Zolpidem Assay?

[jananisoundarya]

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

[Kwet]

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.

[virtu]

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

[Uwe Neue]

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.

[jananisoundarya]

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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