Method Abstract for Post-column Liquid Chromatography 206

Glyphosate (Roundup) And AMPA Analysis
in Crops Method Abstracts
A Simple and Reproducible Extraction and Clean-up
for HPLC Post-column Derivatization

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The recently practiced method1 for analysis of Glyphosate and AMPA in crops suffers from an expensive, time consuming cleanup 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 cleanup followed by pre-column derivatization and GC/MS analysis. We show how this simplified sample preparation is suitable for the classic ionexchange/ post-column analytical protocol.

METHOD

• 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)

• Pickering Laboratories Potassium cation exchange guard column, 3.0 x 20 mm (Cat. No 1953020)

• Cation exchange cleanup column

• 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

• Cleanup column eluant

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 coextractives). 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 (16g 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

1) For crops that absorb large amounts of water, reduce test portion to 12.5 g
    keeping water volume the same.

2) 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.

3) For crops that have high oil content, do the methylene chloride partition twice.

Cation-exchange cleanup

Transfer 1 mL of extract (representing 0.18g normal crop or 0.09g 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 ex: 330 nm em: 465 nm

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