Indian Patents
Title of Invention

METHODS AND COMPOSITIONS FOR DETECTING GLYPHOSATE AND METABOLITES THEREOF
Abstract 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-acetyl glyphosate, 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.
Full Text METHODS AND COMPOSITIONS FOR DETECTING GLYPHOSATE AND
METABOLITES THEREOF
FIELD OF THE INVENTION
The invention relates to the detection of the presence or the amount of
glyphosate and/or its metabolites in a variety of test matrices.
BACKGROUND OF THE INVENTION
Glyphosate (DPX-B2856) is an active ingredient in non-selective herbicides
which are widely used on crops that have been genetically modified for resistance to
the herbicidal activity. Glyphosate inhibits the EPSPS (enolpyruvylshikimate-3-
phosphate synthase) enzyme required for the biosynthesis of aromatic amino acids in
plants. Crop tolerance to glyphosate herbicides has been achieved by genetic
modification of the plant with microbially derived EPSPS (enolpyruvylshikimate-3-
phosphate synthase), GOX (glyphosate oxidoreductase), and glyat (glyphosate N-
acetyltransferase) enzyme variants in plants. EPSPS variants disrupt the activity of
glyphosate on the EPSPS enzyme, leaving intact glyphosate in the plant. GOX
variants detoxify glyphosate by decarboxylation to AMPA. Glyat variants provide a
new mode of glyphosate detoxification through enzymatic acetylation of the
glyphosate molecule to form the non-phytotoxic metabolite N-acetylglyphosate. See,
for example, Castle et al. (2004) Science 304:1151-1154. Regulatory monitoring for
the presence and amount of glyphosate and glyphosate metabolites in a variety of test
matrices including various animal and plant matrices are needed.
BRIEF SUMMARY OF THE INVENTION
The present invention provides methods and compositions which allow for the
determination of the presence or the amount of glyphosate and its metabolites,
derivatives or degradate residues in a variety of test matrices. The relevant glyphosate
metabolites degradates included in the analytical method are N-acetylglyphosate, N-
acetylaminomethylphosphonic acid (N-acetyl AMP A) and aminomethyl phosphonic
acid (AMPA). N-acetylglyphosate and N-acetyl AMPA are novel glyphosate
metabolites found in genetically modified plants containing a glyat (glyphosate N-
acetyltransferase) gene. In one method, determining the presence or amounts 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
a composition comprising the N-acetylglyphosate and/or N-acetyl AMPA from the
test sample; and, detecting the N-acetylglyphosate and/or N-acetyl AMPA in the
composition.
In other methods, the presence or amount of at least one of glyphosate, N-
acetylglyphosate, N-acetyl AMPA and/or AMPA or a metabolite thereof in a test
sample is determined. This 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 a composition comprising at
least one of the glyphosate, N-acetylglyphosate, N-acetyl AMPA or AMPA, or a
metabolite thereof; and, detecting at least one of the glyphosate, N-acetylglyphosate,
N-acetyl AMPA or AMPA or a metabolite thereof. In specific embodiments, the
detection of the glyphosate, N-acetylglyphosate, N-acetyl AMPA or AMPA occurs
without derivatization of the glyphosate, N-acetylglyphosate, N-acetyl AMPA or
AMPA.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides representative glyphosate mass spectra.
Figure 2 provides representative N-acetylglyphosate mass spectra.
Figure 3 provides representative AMPA mass spectra.
Figure 4 provides representative N-acetyl AMPA mass spectra.
Figure 5 provides representative calibration curves.
Figure 6A and B provides representative calibration standard
chromatograms.
Figure 7 provides representative 5 ng/ml glyphosate and AMPA
chromatograms with stable isotope internal standard. Glyphosate and
glyphosate 1,2-13C 15N stable isotope are equivalent at 5 ng/ml and showed
consistent peak area responses (2074, 1885). Since AMPA is 5 ng/ml in
glyphosate equivalents and AMPA 13C 15N is 5 ng/ml, the isotope peak
response was consistent with the molar ratio of AMPA/glyphosate (111/169 =
0.66 ˜ 628/902).
Figure 8 provides representative corn forage chromatograms.
Figure 9 provides representative corn grain chromatograms.
Figure 10 provides representative corn stover chromatograms.
Figure 11 provides representative corn oil chromatograms.
Figure 12 provides representative corn flour chromatograms.
Figure 13 provides representative corn grits chromatograms.
Figure 14 provides representative corn starch chromatograms.
Figure 15 provides representative corn meal chromatograms.
Figure 16 provides representative soybean forage chromatograms.
Figure 17 provides representative soybean seed chromatograms.
Figure 18 provides representative soybean hay chromatograms.
Figure 19 provides representative soybean oil chromatograms.
Figure 20 provides representative soybean meal chromatograms.
Figure 21 provides representative soybean hulls chromatograms.
Figures 22A and B provide representative plum chromatograms.
Figures 23A and B provide representative lime chromatograms.
Figure 24 provides the acid modifier effects on glyphosate LC/MS/MS
response.
Figure 25 provides an LC/MS/MS analysis showing the isotopic purity
evaluation of glyphosate 1,2-13C215N and AMPA 13C15N standards.
Figure 26 provides signal to noise and LOD determinations for soybean meal
LOQ fortification samples.
Figure 27 provides a representative API 4000 LC/MS/MS Chromatogram for
glyphosate 0.050 ppm corn forage fortification sample.
Figure 28 provides a representative API 4000 LC/MS/MS Chromatogram for
N-acetylglyphosate 0.050 ppm corn forage fortification sample.
Figure 29 provides a representative API 4000 LC/MS/MS Chromatogram for
N-acetyl AMPA 0.05 ppm corn forage fortification sample.
Figure 30 provides a representative API 4000 LC/MS/MS Chromatogram for
AMPA 0.05 ppm corn forage fortification sample.
Figure 31 provides a calibration curve for glyphosate, grapes, Trial 1,
Set 2.
Figure 32 provides a calibration curve for N-Acetylglyphosate, grapes,
Trial 1, Set 2.
Figure 33 provides a calibration curve for AMPA, grapes, Trial 1, Set 2.
Figure 34 provides a calibration curve for N-Acetyl AMPA, grapes,
Trial 1, Set2.
Figure 35 provides a representative chromatogram for reagent blank,
glyphosate, grapes, Trial 1, Set 2.
Figure 36 provides a representative chromatogram for 0.500 ng/ml
calibration standard, glyphosate, grapes, Trial 1, Set 2.
Figure 37 provides a representative chromatogram for 100 ng/ml
calibration standard, glyphosate, grapes, Trial 1, Set 2.
Figure 38 provides a representative chromatogram for P1763S02-002,
untreated control sample, glyphosate, grapes, Trial 1, Set 2.
Figure 39 provides representative chromatogram for P1763S02-004,
sample fortified at LOQ, glyphosate, grapes, Trial 1, Set 2.
Figure 40 provides representative chromatogram for P1763S02-009,
sample fortified at 4XLOQ, glyphosate, grapes, Trial 1, Set 2.
Figure 41 provides representative chromatogram for reagent blank,
N-acetylglyphosate, grapes, Trial 1, Set 2.
Figure 42 provides representative chromatogram for 0.500 ng/ml
calibration standard, N-acetylglyphosate, grapes, Trial 1, Set 2.
Figure 43 provides representative chromatogram for 100 ng/ml
calibration standard, N-acetylglyphosate, grapes, Trial 1, Set 2.
Figure 44 provides representative chromatogram for P1763S02-002,
untreated control sample, N-acetylglyphosate, grapes, Trial 1, Set 2.
Figure 45 provides representative chromatogram for P1763S02-004,
sample fortified at LOQ, N-acetylglyphosate, grapes, Trial 1, Set 2.
Figure 46 provides representative chromatogram for P1763S02-009,
sample fortified at 4xLOQ, N-acetylglyphosate, grapes, Trial 1, Set 2.
Figure 47 provides representative chromatogram for reagent blank,
AMPA, grapes, Trial 1, Set 2.
Figure 48 provides representative chromatogram for 0.500 ng/ml
calibration standard, AMPA, grapes, Trial 1, Set 2.
Figure 49 provides representative chromatogram for 100 ng/ml
calibration standard, AMPA, grapes, Trial 1, Set 2.
Figure 50 provides representative chromatogram for P1763S02-002,
untreated control sample, AMPA, grapes, Trial 1, Set 2.
Figure 51 provides representative chromatogram for P1763S02-004,
sample fortified at LOQ, AMPA, grapes, Trial 1, Set 2.
Figure 52 provides representative chromatogram for P1763S02-009,
sample fortified at 4xLOQ, AMPA, grapes, Trial 1, Set 2.
Figure 53 provides representative chromatogram for reagent blank,
N-Acetyl AMPA, grapes, Trial 1, Set 2.
Figure 54 provides representative chromatogram for 0.500 ng/ml
calibration standard, N-Acetyl AMPA, grapes, Trial 1, Set 2.
Figure 55 provides representative chromatogram for 100 ng/ml
calibration standard, N-Acetyl AMPA, grapes, Trial 1, Set 2.
Figure 56 provides representative chromatogram for P1763S02-002,
untreated control sample, N-Acetyl AMPA, grapes, Trial 1, Set 2.
Figure 57 provides representative chromatogram for P1763S02-004,
sample fortified at LOQ, N-Acetyl AMPA, grapes, Trial 1, Set 2.
Figure 58 provides representative chromatogram for P1763S02-009,
sample frortified at 4xLOQ, N-Acetyl AMPA, grapes, Trial 1, Set 2.
Figure 59 provides a representative N-acetylglyphosate mass spectra.
Figure 60 provides a representative glyphosate mass spectra.
Figure 61 providies a representative AMPA mass spectra.
Figure 62 provides a representative N-acetyl AMPA mass spectra.
Figure 63 provides a representative calibration curves.
Figure 64 provides representative calibration standard chromatograms.
Figure 65 provides representative milk chromatograms.
Figure 66 provides representative egg sample chromatograms.
Figure 67A, B and C provides representative chicken muscle sample
chromatograms.
Figure 68 provides representative chicken liver sample chromatograms.
Figure 69 provides representative chicken fat sample chromatograms.
Figure 70 provides representative cow muscle sample chromatograms.
Figure 71 provides representative cow liver sample chromatograms.
Figure 72 provides representative cow kidney sample chromatograms.
Figure 73 provides representative cow fat sample chromatograms.
Figure 74 shows the isotopic purity evaluation of glyphosate 1,2-13C2 15N
and AMPA 13C 15N Standards. Glyphosate impurity (70 area) observed for
glyphosate 1.2-13C2 15N standard represents 70x100/200552 = 0.03% impurity.
AMPA impurity (624 area) observed for AMPA 13C 15N standard represents
624x100/42461 = 1.5% impurity.
Figure 75 provides a signal-to-noise and LOD determination for a milk matrix.
Figure 76 provides a signal-to-noise and LOD determination for an egg
matrix.
Figure 77 provides a signal-to-noise and LOD determination for a liver matrix.
Figure 78 provides a signal-to-noise and LOD determination for a kidney
matrix.
Figure 79 provides a signal-to-noise and LOD determination for a fat matrix.
Figure 80 provides a signal-to-noise and LOD determination for a muscle
matrix.
DETAILED DESCRIPTION OF THE INVENTION
The presently disclosed subject matter now will be described more fully
hereinafter with reference to the accompanying drawings, in which some, but not all
embodiments of the presently disclosed subject matter are shown. Indeed, the
presently disclosed subject matter can be embodied in many different forms and
should not be construed as limited to the embodiments set forth herein; rather, these
embodiments are provided so that this disclosure will satisfy applicable legal
requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the presently disclosed subject
matter set forth herein will come to mind to one skilled in the art to which the
presently disclosed subject matter pertains having the benefit of the teachings
presented in the foregoing descriptions and the associated drawings. Therefore, it is
to be understood that the presently disclosed subject matter is not to be limited to the
specific embodiments disclosed and that modifications and other embodiments are
intended to be included within the scope of the appended claims. Although specific
terms are employed herein, they are used in a generic and descriptive sense only and
not for purposes of limitation.
I. Overview
The present invention provides methods and compositions which allow for the
determination of the presence or the amount of glyphosate and its metabolites and/or
degradate residues in a variety of test matrices. Analytical methods exist for the
analysis of glyphosate and AMPA in plants. See, for example, Cowell et al. (1986) J.
Agric. Food Chem. 34:955-960; Alferness et al. (April 3, 1993) "Touchdown:
Determination of Glyphosate and Aminomefhylphosphonic Acid in Corn Grain, Corn
Forage, and Corn Fodder by Gas Chromatography and Mass-Selective Detection."
Zeneca Ag Products Analytical Method RR 92-042B, available at U.S. EPA
Pesticides: Analytical Methods & Procedures website
(www.epa.gov/oppbeadl/methods/raml2b.htm); and, Method No. 405 in Manual of
Pesticide Residue Analysis Volume I and II available from BfR Federal Institure for
Risk Assessment, official analytical methods for residues of plant protection products
and pesticides (L 00.00 16) (www.bfr.bund.de/cd/1652). Such methods analyze
glyphosate and AMPA only and further require analyte derivatization for detection.
The methods and compositions of the present invention allow for the detection and/or
quantitation of the new glyat metabolites, N-acetylglyphosate and N-acetyl AMPA,
and further provide a combined, highly specific residue method for the quantitative
and confirmatory analysis of glyphosate and relevant metabolites using LC/MS/MS
technology without analyte derivatization. Further provided are novel methods for
purifying glyphosate from various metabolites, such as from AMPA. Moreover, the
methods and compositions of the instant invention further improve the overall
response and linearity of glyphosate detection and/or its metabolites by employing a
weak ion-pairing reagent, such as phosphoric acid, to prepare the final samples and
standard solutions employed in the LC/MS/MS technology. In addition, the methods
can employ extraction methods using an aqueous methanol/dilute acid solution that
allow for an acceptable analyte extraction efficiency and can further employ the use
of mass spectroscopy in positive ion mode. And finally, commercially available
stable isotopes for glyphosate and AMPA can be used in the methods and
compositions of the present invention as internal standards to normalize LC/MS/MS
response for matrix effects. Thus, the novel methods and compositions of the present
invention offer robust quantitative analysis of glyphosate, N-acetylglyphosate, N-
acetyl AMPA and AMPA without matrix effects.
As used herein, an analyte of interest comprises at least one of glyphosate,
N-acetylglyphosate, AMPA, and/or N-acetyl AMPA. As used herein the terms
"glyphosate" or "N-(phosphonomethyl) glycine" refers to a compound having formula
(I) as set forth below.
The term "N-acetylglyphosate" or "N-Acetyl-N-(phosphonomethyl) glycine" refers to
a compound having formula (II) as set forth below.

The term "AMPA" or "aminomethyl phosphonic acid" or "1-aminomethylphosphonic
acid" refers to a compound having formula (III) as set forth below.

The term "N-acetyl AMPA" or "(Acetamidomethyl)phosphonic acid" or
"N-Acetylaminomethylphosphonic acid" refers to a compound having formula (IV) as
set forth below.

