**1. Introduction**

Glyphosate (GLYP) (or, less commonly, but still used, glyphosphate), a broadspectrum herbicide, is one of the most used pesticides in the world [1], nearly \$5 billion in sales and an annual global production about 825,800,000 kg [2]. Glyphosate is a nonselective herbicide; therefore, it is a molecule that eliminates all weeds without distinction.

Glyphosate [IUPAC N-(phosphonomethyl)glycine; CAS registry number 1071- 83-6] is an aminophosphoric analogue of glycine and an important amino acid. It was discovered in the early 1950s by Henri Martin and was patented by Monsanto and sold as a Roundup® product for about 20 years; after 2001 (patent expiration date), free production of glyphosate was legally permitted [3, 4]. As of 2010, more than 750 glyphosate products have been on the market [5, 6]. The first important worldwide warning about the GLYP occurred in 2017: the Canadian Food Inspection Agency (CFIA) confirmed that 36.6% of the Canadian wheat samples had a high

presence of GLYP (3.9% above the legal limits, which in Canada is 5 ppm) [7]. In Canada, GLYP-based products are widely used for improving the wheat ripening and drying. Such occurrence has created a big supply problem in Europe where this practice is prohibited: for instance, Italy imported large amounts of wheat to make flour for pasta from Canada (and from the United States as well).

GLYP inhibits the 5-enolpyruvylshikimate-3-phosphate (EPSP) enzyme produced by plants, which is involved in the synthesis of three essential amino acids such as tyrosine, tryptophan, and phenylalanine. The mechanism of action is absorption through the foliage, and to a small extent through the roots, and transport to growth points. Since this enzyme is present only in the plant kingdom, glyphosate acts only on plant organisms.

GLYP is a leaf herbicide (it is absorbed by the leaves of the plant), systemic (once absorbed, it passes toward the growth points, causing the death of the plant), nonselective (in fact, it is active on all plants, if not genetically modified). Glyphosate-based products are activated by the addition of a surfactant, polyoxyethylene amine (POEA), which promotes penetration through the leaf surface of plants; other additives used are sulfuric acid and phosphoric acid. Its main metabolite is aminomethylphosphonic acid (AMPA). It should be noted that a fraction of AMPA could be due to degradation processes of the detergents/surfactants rather than from glyphosate. GLYP does not penetrate deeply into the soil (maximum 20 cm) and is easily degraded by bacteria. This means that the probability that it reaches the aquifers is very low and that its presence is certainly lower than that of other dangerous pollutants.

The half-life of GLYP in the soil is between 2 and 197 days, a typical half-life of 47 days has been suggested. The soil and climate conditions on the persistence of glyphosate in the soil are very important. The average half-life of GLYP in water varies from few to 91 days. The AMPA metabolite of glyphosate has been found in Swedish forest soils for up to 2 years after a glyphosate application. In this case, the persistence of AMPA has been attributed to frozen soil for most of the year. The adsorption of glyphosate into the soil, and then its release from the soil, varies according to the type of soil. GLYP is generally less persistent in water than in land, with 12–60 days persistence observed in Canadian ponds, although persistence of more than a year has been recorded in American lake sediments.

GLYP (**Figure 1**) is a weak acid commonly used in the form of salt, distributed as a powder or as a water-soluble concentrate. At room temperature, it appears as a colorless crystalline solid, is completely soluble in water, and is highly insoluble in common organic solvents such as benzene and dichloromethane. GLYP is a nonvolatile and photo-resistant molecule, and its dissolution in water generates four chemical equilibria represented by the respective acid dissociation constants (Ka). In logarithmic form, pKa acquires the following values: 2.0, 2.6, 5.6, and 10.6. This aspect makes the molecule highly polar and amphoteric [8].

