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Comparative analysis of detection techniques for glyphosate in urine and in water



Glyphosate is the declared active component of the most extensively used herbicides in the world, and is therefore widely present in the environment. Glyphosate urinary levels represent a relevant biomarker for each individual’s exposure to glyphosate-based herbicides. However, GLY urine level measurement is controversial because different detection methods have led to contradictory results, especially in the cases of enzyme-linked immunosorbent assay (ELISA) versus liquid chromatography coupled to tandem mass spectrometry (LC/MS–MS) for urine, and ELISA versus high-performance liquid chromatography coupled to fluorescence detection (HPLC/Fluo) for water.


We compared the ELISA method to the LC/MS–MS or HPLC/Fluo one by submitting to two laboratories (Biocheck, Germany and Labocéa, France, respectively) identical urine and water samples, spiked or not with precise concentrations of glyphosate, but also with two chemically similar molecules: glycine and aminomethylphosphonic acid, GLY’s analogue and primary metabolite, respectively.


Both laboratories claimed similar glyphosate quantification threshold (LOQ): 0.08 and 0.05 ng/mL, respectively. Each one of the tested methods proved to be specific for glyphosate and therefore did not result in any cross-detection with glycine and aminomethylphosphonic acid. However, these methods showed differences both in reproducibility and reliability depending on the matrix used (water or urine).


While the ELISA method gave less accurate results than the HPLC/Fluo technique when applied to water samples, the glyphosate concentrations measured in urine were much more reliable and reproducible with the ELISA technology than those obtained with the LC/MS–MS one.


Glyphosate-based herbicides (GBHs) are non-selective, broad-spectrum herbicides and the most widely used pesticides in the world [1]. Glyphosate (GLY), N-(phosphonomethyl) glycine, is—as its name suggests—an amino-phosphonic analogue to the natural amino acid glycine. The main biodegradation product of GLY is (aminomethyl) phosphonic acid (AMPA). GLY was patented as a herbicide active ingredient in 1971 (U.S Patent No 3,799,758) by the US firm Monsanto (later acquired by the German company Bayer in 2018). The first GBHs were introduced in the pesticides market in 1974 under the popular trade name Roundup®, and others followed: Glyphogan®, Touchdown®, Glifoglex®. All are mixtures of GLY with other chemicals used to facilitate penetration of GLY through plant epidermis, and increase its stability and activity [2, 3]. GBHs, which represent more than 750 formulations [4], are extensively used in intensive farming, home gardens, forestry, landscaping and wetlands management. Their use has increased dramatically since 1996, especially with the development of Roundup®-tolerant crops [1], which include the vast majority of the genetically modified (GM) plants commercially grown around the world [5]. GBHs’ usage keeps increasing with the emergence of tolerant weeds induced by the widespread application of these herbicides: the proliferation of such weeds requires more frequent spraying and at higher concentrations [1].

Such extensive use of GBHs raises the question of population exposure, both in occupational settings and via drinking water and food. GLY has been detected in the dust of non-agricultural homes [6], and both GLY and AMPA have been found in soils, water, plants, animals and food [7,8,9]. In particular, GLY has been shown to accumulate in Roundup-tolerant GM soybeans [10]. This is more relevant as most animals raised in intensive farming are fed with soycakes made from such GM soybeans [11, 12] mainly grown in the United States, Brazil and Argentina [5]. Moreover, GLY and AMPA may also be dispersed by wind and water erosion [13].

Despite an abundant scientific literature reporting adverse environmental and health outcomes of GLY and GBH (for reviews: [14,15,16]), the adverse impacts of GBHs continue to trigger controversial debates in public, scientific and political spheres [17,18,19]. Ongoing debate has led to scrutiny of evaluation procedures and analytical methodologies [20].

The routes by which GLY enters the body are dermal, oral and inhalation [2, 21, 22]. Even if the dermal route allows a poor absorption (about 2%), it is the main reported route of entry for exposed farmers [23]. Once in the body, GLY accumulates mainly in the detoxification organs (kidneys and liver), colon, small intestine, as well as in bone and bone marrow, and is eliminated mostly via the feces (90%) but also in urine [2]. The detection of GLY in urine is therefore an easy way to assess the exposure and contamination of individuals with GBHs [24, 25]. However, GLY in urine is also controversial since contradictory results have been obtained depending on the detection methods used. A vast campaign was launched in France to detect GLY in urine, and all tests conducted in a laboratory (Biocheck, Germany) using the enzyme-linked immunosorbent assay (ELISA) method were found to be positive [26]. Other studies commissioned by other individuals and carried out in other laboratories (for instance, Labocéa, France) using liquid chromatography coupled with tandem mass spectrometry (LC/MS–MS) gave systematically lower rates or even negative results.

The aim of this study was to carry out a comparative analysis of these two detection techniques for GLY in urine by subjecting identical samples to the two laboratories, supplemented or not with accurate known concentrations of GLY, glycine or AMPA. Supplementation with glycine or AMPA was intended to test a possible cross-detection of chemically similar molecules. Samples of tap water were also submitted to the two laboratories, in order to know if the accuracy of the measured glyphosate levels could be matrix-dependent.