Thus, the various methods and compositions for detecting the presence or quantity of
glyphosate, N-acetylglyphosate, AMPA, and/or N-acetyl AMPA in a test sample are
provided. The methods and compositions of the invention find use in monitoring the
level of glyphosate, N-acetylglyphosate, N-acetyl AMPA, and/or AMPA in a variety
of test samples including crops, crop raw agricultural commodities, and crop process
fractions.
II. Determining the Presence or Amount of at least one of Glyphosate, N-
acetylglyphosate, N-acetyl AMPA, or AMPA in a Test Sample
Methods and compositions are provided for determining the presence or
amount of N-acetylglyphosate and/or N-acetyl AMPA in a test sample. The method
comprises providing a test sample suspected of containing N-acetylglyphosate and/or
N-acetyl AMPA, extracting a composition comprising 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.
Methods and compositions are further provided for determining the presence
or amount of at least one of glyphosate, N-acetylglyphosate, N-acetyl AMPA and/or
AMPA or a metabolite thereof in a test sample. The methods comprise providing the
test sample suspected of containing at least one of glyphosate, N-acetylglyphosate, N-
acetyl AMPA and/or AMPA or a metabolite thereof; extracting from the test sample a
composition comprising at least one of the glyphosate, N-acetylglyphosate, N-acetyl
AMPA or AMPA or a metabolite thereof; and, detecting at least one of the
glyphosate, N-acetylglyphosate, N-acetyl AMPA or AMPA or a metabolite thereof in
the extract, where in specific embodiments, the detection of the glyphosate, the N-
acetylglyphosate, the N-acetyl AMPA or the AMPA occurs without derivatization of
the glyphosate, N-acetylglyphosate, N-acetyl AMPA and/or AMPA. In each matrix
tested, such methods have a target limit of quantitation (LOQ) of the analytes of
interest of 0.050 mg/kg (ppm) or 0.025 mg/kg (depending on the sample matrix) and
are validated at 0.050 mg/kg and 0.50 mg/kg.
As used herein, a "test sample" can include any sample that can contain one or
more of the target analytes of interest. In some embodiments, the test sample
comprises a biological sample or an environmental sample. Thus, test samples can be
from an animal (such as a mammal, including, but not limited to an agricultural
animal, a domestic animal, a dog, a cat, a horse, a human, and the like). In other
embodiments, the test sample is not a bacterium, such as E. coli. In other
embodiments, the test sample is from a plant.
As used herein, the term "plant" includes plant cells, plant protoplasts, plant
cell tissue cultures from which plants can be regenerated, plant calli, plant clumps,
explants, plant tissues, forage, stover, hay, hulls, grits, and plant cells that are intact in
plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers,
branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like.
Grain is intended to mean the mature seed produced by commercial growers for
purposes other than growing or reproducing the species. Accordingly, a sample from
a plant can be derived from any one part or multiple parts of the plant.
Test samples derived from the plant can further comprise raw agricultural
commodities (RAC) or can comprise a sample obtained during the manufacture of a
plant or part thereof. Exemplary corn RACs include, for example, forage, grain and
stover, while exemplary soybean RACs include, for example, forage, seed and hulls.
In other embodiments, a test sample comprises a crop process fraction including solid
process fractions or meal process fractions. Additional test samples can include flour,
starch, grits, oils, refined oils, or meal obtained from the plant. Exemplary corn
process fractions include flour, grits, meal and starch. Exemplary soy process
fractions include meal and hulls. In one embodiment, the test sample is derived from
a plant expressing a glyphosate acetyltransferase (GLYAT) polypeptide. See, for
example, WO 02/36782, WO 2005/012515, U.S. Application Publication
No. 2004/0082770, and U.S. Application No. 11/507,751, all of which are herein
incorporated by reference.
In specific embodiments, the test sample comprises a solid matrix fraction
from a plant. As used herein, the term "solid matrix sample" refers to any matrix in a
solid state, such as, but not limited to, flour, grits, meal, starch, tissue, etc.
Test samples can be obtained from any plant species, including, but not limited
to, monocots and dicots or watery crops or acid crops. Examples of plant species of
interest include, but are not limited to, corn (Zea mays, also referred to herein as
"maize"), Brassica spp. (e.g., B. napus, B. rapa, B.juncea), particularly those Brassica
species useful as sources of seed oil (also referred to as "canola"), flax (Linum spp.),
alfalfa (Medicago sativd), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum
bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet
(Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)),
sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum
aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solatium
tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium
hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea
spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.),
cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado
(Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera
indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium
occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar
beets (Beta vulgaris), sugarcane {Saccharum spp.), oats, barley, vegetables, fruits,
ornamentals (flowers), sugar cane, conifers, Arabidopsis.
Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca
sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas
(Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus),
cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea
(Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus
rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias
(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum.
Any tree can also be employed. Conifers that may be employed in practicing the
present invention include, for example, pines such as loblolly pine (Pinus taeda), slash
pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta),
and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western
hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia
sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies
balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-
cedar (Chamaecyparis nootkatensis). Hardwood trees can also be employed including
ash, aspen, beech, basswood, birch, black cherry, black walnut, buckeye, American
chestnut, cottonwood, dogwood, elm, hackberry, hickory, holly, locust, magnolia, maple,
oak, poplar, red alder, redbud, royal paulownia, sassafras, sweetgum, sycamore, tupelo,
willow, yellow-poplar.
In specific embodiments, test samples are from crop plants (for example, corn
(also referred to as "maize"), alfalfa, sunflower, Brassica, soybean, cotton, safflower,
peanut, sorghum, wheat, millet, tobacco, etc.).
Other plants of interest that test samples can be derived from include grain
plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of
interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-
seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa,
palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar,
locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava
bean, lentils, chickpea, etc.
Test samples can also be derived from other plants of interest including
Turfgrasses such as, for example, turfgrasses from the genus Poa, Agrostis, Festuca,
Lolium, and Zoysia. Additional turfgrasses can come from the subfamily
Panicoideae. Turfgrasses can further include, but are not limited to, Blue gramma
(Bouteloua gracilis (H.B.K.) Lag. Ex Griffiths); Buffalograss (Buchloe dactyloids
(Nutt.) Engelm.); Slender creeping red fescue ( Festuca rubra ssp. Litoralis); Red
fescue (Festuca rubra); Colonial bentgrass (Agrostis tenuis Sibth.); Creeping
bentgrass (Agrostis palustris Huds.); Fairway wheatgrass (Agropyron cristatum (L.)
Gaertn.); Hard fescue (Festuca longifolia Thuill.); Kentucky bluegrass (Poa pratensis
L.); Perennial ryegrass (Loliumperenne L.); Rough bluegrass (Poa trivialis L.);
Sideoats grama (Bouteloua curtipendula Michx. Torr.); Smooth bromegrass (Bromus
inermis Leyss.); Tall fescue (Festuca arundinacea Schreb.); Annual bluegrass (Poa
annua L.); Annual ryegrass (Lolium multiflorum Lam.); Redtop (Agrostis alba L.);
Japanese lawn grass (Zoysia japonica); bermudagrass (Cynodon dactylon; Cynodon
spp. L.C. Rich; Cynodon transvaalensis); Seashore paspalum (Paspalum vaginatum
Swartz); Zoysiagrass (Zoysia spp. Willd; Zoysia japonica and Z. matrella var.
matrelld); Bahiagrass (Paspalum notatum Flugge); Carpetgrass (Axonopus affinis
Chase); Centipedegrass (Eremochloa ophiuroides Munro Hack.); Kikuyugrass
(Pennisetum clandesinum Hochst Ex Chiov); Browntop bent ( Agrostis tenuis also
known as A. capillaris ); Velvet bent (Agrostis canina ); Perennial ryegrass (Lolium
perenne); and, St. Augustinegrass (Stenotaphrum secundatum Walt. Kuntze).
Additional grasses of interest include switchgrass (Panicum virgatum).
In other embodiments, the test sample can be from an animal. Any animal can
be used including, for example, agricultural animals, such as, cops, pigs, poultry
(chickens, ducks, etc.) pigs, sheep, etc. Such samples can be derived from any part of
the animal inlcuding, but not limited to, milk (whole milk, skim milk, cream), eggs
(whole eggs, egg yolks, egg whites), or any meat commodity, such as, muscle, kidney,
liver, fat, etc.
III. Extracting Glyphosate, N-acetylglyphosate, N-acetyl AMP A, AMP A, and Other
Metabolites From the Test Sample
By "extracting" is intended any method that allows for the removal of the
analyte of interest from the sample matrix or a sample derived therefrom. As used
herein, the term "extraction" or derivations thereof does not necessarily refer to the
removal of all materials or constituents other than the analyte(s) of interest from a
sample matrix or a sample derived therefrom. Instead, in some embodiments, the
term "extraction" refers to a procedure that enriches the amount of one or more
analytes of interest relative to one or more other components present in the sample
matrix or in a sample derived therefrom. In some embodiments, an "extraction"
procedure can be used to remove one or more components of a sample that could
interfere with the detection of the analyte, for example, one or more components that
could interfere with detection of an analyte ion by mass spectrometry. In other
embodiments, the extraction procedure is used to remove the analyte of interest from
the test sample matrix, and in still other embodiments, the extraction procedure is
used to purify a first analyte of interest away from a second analyte of interest in a
sample or to purify an analyte of interest away from an interfering substance.
Various extractions can be employed to extract or purify at least one of the
analytes of interest (i.e., glyphosate, AMP A, N-acetyl AMPA and/or N-
acetylglyphosate) from the sample. Various dilute acids can be used including, for
example, solutions comprising dilute formic acid solutions, dilute hydrochloric acid
solutions, or dilute phosphoric acid solutions. In particular embodiments, the
extraction employs, for example, solutions comprising about 96% aqueous 0.1%
formic acid/4% methanol or about 96% aqueous 0.025N hydrochloric acid/ 4%
methanol or about 0.1N HCL in 96% water/4% methanol or about 0.02M phosphoric
acid. In still other embodiments, the extraction solution can comprise a dilute formic
acid solution comprising about 0.01% to about 10% formic acid, about 0.01% to
about 1% formic acid, about 0.1% to about 1% formic acid, or about 0.1% to about
5% formic acid. In still other embodiments, the extraction solution can comprise a
dilute hydrochloric acid solution comprising about 0.0025N to about 0.25N
hydrochloric acid, about 0.025N to about 0.25N hydrochloric acid, about 0.0025N to
about 0.025N hydrochloric acid. In further embodiments, the dilute phosphoric acid
solution can comprise about 0.002M to about 0.2M phosphoric acid, about 0.002M to
about 0.02M phosphoric acid, or about 2M to about 0.02M phosphoric acid. The
extraction solution employed can vary depending on the test sample. For example,
dilute hydrochloric acid can be used instead of formic acid to increase the acidity for
more efficient extraction of -N-acetylglyphosate, glyphosate, AMPA and/or N-acetyl
AMPA from various matrixes, such as corn flour and meal process fractions.
Alternatively, dilute phosphoric acid can be used for an efficient extraction of
N-acetylglyphosate, glyphosate N-acetyl AMPA and/or AMPA from an oil test
sample.
In specific embodiments, the extraction is performed with an aqueous dilute
acid/methanol solution. In further embodiments, the extraction method employed will
be sufficient to allow for an analyte extraction efficiency from the matrix of at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In specific embodiments, the
extraction efficiencies of the various analytes of interest are established through
metabolism studies and not through surface application studies.
Multiple liquid-liquid extractions can be performed to extract the analyte(s) of
interest from the test sample, including 1, 2, 3, 4, 5, or more. As outlined in further
detail in the experimental section, it is further recognized that the extraction volume
employed can vary depending on the moisture content of the matrix. For example,
test samples with lower moisture content (i.e., stover, hay, hull, etc) may need larger
extraction solution volumes and an increased number of extractions.
Following the dilute acidic extraction, an additional extraction step can be
employed to further purify the analytes of interest from various contaminates
remaining in the sample. For example, an aqueous-organic extraction step can be
performed, such as a dilute acid/methylene chloride partition. In such an extraction,
the analytes of interest remain in the aqueous phase which can be subsequently
applied to an extraction column. In specific embodiments, the dilute acid employed
with the methylene chloride extraction comprises a dilute formic acid solution, a
dilute hydrochloric acid solution, or a dilute phosphoric acid solution, as described
above.
One or more of the analytes of interest can further be extracted or purified
from other components of the test sample or from one another by employing a solid
phase extraction (SPE) column. As used herein, an "extraction column" is used to
extract retained materials from non-retained materials and thus allows one to obtain a
"purified" sample for further purification or analysis. Multiple extraction columns
can also be used, including, 1, 2, 3, 4, 5 or more. In specific embodiments, the
extracting or purifying step includes: (a) disposing the sample onto an extraction
column; (b) washing the extraction column under conditions whereby the analyte(s)
of interest is retained on the extraction column; and, (c) eluting the retained analyte(s)
from the extraction column. In some embodiments, one or more of the extraction
columns employed is a hydrophobic silica-based bonded phase extraction column
(i.e., a C-18 column), a cation exchange extraction column (i.e., an m-divinylbenzene
& N-vinylpyrrolidone copolymer, with sulfonic acid substituents) or an anion
exchange extraction column (i.e., m-divinylbenzene & N-vinylpyrrolidone copolymer,
with quaternary amine substituents) or a combination thereof. In one embodiment,
one or more of the analytes of interest are extracted from the sample matrix
employing a liquid-liquid extraction, an extraction column, or any combination
thereof.
Depending on the test sample employed and the analyte of interest, different
extraction columns can be used to extract the analyte of interest (i.e., glyphosate,
AMPA, N-acetyl AMPA and/or N-acetylglyphosate) from the test sample matrix. For
example, for the analysis of solid matrix sample extracts, following an aqueous-
organic liquid-liquid extraction, as described above, a hydrophobic silica-based
bonded phase extraction column can be used for the purification of glyphosate,
AMPA, N-acetyl AMPA and N-acetylglyphosate. In specific embodiments, the
hydrophobic silica-based bonded phase extraction column comprises a C18 extraction
column. Additional extraction columns can be employed following the hydrophobic
silica-based extraction column to further purify the analyte of interest.
For some sample matrices, the use of a solid phase extraction step can occur
with the dilute acid/methanol extraction step. For example, with a meat commodity
matrix, a solid phase extraction sorbent (such as C18) can be added to the
homogenized tissue sample. The homogenized tissue is mixed with the solid phase
extraction sorbent and a dilute acid/methanol solution is added. Steps can be taken to
elute and collect the analytes bound by the solid phase extraction sorbent.
In still other embodiments, for milk and egg commodities, the test samples are
mixed with aqueous 0.1% formic acid/methanol (96/4, v/v). Hexane is then added
followed by centrifugation. A methylene chloride extraction is then performed on the
aqueous layer. Two additional 0.1% formic acid/methanol (96/4, v/v) extractions are
then performed.
In specific embodiments, prior to chromatographically separating the analyte
of interest from the extracted test sample, a sufficient concentration of a weak-ion
pairing reagent is added to the extract. A sufficient concentration of a weak-ion
pairing reagent is a concentration that allows for an improved response and linearity
of glyphosate, N-acetylglyphosate, N-acetyl AMP A and/or AMP A. In specific
embodiments, the weak-ion pairing reagent is phosphoric acid. In specific
embodiments, final extracts are prepared in aqueous 0.02M phosphoric acid and
filtered prior to LC/MS/MS analysis. Glyphosate and AMPA stable isotopes can be
added to final extract solutions as internal standards to normalize for matrix effects.
1. Extracting AMPA from a Solid Matrix or an Animal Commodity
In specific embodiments, an aliquot of the eluate from the hydrophobic silica-
based extraction column is processed for the analysis of AMPA by disposing the
hydrophobic silica-based extraction column eluate onto a cation exchange extraction
column. In specific embodiments, the cation exchange extraction column has a
surface functionality comprising a m-divinylbenzene & Af-vinylpyrrolidone
copolymer with sulfonic acid substituents. Non-limiting examples of cation exchange
extraction columns that can be used include an MCX SPE column (Waters Oasis). As
outlined in further detail elsewhere herein, various methods can be used to dispose the
eluate of the hydrophobic silica-based extraction column onto the cation exchange
extraction column.
Accordingly, methods to extract or purify AMPA from a solid sample matrix
can comprise: (a) extracting a composition comprising the AMPA from the test
sample by liquid-liquid extraction; (b) disposing the composition comprising the
AMPA of step (a) onto a hydrophobic silica-based bonded phase extraction column;
(c) obtaining the elute comprising the AMPA from the extraction column of step (b);
(d) disposing the eluate of step (c) onto a cation exchange column; (e) washing the
extraction column under conditions whereby the AMPA is retained on the extraction
column; and, (f) eluting the retained AMPA from the cation exchange extraction
column. In specific embodiments, the extraction of the AMPA from the solid matrix
sample comprises a dilute acid extraction followed by a methylene chloride partition.
In one non-limiting embodiment, the test sample extraction is performed as
follows. AMPA is extracted from a plant solid matrix into dilute aqueous acid (0.1%
formic acid or 0.025N hydrochloric acid)/methanol (96/4) using a probe homogenizer.
Multiple extractions (1, 2, 3, 4 or more ) are made for quantitative recovery of the
analyte of interest. An aliquot of the filtered aqueous fraction is partitioned with
methylene chloride and the aqueous fraction is recovered and filtered to remove
particulates. An aliquot of the extract is then filtered through a C18 solid phase
extraction (SPE) cartridge. An aliquot of the eluate collected from the C18 SPE
cartridge is diluted and applied to a cation exchange Oasis™ MCX SPE cartridge,
diluted, and filtered.
In another non-limiting embodiment, the test sample comprises a meat tissue
commodity, and the extraction is performed as follows. AMPA is extracted from a
meat tissue commodity by (a) homogenizing the tissue; (b) adding a solid phase
extraction sorbent, such as Cis, to the homogenized tissue; (c) adding 0.1N HCL in
96% water/4% methanol; (d) centrifuging the sample and collecting the C]8 pellet; (e)
eluting the analyte from the solid phase extraction sorbent with water. An aliquiot of
the eluate is then applied to a cation Oasis™ MCX SPE cartridge.
In another non-limiting embodiment, the test sample comprises a milk or egg
commodity and the extraction is performed as follows. An aqueous 0.1% formic
acid/methanol (96/4, v/v) solution is added to the sample. A hexane extraction is
performed. A methylene chloride extraction is performed on the aqueous layer.
Additional formic acid/methanol extractions (at least 1, 2, 3 or more) are performed
on the aqueous layer. An aliquoit of the eluate is then applied to a cation Oasis™
MCS SPE cartridge.
ii. Extracting Glyphosate, N-acetyl AMPA and/or N-acetylglyphosate from a
Solid Matrix or an Animal Commodity
In other embodiments, an aliquot of the eluate from the hydrophobic silica-
based extraction column is processed for the analysis of glyphosate, N-acetyl AMPA
and/or N-acetylglyphosate from a solid matrix. In specific embodiments, an aliquot of
the hydrophobic silica-based extraction eluate is disposed onto an anion exchange
extraction column. The anion exchange extraction column can comprise a surface
functionality comprising a m-divinylbenzene & N-vinylpyrrolidone copolymer with
quaternary amine substituents. Non-limiting examples of such anion exchange
extraction columns include, a MAX SPE column. As outlined in further detail
elsewhere herein, various methods can be used to dispose the eluate of the
hydrophobic sililca-based extraction column onto the anion exchange extraction
column. Briefly, an m-divinylbenzene & N-vinylpyrrolidone copolymer with
quaternary amine substituents extraction column can be employed following an
extract (adjusted to basic pH) in methanol for corn matrices, soybean forage, and hay
matrices. In other methods, an m-divinylbenzene & N-vinylpyrrolidone copolymer
with quaternary amine substituents extraction column following an extract dilution in
water procedure can be used for the analysis of soybean seed, meal, and hull matrices.
Accordingly, methods to extract or purify glyphosate, N-acetyl AMPA and/or
N-acetylglyphosate from a plant solid sample matrix can comprise: (a) extracting a
composition comprising the glyphosate, N-acetyl AMPA and/or N-acetylglyphosate
from the test sample by liquid-liquid extraction; (b) disposing the composition of step
(a) onto a hydrophobic silica-based bonded phase extraction column; (c) obtaining the
elute comprising the glyphosate, N-acetyl AMPA and/or N-acetylglyphosate from the
extraction column of step (b); (d) disposing the eluate of step (c) onto an anion
exchange column; (e) washing the extraction column under conditions whereby the
glyphosate, N-acetyl AMPA and/or N-acetylglyphosate are retained on the extraction
column; and, (f) eluting the retained glyphosate, N-acetyl AMPA and/or N-
acetylglyphosate from the extraction column. In specific embodiments, the aqueous
extraction of the glyphosate, N-acetyl AMPA and/or N-acetylglyphosate from the
solid matrix sample is followed by a methylene chloride partition.
In one non-limiting embodiment, the test sample extraction is performed as
follows. Glyphosate, N-acetyl AMPA and/or N-acetylglyphosate are extracted from a
plant solid matrix or a milk or egg commodity into dilute aqueous acid (0.1% formic
acid or 0.025N hydrochloric acid)/methanol (96/4) using a probe homogenizer.
Multiple extractions (1, 2, 3, 4 or more) are made for quantitative recovery of
analytes. When employing a plant solid matrix sample extract, an aliquot of extract
can be partitioned with methylene chloride and the aqueous fraction is recovered and
filtered to remove particulates. When employing an egg or milk commodity, a hexane
extraction step can be performed before the methylene chloride extraction. An aliquot
of the extract is then filtered through a C18 solid phase extraction (SPE) cartridge. An
aliquot of the eluate collected from the C18 SPE is diluted and applied to an anion
exchange Oasis™ MAX SPE cartridge. The analytes are eluted from the MAX
cartridge with 1% trifluoroacetic acid in methanol/water (9/1) solution following
several solution rinses. The MAX eluate is evaporated to dryness and redissolved in
aqueous 0.02M aqueous phosphoric acid, filtered, and analyzed for glyphosate, N-
acetyl AMPA and/or N-acetylglyphosate. For the analysis of soybean samples (i.e.,
seed and meal) following partitioning, extract samples can be heated in a steam bath
for approximately 15 minutes to precipitate additional material in the extract prior to
particulate filtration.
In another non-limiting embodiment, the test sample is a meat commodity, and
the sample extraction is performed as follows. Glyphosate, N-acetyl AMPA, and/or
N-acetylglyphosate are extracted from the meat commodity by the homogenization of
the tissue and the subsequent addition of the solid phase extraction sorbent, such as
C18. 0. 1N HCL in 96% water/4% methanol is added to the sample. The sample is
centrifudged, and the supernant is removed. The analyte is eluted from the solid
phase extraction sorbent by the addition of water to the pellet. TEA is added to the
sample, followed by an acetonitrile extraction. The sample is then loaded onto an
Oasis™ MAX SPE cartridge. The column is washed and the analytes are eluted from
the column using 1% TFA in methanol/water, 90/10. The MAX eluate is evaporated
to dryness and redissolved in aqueous 0.02M aqueous phosphoric acid.
iii. Extracting Glyphosate, N-acetylglyphosate, N-acetyl AMPA and/or AMP A
from an Oil Sample
In still other embodiments, when the test sample comprises an oil sample, a
single procedure can be employed for the extraction and purification of all analytes of
interest (glyphosate, N-acetylglyphosate, N-acetyl AMPA, and AMPA). In such
embodiments, an extraction column need not be employed. For example, the test
sample can undergo a series of aqueous-organic extractions, such as, with a dilute
acid or a weak ion-pairing reagent (such as 0.02M phosphoric acid) and methylene
chloride. Following a sufficient number of extractions, the aqueous fraction is ready
for the detection of the analyte of interest. The extraction can be repeated 2, 3, 4, 5 or
more times for quantitative transfer of the analytes. Centrifugation can be used to
define phase separation after each partition.
Accordingly, methods are provided to extract glyphosate, N-acetylglyphosate,
N-acetyl AMPA and/or AMPA from an oil sample matrix comprising: (a) performing
an aqueous-organic extraction of the test sample comprising (i) adding a sufficient
concentration of a weak-ion pairing reagent or a dilute acid, such as phosphoric acid,
to the test sample, wherein the sufficient concentration of the weak-ion pairing
reagent or dilute acid allows for the improved response and linearity of glyphosate, N-
acetylglyphosate, N-acetyl AMPA or AMPA; and, (ii) adding an organic solvent; (b)
extracting the analyte of interest from the aqueous phase of the aqueous-organic
partition; and, (c) repeating either of step (a) and/or step (b) a sufficient number of
times to allow for the quantitative transfer of the analytes. In specific embodiments,
the aqueous-organic extraction comprises a phosphoric acid/methylene chloride
partition.
IV. Determining the Presence or Amount of Glyphosate, N-acetylglyphosate, N-acetyl
AMPA, AMPA, and other Metabolites in a Test Sample.
Following the extraction of one or more of the analyte(s) of interest from the
test sample, any method can be employed that allows for the detection of one or more
of the analytes of interest. In one embodiment, detecting one or more of the analytes
of interest comprises separating (and in specific embodiments, chromatographically
separating) at least one of the analytes of interest from the extracted test sample and
analyzing at least one of the chromatographically separated analytes to determine the
presence or amount of at least one of glyphosate, N-acetylglyphosate, N-acetyl AMPA
and/or AMPA in the test sample.
i. Separating at Least One of Glyphosate, N-acetylglyphosate, N-acetyl
AMPA and/or AMPA from the Extracted Test Sample
As used herein, "chromatographically separating" employs an "analytical
column" or a "chromatography column" having sufficient chromatographic plates to
effect a separation of the components of a test sample matrix. Preferably, the
components eluted from the analytical column are separated in such a way to allow
the presence or amount of an analyte(s) of interest to be determined. "Analytical
columns" can be distinguished from "extraction columns," which typically are used to
purify or extract retained materials from non-retained materials to obtain a "purified"
sample for further purification or analysis.
In specific embodiments, each of the analytes of interest (i.e., glyphosate,
AMPA, N-acetyl AMPA or N-acetylglyphosate) is chromatographically separated
from one another prior to detection. It is, however, recognized that depending on the
method of detection employed, it may not be necessary to separate each of the
analytes of interest from one another by chromatography. Such methods of detection
allow each of the analytes of interest to be detected when present as a mixture.
In specific embodiments, following the extraction step described in further
detail above, chromatographically separating the analytes of interest includes: (a)
disposing the composition comprising the extracted analyte(s) onto an analytical
column; and (b) eluting the analyte(s) from the analytical column. In one
embodiment, chromatographically separating the analytes of interest from one another
or other constituents of the test sample comprises the use of a high performance liquid
chromatography (HPLC) column. Any HPLC column that can sufficiently resolve the
analytes of interest and allow for their detection and/or quantitation can be employed.
In specific embodiments, the HPLC column comprises a phenyl phase which employs
a hexyl alkyl linker.
ii. Detecting Glyphosate, N-acetylglyphosate, AMP A, N-acetyl AMPA and
other Metabolites
By "detecting" is intended determining the presence or amount of an analyte
of interest (i.e., glyphosate, N-acetylglyphosate, N-acetyl AMPA and/or AMP A) in a
test sample. The method of detection is not restricted and may be either qualitative or
quantitative. In one embodiment, detecting the glyphosate, N-acetylglyphosate, N-
acetyl AMPA and/or AMPA comprises analyzing the chromatographically separated
analytes by a mass spectrometer. In some non-limiting embodiments, separation
and/or detection does not require the derivatization of the analyte of interest. In
specific embodiments, positive ion mass spectroscopy is performed to detect the
analyte of interest.
The term "mass spectrometry" or "MS" as used herein generally refer to
methods of filtering, detecting, and measuring ions based on their mass-to-charge
ratio, or "m/z." In MS techniques, one or more molecules of interest are ionized, and
the ions are subsequently introduced into a mass spectrographic instrument where,
due to a combination of magnetic and electric fields, the ions follow a path in space
that is dependent upon mass ("m") and charge ("z"). See, e.g., U.S. Pat. No.
6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry," which
is hereby incorporated by reference in its entirety.
In a "quadrupole" or "quadrupole ion trap" mass spectrographic instrument,
ions in an oscillating radio frequency (RF) field experience a force proportional to the
direct current (DC) potential applied between electrodes, the amplitude of the RF
signal, and m/z. The voltage and amplitude can be selected so that only ions having a
particular m/z travel the length of the quadrupole, while all other ions are deflected.
Thus, quadrupole instruments can act as both a "mass filter" and as a "mass detector"
for the ions injected into the instrument.
Further, the resolution of the MS technique can be enhanced by using "tandem
mass spectrometry," or "MS/MS." Tandem mass spectrometry (MS/MS) is the name
given to a group of mass spectrometric methods wherein "parent" ions generated from
a sample are fragmented to yield one or more "daughter" ions, which are subsequently
mass analyzed by a second MS procedure. MS/MS methods are useful for the
analysis of complex mixtures, especially biological samples, in part because the
specificity of MS/MS can minimize the need for extensive sample clean-up prior to
mass spectral analysis. In an example of an MS/MS method, parent ions are
generated from a sample and passed through a first mass filter to select those ions
having a particular mass-to-charge ratio. These ions are then fragmented, typically by
collisions with neutral gas molecules in a suitable ion collision cell, to yield daughter
ions, the mass spectrum of which is recorded by a second mass analyzer. The
daughter ion spectra so produced are indicative of the structure of the parent ion, and
the two stages of mass filtering can eliminate ions from interfering species present in
the conventional mass spectrum of a complex mixture.
The most common type of MS/MS instrument is the triple quadrupole (see, for
example, Yost, Enke in Ch. 8 of Tandem Mass Spectrometry, Ed. McLafferty, pub.
John Wiley and Sons, 1983). Triple quadrupole MS/MS instruments typically consist
of two quadrupole mass filters separated by a fragmentation means (i.e., a colison
cell), (usually a quadrupole mass filter operated in the RF only mode as an ion
containment device and containing a collision gas at a pressure of between 1 and 10
millitorr). Many other types of "hybrid" tandem mass spectrometers are also known,
however, including various combinations of magnetic sector analyzers and
quadrupole filters. These hybrid instruments often comprise high resolution magnetic
sector analyzers (i.e., analyzers comprising both magnetic and electrostatic sectors
arranged in a double-focusing combination) as either or both of the mass filters. Use
of high resolution mass filters is highly effective in reducing chemical noise to very
low levels.
The term "electron ionization" as used herein refers to methods in which one
or more analytes of interest in a gaseous or vapor phase is interacted with a flow of
electrons. Impact of the electrons with the analyte(s) produces analyte ions, which are
then be subjected to a mass spectroscopy technique.
The term "chemical ionization" as used herein refers to methods in which a
reagent gas (e.g., ammonia) is subjected to electron impact, and analyte ions are
formed by the interaction of reagent gas ions and analyte molecules.
The term "fast atom bombardment" as used herein refers to methods in which
a beam of high energy atoms (often Xe or Ar) impacts a non-volatile test sample,
desorbing and ionizing molecules contained in the sample. Samples are dissolved in a
viscous liquid matrix, such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-
crown-6 crown ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and
triethanolamine. The choice of an appropriate matrix for a compound or sample is an
empirical process.
The term "field desorption" as used herein refers to methods in which a non-
volatile test sample is placed on an ionization surface, and an intense electric field is
used to generate analyte ions.
The term "matrix-assisted laser desorption ionization," or "MALDI" as used
herein refers to methods in which a non-volatile sample is exposed to laser irradiation,
which desorbs and ionizes analytes in the sample by various ionization pathways,
including photo-ionization, protonation, deprotonation, and cluster decay. For
MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates
desorption of analyte molecules.
The term "surface enhanced laser desorption ionization," or "SELDI" as used
herein refers to another method in which a non-volatile sample is exposed to laser
irradiation, which desorbs and ionizes analytes in the sample by various ionization
pathways, including photo-ionization, protonation, deprotonation, and cluster decay.
For SELDI, the sample is typically bound to a surface that preferentially retains one
or more analytes of interest. As in MALDI, this process may also employ an energy-
absorbing material to facilitate ionization.
The term "electrospray ionization," or "ESI," as used herein refers to methods
in which a solution is passed along a short length of capillary tube, to the end of
which is applied a high positive or negative electrip potential. Solution reaching the
end of the tube, is vaporized (nebulized) into a jet or spray of very small droplets of
solution in solvent vapor. This mist of droplets flows through an evaporation
chamber which is heated slightly to prevent condensation and to evaporate solvent.
As the droplets get smaller the electrical surface charge density increases until such
time that the natural repulsion between like charges causes ions as well as neutral
molecules to be released.
The term "Atmospheric Pressure Chemical Ionization," or "APCI," as used
herein refers to mass spectroscopy methods that are similar to ESI; however, APCI
produces ions by ion-molecule reactions that occur within a plasma at atmospheric
pressure. The plasma is maintained by an electric discharge between the spray
capillary and a counter electrode. Then ions are typically extracted into the mass
analyzer by use of a set of differentially pumped skimmer stages. A counterflow of
dry and preheated N.sub.2 gas may be used to improve removal of solvent. The gas-
phase ionization in APCI can be more effective than ESI for analyzing less-polar
species.
The term "Atmospheric Pressure Photoionization" ("APPI") as used herein
refers to the form of mass spectroscopy where the mechanism for the photoionization
of molecule M is photon absorption and electron ejection to form the molecular M+.
Because the photon energy typically is just above the ionization potential, the
molecular ion is less susceptible to dissociation. In many cases it may be possible to
analyze samples without the need for chromatography, thus saving significant time
and expense. In the presence of water vapor or protic solvents, the molecular ion can
extract H to form MH+. This tends to occur if M has a high proton affinity. This does
not affect quantitation accuracy because the sum of M+ and MH+ is constant. Drug
compounds in protic solvents are usually observed as MH+, whereas nonpolar
compounds such as naphthalene or testosterone usually form M+. Robb, D. B.,
Covey, T. R. and Bruins, A.P. (2000): See, e.g., Robb et al., Atmospheric pressure
photoionization: An ionization method for liquid chromatography-mass spectrometry.
Anal. Chem. 72(15): 3653-3659.
The term "inductively coupled plasma" as used herein refers to methods in
which a sample is interacted with a partially ionized gas at a sufficiently high
temperature to atomize and ionize most elements.
The term "ionization" and "ionizing" as used herein refers to the process of
generating an analyte ion having a net electrical charge equal to one or more electron
units. Negative ions are those ions having a net negative charge of one or more
electron units, while positive ions are those ions having a net positive charge of one or
more electron units.
The term "desorption" as used herein refers to the removal of an analyte from
a surface and/or the entry of an analyte into a gaseous phase.
In those embodiments, such as MS/MS, where precursor ions are isolated for
further fragmentation, collision-induced dissociation ("CID") is often used to generate
the fragment ions for further detection. In CID, precursor ions gain energy through
collisions with an inert gas, and subsequently fragment by a process referred to as
"unimolecular decomposition." Sufficient energy must be deposited in the precursor
ion so that certain bonds within the ion can be broken due to increased vibrational
energy.
In some embodiments, one or more of the purification or separation steps can
be preformed "online." As used herein, the term "online" refers to purification or
separation steps that are performed in such a way that the test sample is disposed, e.g.,
injected, into a system in which the various components of the system are
operationally connected and, in some embodiments, in fluid communication with one
another. Representative components of such online systems include, but are not
limited to: an autosampler; one or more injection ports; one or more columns,
including but not limited to, an extraction column, including an extraction column,
and in some embodiments, an analytical column; a detection system, e.g., a mass
spectrometer system; one or more pumps; one or more valves; and necessary
plumbing. In such "online" systems, the test sample and/or analytes of interest can be
passed from one component of the system to another without exiting the system, e.g.,
without having to be collected and then disposed into another component of the
system.
In some embodiments, the online purification or separation method can be
automated. In such embodiments, the steps can be performed without the need for
operator intervention once the process is set-up and initiated. One of ordinary skill in
the art also would appreciate that an online, automated procedure can include the use
of software for controlling the various components of the system, including pumps,
valves, autosamplers, and the like. Such software can be used to optimize the
extraction process through the precise timing of sample and solute additions and flow
rate.
In contrast, the term "offline" refers to a purification, separation, or extraction
procedure that is performed separately from subsequent purification or separation
steps and/or analysis steps. In such offline procedures, the analytes of interests
typically are separated, for example, on an extraction column or by liquid-liquid
extraction, from the other components in the sample matrix and then collected for
subsequent introduction into another chromatographic or detector system. Offline
procedures typically require manual intervention on the part of the operator.
V. Specific Embodiments of the Invention
In non-limiting embodiments, determining the presence or amount of
N-acetylglyphosate in a test sample comprises: (a) providing a test sample suspected
of containing N-acetylglyphosate; (b) extracting a composition comprising the
N-acetylglyphosate from the test sample by liquid-liquid extraction; (c) disposing the
composition of step (b) comprising the N-acetylglyphosate onto a C18 extraction
column and eluting the N-acetylglyphosate therefrom; (d) disposing the eluted N-
acetylglyphosate from step (c) onto an anion exchange column having a surface
functionally of m-divinylbenzene and N-vinylpyrrolidone copolymer with quaternary
amines and eluting the N-acetylglyphosate therefrom; (e) adding a sufficient
concentration of phosphoric acid to said eluted N-acetylglyphosate of step (d),
wherein said sufficient concentration allows for an improved response and linearity of
N-acetylglyphosate during detection or wherein said sufficient concentration
comprises a final concentration of about 0.02M phosphoric acid; (f)
chromatographically separating the Af-acetylglyphosate from other constituents in the
composition of step (e) using a phenyl-hexyl high performance liquid chromatography
(HPLC) analytical column; and, (g) analyzing the chromatographically separated
sample of step (f) to determine the presence or amount of the TV-acetylglyphosate in
the test sample by a tandem quadrupole mass spectrometer operated in positive ion
mode. In specific embodiments, the liquid-liquid extraction comprises a dilute acid
and methylene chloride extraction. In further embodiments, the test sample comprises
a solid matrix sample from a plant or is a milk or egg commodity sample.
In non-limiting embodiments, determining the presence or amount of
N-acetylglyphosate in a test sample comprises: (a) providing a test sample suspected
of containing N-acetylglyphosate; (b) mixing the test sample with a solid phase
extraction sorbent under conditions that allow the solid phase extraction sorbent to
bind the N-acetylglyphosate; (c) extracting the solid phase extraction sorbent and the
bound N-acetylglyphosate from the sample by liquid/liquid extraction; (d) eluting
said N-acetylglyphosate from the solid phase extraction sorbent; (e) extracting a
composition comprising the N-acetylglyphosate from the eluate of step (d) by liquid-
liquid extraction; (f) disposing the eluted N-acetylglyphosate from step (e) onto an
anion exchange column having a surface functionally of m-divinylbenzene and
N-vinylpyrrolidone copolymer with quaternary amines and eluting the N-
acetylglyphosate therefrom; (g) adding a sufficient concentration of phosphoric acid
to said eluted N -acetylglyphosate of step (f), wherein said sufficient concentration
allows for an improved response and linearity of N-acetylglyphosate during detection
or wherein said sufficient concentration comprises a final concentration of about
0.02M phosphoric acid; (h) chromatographically separating the N-acetylglyphosate
from other constituents in the composition of step (g) using a phenyl-hexyl high
performance liquid chromatography (HPLC) analytical column; and, (i) analyzing the
chromatographically separated sample of step (h) to determine the presence or amount
of the N-acetylglyphosate in the test sample by a tandem quadrupole mass
spectrometer operated in positive ion mode. In specific embodiments, the liquid-
liquid extraction of step (c) comprises a dilute acid and methanol extraction. In
further embodiments, the test sample comprises a meat tissue commodity.
In other non-limiting embodiments, the method of determining the presence or
amount of glyphosate in a test sample comprises: (a) providing a test sample
suspected of containing glyphosate; (b) extracting a composition comprising the
glyphosate from the test sample by liquid-liquid extraction; (c) disposing the
composition comprising the glyphosate of step (b) onto a C18 extraction column and
eluting the glyphosate therefrom; (d) disposing the eluted glyphosate from step (c)
onto an anion exchange column comprising a surface functionally of m-
divinylbenzene and N-vinylpyrrolidone copolymer with quaternary amines and
eluting the glyphosate therefrom; (e) adding a sufficient concentration of phosphoric
acid to said eluted glyphosate of step (d), wherein said sufficient concentration allows
for an improved response and linearity of glyphosate during detection or wherein the
sufficient concentration comprises a final concentration of about 0.02M phosphoric
acid; (f) chromatographically separating the glyphosate from other constituents of
step (e) using a phenyl-hexyl high performance liquid chromatography (HPLC)
column; and, (g) analyzing the chromatographically separated sample of step (f) 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. In specific
embodiments, the liquid-liquid extraction comprises a dilute aqueous acid/methanol
solution and/or a dilute acid and methylene chloride extraction. In further
embodiments, the test sample comprises a solid matrix sample from a plant or an egg
or a milk commodity.
In non-limiting embodiments, determining the presence or amount of
glyphosate in a test sample comprises: (a) providing a test sample suspected of
containing glyphosate; (b) mixing the test sample with a solid phase extraction
sorbent under conditions that allow the solid phase extraction sorbent to bind the
glyphosate; (c) extracting the solid phase extraction sorbent and the bound glyphosate
from the sample by liquid/liquid extraction; (d) eluting the glyphosate from the solid
phase extraction sorbent; (e) extracting a composition comprising the glyphosate
from the eluate of step (d) by liquid-liquid extraction; (f) disposing the eluted
glyphosate from step (e) onto an anion exchange column having a surface functionally
of m-divinylbenzene and N-vinylpyrrolidone copolymer with quaternary amines and
eluting the glyphosate therefrom; (g) adding a sufficient concentration of phosphoric
acid to said eluted glyphosate of step (f), wherein said sufficient concentration allows
for an improved response and linearity of glyphosate during detection or wherein said
sufficient concentration comprises a final concentration of about 0.02M phosphoric
acid; (h) chromatographically separating the glyphosate from other constituents in
the composition of step (g) using a phenyl-hexyl high performance liquid
chromatography (HPLC) analytical column; and, (i) analyzing the
chromatographically separated sample of step (h) to determine the presence or amount
of the glyphosate in the test sample by a tandem quadrupole mass spectrometer
operated in positive ion mode. In specific embodiments, the liquid-liquid extraction
of step (c) comprises a dilute acid and methanol extraction. In further embodiments,
the test sample comprises a meat tissue commodity.
In other non-limiting embodiments, the method of determining the presence or
amount of aminomethyl phosphonic acid (AMPA) in a test sample comprises: (a)
providing the test sample suspected of containing AMPA; (b) extracting a
composition comprising the AMPA from the test sample by liquid-liquid extraction;
(c) disposing the composition of step (b) onto a C18 extraction column and eluting the
AMPA therefrom; (d) disposing the eluted AMPA from step (c) onto a cation
exchange column comprising a surface functionally of m-divinylbenzene and N-
vinylpyrrolidone copolymer with sulfonic acid substituents and eluting the AMPA
therefrom; (e) adding a sufficient concentration of phosphoric acid to said eluate
AMPA of step (d), wherein said sufficient concentration allows for an improved
response and linearity of AMPA during detection or wherein said sufficient
concentration comprises a final concentration of about 0.02M phosphoric acid;
(f) chromatographically separating the AMPA from other constituents of step (e)
using a phenyl-hexyl high performance liquid chromatography (HPLC) analytical
column; and, (g) analyzing the chromatographically separated sample of step (f) 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 the
AMPA occurs without derivatization of the AMPA. In specific embodiments, the
liquid-liquid extraction comprises a dilute aqueous acid/methanol solution and/or a
dilute acid and methylene chloride extraction. In further embodiments, the test
sample comprises a solid matrix sample from a plant or a milk or egg commodity.
In another non-limiting embodiment, a method of determining the presence or
amount of aminomethyl phosphonic acid (AMPA) in a test sample comprises: (a)
providing the test sample suspected of containing AMPA; (b) mixing the test
sample with a solid phase extraction sorbent under conditions that allow the solid
phase extraction sorbent to bind the AMPA; (c) extracting the solid phase extraction
sorbent and the bound AMPA from the sample by liquid/liquid extraction; (d) eluting
the AMPA from the solid phase extraction sorbent; (e) disposing the eluted AMPA
from step (d) onto a cation exchange column comprising a surface functionally of m-
divinylbenzene and ,/V-vinylpyrrolidone copolymer having sulfonic acid substituents
and eluting the AMPA therefrom; (f) adding a sufficient concentration of phosphoric
acid to said eluated AMPA of step (d), wherein said sufficient concentration allows
for an improved response and linearity of AMPA during detection; (g)
chromatographically separating the AMPA from other constituents in the eluate of
step (f) using a phenyl-hexyl high performance liquid chromatography (HPLC)
analytical column; and, (h) analyzing the chromatographically separated sample of
step (g) to determine the presence or amount of AMPA in the test sample by a tandem
quadrupole mass spectrometer operated in positive ion mode. In further
embodiments, the test sample comprises a meat tissue commodity.
In other non-limiting embodiments, the method of determining the presence or
amount of N-acetyl AMPA in a test sample comprises: (a) providing a test sample
suspected of containing N-acetyl AMPA; (b) extracting a composition comprising the
N-acetyl AMPA from the test sample by liquid-liquid extraction; (c) disposing the
composition of step (b) comprising the N-acetyl AMPA onto a C18 extraction column
and eluting the N-acetyl AMPA therefrom; (d) disposing the eluted N-acetyl AMPA
from step (c) onto an anion exchange column having a surface functionally of m-
divinylbenzene and N-vinylpyrrolidone copolymer with quaternary amines and
eluting the N-acetyl AMPA therefrom; (e) adding a sufficient concentration of
phosphoric acid to said eluted N-acetyl AMPA of step (d), wherein said sufficient
concentration allows for an improved response and linearity of N-acetyl AMPA
during detection or wherein said sufficient concentration comprises a final
concentration of about 0.02M phosphoric acid; (f) chromatographically separating
the N-acetyl AMPA from other constituents in the composition of step (e) using a
phenyl-hexyl high performance liquid chromatography (HPLC) analytical column;
and, (g) analyzing the chromatographically separated sample of step (f) to determine
the presence or amount of the N-acetyl AMPA in the test sample by a tandem
quadrupole mass spectrometer operated in positive ion mode. In specific
embodiments, the liquid-liquid extraction comprises a dilute aqueous acid/methanol
solution and/or a dilute acid and methylene chloride extraction. And in further
embodiments, the test sample comprises a solid matrix sample from a plant or a milk
or egg commodity.
In non-limiting embodiments, determining the presence or amount of N-acetyl
AMPA in a test sample comprises: (a) providing a test sample suspected of
containing N-acetyl AMPA; (b) mixing the test sample with a solid phase extraction
sorbent under conditions to allow the solid phase extraction sorbent to bind the N-
acetyl AMPA; (c) extracting the solid phase extraction sorbent and the bound N-
acetyl AMPA from the sample by liquid/liquid extraction; (d) eluting the N-acetyl
AMPA from the solid phase extraction sorbent; (e) extracting a composition
comprising the N-acetyl AMPA from the composition comprising the N-acetyl AMPA
from step (d) by liquid-liquid extraction; (f) disposing the eluted N-acetyl AMPA
from step (e) onto an anion exchange column having a surface functionally of m-
divinylbenzene and N -vinylpyrrolidone copolymer with quaternary amines and
eluting the N-acetyl AMPA therefrom; (g) adding a sufficient concentration of
phosphoric acid to said eluted glyphosate of step (f), wherein said sufficient
concentration allows for an improved response and linearity of N-acetyl AMPA
during detection or wherein said sufficient concentration comprises a final
concentration of about 0.02M phosphoric acid; (h) chromatographically separating
the N-acetyl AMPA from other constituents in the composition of step (g) using a
phenyl-hexyl high performance liquid chromatography (HPLC) analytical column;
and, (i) analyzing the chromatographically separated sample of step (h) to determine
the presence or amount of the N-acetyl AMPA in the test sample by a tandem
quadrupole mass spectrometer operated in positive ion mode. In specific
embodiments, the liquid-liquid extraction comprises a dilute acid and methylene
chloride extraction. In further embodiments, the test sample comprises a meat tissue
commodity.
In other non-limiting embodiments, a method for determining the presence or
amount of at least one of glyphosate, N-acetylglyphosate, N-acetyl AMPA or AMPA
in an oil test sample comprises: (a) providing the oil 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 N-acetylglyphosate, AMPA, glyphosate, or N-acetyl AMPA during
detection or wherein said sufficient concentration comprises a final concentration of
about 0.02M phosphoric acid; (c) extracting a composition from said test sample
comprising at least one of the AMPA, the N-acetylglyphosate, the N-acetyl AMPA or
the glyphosate by a aqueous-organic extraction; (d) chromatographically separating
at least one of the AMPA, N-acetylglyphosate, N-acetyl AMPA or glyphosate of step
(c) using a high performance liquid chromatography (HPLC) column comprising a
phenyl-hexyl analytical column; and, (e) analyzing the chromatographically
separated sample of step (d) to determine the presence or amount of at least one of
AMPA, glyphosate, N-acetyl AMPA or N-acetylglyphosate in the test sample by a
tandem quadrupole mass spectrometer operated in electrospray ionization mode.
In other non-limiting embodiments, a method of purifying at least glyphosate,
N-acetyl AMPA or N-acetylglyphosate from AMPA is provided. The method
comprises: (a) providing a test sample suspected of containing at least one of the
glyphosate, the N-acetyl AMPA, the N-acetylglyphosate, or the AMPA; (b)
extracting a composition comprising at least one of the N-acetylglyphosate, the
glyphosate, the N-acetyl AMPA or the AMPA from said test sample; (c) disposing
the composition of step (b) onto an anion exchange extraction column and eluting at
least one of the N-acetylglyphosate, N-acetyl AMPA and glyphosate therefrom and
thereby purifying the glyphosate, N-acetyl AMPA and N-acetylglyphosate from the
AMPA; or, disposing the composition of step (b) onto a cation exchange extraction
column and eluting the AMPA therefrom and thereby purifying the glyphosate, N-
acetyl AMPA and the N-acetylglyphosate from the AMPA.
EXPERIMENTAL
Example 1
In one embodiment, an analytical method was developed for the determination
of glyphosate and relevant metabolites or degradate residues in transgenic crop and
crop fraction matrices. The glyphosate metabolites or analytes are N-
acetylglyphosate, aminomethyl phosphonic acid (AMPA) and N-acetyl AMPA. N-
acetylglyphosate is a metabolite associated with transgenic crops containing the
glyphosate N-acetyltransferase (glyat) enzyme. The method target limit of
quantitation (LOQ) in each matrix examined was 0.050 mg/kg (ppm). The method
was validated at 0.050 mg/kg and 0.50 mg/kg using a LC/MS/MS system operating
with an electrospray interface (ESI) in positive ion mode detection. This analytical
method was developed to support residue data collection required for registration of
genetically modified crops.
Raw agricultural and solid process fraction commodities derived from plants
expressing GLYAT were extracted 3 times in acidic (0.1% formic acid or 0.025N
hydrochloric acid) water/methanol (96/4) solution. An aliquot of extract was
partitioned with methylene chloride and the aqueous fraction was recovered and
filtered. An aliquot of the aqueous fraction was filtered through a C18 SPE cartridge.
An aliquot of the eluate from the C18 SPE was diluted and applied to a MAX SPE
cartridge. The analytes were eluted from the MAX sorbent in 1% TFA in
methanol/water (9/1) solution following several solution rinses. The MAX eluate was
evaporated to dryness and redissolved in aqueous 0.02M phosphoric acid, filtered, and
analyzed for glyphosate, N-acetylglyphosate, N-acetyl AMPA. A separate aliquot of
the eluate from the C18 SPE was processed on a MCX SPE cartridge, diluted, filtered,
and analyzed for AMPA.
Processed oil commodities were partitioned with methylene chloride and
aqueous 0.02M phosphoric acid. The solution was centrifuged to resolve organic and
aqueous fractions, and the aqueous fraction was collected. The remaining matrix and
methylene chloride fraction were partitioned again with aqueous 0.02M phosphoric
acid, and the aqueous fraction was collected and combined with the first aqueous
fraction for quantitative recovery of the analytes. The combined aqueous extract was
diluted, filtered, and analyzed for glyphosate, N-acetylglyphosate, N-acetyl AMPA
and AMPA.
Final extracts were analyzed using HPLC with reverse phase chromatography
and a triple quadrupole mass spectrometer with an electrospray source, operating in
positive ion LC/MS/MS (liquid chromatograph/mass spectrometry/mass
spectrometry) mode. Stable isotope standards of glyphosate (1,2-13C2 15N) and
AMPA (13C 15N) were added just prior to LC/MS/MS analysis as needed for internal
standards to normalize response for matrix effects.
The recoveries from samples of various corn and soybean matrices fortified at
0.050 mg/kg (LOQ) and 0.50 mg/kg (10xLOQ) support the satisfactory performance
of this method. Tables 17 A, B and C, 18A and B, 19, 20, and 21 summarize the
average recovery results for each analyte in sample matrices.
Reference Analytical Standards
Reference analytical standards of glyphosate (DPX-B2856-011, 96% pure)
and N-acetylglyphosate, sodium salt (IN-MCX20-000, 67.4% pure as free acid) were
obtained from Sigma Aldrich. Reference analytical standard of AMP A (IN-
YB726-001, 99.53% pure) was obtained from Alfa Aesar. Reference analytical
standard of 7V-acetyl AMPA was synthesized at E. I. du Pont de Nemours and
Company, DuPont Agricultural Products, Wilmington, DE. Characterization data are
archived by DuPont Agricultural Products, E.I. du Pont de Nemours and Company,
Wilmington, Del.
Internal Standards
Glyphosate 1,2-13C2 I5N and aminomethyl phosphonic acid 13C 15N (AMPA)
stable isotope standards were obtained from GmbH for use as internal standards.
Structures and specific information follow.

