Dear Group:

I have an aqueous gel filtration method that uses 0.15 M potassium phosphate buffer for mobile phase. The samples are prepared in water. I accidentally prepared my samples using mobile phase as the diluent. The chromatography is much better using mobile phase as the diluent. In order to change this one aspect of the method (switching from water to mobile phase as the diluent) what needs to be done? A full validation with specificity or an equivalency study? We use an RI detector and using water gives us a huge extraneous peak that is eliminated when I use mobile phase as the diluent. Thanks in advance for your comments.

Since making up samples in the mobile phase is documented good practice anyway, I would just run a few samples, document that results are the same, and put that in the validation file. Makes good sense to me, even FDA would have a hard time quibbling with that, I'd guess.

There is some guidance (minimal) in terms of partial validation, etc, in the May, 2002 guidance documentation on validation of Bioanalytical Method validations. You should look at this. May give some idea as to what the FDA would like to see. I would however tend to agree with Anonymous of Jan 10

PS Why do so many people post as anonymous on this site?

AS Tuco said to Angel Eyes in "The Good, The Bad and The Ugly": 'It's not always wise to use your own name'.

Chris-
To add to Anonymous statement above, several of us on this board work for directly competing companies and while we try to keep our conversations limited to the science, there is always someone who may simply be trying to find out what their competition may be working on. I post as Anonymous for the header but always include my first name only in the text so you know I'm a real person.
-Jeff

I consider the difference between diluting the sample in water or in mobile phase to be a very important part of the chromatographic method.

This is especially valid for gel filtration and depending on the type of packing. You also have to consider the volume injected.

How can this be a validated method? In fact this effect should have been seen and documented during the method development and validation?

What more unexpected effects are to be discovered? I rarely consume pharmaceuticals, but when I do I want to be sure about the quality. I hope FDA have the same opinion.

There is not a consensus (between FDA and
industry) regarding what changes in operating
conditions constitute either an "adjustment" or a
"modification" (method revision vs. complete
revalidation). However, assuming your QA accepts a
change in solvent as an "adjustment"; you could
generate system suitability data, using a retained
sample dissolved in mobile phase, to show that the
existing requirements are met. Having done so,
issue a method revision.

I feel you changing the important parameter of chromatographic method validation. Solubility of drug in diluent will effect your extraction of drug from sample matrix which directly effect your quantification. I feel you do the part method validation. It should covered Recovery, specificity and stability of standard and sample preparation in mobile phase.

I'm trying to implement a method for the determination of sulfonamides in honey, but the five compuonds of interest have similar retention times, and it is not possible to separate them, what should I try?, directly with another mobile phase or is there any other chance?.
thank you in advance.lucia

You need to run experiments in order to make an assesment that the change in sample diluent has no impact in the chromatography and you are still measuring the analyte of interest. Run the new sample diluent vs. the old sample diluent, compare the chromatographic profiles and response factors.