Chromatography Forum

Hi,
I am working on setting up an assay to measure the effects of 6 Beta hydroxy testosterone in the presence of zolpidem.
the problem i am facing is that the 6 B OH testosterone peak overlaps with any of the zolpidems metabolites under all conditions tried?
i couldnot come up with the reason behind this problem?
The columns tried were C8 and C18 Novapak columns
The mobile phases ranged from pH 7.4 , 6.4, 4.6, 2.5 with no success.
Does anyone have any idea on how to overcome this problem

It would be easier to find a solution if we exactly know the co-elutive compound(s) or at least its functional group and pKa if any. However, general advices could apply to your problem:

If pH adjustment is a usual way to change the selectivity, column chemistry and organic solvent type are other to be tested.

Changing acetonitrile to methanol for example can sometimes resolve the co-eluting peaks.

Changing a C18 to C8 column bring small difference in selectivity (even more if you keep the same Nova-Pack chemistry).

On the other hand, selectivity can be more different by changing to a C18 polar embedded group or a phenyl column.

NovaPak is an old silica grade column (type A). I think that a high purity silica column (type B) would give more resolution (and also a change in selectivity). For example, Symmetry and Sunfire are 2 Waters column with type B silica. Perhaps more resolution will be enough to separate your peaks. I don't know what column dimension you're using but if you want more resolution you should go with smaller particles and/or longer column (if your system can afford higher back pressure).

A last point: if you try another column (XBridge for example) you can also try basic pH to enhance the retention of amine group.

Best regards.

Can you show chromatogramms? Can you list used conditions (coloumn, temp., flow rate, composition of mobile phase)?

Did you change your organic solvent? From methanol to acetonitrile or vice versa?

If this does not do any good, I would go for a modern column with an embedded polar group (EPG), such as SymmetryShield RP18. The EPG interacts with the amide group and you will get selectivity changes.

Zolpidem and its metabolites (i think atleast 11 metabolites) all have an amide group. the metabolites are formed by hydrolysis.

Thank you for your help. i will try out your suggestions.

Conditions used

Column: C8 and C18
flow rate: 1ml/min
mobile phase : buffers of different pH

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