Example 2 - Principle of Analytical Method
Glyphosate, N-acetylglyphosate, AMPA, and N-acetyl AMPA were extracted
from plant tissue and solid process fraction matrices of various crops into dilute
aqueous acid (0.1% formic acid or 0.025N hydrochloric acid)/methanol (96/4) using a
probe homogenizer. Dilute hydrochloric acid was substituted for formic acid to
increase acidity for more efficient extraction of N-acetylglyphosate from corn flour
and meal process fractions. Multiple extractions (3) were made for quantitative
recovery of analytes and to eliminate moisture content in the matrix as a recovery
factor. Additional extraction solution volumes were necessary for stover, hay, and
hulls because of the lower moisture content in those matrices.
Purification of glyphosate. N-acetylglyphosate and N-acetyl AMPA in solid
matrix sample extracts:
An aliquot of extract was partitioned with methylene chloride and the aqueous
fraction was recovered and filtered (0.2-1.0 µm) to remove particulates.
Approximately 10 ml of the aqueous fraction was collected following filtration
through a C18 SPE cartridge. An aliquot of the eluate collected from the C18 SPE was
diluted and applied to a MAX SPE cartridge. The analytes were eluted from the
MAX sorbent in 1% TFA in methanol/water (9/1) solution following several solution
rinses. The MAX eluate was evaporated to dryness and redissolved in aqueous 0.02M
aqueous phosphoric acid, filtered, and analyzed for glyphosate and
N-acetylglyphosate. A minor modification was made to this procedure for the
analysis of soybean samples. For soybean seed and meal following partitioning,
extract samples were heated in a steam bath for approximately 15 minutes to
precipitate additional material in the extract prior to particulate filtration.
Purification of AMP A in solid matrix sample extracts:
A second aliquot of the eluate collected from the C18 SPE described above was
processed through a MCX SPE cartridge, diluted, filtered and analyzed for AMPA. A
separate analyte purification procedure was required for AMPA due to low recoveries
using MAX SPE purification.
Analysis of glyphosate, N-acetylglyphosate. AMPA and N-acetyl AMPA in oil
samples:
An aliquot of the sample was diluted with methylene chloride and the analytes
were liquid-liquid partitioned into 0.02M aqueous phosphoric acid. The sample was
partitioned twice for quantitative transfer of the analytes. Centrifugation was used to
define phase separation in each partition.
All final extracts were filtered (0.2 µm) prior to LC/MS/MS analysis to
remove particulates as preventive maintenance measure for the HPLC system. The
use of phosphoric acid in the final sample solution was shown to improve the overall
response and linearity of glyphosate. The use of glyphosate and AMPA stable
isotopes as internal standards in final extract solutions was required to normalize for
matrix effects in the analysis of soybean matrices and is recommended for general use
to improve method ruggedness. The stable isotope internal standards were initially
added to final extracts just prior to LC/MS/MS analysis, but the procedure was
modified for addition just prior to SPE purification to normalize for SPE performance
and matrix effects. The stable isotope forms of glyphosate and AMPA used as
internal standards behave identically to glyphosate and AMPA. The analytes were
resolved by HPLC reverse-phase chromatography using a phenyl-hexyl analytical
column coupled to electrospray ionization in positive ion mode with MS/MS
detection. The same chromatography conditions were used for glyphosate, N-
acetylglyphosate, AMPA, and N-acetyl AMPA analysis. Two molecular ion
transitions were acquired for each analyte, except for AMPA (only 1 molecular ion
transition was available in positive ion mode). Quantitative analysis was
accomplished using a single molecular ion transition for each analyte. With the
exception of AMPA, the relative abundance of the 2 detected MS/MS fragment ions
provides confirmatory evidence for each analyte.
Analytical Procedure
Reasent Solutions
Extraction Solution A: 96% aqueous 0.1% formic acid/4% methanol.
Extraction Solution B: 96% aqueous 0.025N hydrochloric acid/4% methanol.
80% or 95% Methanol in Water Solution
0.1M Acetic Acid in 80% Methanol/Water Solution
0.25% Ammonium Hydroxide in 95% Methanol/5% Water
0.25% Ammonium Hydroxide in Water: Per liter volume, 10 ml of ammonium
hydroxide solution (minimum 25%) was added to small volume of HPLC grade
purified water, then diluted to final volume with HPLC grade purified water. This is a
conditioning solution for MAX SPE (12 ml/sample, dilution in water procedure).
Note: 1 ml of 25% ammonium hydroxide diluted to 100 ml ˜ 0.25% NH4OH.
Elution Solution, 1% TFA in 90% methanol/10% water.
1.0M Phosphoric Acid Solution: Per 10 ml volume, 0.67 ml of concentrated
phosphoric acid (min. 85%) was added to HPLC grade purified water in a 15-ml
polypropylene centrifuge tube and dilute to final volume using gradations on tube
with HPLC grade purified water.
Sample and Standard Final Solution, aqueous 0.02M phosphoric acid
Aqueous 0.2M Formic Acid, aqueous mobile phase
Stock Standard Preparation and Stability
Standards with purity of greater than 95% were used. A minimum of
approximately 10 mg of standard was weighed on an analytical balance that provides
a weight precision to three significant figures, or the amount of standard was
increased to satisfy this condition.
Since residue tolerances are established in glyphosate free-acid equivalents,
stock standard solutions for each analyte are prepared in glyphosate free-acid
equivalents so that fortifications and recoveries can be determined in parent free acid
equivalents. As needed, individual free-acid equivalent stock standards solutions
were prepared were prepared for glyphosate, N-acetylglyphosate, AMPA or N-acetyl
AMPA by adding appropriate amounts of standard to a 100-ml volumetric flask and
diluting to final volume with water (water refers to HPLC grade or equivalent water).
The following calculation and Table 25 provide guidance for the preparation of 100
ml of a 100 µg/ml (ppm) in glyphosate free-acid equivalents for each analyte.

