Methods and compositions for detecting glyphosate and metabolites thereof
The present invention provides various methods and compositions which allow for determining the presence or amount of glyphosate, N-acetylglyphosate, N-acetyl AMPA or aminomethyl phosphoric acid (AMPA) and its various metabolites in a variety of test matrices. In one method, determining the presence or amount of N-acetylglyphosate and/or N-acetyl AMPA in a test sample comprises providing the test sample suspected of containing N-acetylglyphosate and/or N-acetyl AMPA; extracting the N-acetylglyphosate and/or N-acetyl AMPA from the test sample; and, detecting the N-acetylglyphosate and/or N-acetyl AMPA in the extract. In other methods, the presence or amount of at least one of glyphosate, N-acetylglyphosate, N-acetyl AMPA or aminomethyl phosphonic acid (AMPA) or a metabolite thereof in a test sample is determined. The method comprises providing the test sample suspected of containing at least one of glyphosate, N-acetylglyphosate, N-acetyl AMPA or AMPA or a metabolite thereof, extracting from the test sample at least one of the glyphosate, N-acetylglyphosate, N-acetyl AMPA or AMPA; and, detecting at least one of the glyphosate, the N-acetylglyphosate, the N-acetyl AMPA and the AMPA from the test sample; wherein detection of the glyphosate, N-acetylglyphosate, N-acetyl AMPA or AMPA occurs without derivatization of the glyphosate, the N-acetylglyphosate, N-acetyl AMPA or the AMPA.
1. A method of determining the presence or amount of N-acetylglyphosate in a test sample comprising:
a) providing said test sample suspected of containing said N-acetylglyphosate;
b) extracting a composition comprising the N-acetylglyphosate from said test sample;
c) adding a sufficient concentration of phosphoric acid to the composition comprising the N-acetylglyphosate, wherein said sufficient concentration allows for an improved response and linearity of N-acetylglyphosate during detection;
d) chromatographically separating said N-acetylglyphosate from other constituents in said composition by a high performance liquid chromatography (HPLC) column; and,
e) analyzing by a tandem mass spectrometer the chromatographically separated N-acetylglyphosate to determine the presence or amount of the N-acetylglyphosate in the test sample.
2. The method of claim 1 , wherein the mass spectrometer is operated in positive ion mode.
3. The method of claim 1 , wherein prior to chromatographically separating the N-acetylglyphosate from the other constituents in said composition, the composition comprising the N-acetylglyphosate is in aqueous 0.02M phosphoric acid.
4. The method of claim 1 , wherein extracting the composition comprising the N-acetylglyphosate from said test sample comprises the steps selected from the group consisting of:
a) i) extracting the composition comprising the N-acetylglyphosate from the test sample by liquid-liquid extraction; and,
ii) disposing said extracted composition comprising the N-acetylglyphosate onto at least one extraction column and eluting the composition comprising the N-acetylglyphosate therefrom;
or,
b) i) mixing said test sample with a solid phase extraction sorbent under conditions that allow the solid phase extraction sorbent to bind said N-acetylglyphosate; and,
ii) extracting the solid phase extraction sorbent and the bound N-acetylglyphosate from the sample by liquid/liquid extraction; and,
iii) eluting said N-acetylglyphosate from the solid phase extraction sorbent.
5. The method of claim 4 , wherein said liquid-liquid extraction comprises an extraction with an aqueous mixture comprising methanol and a dilute acid.
6. The method of claim 4 , further comprising disposing the eluted composition comprising the N-acetylglyphosate from step 6(a)(ii) or 6(b)(iii) onto an anion exchange extraction column and eluting the N-acetylglyphosate therefrom.
7. The method of claim 1 , wherein derivatization of said N-acetylglyphosate does not occur.
8. The method of claim 1 , further comprising determining the presence or amount of aminomethyl phosphonic acid (AMPA), N-acetyl AMPA or glyphosate in the test sample.
9. The method of claim 1 , wherein said test sample is from an animal or an environmental sample.
10. The method of claim 1 , wherein said test sample is from a plant.
11. The method of claim 10 , wherein said test sample comprises forage, grain, stover, a plant tissue, a solid process fraction matrix, flour, a meal process fractions, hay, hulls, a seed, meal, grits, or starch.
