Chromatography Forum

I'm developing a HPLC-DAD method to quantitate a vitamin in serum and the best I got was 65% recovery. Serum was extracted 3 times. Therefore I assumed an internal standard would be paramount. This way the low recovery would not be important.
Am I right?
Thanks
Matt

That would *only* be true to the extent that the analyte and internal standard both have the same low recovery (which you would have to verify and document).

You have to ask why the recovery is so low. Are you following an established procedure? What was the expected recovery for that procedure? If your recovery is lower, what's different?

If you're not following an established method, but developing your own, you have to look at the chemistry of the vitamin and what's happening to it. Is the extraction procedure quantitative (you you get 100% recovery with just a solution of the vitamin in the absence of serum)? If it's not, then that's the first problem to address. If the recovery is substantially lower with serum, what is the vitamin sticking to? Can you change the extraction (polarity? pH? salt?) to improve that?

Hi
The internal standard should be ok, as it is the published one. Your response pointed out something I think I'm overlooking. I'm changing the organic extractor.
Thanks
Matt

You might be dealing with degradation. Many vitamins are sensitive to heating, exposure to light etc.
An investigation of the above should be a part of the method validation.

Best Regards

As I know, Vit As very sensitive, and Vit Es pretty stable, so if your analyte is a kind of Vit A ,that one probably degraded after your extractoin .