6651 GLYPHOSATE HERBICIDE*

6651 A. Introduction

1. Sources and Significance

Glyphosate [N-(phosphonomethyl)glycine] is a broad-spectrum,

nonselective, postemergence herbicide that has found widespread agricultural and domestic use. It is sold as a terrestrial and

aquatic herbicide under the trade names Roundup and Rodeo.

Because of low mammalian toxicity (LD50  1568 mg/kg rats;

oral) there is less concern about water and food contamination

than with other pesticides, but the nonselectivity of the herbicide

can make nontarget phytotoxicity a problem. Glyphosate’s

(GLYPH) major metabolite is aminomethylphosphonic acid

(AMPA). Contamination of water can occur through runoff and

spray drift.

2. Selection of Method

Several methods for determination of GLYPH and AMPA in

environmental samples have been developed; those using liquid

chromatography are the most precise and accurate. GLYPH and

AMPA are not good chromophores or fluorophores and their

electrochemical or conductometric detection have not been demonstrated. Sensitive and selective detection has been achieved

with the post-column reaction/fluorometric method.1–3 The absence of a sensitive liquid chromatography technique for confirmation necessitates the use of two different stationary phases.

The liquid chromatographic method presented in 6651B is

accurate and precise3 and includes confirmation by using two

columns. Gas chromatography/mass spectrometry confirmation4

has been used when structural confirmation is required, but the

method has not been tested on residues in natural waters.

3. References

1. MOYE, H.A., C.J. MILES & S.J. SCHERER. 1983. A simplified highperformance liquid chromatographic procedure for the determination

of glyphosate herbicide and (aminomethyl)phosphonic acid in fruits

and vegetables employing postcolumn fluorogenic labeling. J. Agric.

Food Chem. 31:69.

2. COWELL, J.E., J.L. KUNSTMAN, P.J. NORD, J.R. STEINMETZ &

G.R. WILSON. 1986. Validation of an analytical residue method for

analysis of glyphosate and metabolite: An interlaboratory study. J.

Agric. Food Chem. 34:955.

3. OPPENHUIZEN, M.E. & J.E. COWELL. 1991. Liquid chromatographic

determination of glyphosate and (aminomethyl)phosphonic acid in

environmental water. J. Assoc. Offic. Anal. Chem. 74:317.

4. DEYRUP, C.L., S.M. CHANG, R.A. WEINTRAUB & H.A. MOYE. 1985.

Simultaneous esterification and acylation of pesticides for analysis by

gas chromatography. 1. Derivatization of glyphosphate and (aminomethyl) phosphonic acid with fluorinated alcohol-perfluorinated anhydrides. J. Agric. Food Chem. 33:944.

4. Bibliography

BARDALAYE, P.C., W.B. WHEELER & H.A. MOYE. 1985. Analytical techniques of glyphosate residue analysis. In E. Grossbard & D. Atkinson, eds. The Herbicide Glyphosate. Butterworths, Woburn, Mass.

6651 B. Liquid Chromatographic Post-Column Fluorescence Method

1. General Discussion

a. Principle: GLYPH and AMPA are separated by anion- or

cation-exchange chromatography and measured by post-column

fluorescence derivatization. The post-column reactions consist of

oxidation of GLYPH (a secondary amine) to glycine (a primary

amine) by hypochlorite solution. Glycine then reacts with an

o-phthalaldehyde (OPA) and mercaptoethanol (MERC) mixed

reagent to form an isoindole that is measured fluorometrically.

AMPA (a primary amine) reacts directly with the OPA/MERC

reagent and is detected (with decreased sensitivity) in the presence of hypochlorite.

b. Interferences: No matrix interferences in water are known.

GLYPH degrades in chlorinated water. GLYPH also is known to

sorb strongly to minerals and glass surfaces.

c. Minimum detectable concentration: Minimum detection

using this method is 25 g/L for GLYPH and AMPA by direct

injection and 0.5 g/L with the concentration step.

2. Sampling and Storage

Collect a 500-mL representative sample in a polypropylene or

amber glass container. Treatment of sample to remove residual

chlorine will prevent glyphosate losses during storage. Destroy

chlorine by adding 100 mg/L sodium thiosulfate. Store samples

at 4°C away from light and analyze within 2 weeks.

3. Apparatus

High-performance liquid chromatograph (HPLC): An analytical system with pump, injector, detectors, and compatible strip

* Approved by Standard Methods Committee, 2005.