Materials and methods

Collection of original samples

A sample of tap water of at least 400 mL was collected in a sterile 500 mL vial. Two samples of morning fasting urine (at least 400 mL each) from two individuals were collected in sterile 500 mL vials.


N-(phosphomethyl) glycine (GLY; CAS: 1071–83-6, also known as “technical glyphosate”, Glycine (CAS: 56–40-6), and (Aminomethyl) phosphonic acid (AMPA; CAS: 1066-51-9) were purchased from Sigma-Aldrich. For each one of these three chemicals, a 1 mg/mL stock solution was obtained by dissolving 10 mg in 10 mL of ultrapure water (Millipore). Each stock solution was then serially diluted (two successive 10–2 dilutions) in ultrapure water to obtain final 0.1 μg/mL solutions.

Protocol design

The protocol design is summarized in Fig. 1. Each one of the three original samples (one of water and two of urine) was distributed evenly into 8 sterile medical grade polypropylene tubes of 50 mL (8 × 50 mL). For each set of 8 tubes, the first (A, I, Q) served as a control (no chemicals added). 50 µL (5 ng), 250 µL (25 ng) and 1000 µL (100 ng) of the final GLY solution was added to the 2nd (B, J, R), 3rd (C, K, S) and 4th (D, L, T) tubes, respectively. 250 µL (25 ng) and 1000 µL (100 ng) of the final glycine solution was added to the 5th (E, M, U) and 6th (F, N, V) tubes, respectively. 250 µL (25 ng) and 1000 µL (100 ng) of the final AMPA solution was added to the 7th (G, O, W) and 8th (H, P, X) tubes, respectively.

Fig. 1
figure 1

Outline of the protocol design. Details for GLY, glycine and AMPA supplementation are described in the “Materials and methods” section

For each series, 40 mL of each one of the eight tubes was then evenly distributed in four tubes (4 × 10 mL): two tubes (identical) destined to the first analysis laboratory (Biockeck, Germany), and two others (identical) destined to the second analysis laboratory (Labocéa, France). All the tubes sent to the same laboratory (16 per series, i.e., per original sample) were randomly numbered so that the laboratory could neither identify them nor detect duplicates. The final tubes were packaged in dry ice and shipped by a transporter specialized in the transport of biological samples. Each package contained a single reading temperature probe.

Experiment supervision and monitoring

This comparative analysis being likely to be cited in the context of legal proceedings, the complete experimental process (from the reception of chemical standards, and collection of the original samples to the shipment) was supervised by a legal chemist expert to the French and European courts, and was followed and photographed by a judicial officer. In addition, the entire experience was filmed in a one-shot time-stamped take. Upon receipt of the packages, each of the two laboratories returned the above-mentioned temperature probe to the judicial officer who read it in order to ensure that there had been no break in the cold chain during transport.

Results and discussion

While Biocheck laboratory used ELISA technology for glyphosate detection both in water and urine, Labocéa laboratory used two different technologies depending on the matrix: high-performance liquid chromatography coupled to fluorescence detection (HPLC/Fluo) for water samples and LC/MS–MS for urine samples.

The results obtained for the water samples are presented in Table 1. Limit of quantification (LOQ) was 0.08 and 0.05–0.1 ng/mL for Biocheck and Labocéa, respectively. The reproducibility (i.e., the results obtained for each pair of identical samples) was very good for both laboratories. Two of the methods used (ELISA and HPLC/Fluo) gave results lower than expected values; the HPLC/Fluo method (Labocéa) produced more accurate results. Results obtained with samples W5 to W8 (supplemented with glycine or AMPA), when compared to those obtained with the control sample (W1), indicated that both detection methods were found to be perfectly GLY-specific, in that the values detected were not increased when the chemically similar molecules (glycine and AMPA) were added in the samples.

Table 1 Measured and expected GLY concentrations ([GLY]) in tap water samples

The results obtained for the urine samples are compiled in Tables 2 and 3. For Biocheck, the LOQ remained unchanged compared to the water matrix, namely 0.08 ng/mL. As for Labocéa, while the LOQ initially claimed was 0.05 ng/mL, it turned out to be 10 times higher (0.5 ng/mL). The rationale given by the laboratory was the need for them to dilute all urine samples 10 times due to a “strong matrix effect” (i.e., “urine was rich in various compounds disturbing analysis by preventing detection of internal labeled standards”). While it is completely understandable to have to dilute samples in order to be within a detection window compatible with the method (and in this specific case, to better detect chromatographic peaks), it is however difficult to understand why this impacts the quantification threshold, which depends on the method and on the target compound [27, 28], but not on the sample. It should therefore be the same, regardless of whether the urine is diluted or not. Why not simply then multiply by 10 the concentration measured in the 10× diluted sample to know the concentration in the original one? Labocéa nevertheless sometimes estimated values lower than the LOQ of 0.5 ng/mL (samples U2, U3, U11, and one of the samples U8, U10, U16) “when a chromatographic peak significantly emerged from the background noise”, but “without the precision of this estimate meeting the requirements of COFRAC” (French accreditation committee).

