Chromatography Forum

Hello,
I'm trying to develop a HPLC-FLD method to determine aflatoxins B1, B2, G1 and G2 in peanut butter and animal feed. I'm dealing low recoveries when I analyse CRMs for both matrices (up to 60-70% for B1 and 50% for B2, G1 and G2 are ok) as well as spiked samples that I prepare (things here get worse with 30-40% recoveries for all four).

Regarding sample preparation
1. Weighting 50-150g sample and adding sodium chloride depending on sample weight.
2. Extracting aflatoxins with pure methanol
3. Mixing for 15 minutes in blender and filtering
4. Taking an aliquot of the extract and dilute with water
5. Passing it all through immunoaffinity columns according to suppliers instructions regarding the column. Eluting the aflatoxins with 2 mL of pure methanol and diluting it with water up to 4 mL
6. Ejecting to HPLC

Chromatography is ok I don't have separation problems and calibration plots are ok. Standard solutions are freshly prepared and stock solutions are checked for degradation with uv spectrometer before preparing standard solutions.

I have tried many variations on sample preparation (e.g. extraction with pure acetonitrile and different proportions of methanol/water, blending time, various sample weight, different immunoaffinity columns from various suppliers etc) and the results are the same.

I can't seem to find what the problem is and any help will be greatly appreciated!

The immunoaffinity columns have an upper concentration limit. Are you certain that the concentration of mycotoxins isn't exceeding the capacity of the column?

Try 80% methanol.

Shake/blend for longer. At least 30 minutes is required for most sample types in my experience.

It is possible to reduce the amount of sample and continue to get representative results. As long as your sampling procedure and homogenization is thorough (probably a bit tough with peanut butter though).

Reading back over your post, I think it might be the amount you are putting on to your column. Do you derivatize your aflatoxins?

I had a small amount of ihop peanut butter left opened in my house for months. I threw it all out but I am very concerned about aflatoxins that could be in the air or touched other dishes. I can be really paranoid sometimes. What do I do? Should I be concerned?

SaraJennifer wrote:
I had a small amount of peanut butter left opened in my house for months. I threw it all out but i am very concerned about aflatoxins that could be in the air or touched other dishes. I can be really paranoid sometimes. What do i do? Should i be concerned?

I'd save my concerns for a driver running a red light while texting.