Presentation on theme: "Glyphosate and AMPA Analysis in Crops"— Presentation transcript:
1 Glyphosate and AMPA Analysis in Crops A simple and reproducible extraction and clean-up for HPLC Post-column derivatizationSaji 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 Clean-up Concentration Analysis Sample extracted with chloroform and 0.1N HClClean-upChelex column, Fe 2+ formAnion exchange columnConcentrationEvaporated to dryness and redissolved in 2 mL DI waterAnalysisHPLC/PCD using OCl-/OPA and FL detector
5 GC/MSD Method Extraction – Water as solvent Clean-up – methylene chloride, CAXMatrix specific modification – for high water, fat or protein contentConcentration – evaporated to dryness and re-dissolved in CAX mobile phaseManual Derivatization – heptaflurobutanol in trifluoroacetic anhydrideAnalysis-GC/MSD
6 HPLC/Post-Column Method Extraction - waterClean-up – methylene chloride, CAXConcentrationAnalysis – HPLC/PCD using OCl-/OPA and FL detector
7 Reagents/Equipment Sample preparation LC Post-column Methylene chlorideAcidic modifier solution(mixture of KH2PO4,, H2O, Methanol, HCl)CAX eluant (mixture of water, HCl and Methanol)CAX column(Pickering or Bio-Rad)LCPotassium eluant (K200), potassium regenerant (RG019)Cation exchange analytical column(Cat.# )Cation exchange guard column(Cat.# )Post-columnReagent 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 ExtractionTo 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 modificationHigh water content – reduce sample amount to 12.5gHigh protein content – add 100mL HCl to 20mL of extract, shake and centrifugeHigh 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 columnWash with two 0.7 mL portions of CAX mobile phase and discard the effluentElute analytes with 12 mL CAX mobile phase
11 ConcentrationEvaporate to dryness using rotary evaporator or a vacuum vortex-type evaporator or lyophylizeRe-dissolve in 2 mL of the CAX mobile phase
12 Instrumentation LC Post-column derivatization unit Fluorescence detector