Hie
i am working currently on diclofenac sodium and misoprostol combination , i need to develope hplc method of analysis for combination formulation but biggest difficulty i am facing is dos as diclofenac is 50 mg and 0.2 mg for misoprostol related substance analysis becomes very much difficult . Any body has idea about it how to proceed for this combi formulation

Wouldn't it be best and more accurate to do the analysis of the related substances separately for diclofenac and misoprostol?

BUT HOW TO DO THAT IS QUESTION AS BOTH ARE VERY CLOSE IN POLARITY AND ONE WILL ELUTE IN EITHER OF BOTH METHOD . AND DUE TO HIGH CONC OF DICLOFENAC ITWILL BE DIFFICULT

Diclofenac and misoprostol are not synthesized in the same pot, right?

For substances with very similar chemical and physical behaviour we develop the CALTREX HPLC phases. There we modify the silica surface with chalice like molecules called Calixarenes. The chromatographic behaviour is not only the separation about different solubility in mobile phase and stationary phase, additional you will be have a discrimination about host guest interaction. And this depends from different structure of both molecules. May be, it can help you. If you like further information please contact me under info@syntrex.de

Ulf, the question was how to handle samples with a 250-fold difference in concentration and probably detector response, not how to do the separation differently.

Increase your theoretical plates (longer column or smaller particles), or

Increase retention (k') by lowering your %B or lenghthening gradient, or

Change your selectivity, by changing stationary phase type or mobile phase modifiers.

Many times that third option is your best hope.

I agree with last mail. If you have a very good selectivity, than you can increase the injection volume for determination of impurities in a range of lower than 0,1 percent from main product.
The widening of the main peak is than no problem.

Cyclodextin are also very useful additives in the mobile phase to modify the selectivity. I suggest to try beta or gamma-cyclodextrin derivatives, methyl or hydroxypropyl cyclodextrins. If you like further information please contact cyclolab@cyclolab.hu

I got very good seperation by applying a tetrabutyl hydrogen sulphate in a mobile phase and adjusting a Ph of 6.7 , my Diclofenc is eluting at a 18 minutes on a C18 25 CM , 5 um Column and misoprostol at 40 minutes . but the peak shape are not at all good for analysis and also on injection 250 mg of diclofenac in a 10 ml to check inteferance its found that it start at 15 minutes and ends at 32 minutes and its taking away my prostol peak . its a real challenging job to do related substance of both , with a conventional technique or what>?

With an injection of 250 mg on a 25 cm x 4.6 mm column, you are overloading the column significantly, however, you still may be able to narrow the peak by maniputating the injection. What is the composition of the sample solvent? If you would inject in water, you may be able to load more onto the column with less peak distortion.

Also, I would stay away from ion pairing, if at all possible, since this type of chromatography overloads easier than standard RP.