During the reactions involving the enzymes glyphosate oxidase and glyphosate N-acetyl transferase, glyphosate can form different metabolites: the main is

**99**

*a As LD50. <sup>b</sup>*

**Table 1.**

Primary eye irritation

Primary skin irritation

*As lethal concentration, 50% (LC50).*

*Relationship between GLYP levels and toxicity.*

*A Review of the Analytical Methods Based on Chromatography for Analyzing Glyphosate in Foods*

considered the amino-methylphosphonic acid (AMPA), whereas the others are glyoxylate, N-acetyl glyphosate, N-acetyl-AMPA, methylphosphonic acid, sarcosine, N-methyl-aminomethylphosphonic acid (MAMPA), hydroxymethylphosphonic acid, and phosphonoformic acid [9]. This behavior is important: these compounds should be considered when toxicity and environmental studies are performed for the risk assessment. Similarly, compounds used as adjuvants in commercial glyphosate formulations should be considered: for instance, polyoxyethylene amine (POEA), used as a surfactant in Roundup [10] or isopropylamine, ammonium and trimesium salts, or formulation impurities such as N-(phosphonomethyl)iminodiacetic acid and bis(phosphonomethyl)amine. This occurrence is really important because the adjuvants can modify the toxicity of pesticides based on glyphosate as active ingredient; so, the result is the need of a novel toxicological evaluation [11]. All these considerations play an important role in the GLYP toxicity. The toxicity of a substance is assessed according to its median lethal dose (lethal dose, 50% – LD50), that is, the dose that causes the death of 50% of the individuals taking the test substance: Class 1, high acute toxicity, LD50 less than 50 mg per kg of live weight; Class 2, moderate toxicity, LD50 between 50 and 500; Class 3, mild toxicity, LD50 between 500 and 5000; and Class 4, harmless, LD50 of over 5000 mg. The GLYP is in Class 3, while in Class 2, we find, for example, caffeine, aspirin, and boiling chloride, and in Class 1, the vitamin D3. In **Table 1**, acute toxicity assess-

Also, important is the concept of daily limit dose (expressed in milligrams per kilogram of body weight considered) definable as the maximum amount of herbicide that can be consumed daily without causing damage. Based on this concept, the glyphosate content of a food or a drink should be correctly evaluated using the milligrams of glyphosate per kilogram of body weight that can be taken per day as a unit of measurement. In this way, the European Food Safety Authority (EFSA) has

A tumor associated with glyphosate would be the non-Hodgkin lymphoma (NHL). In 2013, the German Federal Institute for Risk Assessment (BfR) found that "the available data are contradictory and far from convincing" in terms of the relationship between exposure to glyphosate formulations and the risk of various cancers, including the NHL [13–18]. A meta-analysis published in 2014 identified an increased risk of NHL in workers exposed to glyphosate formulations [19, 20].

**toxicity**

Inhalationb ≤0.05 mg L<sup>−</sup><sup>1</sup> >0.05–0.5 mg L<sup>−</sup><sup>1</sup> >0.5–2.0 mg L<sup>−</sup><sup>1</sup> >2.0 mg L<sup>−</sup><sup>1</sup>

2000 mg kg<sup>−</sup><sup>1</sup>

Corneal involvement (8–21 days)

at 72 hours

**High toxicity Moderate** 

Acute orala ≤50 mg kg<sup>−</sup><sup>1</sup> >50–500 mg kg<sup>−</sup><sup>1</sup> >500–

Corrosive Severe irritation

Dermala ≤200 mg kg<sup>−</sup><sup>1</sup> >200–

Corrosive or corneal involvement of weight per day [12].

**Low toxicity Very low toxicity**

>5000 mg kg<sup>−</sup><sup>1</sup>

>5000 mg kg<sup>−</sup><sup>1</sup>

Minimal effects clearing in 24 hours

Mild or slight irritation at 72 hours

5000 mg kg<sup>−</sup><sup>1</sup>

>2000– 5000 mg kg<sup>−</sup><sup>1</sup>

Corneal involvement (7 days)

Moderate irritation at 72 hours

*DOI: http://dx.doi.org/10.5772/intechopen.92810*

ment is reported.

