While performing recovery test with an extraction method of protein precipitation, the recovery results for all QC levels are always above 100% , the area's of Bio-analytical samples are higher than the aqueous samples . what can be the explanation ?
Hi,
If the actual recovery is 100% then maximum acceptable +15% method error could be a reason. Out of 15% means you have ion enhancement/suppression.
Is this ±15% method error acceptable for the FDA ? I saw a warning letter where the FDA asked in some point an explanation for the Recovery results when it was 110 % .
I think you should have an explanation for each case when you get borderline results. Is the ±10% borderline?! I'm note sure.
How did you precipitate the protein? Which analytical method do you use? UV-VIS?
The protein may be in a salt form with some counterions (e.g. we have trifluoroacetic acid as solvent additive for HPLC purification of peptides - after freeze-dry the TFA salt is generated). This counterions may change pH and therefore the UV absorbance of your sample. You may check the pH value or use a buffer system.
Its an LC-MS/MS method, and we tried the precipitation with ACN , ACN with 0.1% Formic acid, MeOH, MeOH with 0.1% Formic Acid, and in all cases the Recovery is above 100% , always the bio samples' (plasma) areas are higher than the aqueous samples.
PS. The "organic : water" ratio in the final vial is the same for both recovery and analytical samples, so the only difference is the plasma vs water.
There is still a lot of things in plasma, did you measured the water and the plasma without any sample added? You could do a background subtraction then.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry