Determination of Glyphosate and AMPA in Drinking Water Using Ion Chromatography with Pulsed Amperometric Detection

Is glyphosate carcinogenic?

Glyphosate is a broad-spectrum herbicide that is widely used in agriculture across the globe. The chemical is also used as a weed killer in domestic gardens, as well as in private and public spaces such as railway tracks, to prevent vegetal invasion. Since the 1970s, glyphosate has been used in pesticides and until now, it was believed to be safe at usual levels of exposure. However, WHO’s specialized cancer research agency − the International Agency for Research on Cancer (IARC) − published a study in March 2015 stating that glyphosate is likely carcinogenic to humans (Group 2A). As a result, glyphosate attracted a great deal of attention.1 Since then, the use of this chemical has been a topic of much debate.

When the EU market approval of glyphosate expired on June 30, 2016, experts were divided over whether the chemical should be re-approved. This is because it was only recently that the European Food Safety Authority (EFSA) reached the opposed conclusion that glyphosate is unlikely to be genotoxic or represent a carcinogenic threat.2 Glyphosate approval was first extended by 18 months, but as 2017 comes to an end, the question of whether glyphosate use should be continued in the EU will arise again.

Limit values for glyphosate

Since chemicals used in farming can easily seep through the ground and reach ground water, some countries, including the US, have already set limit values for the concentration of glyphosate in drinking water. For instance, the US Environmental Protection Agency (EPA) prohibits any concentrations beyond the limit value of 700 μg/L. Australia specifies a much lower limit value, of 10 μg/L, and in Canada the highest permissible concentration stands at 280 μg/L.

Either HPLC with post-column derivatization and subsequent fluorescence detection (EPA Method 547), or ion chromatography (IC) coupled with a mass-selective detector is used to determine glyphosate and its primary metabolite aminomethylphosphonic acid (AMPA). In the following sections, the initial results of using IC with pulsed amperometric detection to determine glyphosate and AMPA in drinking water in the low μg/L range are shown. For glyphosate and AMPA, the detection limits that were previously achieved with pulsed amperometric detection were approximately ≥ 50 μg/L[3]. Based on this enhanced sensitivity, the technique described here shows a potential method for screening food and water samples for glyphosate and AMPA.


An IC system comprising a 940 Professional IC Vario ONE with an 858 Professional Sample Processor for automatic injection of samples and an IC Amperometric Detector (Figure 1) was used to perform all determinations. On a gold working electrode, FLEXIBLE Integrated Pulsed Amperometric Detection (flexIPAD) was used as a measuring mode in the amperometric detector. This flexIPAD mode is defined by its unique, multi-stage potential profile. Compared with the three-stage potential profile of the regular pulsed amperometric detection (PAD mode), the flexIPAD mode generates a stable signal over a longer period of time in the determination of glyphosate and AMPA. Figure 2 shows the profile of the potential curve created in a single measuring cycle in flexIPAD mode.

Figure 1. Glyphosate and AMPA were determined with the ProfIC IC Vario 1 Amperometry system.

Figure 2. Pulse profile of the flexIPAD method: A measur­ing cycle lasts 0.9 s; measurement of the current is performed during the phase shown in red.

The high-capacity anion separation column - Metrosep Carb 2 - 150/4 - was used to separate the glyphosate and AMPA. The caustic-soda–acetate eluent that was used contains 300 mmol/L sodium acetate and 10 mmol/L sodium hydroxide. Under these conditions, glyphosate elutes after 21.1 minutes and AMPA elutes after 6.4 minutes.


The experiment was carried out to analyze the separation of glyphosate and AMPA in the Metrosep Carb 2 separation column and to clarify the detection through pulsed amperometry and its sensitivity. Carbohydrates, alcohols and sugar alcohols are mainly separated and determined using the Metrosep Carb 2 column. The high capacity of the column along with the eluent’s high pH value (which at about 10 is usual for sugar analysis) lead to a large variation in retention time for glyphosate and AMPA. The reason for this is that all three acid groups have a pH value of 10 and are deprotonated in part of the glyphosate, which means glyphosate is partly present as a trivalent anion while the AMPA metabolite, which lacks the carboxyl group, exists as a divalent anion.

A flow gradient is used to speed up the glyphosate elution; after the elution of AMPA at 6.4 minutes, the flow rate is increased two-fold, from 0.4 mL/minute to 0.8 mL/minute, resulting in a retention time of 21 minutes for the chemical glyphosate. Table 1 summarizes the chromatographic conditions.

The chromatographic conditions are summarized in Table 1.

Table 1. Chromatographic conditions



Metrosep Carb 2 - 150/4.0


10 mmol/L sodium hydroxide

300 mmol/L sodium acetate

Flow rate

0.4 mL/ minute (0–16 minute)

0.8 mL/ minute (16–25 minute)

Injection volume

250 μL

Chromatography duration

25 minute

Column temperature

30 °C

Amperometric detection


Wall-Jet cell

Working electrode


Reference electrode



50 μm


35 °C

Measuring mode


Measured quantity



Shown in Figure 3 is the chromatogram of the determination of glyphosate and AMPA under the conditions described in Table 1. An aqueous standard solution containing 10 μg/L each of both components was injected. Tap water from Herisau (Switzerland) was examined and combined with different quantities of glyphosate and AMPA to assess how suitable the process was for drinking water. Table 2 shows the concentrations and peak areas that were found.

Figure 3. Separation of AMPA and glyphosate: a standard solution containing 10 μg/L of each component in ultrapure water was analyzed. For conditions see Table 1.

Table 2. Investigated drinking water samples and the peak areas found


Peak area of AMPA [nA · min]

Peak area of glyphosate [nA · min]

Tap water

Not detectable

Not detectable

Tap water spiked with 2 μg/L



Tap water spiked with 5 μg/L



Tap water spiked

with 10 μg/L



Using the signal/noise (S/N) ratio (the peak height to baseline noise ratio), the detection limits for both components were established. The S/N ratio was found to be 3 at the detection limit; secured detection is not possible with smaller values. The detection limit for glyphosate was about 1 μg/L, while the detection limit for AMPA was significantly lower than 1 μg/L. The chromatogram of the drinking water combined with 2 μg/L AMPA and glyphosate is shown in Figure 4.

Figure 4. Determination of AMPA and glyphosate in drinking water which was mixed with 2 μg/L of each component. For conditions and results, see Tables 1 and 2.


This is the first time IC with pulsed amperometric detection (flexIPAD) has been used to determine glyphosate and its key metabolite AMPA in drinking water in the low μg/L range. Compared with using HPLC with a mass-selective detector, this process serves as a reliable and low-cost method for determining the concentrations of AMPA and glyphosate in food items and water. With a detection limit of around 1 μg/L, the compliance to limit values for glyphosate can be verified in Australia, the US and Canada, among others.


[1] IARC Monographs Volume 112 (2015). Retrieved from mono112-09.pdf on June 27, 2016.

[2] EFSA press news, 151112 (2015). Retrieved from http:// on June 27, 2016

[3] F. Sanchez-Bayo, R. V. Hyne, and K. L. Desseille (2010) Anal. Chim. Acta, 675 125–131

About Metrohm

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Last updated: Jul 14, 2018 at 6:57 PM

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