12. The method of claim 11 , wherein said test sample comprises a solid matrix sample.
13. The method of claim 10 , wherein said plant is a dicot.
14. The method of claim 10 , wherein said plant is a monocot.
15. The method of claim 1 , wherein said method has a limit of quantitation of about 0.05 mg/kg to about 0.5 mg/kg.
16. A method of determining the presence or amount of N-acetylaminomethylphosphonic acid (N-acetyl AMPA) in a test sample comprising:
a) providing said test sample suspected of containing said N-acetyl AMPA;
b) extracting a composition comprising the N-acetyl AMPA from said test sample;
c) adding a sufficient concentration of phosphoric acid to the composition comprising the N-acetyl AMPA, wherein said sufficient concentration allows for an improved response and linearity of N-acetyl AMPA during detection;
d) separating said N-acetyl AMPA from other constituents in said composition by a high performance liquid chromatography (HPLC) column; and,
e) analyzing by a tandem mass spectrometer the separated N-acetyl AMPA to determine the presence or amount of the in the test sample.
17. The method of claim 16 , wherein the mass spectrometer is operated in positive ion mode.
18. The method of claim 16 , wherein prior to chromatographically separating the N-acetyl AMPA from the other constituents in said composition, the composition comprising the N-acetyl AMPA is in aqueous 0.02M phosphoric acid.
19. The method of claim 16 , wherein extracting the composition comprising the N-acetyl AMPA from said test sample comprises the steps selected from the group consisting of:
a) i) extracting the composition comprising the N-acetyl AMPA from the test sample using an aqueous dilute acid/methanol solution; and,
ii) disposing said extracted composition comprising the N-acetyl AMPA onto at least one extraction column and eluting the composition comprising the N-acetyl AMPA therefrom;
or,
b) i) mixing said test sample with a solid phase extraction sorbent under conditions which allow said solid phase extraction sorbent to bind the N-acetyl AMPA; and,
ii) extracting the solid phase extraction sorbent and the N-acetyl AMPA from the sample by liquid/liquid extraction; and,
iii) eluting said N-acetyl AMPA from the solid phase extraction sorbent.
20. The method of claim 19 , further comprising disposing the eluted composition comprising the N-acetyl AMPA from step 23(a)(ii) or (b)(iii) onto an anion exchange extraction column and eluting the N-acetyl AMPA therefrom.
21. The method of claim 16 , wherein derivatization of said N-acetyl AMPA does not occur.
22. The method of claim 16 , further comprising determining the presence or amount of aminomethyl phosphonic acid (AMPA), glyphosate or N-acetyl glyphosate in the test sample.
23. The method of claim 16 , wherein said test sample is from an animal, a plant, or an environmental sample.
24. The method of claim 23 , wherein said test sample comprises forage, grain, stover, a plant tissue, a solid process fraction matrix, flour, a meal process fractions, hay, hulls, a seed, meal, grits, or starch.
25. The method of claim 24 , wherein said test sample comprises a solid matrix sample.
26. The method of claim 23 , wherein said plant is a dicot.
27. The method of claim 23 , wherein said plant is a monocot.
28. The method of claim 16 , wherein said method has a limit of quantitation of about 0.05 mg/kg to about 0.5 mg/kg.
29. A method of determining the presence or amount of N-acetylglyphosate or N-acetyl AMPA in a test sample comprising:
a) providing said test sample suspected of containing said N-acetylglyphosate or N-acetyl AMPA;
b) extracting the composition comprising the N-acetylglyphosate or N-acetyl AMPA from the test sample, said extracting comprising the steps selected from the group consisting of:
1) i) extracting said N-acetylglyphosate or said N-acetyl AMPA from the test sample by liquid-liquid extraction;
ii) disposing said extracted composition comprising the N-acetylglyphosate or N-acetyl AMPA of step (b) onto a C 18 extraction column and eluting the N-acetylglyphosate or N-acetyl AMPA therefrom;
or,
2) i) mixing said test sample with a solid phase extraction sorbent under conditions which allow said solid phase extraction sorbent to bind the N-acetyl AMPA or N-acetylglyphosate;
ii) extracting the solid phase extraction sorbent and the N-acetyl AMPA or the N-acetylglyphosate from the sample by liquid/liquid extraction; and,
iii) eluting said bound N-acetyl AMPA or said bound N-acetylglyphosate from the solid phase extraction sorbent;
c) disposing the eluted N-acetylglyphosate or N-acetyl AMPA from step (b) onto an anion exchange column comprising a surface functionally of m-divinylbenzene and N-vinylpyrrolidone copolymer having quaternary amines and eluting the N-acetylglyphosate therefrom;
d) adding a sufficient concentration of phosphoric acid to said eluted N-acetylglyphosate of step (c), wherein said sufficient concentration allows for an improved response and linearity of N-acetylglyphosate during detection;
e) chromatographically separating said N-acetylglyphosate or said N-acetyl AMPA from other constituents of step (d) using a phenyl-hexyl high performance liquid chromatography (HPLC) analytical column; and,
f) analyzing the chromatographically separated sample of step (e) to determine the presence or amount of the N-acetylglyphosate or the N-acetyl AMPA in the test sample by a tandem quadrupole mass spectrometer operated in positive ion mode.
