HPLC method for ketoconazole determination

[zokitano]

Could you please help me, I have some doubts about one method for ketoconazole assaying. I wonder if it is right, because I didn't find any logic about it

Here is the HPLC method:
Column: Discovery C8 250x4.6, 5microns
Mobile phase: ACN:3.4g/L tetrabutylammonium hydrogen sulphate = 30:70
Flow: 1 mL/min

This is the structure of ketoconazole:
http://de.wikipedia.org/wiki/Bild:Ketoconazole2.png

It is nonpolar substance, soluble in methanol, insoluble in water. I could not find the pKa values for this compound

Is it ok to use ion pair reagent to separate this compound, when it contains no anion groups. The ion pair reagent is cationic ion pair reagent so I suppose complementary anion species could give with it a electroneutral ion-pair. But I suppose that the pH of the mobile phase is below 7 (the ion pair reaget is hydrogen sulphate salt) and it is not possible (for me) to have negatively ionized groups in the molecule of ketoconazole.

Am I wrong or this could not be appropriate method for determination and assay of ketoconazole.

Please give me some thoughts about this problem

I appreciate your effort and thanks for your time

Zoran

[sassman]

You are correct that your analyte will be positively charged. Positively charged nitrogen species tend to have a strong interaction with the column causing peak tailing. That is why the method calls for TBA. The TBA, because it also has a positively charged nitrogen, blocks the active sites on the column which improves peak shape. Some newer types of columns are less prone to this effect; for those columns TBA may not be necessary. Look for columns made for analysis of basic drugs.

[Uwe Neue]

Ketoconazole is a weak base. The pKa of the imidazole should be around 7.

We have run Ketoconazole at alkaline pH on a pH-stable column (like XTerra) with perfect peak shape and without problems.

[Bruce Hamilton]

If you look at the European Pharmacopoeia, you will see that the method for related substances is similar, even if on a C18 column.
Sample solvent = methanol
Column - C18 150 x4.6 x 3um
Detection 220nm
Flow 2 ml/min
Gradient
T0 1:19 CH3CN : 3.4g/L tetrabutylammonium hydrogen sulfate
T10 1:1 CH3CN : 3.4 g/L tetrabutylammonium hydrogen sulfate
T15 1:1 CH3CN : 3.4 g/L tetrabutylammonium hydrogen sulfate
Obviously, the method is intended to separated related substances from the main compound, and the EP gives relative retentions of typical impurities.

Bruce Hamilton

[zokitano]

That is correct Bruce,

The use of an ion-pair reagent in determination of impurities, here, in this case is useful and obligatory. But, I think that there is no need to use an ion-pair reagent when assaying only the active substance (ketoconazole).
I suppose that the degradation products of ketoconazole are more polar than ketoconazole itself, and that's why the Ph. Eur. HPLC method for determination of impurities include usage of ion-pair, otherwise when nonpolar compound is assaying like ketoconazole, it is, I think, not necessarily to use ion-pair for this purpose (when the method uses reversed-phase column).

Thank you for your help and your thoughts

Best regards

[Bruce Hamilton]

An assay doesn't just need to separate degradation products, but also any residual manufacturing reagents, byproducts, and solvents. The molecule is chiral, and the method does not attempt to resolve the enantiomers.

I'm not disputing your point about the ion-pair reagent, but would suggest that you would have to revalidate any changes, and take into consideration all residual compounds from manufacturing, as well as potential degradation products. You could ask the EP for the reasons why the method was selected and approved for related substances.

There's no reason why you can't change the assay method you have, but then you have to demonstrate that your "improvements" provide at least similar assurance. Not a trivial task, and I would follow a provided assay method, whether it's sensible or not, unless it obviously conflicted with a compendial method for assay or related substances.

Compendial methods are not state of the art ( although this one uses 3 um column, versus 5um for most methods I've seen ), but they are usually robust and appropriate. I'd also be very wary of an assay method that didn't separate compounds shown as related substances.

Please keep having fun,

Bruce Hamilton

[zokitano]

I agree with you, Bruce.

I'm also convinced that the EP method for determination of impurities is OK and there is nothing more to discuss about it.
I just wanted to share my opinions about the physical and chemical characteristics of ketoconazole and to ensure myself (with great help from you) that when you want to detrmine only the ketoconazole (as it is previously extracted from a creme) it is not obligatory to use an ion-pair reagent. Because we all know the advantages of using the ion-pair chromatography, but also we are aware of the disadvantages of using ion-pair reagents (expecially for columns and the shortening of ther lifetime).

Your posts are greatly appreciated, Bruce

Best regards