Aug 24, 2023

Public workspaceC-14 Fluroxypyr Acid Metabolite Extraction

DOI
dx.doi.org/10.17504/protocols.io.kqdg39yopg25/v1
  • 1Colorado State University;
  • 2USDA-ARS
Olivia Todd
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DOI: dx.doi.org/10.17504/protocols.io.kqdg39yopg25/v1
Protocol Citation: Olivia Todd, Scott Nissen 2023. C-14 Fluroxypyr Acid Metabolite Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39yopg25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Other
We have gotten this protocol to work successfully, but haven't needed to repeat it recently.
Created: February 27, 2023
Last Modified: November 08, 2023
Protocol Integer ID: 77644
Keywords: Aqueous phase extraction, Treated Leaf Tissue, C-14 fluroxypyr acid
Abstract
This protocol details C-14 fluroxypyr acid metabolite extraction.
Attachments
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659-1351.pdf
90KB
Materials
Extraction solution:
AB
Water 90%
Acetonitrile 9%
Acetic Acid1%

HPLC Solvent A:
AB
ACN10%
Formic Acid0.01%
Distilled water90%

HPLC Solvent B:
AB
ACN99.99%
Formic Acid0.01%

HPLC Column: C18 - This will be contaminated with radioactivity

Materials:

  • Glass test tubes + glass rod
  • Test tube caps
  • Maxi-Spin Filter Tubes Ciro Tubes
  • 500 uL Pipette
  • Scintillation Vials
  • Liquid N
  • Vacuum Manifold supplies
  • HPLC
  • Sep-Pak C18 Plus Short Cartridge, 360 mg Sorbent per Cartridge, 55 - 105 µm, 50/pk
Safety warnings
This protocol uses radio-labeled herbicides. It should be noted that Radioactivity is extremely harmful and proper training should be taken before using this protocol
Treated Leaf Tissue
Treated Leaf Tissue
Remove the radioactive-herbicide treated leaf from the plant and wash in Amount10 mL of 10% MEOH or ACN plus 0.25% NIS using the vortex mixer in a 20 ml scintillation vial.
Wash
Remove treated leaf from the scintillation vial and combine with the remaining shoot tissue. Add Amount10 mL of scintillation cocktail and count the leaf wash to determine % absorption.
Note
You will need this number to calculate the mass balance.

Pipetting
Tissue Grinding and Preparation
Tissue Grinding and Preparation
Use liquid nitrogen to grind whole plants with a glass rod in a 10mL disposable glass test tube.
Note
Grind plants as fine as possible.

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 Duration00:30:00 .
30m
Add Amount2.5 mL of extraction buffer, grind tissue with glass rod.

Pipetting
Add Amount2.5 mL of extraction buffer to clean glass rod.
Pipetting
Add all Amount5 mL of extraction buffer and plant solution to 0.45um Ciro Nylon Maxi-Spin Filter Tubes. Add Amount2.5 mL + Amount2.5 mL of extraction solution to the original glass tube for rinsing and add rinsate to tube (total solution volume: Amount10 mL ).
Pipetting
Remove Amount10 µL into a scintillation vial for counting on the Liquid Scintillation Counter.

Centrifuge the Ciro tube at ~Centrifigation4700 rpm, 00:10:00 to separate liquid from ground plant material.

10m
Centrifigation
(Dispose of radioactive 10 mL glass vial – save cap to wash)
Critical
Rinse the filter component of the Ciro tube with Amount5 mL extraction buffer.

Wash
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 Amount10 µL from oxidizer solution and run on the Liquid Scintillation Counter, or count entire oxidizer solution.

Solid Phase Extraction
Solid Phase Extraction
30m
30m
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 ~Amount1 mL 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 Amount10 mL .

Collect Amount10 µL from the pulled liquid and count on the Liquid Scintillation Counter.

Switch the vacuum manifold over to the new, labeled tube. Run Amount5 mL 100% ACN through the vacuum manifold to extract fluroxypyr-ester, fluorxypyr-acid and fluroxypyr-metabolites ffrom the column. Amount2.5 mL + Amount2.5 mL

Place these samples in the hood DurationOvernight to allow ACN to evaporate.

30m
Overnight
Sample Preparation and Injection
Sample Preparation and Injection
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 Amount10 µL 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)


Protocol references