30. The method of claim 29 , wherein said liquid-liquid extraction comprises the use of an aqueous dilute acid and methanol solution.
31. A method for determining the presence or amount of glyphosate in a test sample comprising
a) providing the test sample suspected of containing the glyphosate;
b) extracting a composition comprising the glyphosate from said test sample;
c) adding a sufficient concentration of phosphoric acid to the composition comprising said glyphosate of step (b), wherein said sufficient concentration allows for an improved response and linearity of glyphosate during detection;
d) chromatographically separating the glyphosate from other constituents in the composition by a high performance liquid chromatography (HPLC) column; and,
e) analyzing by a tandem mass spectrometer the chromatographically separated glyphosate of step (d) to determine the presence or amount of the glyphosate in the test sample;
wherein detection of said glyphosate occurs without derivatization of said glyphosate.
32. A method for determining the presence or amount of glyphosate in a test sample comprising
a) providing the test sample suspected of containing the glyphosate;
b) extracting a composition comprising the glyphosate from said test sample using a dilute aqueous acid/methanol solution;
c) chromatographically separating the glyphosate from other constituents in the composition by a high performance liquid chromatography (HPLC) column; and,
d) analyzing by a tandem mass spectrometer the chromatographically separated glyphosate of step (c) to determine the presence or amount of the glyphosate in the test sample;
wherein detection of said glyphosate occurs without derivatization of said glyphosate.
33. The method of claim 32 , wherein following step (b) and prior to step (c) a sufficient concentration of phosphoric acid is added to the composition comprising said glyphosate of step (b), wherein said sufficient concentration allows for an improved response and linearity of glyphosate during detection.
34. The method of claim 33 , wherein the mass spectrometer is operated in positive ion mode.
35. The method of claim 33 , wherein extracting the composition comprising the glyphosate from said test sample comprises the steps selected from the group consisting of:
a) i) extracting the composition comprising the glyphosate from the test sample by liquid-liquid extraction; and,
ii) disposing said extracted composition comprising the glyphosate onto at least one extraction column and eluting the composition comprising the glyphosate therefrom;
or,
b) i) mixing said test sample with a solid phase extraction sorbent under conditions which allow the solid phase extraction sorbent to bind the glyphosate; and,
ii) extracting the solid phase extraction sorbent and the bound glyphosate from the sample by liquid/liquid extraction; and,
iii) eluting said glyphosate from the solid phase extraction sorbent.
36. The method of claim 35 , further comprising disposing the eluate of the extraction column or the solid phase extraction sorbent onto an anion exchange extraction column and eluting the glyphosate therefrom.
37. The method of claim 33 , wherein said test sample is from an animal, a plant or an environmental sample.
38. The method of claim 37 , wherein said test sample comprises forage, grain, stover, a plant tissue, a solid process fraction matrix, flour, a meal process fractions, hay, hulls, a seed, meal, grits, or starch.
