C-14 Fluroxypyr Acid Metabolite Extraction

Remove the radioactive-herbicide treated leaf from the plant and wash in 10mL of 10% MEOH or ACN plus 0.25% NIS using the vortex mixer in a 20 ml scintillation vial.

Remove treated leaf from the scintillation vial and combine with the remaining shoot tissue. Add 10mL of scintillation cocktail and count the leaf wash to determine % absorption.

Use liquid nitrogen to grind whole plants with a glass rod in a 10mL disposable glass test tube.

Suspend the ground tissue in extraction solution (recipe in materials) and put on the shaker in a rack, wrapped in diaper paper to prevent spills for 0h 30m 0s.

Add 2.5mL of extraction buffer, grind tissue with glass rod.

Add 2.5mL of extraction buffer to clean glass rod.

Add all 5mL of extraction buffer and plant solution to 0.45um Ciro Nylon Maxi-Spin Filter Tubes. Add 2.5mL + 2.5mL of extraction solution to the original glass tube for rinsing and add rinsate to tube (total solution volume: 10mL).

Remove 10µL into a scintillation vial for counting on the Liquid Scintillation Counter.

Centrifuge the Ciro tube at ~4700rpm to separate liquid from ground plant material.

(Dispose of radioactive 10 mL glass vial – save cap to wash)

Rinse the filter component of the Ciro tube with 5mL extraction buffer.

Remove the entire filter component to dry in an envelope in the drying oven. When it is dry, carefully remove the filter papers and plant material. Before oxidizing these components to count radiation, these samples may be stored at -20C.

Remove 10µL from oxidizer solution and run on the Liquid Scintillation Counter, or count entire oxidizer solution.

Set up vacuum manifold for extraction. Place one Sep-Pak C18 filter on the manifold, and a glass syringe on the Sep-Pak filter. Place 2 labeled glass tubes adjacent to each other in the test tube holder for each sample.

Ensure that the liquid herbicide extract was properly acidified before you run it through the C18 SPE column by testing it with litmus paper.

Precondition the C-18 column with ~1mL of 100% ACN.

Pour the extraction buffer in the Circo tube through the C18 SPE column for each sample. Rinse the Ciro tube with X mL of acidified water to make the volume up to 10mL.

Collect 10µL from the pulled liquid and count on the Liquid Scintillation Counter.

Switch the vacuum manifold over to the new, labeled tube. Run 5mL 100% ACN through the vacuum manifold to extract fluroxypyr-ester, fluorxypyr-acid and fluroxypyr-metabolites ffrom the column. 2.5mL + 2.5mL

Place these samples in the hood 0h 30m 0s to allow ACN to evaporate.

When the samples are evaporated, bring the solution back up in HPLC solvent A and vortex several time to re-suspend everything in the test tube.

Filter the solution through a nylon syringe filter to filter out any particulates. Inject filtrate into a small liquid vial for use with the HPLC.

Take a 10µL subsample before injecting the sample into the HPLC . This will allow you to determine the ratio of parent compound (fluroxypyr-ester) to metabolites.

Metabolites:

• 4-amino-3,5-dichloro-6-fluoro-2-pyridinol (DCP)
• 4-amino-3,5-dichloro-6-fluoro-2-methoxy-pyridine (MP)