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chart recorder. Preferably use a data system for measuring peak

areas and retention times. Use system capable of injecting

200-L portions. See Figure 6651:1.

1. Analytical columns: Use either a cation exchange resin1 or

an anion-exchange resin2 packed in a 4.6-mm 25- to 30-cm

column.* Heat columns to between 50 and 60°C to obtain

maximum efficiency.

b. Post-column reactor: Use system consisting of two separate

pumps capable of delivering reaction solutions at 0.1 to

0.5 mL/min and able to withstand pressures of up to 2000 kPa.

Include two woven 1-mL TFE reaction coils3 (0.5-mm ID 

1.4-mm OD 5 m) with one maintained at 40°C. Turnkey

post-column reactor systems are available commercially.

c. Fluorescence detector: Use filter or grating fluorimeter

capable of sensitively and selectively measuring the isoindole

derivative, with excitation wavelength 230 nm (deuterium),

340 nm (quartz halogen or xenon), and emission wavelength

420 to 455 nm.

4. Reagents

a. Reagent water: See Section 1080.

b. Phosphoric acid, H3PO4, conc.

c. Sulfuric acid, H2SO4, conc. Prepare anion-exchange mobile

phase by adding 26 mL conc H3PO4 and 2.7 mL conc H2SO4 to

5 L water.

d. Hydrochloric acid, HCl, conc.

e. Methanol, CH3OH, tested on HPLC and verified to give no

impurity peaks.

f. Potassium dihydrogen phosphate, KH2PO4. Prepare cationexchange mobile phase by dissolving 0.68 g KH2PO4 in 1 L

methanol-water (4:96). Adjust to pH 2.1 with conc H3PO4. Filter

through a 0.22- or 0.45-m membrane filter and degas.

g. Disodium ethylenediamine tetraacetate dihydrate, EDTA

sodium salt solutions: Prepare a 0.001M solution by dissolving

0.37 g EDTA dihydrate in 1.0 L water and filter through a 0.22-

or 0.45-m filter. Prepare a 0.03M solution by dissolving 11.2 g

EDTA dihydrate in 1.0 L water and filtering through a 0.33- or

0.45-m filter.

h. Sodium chloride, NaCl.

i. Sodium hydroxide, NaOH.

j. Calcium hypochlorite, Ca(OCl)2, 70.9% available chlorine.

k. Oxidation reagent: Dissolve 0.5 g Ca(OCl)2 in 500 mL

water with rapid magnetic stirring for 45 min. In a 1.0-L volumetric flask, dissolve 1.74 g KH2PO4, 11.6 g NaCl, 0.4 g NaOH,

and 10 mL stock Ca(OCl)2 solution. Dilute to volume, mix well,

and filter through a 0.22- or 0.45-m filter.

l. o-Phthalaldehyde, C6H4(CHO)2, OPA.

m. 2-mercaptoethanol, HSCH2CH2OH, MERC.

n. Boric acid powder, H3BO3.

o. Potassium hydroxide, KOH.

p. Fluorogenic labeling reagent: Dissolve 100 g boric acid

and 72 g KOH in about 700 mL water in a 1.0-L flask. This takes

1 to 2 h. Add 0.8 g OPA dissolved in 5 mL methanol. Add

2.0 mL MERC. Mix well.

q. Glyphosate analytical standard, N-(phosphonomethyl) glycine, 99% or greater.

r. Aminomethylphosphonic acid analytical standard, 99% or

greater.

s. Glyphosate and AMPA fortification standards: Prepare a

solution containing both 0.1 mg GLYPH/mL and 0.1 mg

AMPA/mL in water. Make working solutions of 10.0 and

1.0 g/mL by serial dilution of this stock solution. Store in a

refrigerator, in a polypropylene or amber glass container. Prepare fresh monthly.

t. Glyphosate and AMPA HPLC calibration standards: Prepare a solution containing both 0.1 mg GLYPH/mL and 0.1 mg

AMPA/mL in 0.001M disodium EDTA solution. Make working

solutions of 1.00, 0.50, 0.10, 0.05, and 0.025 g/mL by serial

dilution. Store in a refrigerator, in a polypropylene or amber

glass container. Prepare fresh monthly.