Amounts weighed for each analyte were at least 10 mg and, therefore,
individual analyte stock solution concentrations can exceed 100 µg/ml in parent free-
acid equivalents. Stock standard solutions may be prepared at higher concentrations
(not to exceed 1 mg/ml). A minimum standard weight of approximately 10 mg and
final standard volume of at least 10 ml were observed. These solutions were stored at
or below 4°C and are stable for at least 9 months.
Internal Standard Preparation and Stability
Glyphosate 1,2-13C2 15N and aminomethyl phosphonic acid 13C I5N (AMPA)
stable isotope standards were supplied in amber ampules containing 1.1 ml of aqueous
solution at a nominal concentration of 100 mg/L (µg/ml). Each standard solution was
transferred to an individual amber vial and stored at or below 20°C in the dark. The
isotopic purity was verified for each standard by analysis of an approximately
500 ng/ml solution following protocol outlined below.
An intermediate 100 ng/ml internal standard solution containing glyphosate
and AMPA isotopes was prepared by diluting 100 ng/ml stock solution at a rate of
0.1 ml in 100 ml of aqueous 0.02M phosphoric acid or HPLC grade purified water.
Internal standards were included in final extract and calibration standard solutions at a
rate of 50 µL/ml or 5 ng/ml in the final solution volume. Each 100 ml of a 100 ng/ml
standard solution can be used in up to 400 samples when 250 µL of internal standard
is applied in 5 ml of final extract.
The 100 ng/ml standards (Glyphosate 1,2-13C2 15N, 100 ng/ml x 1.1 ml and
Aminomethyl phosphonic acid 13C 15N (AMPA), 100 ng/ml x1.1 ml) were
individually transferred to screwcap amber autosampler vials using glass transfer
pipet. A 0.025ml aliquot of the 100 µg/ml standard was transferred to a 15 ml
polypropylene centrifuge tube and diluted with 5 ml of 0.02M H3PO4 to prepare
separate solutions at 500 ng/ml. A 0.10 ml aliquot of each 100 ng/ml standard was
combined in a single 100 ml volumetric flask and diluted to volume with 0.02M
H3PO4 to prepare a mixed solution at 100 ng/ml. This solution was divided into 2x50
ml polypropylene centrifuge tubes. All standards were stored in refrigerator. The
LC/MS/MS analysis of the 500 ng/ml solutions is shown in Figure 25.
Intermediate and Fortification Standards Preparation and Stability
Fortification solutions were prepared in glyphosate free-acid equivalent
concentrations from dilutions of the individual stock solutions. If glyphosate, N-
acetylglyphosate, AMPA or N-acetyl AMPA fortifications are required at levels
greater than 0.5 mg/kg (10xLOQ), the individual stock standard solutions should be
used to fortify samples. 10.0 ng/ml and 1.0 ng/ml fortification solutions were
prepared for sample fortification at the 10xLOQ and LOQ, respectively. Alternative
concentrations may be prepared as needed for other fortification levels.
10.0 µg/ml Fortification Solution: The stock solution for each analyte was
dilated appropriately into a common volumetric flask, diluted to volume with HPLC
grade purified water, cap, and mix well. For example, 1.00 ml of a 100 ng/ml stock
solution was combined for each required analyte in a 10-ml volumetric flask and
diluted to final volume with water, cap, and mix well.
Stock solutions concentrations will vary and the required volume of each
analyte stock solution used to prepare fortification solution is adjusted for correct final
concentration as determined using the following calculation.

For example, to prepare 50 ml of a 10 µg/ml fortification solution from
glyphosate, N-acetylglyphosate, and AMPA stock solutions prepared respectively at
115 µg/ml, 186 µg/ml, and 206 µg/ml in glyphosate free-acid equivalent
concentrations the following stock solution volumes was added to a 50-ml volumetric
flask.
Glyphosate = 10 µg/ml x 50 ml / 115 µg/ml = 4.35 ml stock solution
N-acetylglyphosate = 10 µg/ml x 50 ml / 186 µg/ml = 2.69 ml stock solution
AMPA = 10 µg/ml x 50 ml / 206 µg/ml = 2.42 ml stock solution
10 ml Fortification Solution: The 10.0 µg/ml fortification solution (preferred) or the
stock solution for each analyte was diluted appropriately with HPLC grade purified
water into a common volumetric flask. For example, 1.0 ml of the 10.0 (µg/ml
fortification solution was transferred to a 10 ml volumetric flask, dilute to volume
with water, cap, and mix well. Store at or below 4°C and replace monthly.
Chromatographic Standard Preparation and Stability
Calibration standards were prepared in glyphosate free-acid equivalent
concentrations from dilutions of fortification standards or individual stock standards.
Glyphosate and AMPA stable isotopes can be used as internal standards in calibration
standards and final extract solutions to normalize recoveries for matrix effects for
sample analysis. A minimum of 5 or more calibration standards over a range from
approximately 50% of LOQ equivalent final concentration to >120% of the highest
expected final sample concentration are recommended for quantification. AMPA
analysis was conducted separately and had an LOQ equivalent final concentration of
2.0 ng/ml. The LOQ equivalent final concentration for glyphosate, N-
acetylglyphosate and N-acetyl AMPA was 1.0 ng/ml.
For example, intermediate standard solutions of 10.0 ng/ml and 100 ng/ml
were prepared from the 1.0 and 10.0 µg/ml fortification solutions, respectively, by
diluting a 250 µL aliquot to final volume of 25.0 ml in standard prep solution
(aqueous 0.02M phosphoric acid). Calibration standards were prepared from serial
dilutions of these intermediate standards with the addition of internal standards as
indicated in the following Table 26.

Calibration standards were kept at or below 4°C and replaced at least biweekly.
Calibration standards use may be extended if supported by stability test data.
Source (& Characterization) of Samples
Corn forage, grain, and stover matrix test samples were collected from
transgenic hybrid maize that contained the glyphosate acetyl transferase gene (glyat).
Corn flour, starch, grits, refined oil (wet milled), meal (dry milled), and refined oil
(dry milled) samples were processed and also collected. Soybean forage, seed, and
hay were from soybean line containing glyat and collected from greenhouse plants.
Soybean hulls were processed and soybean meal from a soybean line containing glyat
was also prepared. Plums and limes were purchased at local supermarkets.
Storaee & Preparation of Samples
All samples were stored at -20 ± 5°C prior to sample preprocessing,
extraction, and analysis. In preparation for analysis, the RAC samples were removed
from frozen storage and ground frozen with dry ice using a Hobart® Food Processor
(Model #84145) for forage and stover and a Quaker Grinding Mill (Model 4E) for
grain. Samples were mixed extensively during the grinding process to ensure
homogeneity. Most of the dry ice was allowed to sublime, and then the samples were
returned to the freezer for storage until extraction and analysis. Process Fraction
samples were homogenous when received and require no further preprocessing.
Sample Fortification Procedure
Untreated matrix control samples are fortified in extraction containers
(250-ml bottles or 50-ml centrifuge tubes) as required in glyphosate free-acid
equivalent concentrations. The 10.0 and 1.0 µg/ml Fortification Standards containing
a mixture of test analytes were used to fortify test samples. Table 27 provides
exemplary fortifications suitable for use with the presently disclosed methods.