set a daily limit dose of 0.5 mg kg<sup>−</sup><sup>1</sup>

**Figure 1.** *Glyphosate [N-(phosphonomethyl)glycine; CAS number 1071-83-6].*

#### *A Review of the Analytical Methods Based on Chromatography for Analyzing Glyphosate in Foods DOI: http://dx.doi.org/10.5772/intechopen.92810*

considered the amino-methylphosphonic acid (AMPA), whereas the others are glyoxylate, N-acetyl glyphosate, N-acetyl-AMPA, methylphosphonic acid, sarcosine, N-methyl-aminomethylphosphonic acid (MAMPA), hydroxymethylphosphonic acid, and phosphonoformic acid [9]. This behavior is important: these compounds should be considered when toxicity and environmental studies are performed for the risk assessment. Similarly, compounds used as adjuvants in commercial glyphosate formulations should be considered: for instance, polyoxyethylene amine (POEA), used as a surfactant in Roundup [10] or isopropylamine, ammonium and trimesium salts, or formulation impurities such as N-(phosphonomethyl)iminodiacetic acid and bis(phosphonomethyl)amine. This occurrence is really important because the adjuvants can modify the toxicity of pesticides based on glyphosate as active ingredient; so, the result is the need of a novel toxicological evaluation [11].

All these considerations play an important role in the GLYP toxicity. The toxicity of a substance is assessed according to its median lethal dose (lethal dose, 50% – LD50), that is, the dose that causes the death of 50% of the individuals taking the test substance: Class 1, high acute toxicity, LD50 less than 50 mg per kg of live weight; Class 2, moderate toxicity, LD50 between 50 and 500; Class 3, mild toxicity, LD50 between 500 and 5000; and Class 4, harmless, LD50 of over 5000 mg. The GLYP is in Class 3, while in Class 2, we find, for example, caffeine, aspirin, and boiling chloride, and in Class 1, the vitamin D3. In **Table 1**, acute toxicity assessment is reported.

Also, important is the concept of daily limit dose (expressed in milligrams per kilogram of body weight considered) definable as the maximum amount of herbicide that can be consumed daily without causing damage. Based on this concept, the glyphosate content of a food or a drink should be correctly evaluated using the milligrams of glyphosate per kilogram of body weight that can be taken per day as a unit of measurement. In this way, the European Food Safety Authority (EFSA) has set a daily limit dose of 0.5 mg kg<sup>−</sup><sup>1</sup> of weight per day [12].

A tumor associated with glyphosate would be the non-Hodgkin lymphoma (NHL). In 2013, the German Federal Institute for Risk Assessment (BfR) found that "the available data are contradictory and far from convincing" in terms of the relationship between exposure to glyphosate formulations and the risk of various cancers, including the NHL [13–18]. A meta-analysis published in 2014 identified an increased risk of NHL in workers exposed to glyphosate formulations [19, 20].


#### **Table 1.**

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

flour for pasta from Canada (and from the United States as well).

glyphosate acts only on plant organisms.

other dangerous pollutants.

presence of GLYP (3.9% above the legal limits, which in Canada is 5 ppm) [7]. In Canada, GLYP-based products are widely used for improving the wheat ripening and drying. Such occurrence has created a big supply problem in Europe where this practice is prohibited: for instance, Italy imported large amounts of wheat to make

GLYP inhibits the 5-enolpyruvylshikimate-3-phosphate (EPSP) enzyme produced by plants, which is involved in the synthesis of three essential amino acids such as tyrosine, tryptophan, and phenylalanine. The mechanism of action is absorption through the foliage, and to a small extent through the roots, and transport to growth points. Since this enzyme is present only in the plant kingdom,

GLYP is a leaf herbicide (it is absorbed by the leaves of the plant), systemic (once absorbed, it passes toward the growth points, causing the death of the plant), nonselective (in fact, it is active on all plants, if not genetically modified). Glyphosate-based products are activated by the addition of a surfactant, polyoxyethylene amine (POEA), which promotes penetration through the leaf surface of plants; other additives used are sulfuric acid and phosphoric acid. Its main metabolite is aminomethylphosphonic acid (AMPA). It should be noted that a fraction of AMPA could be due to degradation processes of the detergents/surfactants rather than from glyphosate. GLYP does not penetrate deeply into the soil (maximum 20 cm) and is easily degraded by bacteria. This means that the probability that it reaches the aquifers is very low and that its presence is certainly lower than that of