5. Procedure

a. HPLC operation: Equilibrate column at 50°C with mobilephase flow rate of 0.5 mL/min (see Figure 6651:1). Use an

approximate flow rate of 0.5 mL/min for the oxidant and 0.3 mL/

min for the OPA-MERC reagent but adjust rates to obtain

maximum response. While GLYPH reaches a maximum response at some flow rate of oxidative solution, the AMPA

response decreases with any addition of this reagent. Thus, an * Aminex, BioRad Labs, A-9 cation exchange and A-27 anion exchange resins, or

equivalent.

Figure 6651:1. Schematic of post-column reaction HPLC system.

GLYPHOSATE HERBICIDE (6651)/Liquid Chromatographic Post-Column Fluorescence Method

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oxidative reagent flow rate that gives an equal response for both

GLYPH and AMPA simultaneously is considered optimum for

simultaneous measurements. Reagent flow rates will differ for

different mobile phases. Establish a standard curve by injecting

calibration standards.

Approximate retention times for GLYPH and AMPA are 13.5

and 10.0 min on the anion-exchange column and 21.5 and

30.0 min on the cation-exchange column.

b. Sample preparation: No concentration is needed for samples containing 25 g/L or more. Fortify a 9.9-mL sample

portion with 0.1 mL 0.10M EDTA, filter through a 0.22- or

0.45-m filter, and inject 200 L. Fortify portions of the same

samples with known amounts of GLYPH and AMPA to determine recovery. Perform duplicate injections on at least 10% of

the samples with measurable GLYPH and AMPA or 10% of

fortified samples to determine precision.

To concentrate samples containing less than the detection

level, transfer 250 mL to a 500-mL round-bottom flask. If

suspended matter is present, filter sample through coarse filter

paper.† For samples used to assess recovery, make known additions. Add 5 mL conc HCl to flask and 5 mL to sample

remaining in original container. Concentrate on a rotary evaporator by slowly increasing temperature from 20 to 60°C. Before

the first portion is completely evaporated, add remaining sample

and two 5-mL rinses of the sample bottle. Evaporate to dryness,

and if necessary, remove final traces of water with a stream of

dry nitrogen. Dissolve residue in 2.9 mL of mobile phase (adjust

pH to 2 if necessary) and 0.10 mL 0.03M EDTA solution. Filter

through 0.45-m filter to a test tube and inject into the HPLC

system.

6. Calculations

Determine concentration of GLYPH and AMPA by regression

analysis of the standard curve. Multiply results for samples that

were concentrated by the concentration factor, 166.7 (500 mL

original sample/3.0 mL), to determine the original water concentration. Report results in milligrams per liter. Report percent

recovery but do not correct for recovery.

7. Quality Control

The quality control practices considered to be an integral part

of each method are summarized in Table 6020:I.

8. Precision and Bias

For six single-operator analyses, the relative standard deviation of duplicate samples (with additions from 0.5 to 5000 g/L)

ranged from 12.1 to 20% with an average of 14.9% for glyphosate. The relative standard deviation for identical AMPA concentrations ranged from 6.5 to 28.8% with an average of 14.5%.4

For six single-operator analyses, recoveries of glyphosate

(with additions from 0.5 to 5000 g/L) ranged from 94.6 to

120% with an average of 104.0%. Recoveries of AMPA ranged

from 86.0 to 100% with an average of 93.1%.4

9. References

1. COWELL, J.E., J.L. KUNSTMAN, P.J. NORD, J.R. STEINMETZ &

G.R. WILSON. 1986. Validation of an analytical residue method for

analysis of glyphosate and metabolite: An interlaboratory study. J.

Agric. Food Chem. 34:955.

2. MOYE, H.A., C.J. MILES & S.J. SCHERER. 1983. A simplified highperformance liquid chromatographic procedure for the determination

of glyphosate herbicide and (aminomethyl)phosphonic acid in fruits

and vegetables employing postcolumn fluorogenic labeling. J. Agric.

Food Chem. 31:69.

3. SELVAKA, C.M., K.S. JAIO & I.S. KRULL. 1987. Construction and

comparison of open tubular reactors for postcolumn reaction detection in liquid chromatography. Anal. Chem. 59:2221.

4. OPPENHUIZEN, M.E. & J.E. COWELL. 1991. Liquid chromatographic

determination of glyphosate and (aminomethyl)phosphonic acid in

† Whatman #1, or equivalent. environmental water. J. Assoc. Offic. Anal. Chem. 74:317.

GLYPHOSATE HERBICIDE (6651)/Liquid Chromatographic Post-Column Fluorescence Method

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