For each solid matrix sample, the appropriate amount was weighed into a
clean 250-ml PPCO centrifuge bottle. For each oil sample, 2.0 ± 0.02 g of sample
was weighed into a clean 50-ml glass or polypropylene centrifuge tube. For dry
commodities (e.g., stover, hay, hulls), the fortification aliquot should be diluted in
5 ml of HPLC grade purified water in a vial or tube prior to fortification for better
dispersion of the analytes in the matrix. After fortification, the solid matrix samples
are allowed to stand in a fume hood for approximately 15 minute to allow the
fortification solution to dissipate.
Example 3 - Analvte Extraction Procedures
Analyte extractions procedures are provided for solid matrices including corn
PvACs (forage, grain, stover), corn process fractions (flour, grits, meal, starch),
soybean RACs (forage, seed, hulls), and soybean process fractions (meal, hulls).
Sample amount, extraction solution, and extraction solution volumes vary by
commodity. Stover, hay, and hulls require 2x sample amount (10.0 g) and extract
volume (200 ml) to compensate for dry commodity. Corn and soybean oil process
fraction procedures are provided elsewhere herein.
Solid Matrix Samples
First Extraction
1.0 Correct amount of sample was weighed into the appropriate container as
indicated above.
2.0 Samples were fortified as indicated above and solid matrices allowed to air dry
in hood for 15 minutes.
3.1 For forage, grain, seed, grits, starch, or soy meal samples, 50 ml of Extraction
Solution A (96% aq. 0.1% formic acid/ 4% methanol) was added to sample.
This step was applied to watery (e.g. plums) and acid (e.g. lime) crop types.
For stover, hay, or hulls, 100 ml of Extraction Solution A (96% aq. 0.1%
formic acid/4% methanol) was added to the sample. The tube or vial used to
dilute the fortification solution to 5 ml was rinsed with the extraction solution
for quantitative transfer of the analytes to the sample during this step for dry
matrix samples.
3.2 For corn meal or flour, 50 ml of Extraction Solution B (96% aq. 0.025N
hydrochloric acid/4% methanol) was added to sample.
4.0 The sample was capped and allowed to stand for 15 minutes so extraction
solution can soak into matrix.
5.0 The sample was uncapped and homogenized using a Tissumizer® for 2
minutes at 40-50% of total motor speed or at a speed that efficiently
homogenizes the samples without overheating or foaming.
6.0 The samples were capped and centrifuged for 15 minutes at 13,000 rpm to
achieve sufficient clarification of supernatant.
7.0 The supernatants were decanted into clean graduated cylinders (100 ml or 250
ml as needed for final volume capacity). The supernatant may be decanted
through paper filters to clarify extract solutions. Hulls were pressed with spoon
or spatula when decanting to recover additional extraction solution (30-40 ml
may be retained in the pellet).
Second Extraction
8.1 For forage, grain, seed, grits, starch, meal, or flour samples, 25 ml of
Extraction Solution A (96% aq. 0.1% formic acid/ 4% methanol) was added to sample.
Apply this step to watery (e.g. plum) and acid (e.g. lime) crop types. Note: the
second and third extractions of meal and flour matrices use Extraction
Solution A.
8.2 For stover, hay, or hulls samples 50 ml of Extraction Solution A (96% aq.
0.1% formic acid/4% methanol) was added to sample.
9.0 Samples were homogenized using a Tissumizer® for 2 minutes at 40-50% of
total motor speed or at a speed that efficiently homogenizes the samples
without overheating or foaming.
10.0 Samples were capped and centrifuged for 15 minutes at 13,000 rpm to achieve
sufficient clarification of supernatant. The supernatant was decanted through
paper filters to clarify extract solutions.
11.0 Supernatants were decanted and combined to respective graduated cylinders.
Third Extraction
12.0 Repeat steps 8.1 thru 11.0
13.1 For forage, grain, seed, grits, starch, meal, or flour samples the final extract
volumes were adjusted to 100 ml with HPLC grade water or determine exact
volume in excess of 100 ml. Apply this step to watery (e.g. plum) and acid
(e.g. lime) crop types.
13.2 For stover, hay, or hulls samples final extract volumes were adjusted to 200 ml
with HPLC grade water or determine exact volume in excess of 200 ml.
14.0 The supernatants were transferred to clean polypropylene bottles. The sample
was poured back and forth from graduated cylinder to bottle to mix solution.
Cap filled bottles. Raw extracts may be stored at or below 4 °C.
Example 4 - Analyte Purification Procedures
For the analysis of solid matrix sample extracts, methylene chloride partition
and C18 SPE filtration procedures (as outlined below) were initially used for the
purification of all analytes. An aliquot of the C18 SPE eluate was processed for the
analysis of AMP A by strong cation exchange MCX SPE (as outlined below). A
separate aliquot of the C18 SPE eluate was processed for the analysis of glyphosate
and N-acetylglyphosate and N-acetyl AMPA by strong anion exchange MAX SPE (as
outlined below). Separate procedures were required due to the characteristics and
behavior of analytes on SPE stationery phases selected. For the analysis of oil
process fractions, a single procedure was used for the extraction and purification of all
analytes (see below).
A. Glyphosate, N-acetylglyphosate, and AMPA and N-acetyl AMPA Initial
Purification Procedure in Solid Matrix Samples
Methylene Chloride Partition
1.0 30 ml sample extract was transferred to a 50-ml polypropylene centrifuge tube
and 10 ml of methylene chloride was added to sample aliquot which was
capped and shook or vortexed for at least 30 seconds (use gradations on tube
for volume measurements). For soybean seed or meal, 20 ml of methylene
chloride was used. Samples were centrifuged 10 minutes at sufficient rpm
(e.g., 3000 rpm) to form distinct layers (aqueous above protein and methylene
chloride). Smaller extract aliquots may be used to accommodate the
individual analyte purification procedures (e.g., 15 ml of extract and 5 ml of
methylene chloride when AMP A analysis not needed or to separate the 2
purification procedures).
2.1 For all solid matrices except soybean seed and meal, as much of the aqueous
fraction was recovered as possible without disturbing the precipitate or
methylene chloride layers and filtered through a 1.0 µm or smaller hydrophilic
filter (nylon or glass microfiber) into a clean 50-ml polypropylene centrifuge
tube. (The original centrifuge tubes were emptied and saved to collect waste
extract in C18 SPE Filtration step 4.0). At least 15 ml of the aqueous fraction
was recovered and filtered to use 14 ml for the following C18 SPE purification.
Processing a smaller volume of extract may be helpful if filtration is difficult.
Filtration is an important precondition for the ion exchange SPE
performance.
2.2 For soybean seed and meal samples, as much of the aqueous fraction was
recovered as possible without disturbing the precipitate or methylene chloride
layers and added to a clean glass tube. (The original centrifuge tubes were
emptied and saved to collect waste extract in C18 SPE Filtration step 4.0). The
aqueous extract samples were placed in a steam bath for at least 15 minutes to
further precipitate matrix, then filtered through a 1.0-µm or smaller
hydrophilic filter (nylon or glass microfiber) into a clean 50-ml polypropylene
centrifuge tube.
C18 SPE Filtration
1.0 Waste collection tubes were installed in vacuum manifold to collect initial
sample load volume. Note: waste collection tubes were used to prevent cross-
contamination in the vacuum manifold during step 3.0.
2.0 C18 SPE (6cc/500mg, Varian #12102052) cartridges were conditioned with 1
ml of methanol, followed by 2 CV's (CV=6 ml) of Extraction Solution A
(96% aq. 0.1% HCOOH/4% MeOH). Vacuum or positive pressure was
applied as needed for slow drip rate (1-2 ml/min). Note: aqueous 0.2M formic
acid may be substituted for Extraction Solution A for C18 SPE conditioning.
3.0 As last of conditioning solution enters the sorbent, 4.0 ml of aqueous sample
extract from Methylene Chloride Partition Purification step 2.0 was added
onto SPE column.
4.0 After dripping stops, the waste collection tubes were moved and clean 15-ml
centrifuge tubes were installed. 10.0 ml of aqueous sample extract from step
2.0 was added, eluted, and eluate collected. Extract solution may be stored at
or below 4 °C.
B. AMP A Purification Procedures for Solid Matrix Samples
Two procedures were used for MCX SPE purification of AMPA. A MCX
SPE Filtration Purification procedure was originally developed for corn matrices to
filter matrix from extract with no retention of AMPA. Subsequently, a MCX SPE
Purification procedure was developed to reduce matrix suppression with soybean
matrices. The MCX SPE Purification Procedure is suitable for use with for all solid
matrix samples. Purification procedure is also used for watery (e.g. plum) and acid
(e.g. lime) crop types.
AMPA MCX SPE Filtration Purification
1.0 Oasis MCX SPE cartridge sequentially was conditioned with 1 CV (CV=6 ml)
of methanol and 1 CV Extraction Solution A (96% Aq. 0.1% HCOOH/
4% MeOH). Slight vacuum can be applied to control elution at slow drip
(1-2 ml/min). Slight vacuum was applied or continued just until dripping
stopped.
2.0 15 ml centrifuge tubes were installed under SPE cartridges in vacuum
manifold and 0.25 ml of 100 ng/ml internal standard was applied to top of
sorbent bed in each SPF cartridge.
3.0 4.0 ml of the C18 filtered extract was applied to MCX SPE cartridge. After
dripping stopped, 4.0 ml of methanol was applied to MCX SPE cartridge.
Slight vacuum may be applied, if necessary. Positive pressure or vacuum can
be applied to recover methanol remaining on SPE cartridge.
4.0 Samples were recovered from vacuum manifold and were evaporated to less
than 4 ml on N-Evap at 45-50°C.
5.0 0.1 ml of aqueous 1M phosphoric acid then diluted to final volume of 5.0 ml
with water. Cap and vortex final extract.
6.0 An aliquot of the final extract for LC/MS/MS analysis was filtered (0.2 µm
nylon). Final solution may be stored at or below 4°C.
AMPA MCX SPE Purification
1.0 4 ml of the C18 filtered extract was diluted to 20 ml with methanol in
50-ml centrifuge tube containing 0.25ml of 100ng/ml internal standard and
applied to MCX SPE cartridge. Slight vacuum may be applied, if necessary.
Note: Precipitate may be observed in the dilute methanol/aqueous extract
solution of some matrices.
2.0 Oasis MCX SPE cartridge was conditioned sequentially with 1 CV (CV=6 ml)
of methanol and 1 CV of Extraction Solution A (96% Aq. 0.1% HCOOH/
4% MeOH):methanol (1:4, v:v) solution. Slight vacuum may be applied to
control elution at slow drip (1 -2 ml/min). Slight vacuum was applied or
continued just until dripping stops.
3.0 The diluate extracted sample (step 1.0) is applied to MCX SPE cartridge.
Slight vacuum may be applied if necessary.
4.0 2 ml of methanol was added to sample tube, mixed and added to SPE cartridge
for quantitative transfer and sorbent rinse. Vacuum or positive pressure was
applied just until dripping stops.
5.0 15-ml centrifuge tubes were installed under SPE cartridges in vacuum
manifold.
6.0 4.0 ml of HPLC grade water was applied to MCX SPE cartridge. After
dripping stopped, 4.0 ml of methanol was applied to MCX SPE cartridge.
Slight vacuum may be applied, if necessary. Positive pressure or vacuum was
applied to recover methanol remaining on SPE cartridge.
7.0 Samples from vacuum manifold were recovered and samples were evaporated
to less than 4 ml on N-Evap at 45-50°C.
8.0 0.1 ml of aqueous 1M phosphoric acid + 0.25 ml of 100 ng/ml internal
standard was added to sample, then diluted to final volume of 5.0 ml with
water. Cap and vortex final extract.
9.0 An aliquot of the final extract was filtered (0.2 µm nylon) for LC/MS/MS
analysis. Final solution may be stored at or below 4°C.
C. Glyphosate and N-acetylglyphosate and N-acetyl AMP A Analyte Purification
Procedures in Solid Matrix Samples
Two procedures were applied to MAX SPE extracts purification for
glyphosate and N-acetylglyphosate and N-acetyl AMPA analysis. A MAX SPE
purification following extract (adjusted to basic pH) dilution in methanol procedure
was generally used for corn matrices and soybean forage and hay matrices. MAX
SPE purification with diulution in methanol can be used for acid (e.g. lime) crop
types. A MAX SPE purification following extract dilution in water procedure was
used for the analysis of soybean seed, meal, and hull matrices. A MAX SPE
purification with dilution in water procedure was used for water (e.g. plum) crop
types.
MAX SPE Purification (Extract Dilution in Methanol Procedure
1.0a Except for acid crops, 2.0 ml of C18 purified extract was transferred (from C18
SPE Filtration, Step 4.0) to a 50-ml graduated centrifuge tube containing 80
µL ammonium hydroxide solution (minimum 25%) and 0.25 ml of 100 ng/ml
internal standard, then dilute to approximately 20 ml with methanol.
1.0b For acid crops, 2.0 ml of C18 purified extract (from C18 SPE Filtration, Step
4.0) was transferred to a 50-ml graduated centrifuge tube containing 0.1 ml
Triethylamine (TEA) and 0.25 ml of 100 ng/ml internal standard, then diluted
to approximately 20 ml with methanol. Note: The volume of extract applied
to the MAX SPE cartridge may be varied over range of 1. 0 to 4.0 ml. The
amount of base added (ammonium hydroxide or TEA) should be adjusted
appropriately and the amount of 100 ng/ml internal standard should be
adjusted for any change to final extract volume in Step 8.0 (0.05 ml internal
standard/ml of final extract volume), but the dilution to 20 ml with methanol,
rinse volumes, and 8 ml elution should not be changed.
2.0a Except for acid crops, MAX SPE (6 cc/500 mg) cartridges were conditioned
with 1 CV (CV = 6 ml) of methanol, followed by 2 CV's of 0.25% ammonium
hydroxide in 95% methanol/water solution. Vacuum was applied as needed to
control flow to 2-5 ml/min.
2.0b For acid crops, MAX SPE (6 cc/500 mg) cartridges were conditioned with 1
CV (CV = 6 ml) of methanol, followed by 2 CV's of 0.1% TEA in
80% methanol/water solution. Vacuum was applied as needed to control flow
to 2-5 ml/min.
3.0 As the last of the conditioning solution enters the sorbent, the base-adjusted
sample extract solution was loaded (Step 1.0). May need to apply slight
vacuum for grain samples, but keep drip rate slow.
4.0 After the last of the sample solution enters the sorbent, sequentially 15 ml of
80% methanol/water, 10 ml of 0.1M acetic acid in 80% methanol/water, and
10 ml of 95% methanol/water was added to rinse the SPE cartridge. Note:
The 15 ml of 80% methanol/water and the 10 ml of 0. 1M acetic acid were
sequentially added to and dispensed from respective emptied sample extract
tubes for quantitative transfer of sample extract to the SPE cartridges. The
final 10 ml of 95% methanol/water rinse were applied directly to the SPE
cartridge in 2 x 5 ml aliquots.
5.0 After dripping stops, vacuum was increased briefly to remove excess solution
from SPE sorbent, and collection vial or tubes in vacuum manifold were
installed. Note: 50-ml glass centrifuge tubes (reused) or 20-ml glass
scintillation vials (discarded after use) were used for sample collection.
Collection container with flat, rounded, or gently sloped bottom for fastest
evaporation were used.
6.0 Analytes were eluted in 2 x 4 ml aliquots of Elution Solution (1% TFA in
methanol/water, 90/10). Elute was performed by gravity feed. At least 5
minutes after first aliquot passes through SPE cartridge was waited before
adding the second aliquot. Positive pressure or vacuum was applied to recover
methanol remaining on SPE cartridge.
7.0 Samples were removed from SPE tank and evaporated to complete dryness on
N-Evap at 45-50°C. Note: An additional 15 minutes of drying to insure TFA
was completely evaporated was allowed.
8.0 5.0 ml of aqueous 0.02M phosphoric acid was added to sample. Sample were
capped, vortex mixed, sonicated at least 5 min, and vortex mixed. Note: If
other volume of C18 extract was used (Step 1.0), the final extract volume and
composition should be adjusted so concentration of analytes are consistent
(e.g., if 1.0 ml of extract from C18 SPE were processed through this
purification procedure, the final extract samples should be reconstituted in
2.5 ml with the addition of O. 125 ml of 100 ng/ml internal standard in
Step 1.0).
9.0 An aliquot of the final extract solutions were filtered (0.2 -µm nylon) into an
autosampler vial for LC/MS/MS analysis.
MAX SPE Purification (Extract Dilution in Water Procedure
1.0 2.0 ml of C18 purified extract was transferred to a 50-ml graduated centrifuge
tube containing 0.25 ml of 100ng/ml internal standard and diluted to
approximately 20 ml with HPLC grade water. Note: The volume of extract
applied to the MAX SPE cartridge may be varied over range of 1.0 to 4.0 ml.
The amount of 100 ng/ml internal standard should be adjusted for any change
to final extract volume in Step 8.0 (0.05-ml internal standard/ml of final
extract volume), but the dilution to 20 ml with water, rinse volumes, and 8-ml
elution should not be changed).
2.0 MAX SPE (6cc/500mg) cartridges were conditioned with 1 CV (CV=6 ml) of
methanol, followed by 2 CV's of 0.25% ammonium hydroxide in HPLC grade
water. Vacuum was applied as needed to control flow to 2-5 ml/min.
3.0 As the last of the conditioning solution entered the sorbent, the sample extract
solution was loaded. May need to apply slight vacuum for grain samples, but
keep drip rate slow.
4.0 After the last of the sample solution entered the sorbent, sequentially 15 ml of
80% methanol/water, 10 ml of 0.1M acetic acid in 80% methanol/water, and
10 ml of 95% methanol/water was added to rinse the SPE cartridge. Note: The
15 ml of 80% methanol/water and the 10 ml of 0. 1M acetic acid should be
sequentially added to and dispensed from respective emptied sample extract
tubes for quantitative transfer of sample extract to the SPE cartridges. The
final 10 ml of 95% methanol/water rinse should be applied directly to the SPE
cartridge in 2 x 5 ml aliquots.
5.0 After dripping stops, vacuum was increased briefly to remove excess solution
from SPE sorbent, then collection vials or tubes were installed in vacuum
manifold. Note: 50-ml glass centrifuge tubes (reused) or 20 ml glass
scintillation vials (discarded after use) were used for sample collection. Use
collection container with flat, rounded, or gently sloped bottom for fastest
evaporation.
6.0 Analytes were eluted in 2 x 4 ml aliquots of Elution Solution (1% TFA in
methanol/water, 90/10). Elution was by gravity feed. At least 5 minutes
passed after first aliquot passed through SPE cartridge before the second
aliquot was added. Positive pressure or vacuum was applied to recover
methanol remaining on SPE cartridge.
7.0 Samples were removed from SPE tank and evaporated to complete dryness on
N-Evap at 45-50°C. Note: Allow additional 15 minutes of drying to insure
TFA is completely evaporated.
8.0 5ml of aqueous 0.02M phosphoric acid was added to sample. Sample was
capped, vortex mixed, sonicated at least 5 min, and vortex mixed. Note: If
other volume of C18 extract was used (step 1.0), the final extract volume and
composition should be adjusted so concentration of analytes are consistent
(e.g, if 1.0 ml of extract from C18 SPE were processed through this purification
procedure, the final extract samples should be reconstituted in 2.5 ml with the
addition of 1.25 ml of 100 ng/ml internal standard).
9.0 An aliquot of the final extract solutions was filtered (0.2 nm nylon) into an
autosampler vial for LC/MS/MS analysis.
D. Glyphosate, N-acetylglyphosate, and AMP A and N-acetyl AMP A Extraction
and Purification Procedure in Oil Matrix Samples
1.0 For each sample 2.0 ± 0.02 g of sample was weighed into a clean
50-ml glass or polypropylene centrifuge tube.
2.0 For Fortified Samples, samples were fortified appropriately as
indicated above.
3.0 15.0 ml of aqueous 0.02M phosphoric acid was added to sample.
4.0 15.0 ml of methylene chloride was added to sample, which was then
capped, vortex mixed for at least 30 seconds.
5.0 Sample was centrifuged at 3000 rpm for approximately 10 minutes.
6.0 Aqueous fraction was transferred to clean 50-ml graduated cylinder.
(As much of the fraction as possible was recovered without disturbing
the organic layer.)
7.0 Another 15.0 ml of aqueous 0.02M phosphoric acid was added to
sample, which was then capped, vortex mixed for at least 30 seconds.
8.0 Sample was centrifuged at 3000 rpm for approximately 10 minutes.
9.0 Aqueous fraction were combined with 1st partition in respective 50-ml
graduated cylinder. (As much of the fraction as possible was
recovered without disturbing the organic layer.
10.0 Aqueous sample was diluted to 40 ml final volume with aqueous
0.02M phosphoric acid.
11.0 Final sample extract was transferred to clean bottle. (Sample was
poured back and forth between graduated cylinder and bottle to insure
homogeneity.)
12.0 If stable isotope glyphosate and AMPA internal standards were used,
the final sample was prepared by combining 4 ml of extract + 0.25 ml
of 100 ng/ml IS + 0.75 ml of aqueous 0.02M phosphoric acid and
sample was filtered (0.2 µm) prior to LC/MS/MS analysis. If stable
isotope internal standards are not used, dilute 4 ml of the extract with
1 ml of aqueous 0.02M phosphoric acid and filter (0.2 µm) prior to
LC/MS/MS analysis.
Example 5 - Detection of Analytes
An Agilent HP 1100 HPLC and a Waters Quattro Premier triple quadrupole mass
spectrometer were used for LC/MS/MS analysis. Typical equipment components and
operating conditions follow:
Agilent HP1100 G1322A vacuum degasser, G1311A quaternary
HPLC: pump, G1367A chilled autosampler, G1330A
chiller, G1316A column compartment
Injection Volume: 25 µL (may be varied to correct for MS
sensitivity)
Guard Column Waters Nova-Pak C18
(optional, preferred): (3.9 mm i.d. x 20 mm, 4 µm diameter particle)
HPLC Column: Phenomenex Luna Phenyl-Hexyl
(15.0 cm x 4.6 mm i.d., 3 µm diameter particle)
Column Temperature: 40°C
Mobile Phases: A = aqueous 0.2M formic acid
B = methanol
Waters Quattro ESI interface, MassLynx Version 4 SP4 software
Premier:
Interface: electrospray (ESI)
Polarity: positive ion
Mode: MRM
The Approximate Analyte Retention Times (ordered by retention time) shown in table
28 are as follows:
AMPA = 4.6 min
glyphosate = 5.3 min
N-acetyl AMPA = 7.1 min
N-acetylglyphosate = 7.4 min
Note: Analyte retention times can shift based on condition of the HPLC column and
formic acid mobile phase. Expect retention times to shorten with column
deterioration. Lower concentration of formic acid in the mobile phase extends the
retention times and broadens the peak shape of the analytes.
Calibration Procedures
Standard mass spectrometer tuning and calibration techniques were used. If
confidence in the mass calibration needed to be established (modern mass
spectrometers under digital control generally do not need frequent mass calibration,
especially for quantitative modes), vendor recommended calibrating solution was
used. Optimization tuning of MS system may be accomplished by infusion of one or
more of the test analytes. This method uses internal and external calibration
standards, prepared as described above.
Instrument calibration was based on the average response factor (analyte peak
area response/analyte concentration) of external calibration standards using Excel®
functions AVERAGE, STDEV (standard deviation), and RSD (relative standard
deviation StDev/mean). For average response factor calibration, a %RSD of less than
or equal to 20% should be observed. The linear regression response of external
calibration standards using Excel® functions SLOPE, INTERCEPT, and RSQ (r-
squared; the square of the Pearson product moment correlation coefficient determined
using Excel® function RSQ) were monitored to establish calibration curve linearity.
Acceptance criteria for valid quantitation are: (1) RSQ value >0.99 for calibration
curve and (2) the %RSD factors. Alternative approaches including linear regression with or without weighting
(e.g., 1/X) may be used if they provide an equivalent or more consistent fit of sample
response to the response of calibration standards.
The nominal LC/MS/MS calibrated range for glyphosate and N-
acetylglyphosate and N-acetyl AMPA was 0.5 - 20.0 ng/ml and for AMPA was 1.0 -
50.0 ng/ml. The LOQ equivalent final extract concentration for glyphosate and N-
acetylglyphosate analysis is 1.0 ng/ml. The LOQ equivalent final extract
concentration for AMPA analysis is 2.0 ng/ml. Generally, 5 calibration solutions
were analyzed for quantitative LC/MS/MS analysis (a minimum of 4 calibration
solutions are required).
Net recoveries may be calculated for fortified samples only (not acceptable for
field samples). Net recoveries may be calculated and reported only when residues in
the control sample are integrable and are >50% of the LOQ, the recovery samples prepared at the LOQ using that control
are invalidated. When the control residues are (mg/kg) found in fortified samples are calculated by subtracting area counts found in
the control from area counts found in fortified samples. If net recoveries are
calculated, those results must be uniquely identified or presented in a separate
spreadsheet column heading for corrected ppm (mg/kg).
Sample Analysis
Preliminary runs of at least 2 calibration standards were routinely made to
demonstrate adequate instrument response and insure the LC/MS/MS system was
equilibrated. If multiple sets were analyzed, a solvent blank injection was made
between the last and first injections of the sets to minimize risk of carryover between
sets. Calibration standard analyses preceded the first sample analysis and follow the
last sample analysis so sample analyses were contained within the external standard
calibration. Generally, the injection sequence was organized from lowest to highest
expected analyte concentrations. Calibration standard runs were intermixed with the
test samples and can be analyzed before and after every 1-3 samples in each
analytical set. Extracts and calibration standards were be refrigerated if stored.
Generally, fortification sample recoveries (70-120%) are required for acceptable
quantitation results in an analysis set.
Calculations
Methods
Glyphosate, N-acetylglyphosate, and AMPA and N-acetyl AMPA residues
were measured as mg/kg (ppm) glyphosate free-acid equivalents in crop matrices.
Quantitation was based on an average response factor determined from the multiple
calibration standards concurrently analyzed with sample extracts. All calculations
were made using unrounded values that were reported to two significant figures.
Fortified sample recoveries were reported to the nearest whole number percentage
(%)•
The calculation to determine mg/kg found in residue samples by average
response factor analysis follows:

With internal standard calibration:

where,
PA is Analyte Peak Area,
FV is Final extract Volume (ml),
XV is total extract Volume (ml),
IS is peak area of Internal Standard in sample extract,
ARFIS is Average Response Factor with Internal Standard

AF is Aliquot Factor (ml of XV diluted to FV),
SW is Sample Weight (5.0 g) of sample aliquot extracted, and
UC Units Conversions
µg/1000 ng x mg/1000 µg x 1000 g/kg = mg g/1000 ng-kg

Examples Without Internal Standard
N-acetylglyphosate in corn grain sample: Grain 10X 062905-1
(Reference: Table 2, Figure 8, Tables 30-40)

Examples With Internal Standard
Glyphosate in soybean seed sample: Soy Seed AX 10X11004
(Reference: Table 10, Figure 16, Table 30-40)

AMP A in soybean seed sample: Soy Seed CX L1 1004
(Reference: Table 10, Figure 16, Table 30-40)

Results
Detector Response
A triple quadrupole mass spectrometer using positive ion ESI and tandem
mass spectrometry detection was used for sample extract analysis. Full-Scan total ion
and MRM spectra for glyphosate, N-acetylglyphosate, and AMPA and N-acetyl
AMPA from analysis of standard solutions are provided in Fig. 1- Fig. 3, respectively.
Calibration standards typically yielded a linear response (r2 >0.99) with %RSD
range of 0.5-20 ng/ml for glyphosate and N-acetylglyphosate or 1.0-50 ng/ml for
AMPA. Representative calibration curves for each analyte were constructed using
calibration standards from validation sets including expanded range of 0.5-100 ng/ml
and are presented in Fig. 4 Representative ion chromatograms of Calibration
Standards are provided in Fig. 6. Representative ion chromatograms of stable isotope
glyphosate and AMPA standards are provided in Fig. 25.
Representative chromatograms of extracts from an untreated control sample, a
0.050 ppm (LOQ) fortification sample, and a 0.50 ppm fortification sample are
provided in Fig. 8-Fig. 21 for corn and soybean matrices.
Controls
No significant matrix interference was observed at the chromatographic
retention times of glyphosate, AMPA, or N-acetylglyphosate elution in
chromatograms of control extracts for corn and soybean matrices. Because
genetically modified plants and glyphosate containing herbicides are widely used in
soybean crop grown in the U.S., commercially available samples (including organic)
generally contain glyphosate and AMPA residues. Soybean control samples used in
this study were untreated controls from field plots in regulatory studies.
Recoveries (Accuracy & Precision)
Recovery results for com matrices are provided in Tables 1 - Table 8 (forage,
grain, stover, oil, flour, grits, starch, and meal, respectively). Recovery results for
soybean matrices are provided in Table 9 - Table 14 (forage, seed, hay, oil, meal, and
hulls, respectively). The average results at the 0.050 mg/kg (LOQ) and 0.50 mg/kg
fortification levels with overall results in corn and soybean matrices are provided in
tables in the Summary section. Representative recovery results from individual
sample set analyses for corn grain, corn oil, soybean seed, and soybean meal are
provided in Tables 30-40.
Extraction Efficiency
In the metabolism study, corn forage, grain, or stover samples were extracted
3 times in 0.1% formic acid/methanol (96/4, v:v) using tissumizer homogenization
and wrist-action shaker. The greatest extract to sample ratio applied in the procedure
was 6 to 1 (150 ml to 25 g), although the ratio varied by commodity and extraction
repetition.
In this residue analytical method, the sample size was reduced to 5.0 g and the
solvent to sample ratio for each commodity and repetition was at least 5 to 1. Table
41 contrasts the sample amounts and ratios used in metabolism and residue methods
for each matrix.

The extraction procedure in this residue analytical method is consistent with or
exceeds the extraction procedure applied to quantitatively extract glyphosate related
residues from crops.
Incurred residue samples from regulatory field studies were analyzed for
glyphosate and AMPA residues by a post-column derivatization fluorescence (PCD-
fluor) method (Cowell et al. (1986) J. Agric. Food Chern. 34:955-960) in the original
studies and by this LC/MS/MS method. The corn in these studies contained the
EPSPS enzyme variant genetic modification and therefore the N-acetylglyphosate
metabolite was not formed. Table 42 summarizes results in mg/kg (ppm) glyphosate
free-acid equivalents for untreated (control) and treated corn grain samples collected
from 2 test sites.

Table 43 summarizes results in mg/kg (ppm) glyphosate free-acid equivalents for
untreated (control) and treated corn grain, forage, and stover samples collected from 2
test sites of the field study.

Analytical results from both methods indicate consistent results for glyphosate
and AMPA in corn and support the extraction efficiency for the analytical method.
Limit of Quantitation (LOQ)
The LOQ validated in this method was 0.050 ppm (mg/kg) for glyphosate, N-
acetylglyphosate, and AMPA in corn and soybean matrices. The LOQ is defined as
the lowest fortification level at which average recoveries of 70-120% and a RSD
consistently represents a signal-to-noise ratio of approximately 5-20 to 1 for the least
responsive analyte, AMPA.
Background Evaluation
Background levels experienced in tandem mass spectrometry analyses are
minimal. Generally, the chromatographic profiles of a sample extract solution and a
calibration standard solution appear the same. The control sample chromatograms for
each matrix tested are provided in Figure 9-Figure 21.
Limit of Detection (LOD)
The LOD is defined as the analyte concentration in matrix with a response
equivalent to a signal-to-noise ratio (s/n) of approximately 3 to 1. The LOD was
estimated from the s/n response determined in a LOQ fortification sample using the
following equation for each analyte.

The LOD estimates for were 0.004 mg/kg for glyphosate, 0.006 mg/kg for N-
acetylglyphosate, and 0.007 mg/kg for AMPA and 0.006 mg/kg for N-acetyl AMPA.
The individual chromatograms showing s/n determination and calculated estimates for
each analyte are provided in Figure 26. Variation in the LOD was observed and each
lab using this method should estimate an LOD value.
Two independent MS/MS transitions of the molecular ion for glyphosate and
N-acetylglyphosate were monitored. The relative response ratios of the two fragment
ions (base peak/secondary peak) were determined from calibration standard responses
for confirmation of analyte in matrix samples. Acceptable confirmation criteria are a
co-eluting peak (+ 5%) and equivalent ion ratio (± 30%) compared to the average
response observed in calibration standards at or above the LOQ equivalent
concentration concurrently analyzed with the samples. The calculated response ratios
and retention times for each analyte determined in an analysis set are shown in Tables
43-48. Confirmation for glyphosate and AMPA can be determined using existing
regulatory methods. See, for example, Cowell et al. (1986) J. Agric. Food Chem. 34:
955-960; Alferness et al. (April 3, 1993) "Touchdown: Determination of Glyphosate
and Aminomethylphosphonic Acid in Corn Grain, Corn Forage, and Corn Fodder by
Gas Chromatography and Mass-Selective Detection". Zeneca Ag Products Analytical
Method RR 92-042B, Available at U.S. EPA Pesticides: Analytical Methods &
Procedures website rwww.epa.gov/oppbeadl/methods/raml2b.htm): and, Method No.
405 in Manual of Pesticide Residue Analysis Volume I and II available from BfR
Federal Institure for Risk Assessment, Official analytical methods for residues of
plant protection products and pesticides (L 00.00 16) (www.bfr.bund.de/cd/1652).
Additional validation trials for this method were conducted. Glyphosate,
Af-acetylglyphosate, and AMPA were fortified at 0.050 and 0.50 ppm levels in corn
(forage, grain, and stover) and soybean (forage, seed, and hay) matrices. A
VirTishear™ (Virtis Company Inc., Gardiner, NY) homogenizer was used instead of a
Tissumizer™ homogenizer to macerate tissues during extraction. An Applied
Biosystems/MDS SCIEX API 4000 mass spectrometer was used instead of the
Quattro Premier. Injection volumes were increased to compensate for decreased mass
spectrometer sensitivity. Acceptable results were found. Data not shown. The API
4000 LC-MS/MS System instrumental conditions are provided in Table 49 and Figure
27-30.
In summary, the analytical methods presently disclosed are suitable for the
quantitation of glyphosate, N-acetylglyphosate, and AMPA and /V-acetyl AMPA
residues in corn and soybean matrices. The results support an LOQ of 0.050 mg/kg
(ppm) with estimated LOD values of 0.004 mg/kg for glyphosate, 0.006 mg/kg for iV-
acetylglyphosate, and 0.007 mg/kg for AMPA.
This analytical method procedure was successfully applied to plum and lime
matrices to demonstrate suitability in watery and acidic crop matrices, respectively,
even though these crop types do not contain the gat trait and N-acetylglyphosate.
Overall average recoveries for each analyte and matrix in the validation trials
ranged from 75% (glyphosate in soybean hulls) to 109% (AMPA in soybean oil) with
maximum RSD of 19% (AMPA in corn oil).
Residue confirmation for glyphosate and N-acetylglyphosate was
demonstrated at 0.050 mg/kg (LOQ) and 0.50 mg/kg fortification levels based on
retention time and the relative ratios of two MS/MS parent-to-fragment ion transitions
detected during sample analysis.
Example 6.
Summary
/V-acetylglyphosate, aminomethyl-phosphonic acid (AMPA), and N-acetyl
aminomethylphosphonic acid (/V-acetyl AMPA) were examined. This study was
designed to demonstrate the utility, ruggedness, and efficiency of the method. The
method is designed to measure glyphosate and metabolites in plant matrices with a
limit of quantitation (LOQ) of 0.05 ppm. The method's reported LOQ of 0.05 ppm in
glyphosate equivalents for both glyphosate and metabolites was confirmed by
obtaining individual recoveries within the acceptable range of 70 to 120% for controls
fortified five times at 0.05 ppm in grapes and soybean seed.
Performance of the subject method was successfully validated for the
quantitation of glyphosate and metabolites in grapes and soybean seed at the method
LOQ and respective tolerance levels for each matrix. The method was successfully
validated in grapes with one trial. Two trials were required for successful validation
of soybean seed. A summary of the results is presented in the following table.

