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Method Abstract 206

206 / GLYPHOSATE AND AMPA ANALYSIS IN CROPS

A SIMPLE AND REPRODUCIBLE EXTRACTION AND CLEAN-UP2 FOR HPLC POST-COLUMN DERIVATIZATION

A Simple and Reproducible Extraction and Clean-up2 For HPLC Post-column Derivatization The recently practiced method1 for analysis of Glyphosate and AMPA in crops suffers from an expensive, time consuming clean-up procedure that has less than ideal recoveries. Although the analysis (after clean-up) by ion-exchange chromatography with post-column derivatization is rugged and sensitive, a new method was sought to improve the sample preparation. This resulted in AOAC Method 2000.522 which has a streamlined clean-up followed by pre-column derivatization and GC/MS analysis. We show how this simplified sample preparation is suitable for the classic ion-exchange/post-column analytical protocol.
 

METHOD

Equipment:

  • LC with a binary pump
  • Fluorescence detector
  • Pickering Laboratories Vector PCX or Pinnacle PCX Post-column Derivatization instrument
  • Pickering Laboratories Potassium cation exchange column, 4.0 x 150 mm (Cat. No. 1954150)
  • Cation-exchange GARD™ Column Protection System (Cat. No. 1700-3102)
  • Cation exchange clean-up column (Cat. No 1705-0001)

Reagents:

  • Potassium eluant (Cat. No.K200)
  • Potassium regenerant (Cat. No. RG019)
  • Hypochlorite diluent (Cat. No. GA116)
  • o-Phthalaldehyde diluent (Cat. No. GA104)
  • Thiofluor (Cat. No. 3700-2000)
  • o-Phthalaldehyde (Cat. No. O120)
  • 5 % Sodium hypochlorite solution
  • Methylene chloride
  • Acidic modifier solution
  • Clean-up column eluant

Sample Preparation

Extraction:

To 25 g of a homogenous sample, add enough water (after estimation of moisture content) to make the total volume of water 125 mL. Blend at high speed for 3–5 min. and centrifuge for 10 min. Transfer 20 mL of the aqueous extract into a centrifuge tube and add 15 mL of methylene chloride (to remove nonpolar co-extractives). Shake for 2–3 min. and centrifuge for 10 min. Transfer 4.5 mL of the aqueous layer into a vial and add 0.50 mL acidic modifier solution (16 g KH2PO4, 160 mL H2O, 40 mL Methanol, 13.4 mL HCl). Shake and centrifuge for 10 min.

Matrix Specific Modification:

Plants with high: 1) Water 2) Protein 3) Fat Content

  • For crops that absorb large amounts of water, reduce test portion to 12.5 g keeping water volume the same.
  • For crops that have high protein content add 100 μL HCl to 20 mL aliquot of crude extract. Cap, shake and centrifuge for 10 min.
  • For crops that have high oil content, do the methylene chloride partition twice.

Cation-Exchange Clean-up:

Remove top cap and then the bottom cap of the cation exchange clean-up column. Place the column into the manifold and drain solution in the column to the top of the resin lid.

Transfer 1 mL of extract (representing 0.18 g normal crop or 0.09 g dry crop) to the column reservoir and elute to the top of the resin bed. Add 0.70 mL of the elution solution (160 mL H20, 2.7 mL HCl, 40 mL Methanol) and discard the effluent. Repeat with a second 0.70 mL portion and discard effluent. Elute with 12 mL of the elution solution and collect in a round-bottomed flask. Evaporate to dryness in a water bath set at 40 ˚C using a rotary evaporator. Or collect in a centrifuge tube and evaporate using a vacuum
vortex evaporator. Dissolve residue in 2.0 mL of the elution solution (use 1.5 mL for dry crops). Extracts before evaporation can be stored refrigerated for up to 7 days.

LC Conditions

LC Column Temperature: 55 ˚C
Sample Injection Volume: 100 μL
LC Flow Rate: 0.40 mL/min
Mobile Phases:  K200, Potassium Eluant
                         RG019, Regenerant

Post-column Conditions

Post-column System: Vector PCX or Pinnacle PCX
Reactor Volume: 0.5 mL
Reactor Temperature: 36 ˚C
Reagent 1: 100μL of 5 % NaOCl (Bleach) in GA116 Diluent
Reagent 2: 100 mg o-Phthalaldehyde and 2g Thiofluor in 950 mL GA104 Diluent
Flow rate: 0.3 mL/min

Detection:

Fluorescence detector λex: 330 nm, λem: 465 nm
 

STEP

TIME (Min)

INTERVAL (Min)

K200 %

RG019 %

0

0

100

0

1

0-15

15

100

0

2

15.1-17

1.9

0

100

3

17.1-25

7.9

100

0

NOTE: Ensure that the auto sampler injection valve is fitted with a Tefzel or PEEK rotor seal for compatibility with the high-pH column regenerant.
Heavy-metal ions, especially iron can cause loss of sensitivity. Contamination of the guard and column can be removed by using RESTORE (Cat. No. 1700-0140) reagent.

 

chart-m-206

ACKNOWLEDGEMENT:
We thank The Montana Department of Agriculture, Laboratory Bureau for extracting alfalfa, tomato and other samples.

REFERENCES:
1) “Validation of an Analytical Residue Method for Analysis of Glyphosate and Metabolite: An Interlaboratory Study.” J. Agric. Food Chem. 34, (1986) 955–960.
2) P.L. Alferness and L.A. Wiebe, “Determination of Glyphosate and Aminomethylphosphonic Acid in Crops by Capillary Gas Chromatography with Mass-Selective Detection: Collaborative Study.” Journal of AOAC International, 2001 84, 823–846.