TITLE: C-14 Fluroxypyr Acid Metabolite Extraction
AUTHORS: olivia.todd, Scott Nissen

[DESCRIPTION]
This protocol details C-14 fluroxypyr acid metabolite extraction.

[STEPS]
SECTION: Treated Leaf Tissue
1. Remove the radioactive-herbicide treated leaf from the plant and wash in 10 mL of 10% MEOH or ACN plus 0.25% NIS using the vortex mixer in a 20 ml scintillation vial.
SECTION: Treated Leaf Tissue
2. Remove treated leaf from the scintillation vial and combine with the remaining shoot tissue. Add 10 mL of scintillation cocktail and count the leaf wash to determine % absorption. 
 

SECTION: Tissue Grinding and Preparation
3. Use liquid nitrogen to grind whole plants with a glass rod in a 10mL disposable glass test tube. 
 

SECTION: Tissue Grinding and Preparation
4. 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 30 min. 
SECTION: Tissue Grinding and Preparation
4.1. Add 2.5 mL of extraction buffer, grind tissue with glass rod.

SECTION: Tissue Grinding and Preparation
4.2. Add 2.5 mL of extraction buffer to clean glass rod.
SECTION: Tissue Grinding and Preparation
5. Add all 5 mL of extraction buffer and plant solution to 0.45um Ciro Nylon Maxi-Spin Filter Tubes. Add 2.5 mL + 2.5 mL of extraction solution to the original glass tube for rinsing and add rinsate to tube (total solution volume: 10 mL).
SECTION: Tissue Grinding and Preparation
5.1. Remove 10 µL into a scintillation vial for counting on the Liquid Scintillation Counter.

SECTION: Tissue Grinding and Preparation
6. Centrifuge the Ciro tube at ~4700 rpm, 10 min to separate liquid from ground plant material.

SECTION: Tissue Grinding and Preparation
6.1. (Dispose of radioactive 10 mL glass vial – save cap to wash)
8. Ensure that the liquid herbicide extract was properly acidified before you run it through the C18 SPE column by testing it with litmus paper.
SECTION: Tissue Grinding and Preparation
6.2. Rinse the filter component of the Ciro tube with 5 mL extraction buffer.

SECTION: Tissue Grinding and Preparation
6.3. 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.


SECTION: Tissue Grinding and Preparation
6.4. Remove 10 µL from oxidizer solution and run on the Liquid Scintillation Counter, or count entire oxidizer solution.

SECTION: Solid Phase Extraction
7. 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.
9. Precondition the C-18 column with ~1 mL of 100% ACN.
10. 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 10 mL. 

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

12. Place these samples in the hood 30 min to allow ACN to evaporate.

SECTION: Sample Preparation and Injection
13. 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.
SECTION: Sample Preparation and Injection
14. 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.
SECTION: Sample Preparation and Injection
14.1. Take a 10 µL subsample before injecting the sample into the HPLC . This will allow you to determine the ratio of parent compound (fluroxypyr-ester) to metabolites.

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

SECTION: Sample Preparation and Injection
15. Metabolites:
4-amino-3,5-dichloro-6-fluoro-2-pyridinol (DCP)
4-amino-3,5-dichloro-6-fluoro-2-methoxy-pyridine (MP)