A minor method modification made during the course of this method was to
dilute the final soybean seed extracts of the 20 ppm fortification samples 100-fold
prior to LC/MS/MS analysis to adjust residue concentrations within the calibration
curve. Instrument parameters were also adjusted to increase sensitivity.
This LC/MS/MS method with multiple reaction monitoring (MRM) detection
was free of interferences at the retention times corresponding to glyphosate and
metabolites in unfortified samples. Acceptable recoveries (70 to 120%) were
achieved for five replicate analyses of control samples fortified at two levels in grapes
and soybean seed. One recovery of 126% of N-acetyl AMPA in soybean seed was
accepted on the basis that recoveries for soybean seed were consistent and average
recoveries of this metabolite at each fortification level were within the acceptable
range of 70 to 120%.
For all matrices, unfortified control samples showed no detectable residues of
glyphosate or metabolites.
Fortification levels in this study were chosen to provide method performance
data at the method LOQ and at the proposed tolerance levels. The LOQ was 0.050
ppm in glyphosate equivalents for all analytes in all matrices. Grapes were fortified
with all analytes at the proposed tolerance level of 0.20 ppm in glyphosate equivalents
while soybean seed was fortified at the proposed tolerance level of 20.0 ppm in
glyphosate equivalents. Results presented in the summary tables reflect the actual
concentration of each compound; not glyphosate equivalents.
The analytical method was performed without any significant modifications.
Successful validation sets were achieved on the second attempt for soybean seed and
the first attempt for grapes. This independent laboratory validation study
demonstrated that the analytical method is acceptable for the quantitation of
glyphosate and metabolites in soybean seed and grapes.
Test System
The subject method is applicable for the quantitation of glyphosate and
glyphosate metabolites in various crop matrices. Grapes and soybean seed were
chosen to validate the analytical method. The grape control matrix was purchased
from an outside source (organic food store). The sample was stored frozen and
processed prior to being analyzed to verify that the control was free of interferences at
the appropriate retention times. The soybean seed control matrix was provided by
ABC Laboratories, Inc., 7200 E. ABC Lane, Columbia, MO.
Equipment
The following equipment items were used in the conduct of this independent
laboratory validation.
Instrumentation/Chromatography:
MDS Sciex API 4000 LC-MS/MS System, comprised of:
MDS Sciex API 4000 MS/MS, Serial No. V04560403 (Applied
Biosystems Group, Foster City, CA), equipped with a TurboIonSpray
interface and Analyst software version 1.4
HPLC Column: 4.6 mm i.d. x 150 mm, Phenomenex Luna Phenyl Hexyl,
Serial No. 208752-1,3-µm diameter packing Part No. 00F-4256-E0
(Phenomenex, Torrance, CA)
Ten Port Electrically Actuated Valve, Serial No. EM2M06183
(Valco Instruments Co. Inc., Houston, TX)
Shimadzu LC-10ADVP HPLC pumps, Serial Nos. C2096 41 53748US
and C2096 41 53747 US (Shimadzu US Manufacturing Inc., Columbia,
MD)
Shimadzu SiL-HTC Autosampler, Serial No. L2002 42 50137US
(Shimadzu US Manufacturing Inc., Columbia, MD)
Shimadzu CTO-10AVP Column Oven, Serial No. C2102 41 50408
(Shimadzu US Manufacturing Inc., Columbia, MD)
Shimadzu DGU-14A Degasser, Serial No. SS132668
(Shimadzu US Manufacturing Inc., Columbia, MD)
Phenomenex C18 Guard Column, 4x3 mm, Part No. AJO-4287
Solid-Phase Extraction Equipment/Supplies:
24-port SPE vacuum manifold (Burdick and Jackson, Muskegon, MI)
Bond Elut SPE cartridges: C18, 500 mg/6 cc, Cat No. 12102052, Lot No.
0723704 (Varian, Inc. Palo Alto, CA)
Oasis MAX SPE cartridges, 500 mg/6 ml, Cat. No. 1860000865, Lot No.
001336341A (Waters Corporation, Millford, MA)
Oasis MCX SPE cartridges, 500 mg/6 ml, Cat. No. 1860000776, Lot No.
002236322A (Waters Corporation, Millford, MA)
Labware
15 ml Polypropylene Centrifuge Tubes, Part No. 20171-024
(VWR, West Chester, PA 19380)
50 ml Polypropylene Centrifuge Tubes, Part No. 89004-367
(VWR, West Chester, PA 19380)
Borosilicate glass scintillation vials with cap, 20ml Part No. 986546
(Wheaton, Millville NJ 08332)
HPLC vials, 2 ml, Part No. 5182-0716
(Agilent Technologies, Palo Alto, CA 94306)
HPLC vial caps, Part No. 5182-0717
(Agilent Technologies, Palo Alto, CA 94306)
Disposable Transfer Pipettes, 3 ml, Part No. 16001-176
(VWR, West Chester, PA 19380)
Pyrex graduated cylinders, 100 ml, with stoppers, Part Nos. CLS2982250
and CLS3022250 (Sigma-Aldrich, St. Louis, MO 63103)
HDPE widemouth polypropylene bottles, 250 ml, with linerless cap,
Part No. 209548SP (Wheaton, Millville, NJ 08332)
Syringe filter, Nylon 0.45 µm, 30-mm diameter filter unit, Part. No.
F2500-1
(National Scientific, Rockwood, TN 37854)
Syringe filter, Nylon 0.20 µm, 17-mm diameter filter unit, Part. No.
F2513-2
(National Scientific, Rockwood, TN 37854)
Reagents
Acetone - HPLC-grade, Catalog No. AX0115-1, EMD
(Chemicals, Gibbstown, NJ)
Acetic Acid - Glacial, Catalog No. 9515-03 (J.T. Baker, Philipsburg, NJ)
Acetonitrile - HPLC-grade, Catalog No. AX0145-1 (used for LC/MS/MS
needle rinse only) (EMD Chemicals, Gibbstown, NJ)
Ammonium Hydroxide - 28% NH3 in water 99.99+% pure (Sigma-
Aldrich, St. Louis, MO 63103)
Methylene Chloride - HPLC-grade, Catalog No. DX0838-1 (EMD
chemicals, Gibbstown, NJ)
Formic acid, 99.0 % pure, Fluka, Catalog No. 06440 (Sigma-Aldrich, St.
Louis, MO 63103)
Methanol-HPLC-grade Catalog No. MX0475-1 (EMD Chemicals,
Gibbstown, NJ)
Phosphoric acid - Baker Analyzed 86.0 % pure, Catalog No. 7664-38-2
(J.T. Baker, Philipsburg, NJ)
Trifluoroacetic acid, 99.0 % pure, Fluka, Catalog No. 91703 (Sigma-
Aldrich, St. Louis, MO 63103)
Water - Ultra high purity, obtained from Purelab Classic UV UHP
Water System
Principles of the Analytical Method
Glyphosate, N-acetylglyphosate, AMPA, and N-acetyl AMPA were extracted
from grape and soybean seed samples into dilute aqueous acid/methanol (96/4, v/v)
using a probe homogenizer. Three extractions were made for quantitative recovery of
analytes and then continued with purification and analysis.
Purification of glyphosate and N-acetylglyphosate: An aliquot of extract was
partitioned with methylene chloride and the aqueous fraction was recovered and
filtered to remove particulates. Approximately 10 ml of the aqueous fraction was
collected following filtration through a C18 SPE cartridge. An aliquot of the eluate
collected from the C18 SPE was diluted and applied to a MAX SPE cartridge. The
analytes were eluted from the MAX sorbent in 1% TFA in methanol/water (90/10)
solution following several solution rinses. The MAX eluate was evaporated to
dryness and redissolved in aqueous 0.02 M aqueous phosphoric acid, filtered, and
analyzed for glyphosate and N-acetylglyphosate. Following partitioning, soybean
seed samples were subjected to a steam bath for approximately 15 minutes to
precipitate additional material in the extract prior to particulate filtration.
Purification of AMP A and N-acetyl AMPA: A second aliquot of the eluate,
collected from the C18 SPE described above, was processed through a MCX SPE
cartridge. Samples were applied to the cartridge, and then eluted with water and
methanol. An N-Evap is used to blow down solvents; a concentrated aqueous
solution of phosphoric acid was added so that the final solution equals 0.02 M
aqueous phosphoric acid. The samples were then analyzed for AMPA and N-acetyl
AMPA. Soybean seed samples were diluted in methanol before application to the
cartridge.
Modifications, Interpretations, and Critical Steps
A minor method modification was made for the validation of glyphosate in
soybean seed. The final extracts were diluted 100-fold prior to LC/MS/MS analysis
to incorporate residues within the calibration curve.
Some API 4000 LC/MS/MS instrumental parameters were modified to
optimize sensitivity. The modifications included: increasing source temperature,
adjusting collision energy potential and adjusting gas flow settings.
In Step 2.2 of example 4A of the method, the temperature for the steam bath
was not specified. During correspondence with the Sponsor, a temperature of 85 °C
was recommended to more effectively precipitate protein from the matrix.
In Step 5.0 of example 4C, use of a 50 ml or 20 ml collection container with a
flat or gently sloped bottom was recommended. From this, it was assumed that a
15-ml centrifuge tube would be adequate. However, based upon the low recoveries
obtained in soybean seed Trial 1, Set 3, it was determined that a 20-ml scintillation
vial, which has a flatter bottom and more surface area, is necessary for effective
evaporation.
Instrumentation
Chromatography: Reversed-phase liquid chromatography was used to
separate glyphosate and its metabolites from co-extractants. A Phenomenex Luna
Phenyl Hexyl column was selected.

LC/MS/MS Analysis
Analysis of glyphosate and its metabolites was performed using a MDS Sciex
API 4000 LC/MS/MS, equipped with a TurboIonSpray source, and operated in MRM,
positive ion mode. Quantitation was based on the integrated area of a single ion
transition for each analyte using a linear regression with a 1/x weighting on Analyst
software version 1.4. A summary of representative experimental conditions is
provided in Table 56.

Calibration Procedure
Calibration standards were embedded with samples in each set and typically
progressed from low to high concentrations. The response factor of each calibration
standard was calculated by dividing the analyte peak area of each standard by the
analyte concentration for that standard. The average response was calculated for
calibration standards injected with each set.
Results and Discussion
Detector Response
Calibration standards were analyzed over the range of 0.5 to 100 ng/ml. The
response of the MS/MS detector was linear with 1/x weighting over the range of
standards analyzed. Detector response was stable throughout the course of each
analytical run as was demonstrated by standard accuracy values.
Control Samples
There were no interference peaks detected at the retention time for either
glyphosate or its metabolites in duplicate unfortified control samples analyzed
concurrently with each validation trial.
Trial 1, Soybean Seed
The first trial for soybean seed (Set 3) failed due to low recoveries observed
for glyphosate and each metabolite. Results of Trial 1 are summarized in table 57.
The first trial for soybean seed was analyzed according to the procedure given
in the analytical method, except that glyphosate and AMPA stable isotopes were not
used as internal standards and the collection vessel for MAX SPE was not consistent
with the larger volume and dimensions recommended in the method.
Following the analysis of the first trial for soybean seed it was determined that
the low recoveries of glyphosate were likely due to samples not being completely dry
from blow down, and the lack of internal standard. The no detection of N-acetyl
AMPA was likely related to retention time shift from residual TFA in final solution
due to samples not being completely dry from blow down. Further all method
development and validation work had been performed at temperatures of at least 80°C
during the steam bath steps. The temperature of the steam bath in the first trial for
soybean seed was 45°C. This may have not promoted proteins within the matrix to
precipitate out, which could have suppressed the analytes during LCMS/MS analysis.
The addition of glyphosate and AMPA internal standards would also adjust for
suppression and drift during LCMS/MS analysis.
Trial 2, Soybean Seed
The second soybean seed trial (Set 4) was analyzed according to the procedure
given in the analytical method with clarifications in correspondence following the
failure of the first trial. A set of new calibration standards, with the internal standards
incorporated, were prepared for analysis of the second tryout. The glyphosate extracts
were blown down in 20-ml glass scintillation vials to ensure complete dryness.
Internal standard was added to all samples before analysis.
Recoveries for glyphosate, AMPA, and N-acetylglyphosate were within the
acceptable range of 70 to 120%. N-acetyl AMPA had one recovery of 126%, which
is above the acceptable range of 70 to 120%. However, these results were accepted
on the basis that they were consistent, and the average recovery values for each
fortification level were within the acceptable range of 70 to 120%, thus establishing
successful method validation for the analysis of glyphosate and metabolites in
soybean seed. Calibration data and exemplary chromatograms are not shown. The
recovery data for glyphosate and metabolites are presented in table 58.

Trial 1, Grapes
Successful recovery data were obtained for grapes in the first trial (Set 2).
Refer to Figure 31 though Figure 34 for plots of calibration data and Figure 35
through Figure 58 for example chromatograms from Trial 1 for grapes. The recovery
data for these validation trials are presented in table 59.

Conclusion
This study demonstrated that the analytical method is acceptable for the
quantitation of glyphosate and metabolites in soybean seed and grapes. The method's
stated LOQ of 0.05 ppm was demonstrated by acceptable recovery values from
controls fortified at this level in both matrices. Acceptable method performance at the
proposed tolerance limits for glyphosate in grapes (0.2 ppm) and soybean seed
(20 ppm) was demonstrated by acceptable recoveries obtained from control samples
fortified at these levels in each matrix. A summary of the data appears in Tables 50-
67.
Example 5. Analytical Method for the Determination of N-acetylglyphosate
and Other Analytes in Various Matrices using LC/MS/MS
Summary
An analytical method was developed for the determination of N-
acetylglyphosate, glyphosate, AMPA, and N-acetyl AMPA in animal matrices
including milk, eggs, muscle, kidney, liver, and fat. The method target limit of
quantitation (LOQ) in glyphosate equivalents for each analyte was 0.025 mg/kg in
egg, milk, and muscle matrices and 0.050 mg/kg in kidney, liver, and fat matrices.
The method was validated at the respective LOQ and 10xLOQ level. Milk and egg
matrices were validated at 0.025 mg/kg (LOQ), 0.050 mg/kg (2xLOQ), and 0.5 mg/kg
(20xLOQ). For each matrix using a LC/MS/MS system operating with an
electrospray interface (ESI) in positive or negative ion mode detection. This
analytical method was developed to support residue data collection in livestock
feeding studies required for registration of genetically modified crops.
For milk and egg matrices, matrix samples were diluted in aqueous 0.1%
formic acid/methanol (96/4, v/v) and shaken to dilute sample in aqueous medium.
Samples may be stored frozen or extracted promptly without freezing. The dilute
sample was partitioned with hexane (after thawing if frozen) and the hexane layer
discarded. The remaining aqueous fraction was partitioned with methylene chloride
and the aqueous layer was collected. The methylene chloride fraction was back
extracted with additional 0.1% formic acid/methanol (96/4, v/v) for quantitative
recovery of analytes. The aqueous fractions were combined and diluted to final
volume 50 mL. An aliquot of the aqueous fraction was filtered through a C18 SPE
cartridge. The C18 filtered extract was further purified by solid phase extraction
using polymeric anion exchange (MAX) SPE cartridge and/or polymeric cation
exchange (MCX) SPE cartridge, depending on matrix and analytes to be examined.
Glyphosate and/or AMPA stable isotope standards used as internal standards were
added to extracts prior to ion exchange SPE purification. Final extracts were filtered
prior to LC/MS/MS analysis.
For animal tissue matrices, matrix samples were initially blended with C18
sorbent material (matrix solid phase dispersion) prior to extraction in 0. 1N HCl
solution (96% water/4% methanol) followed by water for final extract volume of 50
mL. An aliquot of the extract was diluted in acetonitrile and methanol to precipitate
proteins, then purified by solid phase extraction using polymeric anion exchange
(MAX) SPE cartridge and/or polymeric cation exchange (MCX) SPE cartridge,
depending on matrix and analytes to be examined. Glyphosate and/or AMPA stable
isotope standards used as internal standards were added to extracts prior to ion
exchange SPE purification. Final extracts were filtered prior to LC/MS/MS analysis.
Final extract and calibration standard solutions were adjusted to 0.02M
phosphoric acid. Samples and standards were analyzed using HPLC with reverse
phase chromatography and a triple quadrupole mass spectrometer with an electrospray
source, operating in positive ion LC/MS/MS mode.
The recoveries from matrix samples fortified at the respective LOQ and higher
levels support the satisfactory performance of this method. Tables 68-71 summarize
the average recovery results for N-acetylglyphosate, glyphosate, N-acetyl AMPA, and
AMPA in sample matrices. Liver, fat, and muscle include samples from cow and
chicken. Cow was source of kidney samples.
Reference Analytical Standards
Reference standards were supplied by DuPont Crop Protection, E. I. du Pont
de Nemours and Company, Wilmington, DE. Information pertaining to the
characterization and stability of the reference standards along with chemical retain
samples are archived by E. I. du Pont de Nemours and Company, DuPont Crop
Protection, Newark, Delaware. The reference standards in solid form were stored at
room temperature in a dessicator typically in the presence of desiccant. Stable isotope
standards of glyphosate and AMPA used for internal standards were acquired from
Dr. Ehrenstorfer GmbH (Atlanta, Georgia).
Principle of the Analytical Method
For milk and egg commodities, matrix samples (2 g) were diluted in aqueous
0.1% formic acid/methanol (96/4, v/v) and shaken to dilute sample in aqueous
medium. The dilute sample was partitioned with hexane and the hexane layer
discarded. The remaining aqueous fraction was partitioned with methylene chloride
and the aqueous layer was collected. The methylene chloride fraction was back
extracted with additional 0.1% formic acid/methanol (96/4, v/v) for quantitative
recovery of analytes. The aqueous fractions were combined and diluted to final
volume of 50 mL. An aliquot of the aqueous fraction was filtered through a C18 SPE
cartridge. The C18 purified extract was further purified by solid phase extraction
using polymeric anion exchange (MAX) SPE cartridge and/or polymeric cation
exchange (MCX) SPE cartridge, depending on matrix and analytes to be examined.
For MAX SPE, an aliquot of the C18 eluate and internal standard were diluted to 20
mL with water and applied to a conditioned SPE cartridge. The MAX sorbent is
sequentially rinsed with methanol/water (80/20), 0.1M acetic acid in methanol/water
(80/20) and methanol/water (95/5). The analytes were eluted in 1% TFA in 90%
methanol/10% water solution and the eluate was evaporated to dryness then restored
in final solution for LC/MS/MS following mixing and filtration for the analysis of
glyphosate and N-acetylglyphosate and/or N-acetyl AMPA. For MCX SPE, an
aliquot of the C18 eluate and internal standard were eluted through a conditioned SPE
cartridge followed by methanol rinse. The load and methanol rinse from the MCX
SPE cartridge were collected and the methanol was evaporated, then extract was
diluted to final volume containing 0.02M phosphoric acid, mixed, and filtered for
LC/MS/MS for the analysis N-acetylglyphosate, AMP A, and/or N-acetyl AMPA
depending on the sample matrix.
For animal tissue commodities, samples (2 g) were blended with C18 sorbent
material (4 g) until tissue was macerated and homogenized (matrix solid phase
dispersion). Prepped samples were extracted in 25 mL of 0. 1N HClsolution (96%
water/4% methanol) using vortexing and mechanical shaking. The extraction solution
was decanted, from sample after centrifugation, then sample was re-extracted with
water for quantitative transfer of analytes to final extract volume of 50 mL. Aliquots
of the extract were purified by solid phase extraction using polymeric anion exchange
(MAX) SPE cartridge and/or polymeric cation exchange (MCX) SPE cartridge,
depending on matrix and analytes to be examined. For MAX SPE purification, an
aliquot of the extract was diluted in acetonitrile and methanol in the presence of
triethylamine (adjusts pH basic to promote protein precipitation and prepare analytes
for loading on anion exchange medium). Following centrifugation to isolate
precipitants in a pellet, the extract solution was diluted with methanol to
approximately 20 mL and loaded onto a conditioned MAX SPE cartridge. The MAX
sorbent was sequentially rinsed with methanol/water (80/20), 0.1M acetic acid in
methanol/water (80/20) and methanol/water (95/5). The analytes were eluted in 1%
TFA in 90% methanol/10% water solution and the eluate was evaporated to dryness
then restored in final solution for LC/MS/MS following mixing and filtration for the
analysis of glyphosate and N-acetylglyphosate and/or N-acetyl AMPA. For MCX
SPE, an aliquot of the extract was diluted in acetonitrile and methanol to promote
protein precipitation. Following centrifugation to isolate precipitants in a pellet, the
extract solution was diluted with methanol to approximately 20 mL and loaded onto a
conditioned MCX SPE cartridge. The MCX sorbent was rinsed with methanol, then
the analytes were eluted in water (4 mL) followed by methanol (4 mL) for
quantitative recovery. The methanol in the collected eluate was evaporated and
solution was: adjusted to final volume containing 0.02M phosphoric acid, mixed, and
filtered for LC/MS/MS for the analysis AMPA.
Glyphosate and/or AMPA stable isotope standards used as internal standards
were added to extracts prior to ion exchange SPE purification. Final extracts were
prepared in aqueous 0.02M phosphoric acid and filtered (0.2 µm) prior to LC/MS/MS
analysis to remove particulates as preventive maintenance measure for the HPLC
system. Phosphoric acid acts a weak ion-pairing agent on HPLC polymeric stationery
phase and was used as the final solution to improve glyphosate LC/MS/MS
performance (response and linearity). The analytes were resolved by HPLC reverse-
phase chromatography using a phenyl-hexyl column coupled to electrospray
ionization in with MS/MS detection to acquire 2 molecular ion transitions (only 1 ion
transition is monitored for AMPA in positive ion mode). Quantitative analysis was
accomplished using a single molecular ion transition. The relative abundance of the 2
MS/MS fragment ions provided confirmatory evidence for glyphosate,
iV-acetylglyphosate, N-acetyl AMPA, and AMPA (negative mode).
Analytical Procedure
Preparation & Stability of Reasent Solutions
The following procedures may be adjusted to prepare different volumes.
Aqueous 0.1% formic acid/methanol (96/4, v/v) Extraction Solution: Per
liter volume, add 40 mL of methanol followed by 0.96 mL of formic acid to a
1-L graduated cylinder and dilute to final volume with HPLC grade purified water.
Transfer solution to a clean bottle and cap. The solution may be stored at room
temperature and should be prepared at least monthly.
0.1N HCl in water/methanol (96/4, v/v) Extraction Solution: Per liter
volume, add water in a 1-L graduated cylinder to at least half volume followed by 8.3
mL of concentrated HCl. Add 40 mL of methanol and dilute to final volume of 1-L
with HPLC grade purified water. Transfer solution to a clean bottle and cap. The
solution may be stored at room temperature and should be prepared at least weekly.
80% or 95% Methanol in Water Solution: Per liter volume, add 800 mL or
950 mL, respectively, of methanol to a 1-L graduated cylinder and dilute to final
volume with HPLC grade purified water. Transfer solution to a clean bottle and cap.
The solution may be stored at room temperature and should be prepared at least
monthly.
0.1 M Acetic Acid in 80% Methanol/Water Solution: Per liter volume, add
5.73 mL acetic acid to 200 mL of HPLC grade purified water in a 1-L graduated
cylinder and dilute to final volume with methanol. Transfer solution to a clean bottle
and cap. The solution may be stored at room temperature and should be prepared at
least monthly.
0.25% Ammonium Hydroxide in Water: Per liter volume, add 10 mL of
ammonium hydroxide solution (minimum 25%) to small volume of HPLC grade
purified water, then dilute to final volume with HPLC grade purified water. This is a
conditioning solution for egg and milk MAX SPE (12 mL/sample, dilution in water
procedure). The solution may be stored at room temperature and should be prepared
at least monthly. Note: 1 mL of 25% ammonium hydroxide diluted to 100 mL ˜
0.25% NH4OH
0.1% Triethylamine (TEA) in methanol/acetonitrile (75/25, v/v): Per liter
volume, add 1.0 mL of TEA to 750-mL methanol in a 1L graduated cylinder and
dilute to final volume with acetonitrile. This is a conditioning solution for tissues
MAX SPE (12 mL/sample, dilution in methanol procedure). The solution may be
stored at room temperature and should be prepared at least monthly.
Elution Solution, 1% TFA in 90% methanol/10% water: Prepare sufficient
volume needed for analysis. For 100 mL volume, add 1 mL of trifluoroacetic acid to
about 10 mL of methanol in a 100 mL graduated cylinder, followed by 10 mL of
HPLC grade purified water, then dilute to final volume with methanol. Transfer
solution to a clean bottle and cap. 8 mL of the Elution Solution is required for each
sample (100 mL preparation is consistent with 12 samples requiring 96 mL). Prepare
as needed, do not store.
1.0M Phosphoric Acid Solution: Per 10 mL volume, add 0.67 mL of
concentrated phosphoric acid (min. 85%) to HPLC grade purified water in a 15-mL
polypropylene centrifuge tube and dilute to final volume using gradations on tube
with HPLC grade purified water. The solution may be stored at room temperature
and should be prepared at least monthly.
Sample and Standard Final Solution, aqueous 0.02Mphosphoric acid: Per
liter volume, add 1.34 mL concentrated phosphoric acid (min. 85%) to HPLC grade
purified water in a 1-L graduated cylinder and dilute to final volume of 1000 mL with
HPLC grade purified water. Transfer solution to a clean bottle and cap. The solution
may be stored at room temperature and should be prepared at least monthly.
Aqueous 0.2M Formic Acid, aqueous mobile phase: Per liter volume, add 8.3
mL concentrated formic acid (98%) to HPLC grade purified water in a 1-L graduated
cylinder and dilute to final volume of 1000 mL with HPLC grade purified water.
Transfer solution to a clean bottle and cap. The solution may be stored at room
temperature and should be prepared at least monthly.
Stock Standard Preparation and Stability
If possible standards with purity greater than 95% were used. A minimum of
approximately 10 mg of standard was weighed on an analytical balance that provides
a weight precision to three significant figures, or the amount of standard should be
increased to satisfy this condition.
Since residue tolerances were established in glyphosate free-acid equivalents,
stock standard solutions for each analyte were prepared in glyphosate free-acid
equivalents so that fortifications and recoveries can be determined in parent free acid
equivalents. As needed, individual parent free-acid equivalent stock standards
solutions for N-acetylglyphosate, glyphosate, N-acetyl AMPA, or AMPA were
prepared by adding appropriate amounts of standard to a 100-mL volumetric flask and
diluting to final volume with water. Water refers to HPLC grade or equivalent water.
The following calculation and example provide guidance for the preparation of 100
mL of a 100 (µg/mL stock solution in glyphosate equivalents for each analyte.

