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Glyphosate and AMPA Analysis in Crops A simple and reproducible extraction and clean-up for HPLC Post-column derivatization Saji George, Lazslo Torma,

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Presentation on theme: "Glyphosate and AMPA Analysis in Crops A simple and reproducible extraction and clean-up for HPLC Post-column derivatization Saji George, Lazslo Torma,"— Presentation transcript:

1 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

2 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.

3 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)

4 Monsanto Method Extraction 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

5 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

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

7 Reagents/Equipment Sample preparation Methylene chloride Acidic modifier solution(mixture of KH 2 PO 4,, H 2 O, 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.# ) Cation exchange guard column(Cat.# ) 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.# ) in 950 mL of OPA diluent(GA104 )

8 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 100  L HCl to 20mL of extract, shake and centrifuge High fat content – do the methylene chloride partition twice.

9 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.

10 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

11 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

12 Instrumentation LC Post-column derivatization unit Fluorescence detector

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

14 Two-reagent Post-column System

15 Post-column Derivatization Reaction

16 Calibration curve

17 Alfalfa Sample IDSpiked Conc.(ppm)AreaCal. Conc.(ppm)% recovery A A

18 Alfalfa glyphosate AMPA 0.250ppm blank ppm

19 Broccoli Sample IDSpiked Conc.(ppm)AreaCal. Conc.(ppm)% recovery B B B B B

20 Broccoli glyphosate AMPA ppm blank ppm

21 Strawberry Sample IDSpiked Conc.(ppm)AreaCal. Conc. (ppm)% recovery S S S S S

22 Strawberry glyphosate AMPA blank ppm ppm

23 Pickering CAX Column Sample IDSpiked Conc.(ppm)AreaCal.Conc.(ppm)% recovery A2P S2P S4P B2P B4P

24 Calibration curve

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

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


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