Glyphosate and AMPA Analysis in Crops A simple and reproducible extraction and clean-up for HPLC Post-column derivatization Saji George, Lazslo Torma, Wendy Rasmussen, Maria Ofitserova Ph.D., Michael Pickering Ph. D., Pickering Laboratories

Current Use of Glyphosate Glyphosate is a nonselective herbicide and is used to control a great variety of annual, biennial, and perennial grasses, sedges, broad leafed weeds and woody shrubs. It is used in fruit orchards, vineyards, conifer plantations and many plantation crops(coffee. tea, bananas). It is used on non-crop areas(road shoulders and right of way) to control weeds and in forestry, gardening and horticulture. Genetically modified, glyphosate tolerant crops increase the use of glyphosate and possible misapplication.

Commonly Used Methods for Glyphosate Analysis in Crops Monsanto HPLC post-column method (J.Agric. Food Chem. 1986: 34, 955 – 960) GC/MSD (J.AOAC International. 2001: (84), 823 – 846)

Monsanto Method Extraction Clean-up Concentration Analysis Sample extracted with chloroform and 0.1N HCl Clean-up Chelex column, Fe 2+ form Anion exchange column Concentration Evaporated to dryness and redissolved in 2 mL DI water Analysis HPLC/PCD using OCl-/OPA and FL detector

GC/MSD Method Extraction – Water as solvent Clean-up – methylene chloride, CAX Matrix specific modification – for high water, fat or protein content Concentration – evaporated to dryness and re-dissolved in CAX mobile phase Manual Derivatization – heptaflurobutanol in trifluoroacetic anhydride Analysis-GC/MSD

HPLC/Post-Column Method Extraction - water Clean-up – methylene chloride, CAX Concentration Analysis – HPLC/PCD using OCl-/OPA and FL detector

Reagents/Equipment Sample preparation LC Post-column Methylene chloride Acidic modifier solution(mixture of KH2PO4,, H2O, Methanol, HCl) CAX eluant (mixture of water, HCl and Methanol) CAX column(Pickering or Bio-Rad) LC Potassium eluant (K200), potassium regenerant (RG019) Cation exchange analytical column(Cat.#1954150) Cation exchange guard column(Cat.# 1953020) Post-column Reagent 1 – 100uL of 5% sodium hypochlorite solution in 950 mL of hypochlorite diluent(GA116) Reagent 2 – 100 mg of OPA(Cat.#O120) and 2 g Thiofluor(Cat.#3700-2000) in 950 mL of OPA diluent(GA104)

Extraction To 25g of a homogenous sample add enough water (after estimation of moisture content) to make the total volume of water 125 mL. Blend and Centrifuge. Matrix specific modification High water content – reduce sample amount to 12.5g High protein content – add 100mL HCl to 20mL of extract, shake and centrifuge High fat content – do the methylene chloride partition twice.

Clean-up – methylene chloride partition To 20 mL of aqueous extract add 15 mL methylene chloride, shake for 2-3 min and centrifuge. To 4.5 mL of aq layer add 0.5 mL acidic modifier solution. Shake and centrifuge.

CAX clean-up Prepare CAX column Transfer 1 mL of the extract and elute to top of column Wash with two 0.7 mL portions of CAX mobile phase and discard the effluent Elute analytes with 12 mL CAX mobile phase

Concentration Evaporate to dryness using rotary evaporator or a vacuum vortex-type evaporator or lyophylize Re-dissolve in 2 mL of the CAX mobile phase

Instrumentation LC Post-column derivatization unit Fluorescence detector

LC and Post-Column Conditions LC conditions Column temperature: 55oC Injection volume: 100mL Flow rate: 0.40 mL/min Mobile phases: Potassium eluent (K200), Regenerent (RG019) Post column conditions Reactor volume: 0.5 mL Reactor temperature: 36oC Reagent 1: hypochlorite solution Reagent 2: OPA solution Flow rates: 0.3 mL/min Detection: fluorescence; lex 330nm, lem 465 nm

Two-reagent Post-column System

Post-column Derivatization Reaction

Calibration curve

Alfalfa Sample ID Spiked Conc.(ppm) Area Cal. Conc.(ppm) % recovery A1 0.250 31669 0.267 107 29005 0.245 98 A2 0.500 72112 0.678 135 71910 0.676

Alfalfa glyphosate AMPA 0.500 ppm blank 0.250ppm

Broccoli Sample ID Spiked Conc.(ppm) Area Cal. Conc.(ppm) % recovery 0.25 30369 0.257 103 31735 0.268 107 B1 1.25 102766 0.96 77 99554 0.93 74 B2 110110 1.03 82 107947 1.10 88 B3 2.50 211558 2.14 86 210983 B4 206927 2.10 84 203774 2.06 83

Broccoli glyphosate AMPA 0.500 ppm blank 0.250 ppm

Strawberry Sample ID Spiked Conc.(ppm) Area Cal. Conc. (ppm) % recovery S5 0.25 31981 0.271 110 32334 0.274 111 S1 1.25 117539 1.08 87 117941 1.09 S2 105826 0.98 79 108658 1.01 81 S3 2.50 192026 1.95 78 193789 1.96 S4 224064 2.27 91 224965 2.28

Strawberry glyphosate AMPA blank 0.500 ppm 0.250 ppm

Pickering CAX Column Sample ID Spiked Conc.(ppm) Area Cal.Conc.(ppm) % recovery A2P 0.500 68646 0.646 129 61700 0.580 116 S2P 1.25 112498 1.05 84 110597 1.03 82 S4P 2.5 193554 1.96 78 193619 B2P 107347 1.09 88 101323 87 B4P 238655 2.42 97 233830 2.37 95

Calibration curve

Advantages of Proposed Method Simple and faster extraction and clean-up Availability of post-column systems Time consuming derivatization for GC/MSD

Acknowledgements Saji George, Wendy Rasmussen, Maria Ofitserova and Michael Pickering – Pickering Laboratories Montana State Department of Agriculture