The half-life of GLYP in the soil is between 2 and 197 days, a typical half-life of 47 days has been suggested. The soil and climate conditions on the persistence of glyphosate in the soil are very important. The average half-life of GLYP in water varies from few to 91 days. The AMPA metabolite of glyphosate has been found in Swedish forest soils for up to 2 years after a glyphosate application. In this case, the persistence of AMPA has been attributed to frozen soil for most of the year. The adsorption of glyphosate into the soil, and then its release from the soil, varies according to the type of soil. GLYP is generally less persistent in water than in land, with 12–60 days persistence observed in Canadian ponds, although persistence of

GLYP (**Figure 1**) is a weak acid commonly used in the form of salt, distributed as a powder or as a water-soluble concentrate. At room temperature, it appears as a colorless crystalline solid, is completely soluble in water, and is highly insoluble in common organic solvents such as benzene and dichloromethane. GLYP is a nonvolatile and photo-resistant molecule, and its dissolution in water generates four chemical equilibria represented by the respective acid dissociation constants (Ka). In logarithmic form, pKa acquires the following values: 2.0, 2.6, 5.6, and 10.6. This

During the reactions involving the enzymes glyphosate oxidase and glyphosate N-acetyl transferase, glyphosate can form different metabolites: the main is

more than a year has been recorded in American lake sediments.

aspect makes the molecule highly polar and amphoteric [8].

*Glyphosate [N-(phosphonomethyl)glycine; CAS number 1071-83-6].*

**98**

**Figure 1.**

*Relationship between GLYP levels and toxicity.*

In March 2015, the International Agency for Research on Cancer (IARC) classified glyphosate "probably carcinogenic to humans" (Group 2a) based on epidemiological studies, animal studies, and in vitro studies: in particular, GLYP has been defined genotoxic through at least two mechanisms known to be associated with human carcinogens [21–23]. In contrast, EFSA concluded in November 2015 that "the substance is unlikely to be genotoxic (i.e., harmful to DNA), or pose a threat to humans." Subsequently, EFSA itself states that while there may be formulations containing glyphosate that are carcinogenic, studies relating only to glyphosate as an active ingredient do not show this effect [24, 25]. The European Chemicals Agency (ECHA), on the basis of "the scientific evidence available at the moment," classified GLYP, according to the CLP Regulation, as a chemical causing eye damage (H318) and being toxic to aquatic life with long-lasting effects (H411), but "the available scientific evidence did not meet the criteria in the CLP Regulation to classify glyphosate for specific target organ toxicity, or as a carcinogen, as a mutagen or for reproductive toxicity" [26]. The United States Environmental Protection Agency (US EPA) has classified glyphosate as a Group E chemical, meaning the agency has determined that there is "evidence of noncarcinogenicity to humans" [27, 28]. In any case, US EPA has established tolerances for GLYP residues in different commodities [29]. The difference of point of views depends on the fact that IARC and US EPA have analyzed different studies and applied different statistics. Further, EFSA analyses concern only the glyphosate molecule, whereas the studies considered by IARC also concern glyphosate-based products placed on the market [30].

This brief analysis shows that, in any case, international pesticide regulatory agencies and scientific organizations agree that there is no evidence that GLYP as an active substance is carcinogenic to humans, only IARC has classified glyphosate as "probably carcinogenic."

Finally, it should be considered an interesting hypothesis by Samsel and Seneff [31]: they propose a relationship between celiac disease and imbalances in gut bacteria generated by the known GLYP effects on them.

The EFSA has renewed the authorization for GLYP, establishing the acute reference dose (ARfD) at 0.5 mg kg<sup>−</sup><sup>1</sup> of body weight, while the acceptable operator exposure level (AOEL) was set at 0.1 mg kg<sup>−</sup><sup>1</sup> body weight per day and the acceptable daily intakes (ADIs) for consumers are in line with the ARfD threshold, 0.5 mg kg<sup>−</sup><sup>1</sup> body weight per day.

