Detect aflatoxins in urine

[Nguyen Quang Thieu]

Hello Friends,

I am trying to detect aflatoxins in pig urine by use HPLC system but the recovery is low and variation so much. Here is the protocol: Pig urine is incubate with beta glucuronidase in sodium acetate 0.2 M pH 5.5 for 16h, after incubation add 10 ml of phosphate buffer ph 7 then it will be extract by extrelude column with 60 ml ethyl acetate: acetinitril (9:1) to round botton flask. Evaporate the extract to dryness in rotar evaporator, dissolve residue in 1 ml of ethyl acetate-acetonitril (9:1) and transfere to 10 ml vial. Rine flask with additional portions of ethyylacetate followed by methanol. Evaporate combined residue and rinses to dryness under nitrogen in heated dry block. Dissolve the urine sample extract in 1 ml methanol-water (8:2), dilute with 15 ml PBS and mix. Pass the sample solution through the Immunoaffinity column (Rhone Easi Extract Afla) and let it pass at a speed of 1 drop per 2 seconds by gravity. Wash the column with 15 ml of PBS and dry by passing air through the immunoaffinity column. Elute the aflatoxins in a three steps by apply 0.5ml acetonitrile on the column and let it pass through by gravity (1 drop/2-3 seconds). Collect most of the elution solvent by passing air through. Evaporate the extract under nitrogen to dryness and derivatizate with 100µl trifluoroacetic acid in 5 minuses and dissolve with 900 ml of acetonitril-water (1:9) and put in HPLC. The mobile phase was acetonitril:water (2:8). LC column: Novarpack C18 4µ, 150x4mm, Water. Guard-pak column Novapark C18 4µ, Waters.
Fluoresence detection with Exitation 365 nm and Emission 435 nm.
I was also silanized all round bottom flasks and vials by dichlorodimethylsilane in 20 minuses.

If you have any ideal, please let me know where can I improve my procedure.

Best regards

Ngueyn Quang Thieu

[Uwe Neue]

The method is very complex, and there are many things that could go wrong.
My suggestion is to think about what you need and what you do not need, and then simplify.

As a starter, I would inject the sample after the initial incubation and before the extraction with Extrelute. You will have a problem with the glucuronidase, which will stick to the guard column. Have a spare guard column on hand. At this point, you should have a complete recovery of aflatoxin standard. Next, test the extraction through the Extrelute column and inject this sample. Do not bother with any of the other steps.

If you have complete recovery until this step and if your chromatogram looks good, stick with this and do not bother with the other steps.

If it does not look good, the problem is the extraction. Report back what you see and we work out a better extraction protocol, if necessary.

Aflatoxins can stick to all kinds of things. So the more steps you have, the more things can go wrong...

[JA]

My thoughts essentially echo Uwe's comments in that I was curious as to whether all of these preparation steps are necessary. Whether the recovery of the procedure had been verified with standards through each stage.

What is the purpose of the glucuronidase? Is it to free up the aflatoxins or some how assist in cleaning out other constituents of the urine? Maybe without it there is a good chance of isolating the target compounds (from the contaminants which presumably remain glucuronidated) on hydrophicity alone.

Is the recovery of the targets from the Immuno-column quantitative with only 1.5 ml acetonitrile?... etc.

Is the TFA derivitisation quantitative? This could be cross checked with UV detection.

[mbicking]

Which of the aflatoxins are you interested in? All or just some? Are recoveries bad for all, or just some of the compounds?

I agree with the other comments that the method is too complex. There is a high probability that you are actually decomposing the analytes during the evaporation steps. There is probably not anything left to derivatize. These molecules are sensitive to a number of chemical reagents, including acids, bases, and oxidizing agents.

As Uwe suggested, try to work with only some of the steps. Prepare a high concentration sample in water/solvent only and process it through each of the steps to determine if the problems occur at one step, or all.

[Nguyen Quang Thieu]

Dear friends,

Thanks for your all ideals, I will try with your suggestions.
The enzyme is to free up some of afla metabolites.
I work with aflatoxins: M1, B1, B2, G1 and G2. The recovery is a little bit better for B1 and b2, but still below 60%.
Best regards