We are doing UV detection and would like to get a reliable method that can see small levels of Tylosin. Any thoughts on how best to go about this and what columns might work well would be greatly appreciated.

You can try few approaches.

1. Mixed mode approach. You antibiotic will retain based on reverse phase and ion-exchange mechanism, similar to amino sugars in the following application:

http://www.sielc.com/compound_003.html

You will need to do some basic protein removal to extend life of the column

2. Our new HILIC/Ion-exchange/Mixed Mode approach (see p 5-6):

http://www.sielc.com/pdf/SIELC_Obelisc_Intro.pdf


I don't have examples yet, but can imagine that it will work. Let me see if I can find another antibiotic with amino groups to run it in the lab

3. Pure HILIC approach (there are experts on this board for HILIC)