Amounts weighed for each analyte should be at least 10 mg. Individual analyte stock
solution concentrations can exceed 100 ng/mL in glyphosate equivalents. Stock
standard solutions may be prepared at higher concentrations (not to exceed 1 mg/mL).
A minimum standard weight of approximately 10 mg and final standard volume of at
least 10 mL should be observed.
Internal Standard Preparation and Stability
Glyphosate 1,2-13C2 15N and aminomethyl phosphonic acid 13C 15N (AMPA)
stable isotope standards were supplied in amber ampules containing 1.1 mL of
aqueous solution at a nominal concentration of 100 mg/L (µg/mL). Each standard
solution was transferred to a 15 mL polypropylene centrifuge tube and diluted to 5
mL volume with HPLC grade water rinses of the original container for quantitative
transfer of the standard material. Final concentration for stock standard solution was
approximately 20 µg/mL. The isotopic purity was verified for each standard by
analysis of an approximately 500 ng/mL solution. See, figure 74.
An intermediate 100 ng/mL internal standard solution containing glyphosate
and AMPA isotopes was prepared by diluting 100 ng/mL stock solution at a rate of
0.1 mL in 100 mL of aqueous 0.02M phosphoric acid or HPLC grade purified water.
Internal standards were included in final extract and calibration standard solutions at a
rate of 50 µL/mL. Each 100 mL of a 100 ng/mL standard solution can be used in up
to 400 samples when 250 µL of internal standard is applied in 5 mL of final extract.
Specifically, individually transfer the 100 ng/mL standards to a 15 mL
polypropylene centrifuge tube and dilute to 5 mL volume with HPLC grade water
rinses of the original container for quantitative transfer of the standard material. Final
concentration for stock standard solution were approximately 20 µg/mL. For each
stock solution, 0.025 mL of the 20 µg/mL standard solution was transferred to a
autosampler vial and dilute to 1 mL volume with 0.975 mL of 0.02M H3P04 to
prepare separate solutions at approximately 500 ng/mL for purity evaluation.
Next, 1.00 mL aliquot of each 20 µg/mL standard stock solution was
combined in a single 250 mL graduated cylinder and diluted to final volume with
0.02M H3PO4 to prepare a mixed solution at 100 ng/mL. 100 ng/mL IS solution was
transferred to a 250 mL bottle. Solution was store refrigerated (~4°C).
Intermediate and Fortification Standards Preparation and Stability
Stock solution for each analyte was diluted appropriately into a common
volumetric flask, dilute to volume with HPLC grade purified water, cap, and mix
well. For example, 1.00 mL of a 100 µg/mL stock solution for each required analyte
was combined in a 10-mL volumetric flask and diluted to final volume with water,
cap, and mix well. It was store at or below 4°C and replaced monthly.
Stock solutions concentrations will vary and the required volume of each
analyte stock solution used to prepare fortification solution was adjusted for correct
final concentration as determined using the following calculation.

For example, to prepare 50 mL of a 10 µg/mL fortification solution from
glyphosate, N--acetylglyphosate, N-acetyl AMP A, and AMPA stock solutions
prepared respectively at 115 µg/mL, 186 µg/mL, 143 µg/mL, and 206 µg/mL in
glyphosate free-acid equivalent concentrations the following stock solution volumes
were added to a 50-mL volumetric flask.
Glyphosate = 10 µg/mL x 50 mL /115 µg/mL = 4.35 mL stock
solution
N-acetylglyphosate =10 µg/mL x 50 mL /186 µg/mL = 2.69 mL stock
solution
N-acetyl AMPA = 10 µg/mL x 50 mL /143 µg/mL = 3.50 mL stock
solution
AMPA = 10 µg/mL x 50 mL / 206 µg/mL = 2.42 mL stock solution
10.0 ng/mL Fortification Solution
Each stock solution was diluted appropriately with water into a volumetric
flask, cap and mix well. For example, 1.00 mL of a 100 µg/mL stock solution was
combined in a 10-mL volumetric flask and diluted to final volume with water, cap and
mix well. It was stored at or below 4°C and replaced monthly.
1.0 µg/mL Fortification Solution
10.0 µg/mL fortification solution (preferred) or the stock solution for each
analyte was diluted appropriately with water into a volumetric flask. For example,
1.0 mL of the 10.0 µg/mL fortification solution was transferred to a 10-mL volumetric
flask, diluted to volume with water, caped and mixed well. It was stored at or below
4°C and replaced monthly.
Chromatographic Standard Preparation and Stability
Calibration standards were prepared from dilutions of fortification standards
or individual stock standards. A minimum of 5 calibration standards over a range
from approximately 50% of LOQ equivalent final concentration to >120% of the
highest expected final sample concentration is recommended for quantification. The
LOQ equivalent final concentration for N-acetylglyphosate was 1.0 ng/mL.
For example, calibration standards of 10.0 ng/mL and 100 ng/mL were
prepared from the 1.0 and 10.0 µg/mL fortification solutions, respectively, by diluting
a 100 µL aliquot to final volume of 10.0 mL in aqueous 0.02M phosphoric acid (this
simulates sample fortification). Calibration standards were prepared concurrently
with sample fortifications using this procedure.
Source (& Characterization) of Samples
Raw milk was obtained from the University of Delaware Agricultural Farm.
Milk commodities (whole, skim, and heavy cream), and eggs were obtained from
local supermarket. Beef liver, kidney, fat, and muscle were obtained from a cattle
feeding study. Poultry liver, fat, and muscle were obtained from a laying hen feeding
study.
Storage & Preparation of Samples
Whole eggs were beaten prior to subsampling. Egg whites and yolks were
separated and beaten prior to subsampling. Egg commodity subsamples (2 g) were
weighed into tared 50-mL polypropylene centrifuge tubes. If subsamples were not
analyzed promptly, 25 mL of aqueous 0.1% formic acid/methanol (96/4, v/v) was
added, capped, mixed, and the sample was frozen.
Raw, whole, skim, or heavy cream milk subsamples (2 g) were weighed into
tared 50-mL polypropylene centrifuge tubes. If subsamples were not analyzed
promptly, 25 mL of aqueous 0.1% formic acid/methanol (96/4, v/v) was added,
capped, mixed, and the sample was frozen.
Animal tissue samples were ground with dry ice using a Waring commercial
food processor (Lab Micronizer Model 31FP93) prior to freezer storage. Samples
were mixed extensively during the grinding process to ensure fine maceration and
homogeneity. The dry ice was allowed to sublime in a fume hood or overnight in
freezer prior to subsampling and freezer storage.
All frozen samples were stored at -20 ± 5°C prior to sample extraction, and
analysis.
Sample Fortification Procedure
Untreated matrix control samples in extraction containers (50-mL centrifuge
tubes) were fortified as required. The 10.0 and 1.0 µg/mL Fortification Standards
(see above) were used to fortify test samples. Table 75 is provided as an example for
the fortifications made in this study.
For each matrix sample, 2.0 ± 0.1 g was weighed into a clean 50-mL
centrifuge tube. For milk and egg commodities, the sample was gently swirled or
vortexed after fortification to disperse the analyte in the sample. For animal tissue
commodities, samples were allowed to stand in fume hood for approximately 15
minutes to allow fortification solution to dissipate. Following fortification, milk and
egg samples were diluted in 25 mL of aqueous 0.1% formic acid/methanol (96/4, v/v),
frozen, and stored prior to extraction and analysis.
Analyte Extraction Procedures
Separate analyte extraction procedures are provided for milk/eggs and animal
tissue commodities.
Milk and Egg Commodities
1.0 For freshly prepared samples, the samples were fortified as appropriate
and gently swirledor vortexed mixed after fortification to disperse the
analyte in the sample.
2.0 For freshly prepared samples, 25 mL of aqueous 0.1% formic
acid/methanol (96/4, v/v) was added to each sample, capped, vortex
mixed for approximately 5 seconds. Samples can be stored frozen for
later analysis.
3.0 If samples were stored frozen, sample were thawed in an ultrasonic
bath (approximately 15 min). Note: Samples may be shaken together
in a sample rack by holding a cover in place over tops of the tubes.
4.0 20 mL of hexane was added to sample, capped, and shaken gently for
at least 30 seconds.
5.0 Samples were centrifuged for 10 minutes at speed sufficient to resolve
partitions (upper hexane, aqueous, and formed precipitates).
6.0 As much of the upper hexane fraction was pipetted as possible without
disturbing the lower aqueous layer and discard (remaining hexane will
be incorporated in the methylene chloride fraction in the next step).
7.0 20 mL of methylene chloride was added to remaining sample, capped,
and shook gently for at least 30 seconds.
8.0 Samples were centrifuged for 10 minutes at speed sufficient to resolve
partitions (upper aqueous, formed precipitates, and lower methylene
chloride).
9.0 As much of the upper aqueous fraction was transferred to a clean 50
mL graduated cylinder (TC) as possible without disturbing the lower
precipitates and methylene chloride fraction.
10.0 20 mL of aqueous 0.1% formic acid/methanol (96/4, v/v) was added to
each sample, capped, and shaken gently for at least 30 seconds.
11.0 Steps 8.0 and 9.0 were repeated and extract was combined with first
extract in 50 mL graduated cylinder.
12.0 The combined extract was diluted to 50 mL with aqueous 0.1% formic
acid/methanol (96/4, v/v) in graduated cylinder or if extract volume
exceeds 50 mL, the volume was adjusted to mL reading and final
volume was recoreded.
13.0 The aqueous extracts were transferred to a clean 50-mL tube or bottle,
by poring extract back and forth and finally to clean container to mix.
Meat Tissue Commodities
1.0 2.0 ± 0.1 g of homogenized tissue was weighed in a tared 50-mL
polypropylene centrifuge tube.
2.0 Approximately 4 g of C18 sorbent was added to sample tube (4 g
sorbent equivalent to 7 mL fill in a 15-mL centrifuge tube).
3.0 Sample and C18 sorbent were mixed well with spatula for solid phase
dispersion of matrix.
4.0 25 mL of 0.1N HCl in 96% water/4% methanol was added, vortexed,
and shaken on wrist action shaker for 15 min.
5.0 10 min (3500 rpm minimum) of centrifugation was performed and
supernatant was decanted through reservoir equipped with
polyethylene frit into 50 mL polypropylene centrifuge tube. Vacuum
was briefly after all extracts have been added to start filtration then
vacuum was broken.
6.0 20 mL of water was added to sample pellet, the sample was vortexed to
re-suspend sample, centrifuged, then the sample solution was decanted
through reservoir with paper frit into respective 50 mL polypropylene
centrifuge tube. If needed, vacuum was applied briefly after all
extracts have been added to start filtration then vacuum was broken.
7.0 Common volume of water was added to each sample pellet to achieve
final extract near, but less than 50 mL for all extracts (generally
8-10 mL). The sample was vortexed and poured into reservoir,
vacuum was applied to collect final rinse. Sample collection tube was
removed and final volume was adjusted to 50 mL with water. Sample
extract can be centrifuged and decanted into reservoir after vortexing
to prevent frit plugging. This option is recommended for muscle
extracts.
Analyte Purification Procedures
C18 SPE filtration, MAX SPE and MCX SPE filtration procedures were
applied to milk (except skim) and egg commodity sample extracts.
C18 SPE Filtration (whole milk, cream, & egg commodities)
1.0 Waste collection tubes were installed in vacuum manifold to collect
conditioning solutions and initial sample load volume (waste collection
tubes were used to contain analyte containing eluate and prevent cross-
contamination).
2.0 C18 SPE (6cc/500mg, Varian #12102052) cartridges was conditioned
with ~1 mL of methanol, followed by 2 CV's (CV ˜ 6 mL) of aqueous
0.1% formic acid/methanol (96/4, v/v). Vacuum or positive pressure
was applied as needed for slow drip rate (1-2 mL/min).
3.0 As last of conditioning solution entered the sorbent, 4.0 mL of aqueous
sample extract from Analyte Extraction Procedure step 13.0 was added
onto SPE column.
4.0 After dripping stops, waste collection tubes were removed and clean
15 mL centrifuge tubes were installed. 10 mL of aqueous sample
extract from Analyte Extraction Procedure step 13.0 was added, eluted,
and eluate collected. Extract was capped and vortex mixed. Sample
can be centrifuged to partially clarify the solution (fine particulates
require higher centrifugation speed, e.g., 7000 rpm).
MAX SPE Purification (milk and egg commodities)
Analysis of glyphosate, N-acetylglyphosate, and N-acetyl AMPA in egg
matrices and skim milk. Analysis of glyphosate only in whole milk or cream.
1.0 0.25 mL of internal standard + 2.5 mL of C18 purified extract was
transferred to a 50-mL graduated centrifuge tube and diluted to
approximately 20 mL with HPLC grade water.
2.0 MAX SPE (6cc/500mg) cartridges were conditioned with 1 CV
(column volume, ~6 mL) of methanol, followed by 2 CV's of 0.25%
ammonium hydroxide in HPLC grade water.
3.0 As the last of the conditioning solution enters the sorbent, samle
extract solution was loaded. A slight vacuum may need to be applied,
but the drip rate is kept slow.
4.0 After the last of the sample solution enters the sorbent, sequentially
10 mL of 80% methanol/water, 10 mL of 0.1M acetic acid in 80%
methanol/water, and 10 mL of 95% methanol/water was added to rinse
the SPE cartridge. Note: The 10 mL of 80% methanol/water and the
10 mL of 0.1M acetic acid was sequentially added to and dispensed
from respective emptied sample extract tubes for quantitative transfer
of sample extract to the SPE cartridges. The final 10 mL of 95%
methanol/water rinse was applied directly to the SPE cartridge in 2x5
mL aliquots.
5.0 After dripping stops, the vacuum was increased briefly to remove
excess solution from SPE sorbent, then collection vial or tubes in
vacuum manifold was installed.
6.0 Analytes were eluted in 2 x 4 mL aliquots of Elution Solution (1%
TFA in methanol/water, 90/10) by gravity feed. At least 5 minutes
were waited after first aliquot passes through SPE cartridge before
adding the second aliquot. Positive pressure or vacuum was applied to
recover methanol remaining on SPE cartridge.
7.0 Samples were removed from SPE tank and evaporated to complete
dryness on N-Evap at 45-50°C. Note: An aadditional 15 minutes of
drying was alloed to insure TFA is completely evaporated.
8.0 5.0 mL of aqueous 0.02M phosphoric acid to sample was added.
Capped, vortex mixed, sonicated at least 5 min, and vortex mixed.
9.0 An aliquot of the final extract solutions was added into a autosampler
vial for LC/MS/MS analysis.
MAX SPE Purification (Meat Tissue Commodities)
Analysis of glyphosate, N-acetylglyphosate, and N-acetyl AMP A in liver and
kidney sample extracts. Analysis of glyphosate and N-acetylglyphosate in muscle
sample extracts.
1.0 1.25 mL (fat, kidney, or liver) or 2.5 mL (muscle) extract was tranfered
to a 15-mL polypropylene centrifuge tube and 0.1 mL of triethylamine
(TEA) was added to the sample. Note: For fat and kidney samples
Steps 1.0-3.0 can be combined and all dilutions performed in the 50
mL tube (Step 3.0) since little or no precipitate forms for these
matrices.
2.0 5 mL of acetonitrile was added to extract aliquot, capped, and vortexed
solution. 5 mL of methanol was added to sample solution, capped,
vortexed, and sample was rested for 10 min.
3.0 Samples were centrifuged 10 min at a speed sufficient to form sample
pellet and decanted into clean 50 mL polypropylene centrifuge tube
containing 0.125 mL of internal standard. Original tube was rinsed
with 7-8 mL of methanol and combined with extract in 50 mL tube for
a final volume of approximately 20 mL.
4.0 MAX SPE (6cc/500mg) cartridges were conditioned with 1 CV
(column volume, ~6 mL) of methanol, followed by 2 CV's of 0.1%
TEA in methanol/acetonitrile (75/25). Note: Approximately 12
mL/sample of the TEA solution was required.
5.0 As the last of the conditioning solution enters the sorbent, loading of
the sample extract solution began. Gravity elution was preferred, but
slight vacuum may need to be applied for slow drip.
6.0 After the last of the sample solution enters the sorbent, 10 mL of 80%
methanol/water, 10 mL of 0.1M acetic acid in 80% methanol/water,
and 10 mL of 95% methanol/water was sequentially added to rinse the
SPE cartridge. Note: The 10 mL of 80% methanol/water and the 10
mL of 0.1 M acetic acid should be sequentially added to and dispensed
from respective emptied sample extract tubes for quantitative transfer
of sample extract to the SPE cartridges. The final 10 mL of 95%
methanol/water rinse was applied directly to the SPE cartridge in 2x5
mL aliquots.
7.0 After dripping stops, vacuum is briefly increased to remove excess
solution from SPE sorbent, then collection vial or tubes was installed
in vacuum manifold. A flat, rounded, or gently sloped bottom can be
used for fastest evaporation.
8.0 Analytes in 2 x 4 mL aliquots of Elution Solution (1% TFA in
methanol/water, 90/10) were eluted by gravity feed. At least 5 minutes
was waited after first aliquot passes through SPE cartridge before
adding the second aliquot. Positive pressure or vacuum was applied to
recover methanol remaining on SPE cartridge.
9.0 Samples from SPE tank were removed and evaporated to complete
dryness on N-Evap at 45-50°C. Note: An additional 15 minutes of
drying was allowed to insure TFA is completely evaporated.
10.0 2.5 mL of aqueous 0.02M phosphoric acid to sample was added. The
sample was capped, vortex mixed, sonicated at least 5 min, and vortex
mixed.
11.0 An aliquot of the final extract solutions was filtered (0.2 urn nylon)
into an autosampler vial for LC/MS/MS analysis.
MCX SPE Filtration Purification
Analysis of AMP A in eggs and skim milk. Analysis of AMP A,
N-acetylglyphosate, and N-acetyl AMP A in whole milk or cream.
1.0 Oasis MCX SPE cartridge (6cc/500mg, Waters#186000776) was
conditioned sequentially with 1 CV (CV ˜ 6 mL) of methanol and 1
CV aqueous 0.1% formic acid/methanol (96/4, v/v). Slight vacuum
can be applied to control elution at slow drip (1-2 mL/min). Vacuum
was applied or continued just until dripping stops.
2.0 15 mL graduated centrifuge tubes was installed under SPE cartridges
in vacuum manifold.
3.0 0.25 mL of internal standard (only for AMPA analysis) + 4.0 mL of
the C18 filtered extract was applied to MCX SPE cartridge. After
dripping stops, 4.0 mL of methanol was applied to MCX SPE
cartridge. A slight vacuum can be applied, if necessary. Positive
pressure or vacuum was applied to recover methanol remaining on SPE
cartridge.
4.0 Samples were recovered from vacuum manifold and sample was
evaporated to less than 4 mL on N-Evap at 45-50°C.
5.0 0.1 mL of aqueous 1.0 M phosphoric acid was added and diluted to
final volume of with 5 mL water. Final extract was capped and
vortexed. Final volume may be adjusted to meet sensitivity
requirements for instrument, e.g., final volume of 4 mL including 0.08
mL of aqueous 1.0 M phosphoric acid with earlier addition of 0.20 mL
of 100 ng/mL internal standard would adjust final concentration at
LOQ to 2.0 ng/mL.
6.0 An aliquot of the final extract for LC/MS/MS analysis was filtered (0.2
nm nylon). Final solution may be stored at or below 4°C.
MCX SPE Purification (meat tissue commodities)
Analysis of AMPA in muscle, liver, kidney, or fat commodities.
1.0 2.5 of extract were transferred to a 50-mL polypropylene centrifuge
tube. 5 mL of acetonitrile was added to extract aliquot and the sample
was capped, and vortexed. The sample was centrifuged for 10 min.
Note: No significant precipitation may be observed in this solution
because the solution is not adjusted to basic pH.
2.0 For muscle: 0.125 mL of internal standard (only for AMPA analysis)
was added and diluted to approximately 20 mL with methanol.
For liver, kidney, or fat: 0.25 mL of internal standard (only for
AMPA analysis) was added and dilute to approximately 20 mL with
methanol.
3.0 Oasis MCX SPE cartridge (6cc/500mg, Waters# 186000776) was
conditioned sequentially with 1 CV (CV ˜ 6 mL) of methanol and 1
CV aqueous 0. 1N HCl in 96% water/4% methanol. Slight vacuum
may be applied to control elution at slow drip (1-2 mL/min). The
slight vacuum can be applied just until dripping stops.
4.0 Dilute sample was applied to MCX SPE cartridge. Vacuum may be
applied, if necessary.
5.0 2 mL of methanol was added to sample tube, mixed and added to SPE
cartridge for quantitative transfer and sorbent rinse. Vacuum or
positive pressure was applied just until dripping stops.
6.0 15-mL centrifuge tubes were installed under SPE cartridges in vacuum
manifold.
7.0 4.0 mL of HPLC grade water to MCX SPE cartridge was added. After
dripping stops, 4.0 mL of methanol to MCX SPE cartridge was
applied. Slight vacuum may be applied, if necessary. Positive
pressure or vacuum was applied to recover methanol remaining on SPE
cartridge.
8.0 For muscle: Samples from vacuum manifold were recovered and
sample was evaporated to less than 2.5 mL on N-Evap at 45-50°C.
For liver, kidney, or fat: Samples from vacuum manifold were
recovered and sample was evaporated to less than 4 mL on N-Evap at
45-50°C.
9.0 For muscle: 0.05 mL of aqueous 1M phosphoric acid was added and
sample was diluted to final volume of 2.5 mL with water. Final extract
was capped and vortexed.
For liver, kidney, or fat: 0.1 mL of aqueous 1M phosphoric acid was
added and sample was diluted to final volume of 5.0 mL with water.
Final extract was capped and vortexed.
10.0 An aliquot of the final extract for LC/MS/MS analysis was filtered (0.2
µm nylon). Final solution may be stored at or below 4°C.
Instrumentation
An Agilent HP 1100 HPLC and a Waters Quattro Premier or AB Sciex API
5000 triple quadrupole mass spectrometer were used for LC/MS/MS analysis.
Typical equipment components and operating conditions are as follows:
Agilent HP1100 HPLC: G1322A vacuum degasser, G1311A quaternary
pump, G1367A chilled autosampler, G1330A chiller,
G1316A column compartment
Injection Volume: 25 µL (may be varied to correct for MS sensitivity)
HPLC Column: Phenomenex Luna Phenyl-Hexyl
(15.0 cm x 4.6 mm i.d., 3 µm diameter particle)
Column Temperature: 40°C
Mobile Phases: A = aqueous 0.2M formic acid (positive ion) or
0.05% formic acid (negative ion)
B = methanol
Waters Quattro Premier: ESI interface, MassLynx Version 4 SP4 software
AB Sciex API 5000: ESI interface, Analyst Version 1.42 software
Interface: electrospray (ESI)
Polarity: Positive or negative ion
Mode: MRM
Calibration Procedures
Standard mass spectrometer tuning and calibration techniques were used. If
confidence in the mass calibration needs to be established (modern mass
spectrometers under digital control generally do not need frequent mass calibration,
especially for quantitative modes), use vendor recommended calibrating solution.
Optimization tuning of MS system may be accomplished by infusion of the test
analyte. This method uses external standards, prepared as described above.
Instrument calibration was based on the average response factor (analyte peak
area response/analyte concentration) of external calibration standards using Excel®
functions AVERAGE, STDEV, and RSD. For average response factor calibration,
a %RSD of less than or equal to 20% should be observed. The linear regression
response of external calibration standards using Excel® functions SLOPE,
INTERCEPT, and RSQ were monitored to establish calibration curve linearity.
Acceptance criteria for valid quantitation are: (1) RSQ value >0.99 for calibration
curve and (2) the %RSD factors. Alternative approaches including linear regression with or without weighting
(e.g., 1/x) may be used if they provide an equivalent or more consistent fit of sample
response to the response of calibration standards.
The LC/MS/MS calibrated range was 0.25 ng/mL to 50.0 ng/mL for
N-acetylglyphosate, glyphosate, and N-acetyl AMPA. The LC/MS/MS calibrated
range was 0.5 ng/mL to 50.0 ng/mL for AMPA. Generally, a minimum of
5 calibration solutions were analyzed for quantitative LC/MS/MS analysis.
Net recoveries may be calculated for fortified samples only (not acceptable for
field samples). If residues in the control sample can be integrated with signal-to-noise
response of at least 3-to-l, then net recoveries may be calculated by subtracting mg/kg
found in the control sample from mg/kg found in the fortified samples. When the
control residues are >50% of the LOQ, the recovery samples prepared at the LOQ
using that control are invalidated. If net recoveries are calculated, those results must
be uniquely identified or presented in a separate spreadsheet column heading for
corrected mg/kg.
Sample Analysis
Preliminary runs of at least 2 calibration standards were routinely made to
demonstrate adequate instrument response and insure the LC/MS/MS system was
equilibrated. If multiple sets were analyzed, a solvent blank injection was made
between the last and first injections of the sets to minimize risk of carryover between
sets. Calibration standard analyses preceded the first sample analysis and follow the
last sample analysis so sample analyses were contained within the external standard
calibration. Generally, the injection sequence was organized from lowest to highest
expected analyte concentrations. Calibration standard runs were intermixed with the
test samples and were analyzed before and after every 1-3 samples in each analytical
set. Extracts and calibration standards were refrigerated if stored. Generally,
fortification sample recoveries (70-120%) were required for acceptable quantitation
results in an analysis set.
Calculations
Methods
N-acetylglyphosate, glyphosate, N-acetyl AMPA, and AMPA residues were
measured as ppm (mg/kg) in glyphosate free acid equivalents for animal matrices.
Quantitation was based on an average response factor determined from the multiple
calibration standards concurrently analyzed with sample extracts. All calculations
were made using unrounded values that were reported to two significant figures.
Fortified sample recoveries are reported to the nearest whole number percentage (%).
The calculation to determine mg/kg found in residue samples by average
response factor analysis follows:
Without internal standard calibration:

