Separation and impurity profiling of nucleosides using LCMS

[TGH]

I need suggestions for developing a gradient LCMS method suitable for complete impurity profiling in modified nucleosides and amino nucleosides (MW 200-600). Soluble in water and dilute ion pair buffers (water pH >7 for the aminos). The trouble is the product, hence impurities are not retained, e.g. I have used RP 5-10 mM TEAA and ACN Eclipse RP column and Prodigy ODS column with dilute ammonium formate ph 3.5 + ammonium formate 10% ACN. MeOH/H2O and KH2PO4 system may work for the LC end with a hypersil type column although I haven't tried it and MS will suffer. Is it OK to go to a lower pH usng a simple acetic acid/ACN/water system? Please keep in mind these compounds are modified at the 3' and can be base protected so they have multiple functionalities.

Ideas and suggestions would be appreciated.

[Uwe Neue]

You need to start with a mobile phase that does not contain any organic solvent to get adequate retention. The columns that you are working with do not allow you to do that. You need to work with a column that is completely compatible with a completely aqueous mobile phase and start your gradient with 100% water. Most C18s can't do that. I recommend to use Waters Atlantis dC18 column, which has been designed exactly for your type of application. As an alternative, you can use phases with an embedded polar group (such as SymmetryShield RP18) for the same type of application.

[SIELC_Tech]

I think that we can help you in developing a method. PLease provide me with your email and I will send you some links and examples (mail@silec.com)

regards,

[SIELC_Tech]

sorry it is mail@sielc.com, It is Friday and my mind is somewhere else.

[TGH]

Thanks Uwe, As you say, I have tried to retain using the columns I have on hand with water. I have read the dC18 lit and this column should be fine for the application.

These nucleosides can have modifications at the 3' position, do you expect any problems with amine functionality/pH? What are best options for gradient? acetic acid, 10 mM ion pair?

Thanks for your time.

[SIELC_Tech]

Here are few links which might be useful in terms of mechnisms of retention for nucleocides (ion-exchange /reverse phase alternative instead of ion-pairing reagent or non-organic mobile phases))

Nucleic bases
http://allsep.com/makeChr.php?chr=Chr_013

Nucleic acids
http://allsep.com/makeChr.php?chr=Chr_014

Other Similar Application:
http://allsep.com/makeChr.php?chr=Chr_068

regards,

[TGH]

Thanks for the additional links. Any information on functionally modified nuclesides would be very useful. e.g. base protected and 3' modified ones.

Cheers

[SIELC_Tech]

We can do free method development for you, we just need compounds (2-5 mg each). Contact us if you are interested. Although I want to let you know that we are going to use our Primesep mixed mode columns, there is no obligation to purchase columns after we done with the method. If compounds are proprietary we can do it under Secrecy Agreement. Usually we can do this in one or two days.

Regards,

[Einar Ponten]

The ZIC®-pHILIC column is very well suited for this application.

The mobile phase and gradient consists of a high ratio of acetonitrile that will enable sensitive MS detection and the polymeric column may be used in a wide pH range that is profitable for selectivity reasons.

We can provide you with more data and details <info@sequant.com>.