Chromatography Forum

Hi
I am developing an assay for the analysis of nebulised DNASE.
The nebulised dnase for hplc analysis was collected using a respirgard II filter and extracted from the filter by centrifugation before analysis by hplc

OUR result shows that we are recovering betweem 85% to 101% of the dnase sample.
Is this an acceptable range to validate the assay with or should we try and change the extraction method so as to recover 98-102% of our sample from the filter which is the recommeded range from the ICH?
I have tried several extraction methods but this was the best recovery we could achieve.

I don't think the recovery is as important as the variation in the recovery recovery.

Work the problem backward: how precisely do you need to know the DNASE concentration?

THANKS TOM,

I have decided to set the recovery range between 85-103% because of the difficulting of extracting the drug from the filter.
The main purpose of the assay was to investigate the feasibility of delivery dnase to the lung via a nebuliser. So i guest recovering more than 85% of the drug from the filter is suitable.
Thanks again for your help.

As far as you use IS or standard addition, the variation in recovery is not a critical issue...

This kind of recovery is acceptable for biological samples but you need to factor in low recovery when reporting the results or as indicated by Rafael use internal standard and use response ratio.

JM