39. The method of claim 38 , wherein said test sample comprises a solid matrix sample.
40. The method of claim 37 , wherein said plant is a dicot.
41. The method of claim 37 , wherein said plant is a monocot.
42. A method of determining the presence or amount of glyphosate in a test sample comprising:
a) providing said test sample suspected of containing glyphosate;
b) extracting the composition comprising the glyphosate from the test sample, said extracting comprising the steps selected from the group consisting of:
1) i) extracting said glyphosate from the test sample by liquid-liquid extraction;
ii) disposing said extracted composition comprising the glyphosate of step (1)(i) onto a C 18 extraction column and eluting the glyphosate therefrom; or,
2) i) mixing said test sample with a solid phase extraction sorbent under conditions which allow the solid phase extraction sorbent to bind said glyphosate;
ii) extracting the solid phase extraction sorbent and the bound glyphosate from the sample by liquid/liquid extraction; and,
iii) eluting said glyphosate from the solid phase extraction sorbent;
c) disposing the eluted glyphosate from step (b) onto an anion exchange column comprising a surface functionally of m-divinylbenzene and N-vinylpyrrolidone copolymer having quaternary amines and eluting the glyphosate therefrom;
d) adding a sufficient concentration of phosphoric acid to said eluted glyphosate of step (c), wherein said sufficient concentration allows for an improved response and linearity of glyphosate during detection;
e) chromatographically separating said glyphosate from other constituents in the eluate of step (d) using a phenyl-hexyl high performance liquid chromatography (HPLC) analytical column; and,
f) analyzing the chromatographically separated sample of step (e) to determine the presence or amount of glyphosate in the test sample by a tandem quadrupole mass spectrometer operated in positive ion mode;
wherein detection of said glyphosate occurs without derivatization of said glyphosate.
43. A method for determining the presence or amount of aminomethyl phosphonic acid (AMPA) in a test sample comprising
a) providing the test sample suspected of containing the AMPA;
b) extracting a composition comprising at least one of said AMPA from said test sample;
c) adding a sufficient concentration of phosphoric acid to the composition comprising the AMPA, wherein said sufficient concentration allows for an improved response and linearity of AMPA during detection;
d) chromatographically separating the AMPA from other constituents in the composition by a high performance liquid chromatography (HPLC) column; and,
e) analyzing by a tandem mass spectrometer at least one of the chromatographically separated AMPA of step (c) to determine the presence or amount AMPA in the test sample;
wherein detection of the AMPA occurs without derivatization of said AMPA.
44. The method of claim 43 , wherein the mass spectrometer is operated in positive ion mode.
45. The method of claim 43 , wherein prior to chromatographically separating the AMPA from the constituents in said composition, the composition comprising said AMPA is in aqueous 0.02M phosphoric acid.
46. The method of claim 43 , wherein extracting the composition comprising the AMPA from said test sample comprises the steps selected from the group consisting of:
a) i) extracting the composition comprising the AMPA from the test sample by liquid-liquid extraction; and,
ii) disposing said extracted composition comprising the AMPA onto at least one extraction column and eluting the composition comprising the AMPA therefrom;
or,
b) i) mixing said test sample with a solid phase extraction sorbent under conditions which allow the solid phase extraction sorbent to bind the AMPA; and,
ii) extracting the solid phase extraction sorbent and the bound AMPA from the sample by liquid/liquid extraction; and,
iii) eluting said AMPA from the solid phase extraction sorbent.
47. The method of claim 46 , wherein said liquid-liquid extraction comprises an extraction with a dilute aqueous acid/methanol solution.
48. The method of claim 46 , further comprising disposing the eluate of the extraction column onto a cation exchange extraction column and eluting the AMPA therefrom.
49. The method of claim 43 , wherein said test sample is from an animal, a plant, or an environmental sample.
50. The method of claim 49 , wherein said test sample comprises forage, grain, stover, a plant tissue, a solid process fraction matrix, flour, a meal process fractions, hay, hulls, a seed, meal, grits, or starch.