There are several exposure sources of humans to GLYP in the environment, for example, air, water, application to crops and target weeds, and food [32–34]. Solomon deeply reviewed the exposure data from the literature (PubMed and Google Scholar) and unpublished reports in different papers [35, 36]: in both papers, he reaches a similar conclusion: "In all cases, measured and estimated systemic exposures to glyphosate in humans and animals were less than the ADIs and the RfD. Based on this large dataset, these exposures represent a *de minimis* risk." The conclusion reached by Gillezeau et al. [33] is instead intermediate by reviewing the same literature (PubMed and Google Scholar): they state that "additional studies are urgently needed to evaluate levels of glyphosate and related metabolites in the general population and in workers." Further, they observe the great differences in the analyzed papers: they detected some bias such as the few studies on potential occupational GLYP exposure, or no study designed to address the hypothesis of seasonality in exposure, or the use of a few populations of farmers and relative collection of one-time spot urine. They rise serious doubts about the data generalizability, which they consider rather limited.

This paper would like to critically revise the literature on chromatographic methods developed for analyzing GLYP and AMPA in food matrices, specifically grains (e.g., rice, wheat, soybean, and maize), honey, olive and oil, vegetables,

**101**

*A Review of the Analytical Methods Based on Chromatography for Analyzing Glyphosate in Foods*

Starting from the Canadian study performed in 2017, the scientific attention on GLYP has become stronger, and several papers are annually published dealing the determination of such compound, along with its main metabolite AMPA, on different agricultural and food matrices. For avoiding dispersive information due to the big amount of studies aimed to this determination, the authors have focused their attention on the main innovative analytical methods based on chromatographic methods for determining both compounds in such matrices. It is also necessary to advise the reader that different matrices could be determined with same analytical protocols, at least showing different analytical parameters (multiresidue analyses), as well as in literature are present papers dealing important toxicological studies

Before approaching the discussion on the different analytical methodologies developed for analyzing GLYP and AMPA in agricultural and food matrices, it should be necessary to resume some toxicological information on it along with some chemical characteristics to be taken into account for evaluating the analytical

First, a maximum residue level (MRL) is defined as the highest level of a pesticide residue legally tolerated in or on food or feed when pesticides are applied correctly [37]. For each product, an MRL of GLYP has been determined [38]. An

A preliminary important information comes from the EU Reference Laboratories for Residues of Pesticides (EURL-SRM): for all the analytical steps, it is highly recommended the use of plastic vials because there is an interaction between the pesticide and the glass surface, especially when aprotic solvents are used. These interactions greatly affect the precision and accuracy, especially at low GLYP concentration. This statement is important because it influences its stability and degradation as well. Among the different solvents, water with 10% acetonitrile is considered a good storage solvent, whereas the compound is not stable in water and methanol. At room temperature, the degradation is very low within 14 days, whereas if extract is stored in the refrigerator, it is stable over 7 months [39]. Finally, the authors would like to remember some definitions regarding the parameter of an analytical method. Recovery is the term used in analytical and preparative chemistry to denote the fraction of the total quantity of a substance recoverable following a chemical procedure [40]. Accuracy is the difference between the mean of some measurements and the value considered as the true or correct value for the quantity measured, whereas precision is the measurement reproducibility, that is, the dispersion around a central value. In regard to the chromatographic separation, a signal-to-noise (S/N) ratio of 3 is acceptable for determining the limit of detection (LOD), that is, the lowest amount of analyte in a sample, which can be detected, whereas a ratio of 10 for the limit of quantification (LOQ ), that is, the lowest amount of analyte in a sample, which can be quantitatively determined with precision and accuracy [41–44]. The S/N definition for chromatography is the ratio

fruit, beverages (e.g., drinking water, milk, tea, and coffee), cheese, and meat/ fish products. In literature (source: Scopus database), there are 2666 papers using keywords "glyphosate" and "analysis" by the end of April 2020 and 361 using

**2. Glyphosate determination in different food matrices**

*DOI: http://dx.doi.org/10.5772/intechopen.92810*

"chromatography" as third keyword.

with no analytical information.

process.

**2.1 Approaching the determination**

example of this database is reported in **Table 2**.

#### *A Review of the Analytical Methods Based on Chromatography for Analyzing Glyphosate in Foods DOI: http://dx.doi.org/10.5772/intechopen.92810*

fruit, beverages (e.g., drinking water, milk, tea, and coffee), cheese, and meat/ fish products. In literature (source: Scopus database), there are 2666 papers using keywords "glyphosate" and "analysis" by the end of April 2020 and 361 using "chromatography" as third keyword.
