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 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

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 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.# )
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 100mL 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: 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

14. Two-reagent Post-column System

15. Post-column Derivatization Reaction

16. Calibration curve

17. 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

18. Alfalfa
glyphosate
AMPA
0.500 ppm
blank
0.250ppm

19. 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

20. Broccoli
glyphosate
AMPA
0.500 ppm
blank
0.250 ppm

21. 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

22. Strawberry
glyphosate
AMPA
blank
0.500 ppm
0.250 ppm

23. 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

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