Table 2 Measured and expected GLY concentrations ([GLY]) in samples of individual’s urine 1
Table 3 Measured and expected GLY concentrations ([GLY]) in samples of individual’s urine 2

Besides this LOQ issue, making it difficult to interpret some of Labocéa’s values, it seems that the Biocheck's method (ELISA) resulted in an overall better reproducibility, with the exception of a single pair of identical samples (U4). In contrast, several duplicate samples returned relatively distant values in the case of Labocéa (U4, U7, U10, U12). Moreover, the Biocheck’s measured values were much closer to the expected concentrations than those of Labocéa’s, even when the measured values were greater than 0.5 ng/mL (Labocéa's LOQ)—and therefore met the COFRAC requirements—(samples U4 and U12). Finally, as in the case of water samples, it is clear from our results that neither of the two methods (ELISA and LC/MS–MS) showed the slightest cross-reaction or cross-detection with glycine or AMPA. However, Labocéa also measured AMPA concentration in all samples. Surprisingly, the concentration of AMPA was overestimated in the samples supplemented with GLY (when compared with the control samples), indicating that even though the LC/MS–MS method allowed a specific detection of GLY, the latter still interfered with AMPA detection (data not shown).

The ELISA method for the detection of GLY was developed on water samples [29]. The GLY concentrations determined by ELISA in water samples then correlated perfectly (correlation coefficient of 0.99) with those determined by HPLC [29]. Such a similar performance between the two techniques for water samples was later confirmed by another comparison analysis [30]. This slight discrepancy with our results could be explained by the different GLY concentration ranges between their samples and ours. Indeed, the main differences that we obtained between the two techniques for the water samples concerned those which had been spiked with GLY. Therefore, ELISA technique would prove to be less efficient than the HPLC/Fluo for GLY detection in water only for the most concentrated samples. Another difference with the original work of Clegg et al. [29] concerns the specificity of the ELISA method towards GLY: unlike us, they observed that the GLY polyclonal antisera did cross-react with AMPA. This indicates that the ELISA kits used by Biocheck [31] were more specific than the original one.

Urine is a much more complex matrix than water. In particular, urine can contain a large number of metabolites such as amino acids including glycine [32]. Therefore, the transposition of the ELISA method for the detection of GLY in urine could have been limited by possible cross-reactions with molecules structurally similar to GLY, and especially glycine. Such a cross-reaction would lead to a general overestimation of the GLY urine levels, and even to false positives. Our data clearly showed that this is not the case.


For the determination of the GLY levels in water, the HPLC/Fluo technique used by Labocéa gave more precise results than the ELISA technique used by Biocheck.

Conversely, for the determination of the GLY urine levels, the Biocheck ELISA technology gave much more reliable and reproducible results than the LC/MS–MS method used by Labocéa.

None of the three techniques applied for the detection of GLY, whether in water or in urine, showed a cross-reaction or cross-detection with the chemically similar molecules glycine and AMPA.

Labocéa's LC/MS–MS method required dilution of the first morning urines due to a "strong matrix effect", implying—according to Labocéa—an increase in their quantification threshold (LOQ) which had to be multiplied by the dilution factor of the samples. Therefore, when urinary GLY levels are given by Labocéa with a LOQ of 0.05 ng/mL (the originally stated LOQ), this implies that the urine samples analyzed had not required dilution, and therefore that these urines were probably not first morning urines, as recommended by the laboratory, but later and clearer urines which, of course, were not representative of the real GLY levels to which the body has been exposed.

Availability of data and materials

Please contact the authors for data requests.



(Aminomethyl) phosphonic acid


Enzyme-linked immunosorbent assay




Glyphosate-based herbicides


High-performance liquid chromatography coupled to fluorescence detection


Liquid chromatography coupled to tandem mass spectrometry


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We thank Guillaume de Crop for technical assistance and the production of the experiment movie. We are grateful to Marcel Lamour, Morgane Michel and Dominique Berlet-Noel for proofreading the English.


This work was supported by the Foundation Un Monde par Tous (Grant n°. 00081254).

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CV and JSdV designed the protocol. CV conducted the experiment. FP, as a legal chemist expert, supervised procedures followed during the experiment and ensured that they complied perfectly with the pre-established protocol. CV wrote the first version, and revised the manuscript based on feedback from the co-authors. All authors read and approved the manuscript.

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Correspondence to Christian Vélot.

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Non applicable (urine samples were provided by two of the co-authors: CV and JSdV).

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Vélot, C., Poitou, F. & de Vendômois, J.S. Comparative analysis of detection techniques for glyphosate in urine and in water. Environ Sci Eur 34, 59 (2022).

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  • Glyphosate
  • LC/MS–MS
  • Limit of quantification (LOQ)
  • Urine