where,
PA is Analyte Peak Area,
FV is Final extract Volume (mL),
XV is total extract Volume (mL),
ARF is Average Response Factor from standards in the analytical set,
IS is peak area of Internal Standard in sample extract,
ARFIS is Average Response Factor with Internal Standard from standards
in the analytical set,
AF is Aliquot Factor (mL of XV diluted to FV),
SW is Sample Weight (2.0 g) of sample aliquot extracted, and
UC Units Conversions
Hg/1000 ng x mg/1000 µg x 1000 g/kg = mg.g/1000 ng-kg
Percent recoveries (reported to the nearest whole number) from fortified
samples were calculated as follows:

Examples without internal standard
N-acetylglyphosate in Whole Milk LOQ Fortification, MCX cleanup
(RML1 MCX012307)

N-acetyl AMPA in Whole Eggs LOQ Fortification, MAX cleanup
(EG-0.025-3)

Example with internal standard
Glyphosate in Whole Milk LOQ Fortification, MAX cleanup

Results and Discussion
Method Validation Results
Detector Response
A triple quadrupole mass spectrometer using positive or negative ion ESI and
tandem mass spectrometry detection was used for sample extract analysis. Full-Scan
total ion chromatograms and spectra for N-acetylglyphosate, glyphosate, AMPA, and
N-acetyl AMPA from analysis of standard solutions are provided in Figure 59 through
Figure 62, respectively.
Calibration standards typically yielded a linear response (r-squared >0.99)
with %RSD over the range of 0.25-20 ng/mL for Glyphosate, N-acetylglyphosate and N-acetyl
AMPA or 0.5-50 ng/mL for AMPA. Representative calibration curves for each
analyte were constructed using calibration standards from a validation set and are
presented in Figure 63. Representative ion chromatograms of Calibration Standards
are provided in Figure 64. Representative ion chromatograms of extracts from a
Control a LOQ fortification, and 10xLOQ fortification sample extracts for milk, egg,
and tissue matrices are provided in Figure 65 through Figure 73, respectively.
Controls
No significant matrix interference was observed in the regions of N-
acetylglyphosate, glyphosate, or N-acetyl AMPA elution in chromatograms of control
extracts from matrix samples. AMPA was found in control chicken liver (0.011
mg/kg) and muscle (0.003 mg/kg) samples.
Recoveries (Accuracy & Precision)
Representative recovery results are provided in Table 80 through Table 82 for
milk matrices, Table 83 through Table 85 for egg matrices, and Table 86 through
Table 89 for animal tissue matrices. Average recovery results at each fortification
level with overall recovery results for each matrix are provided in the summary.
Representative results from individual sample set analyses for each commodity
including calibration standards statistics are provided in Tables 90-101.
Extraction Efficiency
Chicken liver, fat, and muscle samples treated with 14C N-acetylglyphosate in
a poultry metabolism study were extracted using procedures in described herein for
radiochemical validation of the extraction efficiency. Duplicate 2 g subsamples from
each matrix were extracted and aliquots from each extract were analyzed by liquid
scintillation counting (LSC) for radioactive recovery. Radioactive recovery results
(dpm/g) were converted using specific activity of original I4C N-acetylglyphosate test
substance (13.83 µCi/mg = 30703 dpm/(µg) to ng/g concentrations for comparison
with reported recoveries in the poultry metabolism study. The respective recovery
results for liver, fat, and muscle were 98%, 88%, and 104% of the µg/g concentrations
found in the metabolism study extractions. LSC data and results are provided in
Table 102.
Milk and egg samples are generally liquid matrices that were diluted in
aqueous solution prior to purification procedures of partitioning and solid phase
extraction SPE. No extraction efficiency testing was conducted for these matrices.
Limit of Quantitation (LOQ)
The LOQ determined in this method was 0.025 mg/kg (ppm) in milk, egg, and
muscle matrices and 0.050 mg/kg in kidney, liver, and fat matrices for the analysis
of glyphosate, N-acetylglyphosate, AMPA, and N-acetyl AMPA. The LOQ is defined
as the lowest fortification level at which average recoveries of 70-110% and a RSD
consistently represents a signal-to-noise ratio of approximately 5-20 to 1 for
N-acetylglyphosate.
Background Evaluation
Background levels experienced in tandem mass spectrometry analyses are
minimal. Generally, the chromatographic profiles of a sample extract solution and a
calibration standard solution appear the same. Representative matrix chromatograms
are provided in Figure 65 through Figure 73.
Limit of Detection (LOD)
The LOD is defined as the analyte concentration in matrix with a response
equivalent to a signal-to-noise ratio (s/n) of approximately 3 to 1. The LOD was
estimated from the s/n response determined in a LOQ fortification sample using the
following equation for each analyte.

The individual chromatograms showing s/n determination and calculated estimates for
each analyte are provided in Figures 75-80. The LOD estimates for each analyte and
matrix examined with are summarized in the table 79.
Variation in the LOD was observed and each lab using this method should
estimate LOD values. Sample extracts stored frozen should be thawed and sonicated
approximately 15 minutes prior to re-purification or analysis. Routinely, a
LC/MS/MS end run was setup so that high percentage organic solvent (99%
methanol) would continue overnight at a low flowrate (0.025 mL/min) after the final
analysis to keep the HPLC pump and column in good condition.
The stability of the analyte stock standards prepared in distilled water and
stored at approximately 4°C were shown to be stable for more than a year. Data not
shown. Fortification Standards, also prepared in distilled water, should be stable for
same time period as the stock solutions. Calibration standards were prepared in
aqueous 0.02M phosphoric acid or 80% control matrix/20% aqueous 0.02M
phosphoric acid were shown to be stable for at least 2.7 months when stored at
approximately 4°C.
No interferences attributable to glassware, reagents, or matrices were observed
to co-elute with test analytes. A peak was observed in the m/z 212?88 MRM
transition channel eluting just after N-acetylglyphosate that was attributable to MAX
SPE sorbent.
Confirmatory Procedure
Two independent MS/MS transitions of the molecular ion for
N-acetylglyphosate, glyphosate, and N-acetyl AMPA were monitored in ESI positive
or negative ion mode. Two independent MS/MS transitions of the molecular ion for
AMPA were monitored in ESI negative mode only. The relative response ratios of
the two fragment ions (base peak/secondary peak) were determined from calibration
standard responses for confirmation of analyte in matrix samples. Acceptable
confirmation criteria are a co-eluting peak (± 5%) and equivalent ion ratio (± 30%)
compared to the average response observed in calibration standards at or above the
LOQ equivalent concentration concurrently analyzed with the samples.
Representative confirmatory analyses showing calculated response ratios and
retention times for each analyte are provided in tables 103-106.
Conclusions
This analytical method is suitable for the quantitation of glyphosate,
N-acetylglyphosate, AMPA, and N-acetyl AMPA residues in milk, egg, and animal
tissue matrices. The results support an LOQ of 0.025 mg/kg glyphosate equivalents
in milk, egg, and muscle matrices, and an LOQ of 0.050 mg/kg in liver, kidney, and
fat matrices. The estimated LOD values for all analytes were less than or equal to
0.008 mg/kg glyphosate equivalents in milk, egg, and muscle matrices, and less than
or equal to 0.019 mg/kg glyphosate equivalents in liver, kidney, and fat matrices. The
estimated LOD for glyphosate in all matrices examined was less than or equal to
0.009 mg/kg glyphosate equivalents.
The mean recoveries for individual fortification levels of each analyte and
matrix in the validation trials ranged from 76% (AMPA in skim milk at 0.50 mg/kg)
to 110% (AMPA in Egg Yolks at 0.025 mg/kg). The maximum standard deviation for
individual fortification levels was 16% (iV-acetyl AMPA in liver at 0.050 mg/kg).
Residue confirmation for glyphosate, N-acetylglyphosate, AMPA, and N-
acetyl AMPA was demonstrated at the LOQ and lOxLOQ fortification levels based
on retention time and the relative ratios of two MS/MS parent-to-fragment ion
transitions detected during sample analysis.
The articles "a" and "an" are used herein to refer to one or more than one (i.e.,
to at least one) of the grammatical object of the article. By way of example, "an
element" means one or more element. As used herein, the term "about," when
referring to a value is meant to encompass variations of in some embodiments ± 20%,
in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±
1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the
specified amount, as such variations are appropriate to perform the disclosed methods
or employ the disclosed compositions.
All publications and patent applications mentioned in the specification are
indicative of the level of those skilled in the art to which this invention pertains. All
publications and patent applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was specifically and
individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be obvious
that certain changes and modifications may be practiced within the scope of the
appended claims.
THAT WHICH IS CLAIMED:
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) chromatographically separating said N-acetylglyphosate from
other constituents in said composition; and,
d) analyzing 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 analyzing the chromatographically
separated N-acetylglyphosate occurs by a mass spectrometer.
3. The method of claim 1 or 2, wherein chromatographically separating
the N-acetylglyphosate from the test sample comprises a high performance liquid
chromatography (HPLC) column.
4. The method of claim 2, wherein prior to chromatographically
separating the N-acetylglyphosate from the other constituents in said composition, a
sufficient concentration of phosphoric acid is added to the composition comprising
the N-acetylglyphosate, wherein said sufficient concentration allows for an improved
response and linearity of N-acetylglyphosate during detection.
5. The method of claim 2, 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 for said N-acetylglyphosate to be bound by said solid
phase extraction sorbent; 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.
6. The method of claim 6, further comprising disposing the eluted
composition comprising the N-acetylglyphosate from claim 5 step (a)(ii) or (b)(iii)
onto an anion exchange extraction column and eluting the N-acetylglyphosate
therefrom.
7. The method of any one of claims 1-6, wherein derivatization of said N-
acetylglyphosate does not occur.
8. The method of any one of claim 1 -7, further comprising determining
the presence or amount of aminomethyl phosphonic acid (AMPA), N-acetyl AMPA or
glyphosate in the test sample.
9. A method of determining the presence or amount of
N-acetylaminomethylphosphonic acid (N-acetyl AMP A) 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) separating said N-acetyl AMPA from other constituents in said
composition; and,
d) analyzing the separated N-acetyl AMPA to determine the
presence or amount of the in the test sample.
10. The method of claim 9, wherein separating said N-acetyl AMPA
comprises chromatographically separating said N-acetyl AMPA from the other
constituents in said composition and analyzing the chromatographically separated N-
acetyl AMPA by a mass spectrometer.
11. The method of claim 10, wherein chromatographically separating the
N-acetyl AMPA from the test sample comprises a high performance liquid
chromatography (HPLC) column.
12. The method of claim 10, wherein prior to chromatographically
separating the N-acetyl AMPA from the other constituents in said composition, a
sufficient concentration of phosphoric acid is added 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.
13. The method of any one of claims 9-12, 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 that allow for said N-acetyl AMPA to be bound by said solid phase
extraction sorbent; and,
ii) extracting the solid phase extraction sorbent and the bound
N-acetyl AMPA from the sample by liquid/liquid extraction; and,
iii) eluting said N-acetyl AMPA from the solid phase
extraction sorbent.
14. The method of claim 13, further comprising disposing the eluted
composition comprising the N-acetyl AMPA from step 13(a)(ii) or 13(b)(iii) onto an
anion exchange extraction column and eluting the N-acetyl AMPA therefrom.
15. The method of any one of claim 9-14, wherein derivatization of said N-
acetyl AMPA does not occur.
16. The method of any one of claim 9-15, further comprising determining
the presence or amount of aminomethyl phosphonic acid (AMPA), glyphosate or N-
acetyl glyphosate in the test sample.
17. 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 C18
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 that allow for said N-acetyl
AMPA or the N-acetylglyphosate to be bound by said
solid phase extraction sorbent;
ii) extracting the solid phase extraction sorbent and the
bound N-acetyl AMPA or the bound N-acetylglyphosate
from the sample by liquid/liquid extraction; and,
iii) eluting said N-acetyl AMPA or said N-acetylglyphosate
from the solid phase extraction sorbent;
c) disposing the eluted N-acetylglyphosate or N-acetyl AMPA
from step (b)(l)(ii) or (b)(2)(iii) 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.
18. 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; and,
e) analyzing 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.
19. 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; and,
d) analyzing 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.
20. The method of claim 19, 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.
21. The method of claim 19 or 20, wherein analyzing the
chromatographically separated glyphosate occurs by a mass spectrometer.
22. The method of any one of claims 19-21, wherein chromatographically
separating said glyphosate from the composition comprises a high performance liquid
chromatography (HPLC) column.
23. The method of any one of claimd 19-22, 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 that allow for said glyphosate to be bound by said solid phase
extraction sorbent; 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.
24. The method of claim 23, 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.
25. 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 (l)(i) onto a C18 extraction column and eluting the
glyphosate therefrom;
or,
2) i) mixing said test sample with a solid phase extraction
sorbent under conditions that allow for said glyphosate to be
bound by said solid phase extraction sorbent;
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)(1)(H) or (b)(2)(iii)
onto an anion exchange column comprising a surface functionally of m-
divinylben2;ene 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.
26. 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) chromatographically separating the AMPA from other
constituents in the composition; and,
d) analyzing 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.
27. The method of claim 26, wherein analyzing the chromatographically
separated AMPA occurs by a mass spectrometer.
28. The method of any one of claims 26-28, wherein chromatographically
separating said AMPA from the composition comprises a high performance liquid
chromatography (HPLC) column.
29. The method of any one of claims 26-28, wherein prior to
chromatographically separating the AMPA from the constituents in said composition,
a sufficient concentration of phosphoric acid is added to the composition comprising
said AMPA, wherein said sufficient concentration allows for an improved response
and linearity of AMPA during detection.
30. The method of any one of claims 26-29, 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 that allow for said AMPA to be bound by said solid phase extraction
sorbent; 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.
31. The method of claim 30, further comprising disposing the eluate of the
extraction column or solid phase extraction sorbent onto a cation exchange extraction
column and eluting the AMPA therefrom.
32. 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 C18 extraction column and eluting the
AMPA therefrom;
or,
2) i) mixing said test sample with a solid phase extraction
sorbent under conditions that allow for said AMPA to be bound
by said solid phase extraction sorbent;
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)(l)(ii) or (b)(2)(iii)
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.
33. A method for determining the presence or amount of at least one of
glyphosate, N-acetylglyphosate, N-acetylaminomethylphosphonic acid (N-acetyl
AMP A) or aminomethyl phosphonic acid (AMP A) 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.
34. The method of claim 33, 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; and,
(b) analyzing 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.
35. The method of claim 33 or 34, wherein said aqueous-organic
extraction comprises a methylene chloride and phosphoric acid partition.
36. The method of claim 34, wherein analyzing the chromatographically
separated glyphosate, N-acetylglyphosate, N-acetyl AMPA or AMPA occurs by a
mass spectrometer.
37. The method of claim 34 or 36, wherein chromatographically separating
said N-acetylglyphosate, the AMPA, the N-acetyl AMPA or the glyphosate from the
composition comprises a high performance liquid chromatography (HPLC) column.
38. A method for determining the presence or amount of at least one of
glyphosate, N-acetylglyphosate, N-acetylaminomethylphosphonic acid (N-acetyl
AMP A) or aminomethyl phosphonic acid (AMP A) 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 AMP A;
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.
39. A method of purifying at least glyphosate, N-acetyl AMPA or
N-acetylglyphosate from AMPA comprising:
a) providing a test sample suspected of containing at least one of
the glyphosate, the N-acetyl AMPA, the N-acetylglyphosate, or the AMPA;
b) extracting a composition comprising at least one of the N-
acetylglyphosate, the glyphosate or the AMPA from said test sample; and,
c) disposing said composition of step (b) onto an anion exchange
extraction column and eluting at least one of the N-acetylglyphosate, N-acetyl AMPA
and glyphosate therefrom and thereby purifying said glyphosate, N-acetyl AMPA and
N-acetylglyphosate from said AMPA; or,
disposing said composition of step (b) onto a cation exchange extraction
column and eluting said AMPA therefrom and thereby purifying said glyphosate, N-
acetyl AMPA and said N-acetylglyphosate from said AMPA.
40. The method of any one of claims 2, 10, 21, 27, or 36, wherein the mass
spectrometer is operated in positive ion mode.
41. The method of any one of claims 5, 17 or 30, wherein said liquid-
liquid extraction comprises an extraction with an aqueous mixture comprising
methanol and a dilute acid.
42. The method of any one of claim 1-41, wherein said test sample is from
an animal or an environmental sample.
43. The method of claim 42, wherein said test sample is from a plant.
44. The method of claim 43, 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.
45. The method of any one of claim 1-44, wherein said test sample
comprises a solid matrix sample.


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 containingN-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-acetyl glyphosate, 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.

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Patent Number 279579
Indian Patent Application Number 3452/KOLNP/2009
PG Journal Number 05/2017
Publication Date 03-Feb-2017
Grant Date 25-Jan-2017
Date of Filing 05-Oct-2009
Name of Patentee E.I. DU PONT DE NEMOURS AND COMPANY
Applicant Address 1007 MARKET STREET WILMINGTON, DE 19898 UNITED STATES OF AMERICA
Inventors:
# Inventor's Name Inventor's Address
1 PENTZ, ANNE, M. 108 PEACEDALE ROAD, LANDENBERG, PA 19350 UNITED STATES OF AMERICA
2 BRAMBLE, FREDERICK, Q., JR. 111 CORNWALL ROAD, REHOBOTH BEACH, DE 19971 UNITED STATES OF AMERICA
PCT International Classification Number A01N57/00; C07F9/38; G01N30/00
PCT International Application Number PCT/US2008/058104
PCT International Filing date 2008-03-25
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/908,336 2007-03-27 U.S.A.
2 12/051,220 2008-03-19 U.S.A.