51. The method of claim 50 , wherein said test sample comprises a solid matrix sample.
52. The method of claim 49 , wherein said plant is a dicot.
53. The method of claim 51 , wherein said plant is a monocot.
54. A method of determining the presence or amount of aminomethyl phosphonic acid (AMPA) in a test sample comprising:
a) providing said test sample suspected of containing AMPA;
b) extracting the composition comprising the AMPA from the test sample, said extracting comprising the steps selected from the group consisting of:
1) i) extracting said AMPA from the test sample by liquid-liquid extraction;
ii) disposing said extracted composition comprising the AMPA of step (1)(i) onto a C 18 extraction column and eluting the AMPA therefrom;
or,
2) i) mixing said test sample with a solid phase extraction sorbent under conditions which allow the solid phase extraction sorbent to bind the AMPA;
ii) extracting the solid phase extraction sorbent and the bound AMPA from the sample by liquid/liquid extraction; and,
iii) eluting said AMPA from the solid phase extraction sorbent;
c) disposing the eluted AMPA from step (b) onto a cation exchange column comprising a surface functionally of m-divinylbenzene and N-vinylpyrrolidone copolymer having sulfonic acid substituents and eluting the AMPA therefrom;
d) adding a sufficient concentration of phosphoric acid to said eluated AMPA of step (c), wherein said sufficient concentration allows for an improved response and linearity of AMPA during detection;
e) chromatographically separating said AMPA from other constituents in the eluate of step (d) using a phenyl-hexyl high performance liquid chromatography (HPLC) analytical column; and,
g) analyzing the chromatographically separated sample of step (e) to determine the presence or amount of AMPA in the test sample by a tandem quadrupole mass spectrometer operated in positive ion mode;
wherein detection of said AMPA occurs without derivatization of said AMPA.
55. A method for determining the presence or amount of at least one of glyphosate, N-acetylglyphosate, N-acetylaminomethylphosphonic acid (N-acetyl AMPA) or aminomethyl phosphonic acid (AMPA) in an oil test sample comprising
a) providing the oil test sample suspected of containing at least one the glyphosate, the N-acetylglyphosate, the N-acetyl AMPA or the AMPA;
b) adding a sufficient concentration of phosphoric acid to said test sample of (a) wherein said sufficient concentration allows for an improved response and linearity of the glyphosate, the N-acetylglyphosate, the N-acetyl AMPA or the AMPA during detection;
c) extracting from said test sample a composition comprising at least one of said AMPA, said N-acetylglyphosate, said N-acetyl AMPA or the glyphosate by an aqueous-organic extraction; and,
d) detecting at least one of said AMPA, said N-acetylglyphosate, said N-acetyl AMPA or said glyphosate,
wherein said detecting comprises
(a) chromatographically separating at least one of the AMPA, the N-acetylglyphosate, the N-acetyl AMPA or the glyphosate from the composition by a high performance liquid chromatography (HPLC) column; and,
(b) analyzing by a tandem mass spectrometer at least one of the chromatographically separated glyphosate, N-acetylglyphosate, N-acetyl AMPA or AMPA to determine the presence or amount of at least one of the glyphosate, the N-acetylglyphosate, the N-acetyl AMPA or the AMPA in the test sample.
56. The method of claim 55 , wherein said aqueous-organic extraction comprises a methylene chloride and phosphoric acid partition.
57. The method of claim 55 , wherein the mass spectrometer is operated in positive ion mode.
58. The method of claim 55 , wherein said test sample is from an animal, a plant, or an environmental sample.
59. The method of claim 58 , wherein said plant is a dicot or a monocot.
60. A method for determining the presence or amount of at least one of glyphosate, N-acetylglyphosate, N-acetylaminomethylphosphonic acid (N-acetyl AMPA) or aminomethyl phosphonic acid (AMPA) in an oil test sample comprising
a) providing the test sample suspected of containing at least one of the glyphosate, the N-acetylglyphosate, the N-acetyl AMPA or the AMPA;
b) adding a sufficient concentration of phosphoric acid to said test sample of (a) wherein said sufficient concentration allows for an improved response and linearity of the glyphosate, the N-acetylglyphosate, the N-acetyl AMPA or the AMPA during detection;
c) extracting a composition from said test sample comprising at least one of said AMPA, said N-acetylglyphosate, the N-acetyl AMPA or the glyphosate by an aqueous-organic partition;
d) chromatographically separating at least one of said AMPA, said N-acetylglyphosate, the N-acetyl AMPA or said glyphosate from the composition of step (c) using a phenyl-hexyl high performance liquid chromatography (HPLC) analytical column; and,
e) analyzing the chromatographically separated sample of step (d) to determine the presence or amount of at least one of the AMPA, the glyphosate, the N-acetyl AMPA, or the N-acetylglyphosate in the test sample by a tandem quadrupole mass spectrometer operated in positive ion mode.