Glyphosate Analysis in Soy Beans, Corn, and Sunflower Seeds by HPLC with Post-Column Derivatization and Fluorescence Detection

Glyphosate is a broad-spectrum herbicide widely used around the world. Monitoring of glyphosate in crops and water is mandated in many countries. We describe a sensitive and robust HPLC method for analysis of glyphosate in soy beans, corn, and sunflower seeds. This method utilizes a simplified sample preparation procedure that has proven to be effective even for challenging matrices.

Method

Analytical Conditions

Column: Cation-exchange column, K+ form, P/N 1954150
Guard column: Cation-exchange GARD™ Column
Protection System or Cation-exchange guard column P/N 1953020
Column temperature: 55 °C
Flow rate: 0.4 mL/min
Mobile phase: K200, RG019
Injection volume: 100 μL

Post-Column Conditions

Post-column system: Pinnacle PCX or Vector PCX
Heated reactor volume: 0.5 mL
Temperature: 36 °C
Ambient reactor: 0.1 mL
Reagent 1: 100 μL of 5% NaOCl (Bleach) in 950 mL of GA116 Diluent
Reagent 2: 100 mg of OPA and 2 g of Thio- fluor in 950 mL of GA104 Diluent
Reagent flow rate: 0.3 mL/min each reagent
Detection: λ_EX 330 nm, λ_EM 465 nm

Supplies for Sample Preparation

Methylene chloride, HPLC grade
Acidic modifier solution (16 g KH₂PO₄, 160 mL of water, 40 mL of methanol, 13.4 mL of conc. HCl)
Elution solution (160 mL of water, 40 mL of methanol, 2.7 mL of HCl)
RESTORE™
SPE sample clean-up cartridges P/N 1705-0001

Figure 1: Chromatogram of soy beans sample spiked with glyphosate at 0.1 ppm level.

Sample Preparation

Extraction

To 25 g of homogenized sample, add enough water (after estimating moisture content) such that the total volume of water is 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. Shake for 2–3 min and centrifuge for 10 min. Transfer 4.5 mL of aqueous layer to another centrifuge tube and add 0.5 mL of acidic modifier solution. Shake and centrifuge for 10 min. Filter through a 0.45 μm filter.

Figure 2: Chromatogram of corn sample spiked with glyphosate at 0.1 ppm level.

Matrix-Specific Modifications

Matrix with high 1) Water; 2) Protein; 3) Fat Content:

1) For samples that absorb large amounts of water, reduce test portion to 12.5 g while keeping water volume the same.
2) For samples with high protein content, add 100 μL of concentrated HCl to 20 mL of crude extract. Shake and centrifuge for 10 min.
3) For samples with high fat content, do the methylene chloride partitioning twice.

Figure 3: Chromatogram of sunflower seeds sample spiked with glyphosate at 0.1 ppm level.

SPE Cleanup

Remove the top cap first, then the bottom cap of the SPE columns and place them into the manifold. Drain the solution to the top of the resin bed. Transfer 1 mL of extract into the column and elute to the top of the resin bed. Add 0.7 mL of the elution solution and discard the effluent. Repeat with a second 0.7 mL portion of the elution solution and discard the effluent. Elute glyphosate with 12 mL of the elution solution and collect the effluent in a round bottom flask. Evaporate to dryness at 40 °C using a rotary evaporator. Dissolve the residue in 2.0 mL of a solution of 10% RESTORE™ in water (use 1.5 mL for dry samples), filter through a 0.45 μm syringe filter and inject onto the HPLC column. Extracts can be stored refrigerated for up to seven days before the evaporation step.

Table II: Recoveries for glyphosate

Pickering Laboratories
1280 Space Park Way, Mountain View, CA 94043
tel. (800) 654-3330, (650) 694-6700, fax: (650) 968-0749
Website: www.pickering.com