**2.7 Beverages**

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

47 pesticides in 33 honey samples collected from beehives of Estonia. The paper was focused on the detection of the pesticide concentration and the relative maximum residue levels and the possible impact of the agriculture on the product. In any case, the authors largely used the analytical methodology based on using QuEChERS (acronym of Quick, Easy, Cheap, Effective, Rugged and Safe) extraction methodology followed by detection using GC-MS and ultra-high-performance liquid chromatography-MS/MS (UHPLC-MS/MS): the method shows recovery between 78 and 115%, repeatability from 3.0 to 16%, LOQ for GLYP of 0.050 mg kg<sup>−</sup><sup>1</sup>

In 2018, Zoller et al. found GLYP at very low levels in 15 of 16 honey samples analyzed; in addition, they also analyzed pulses (tofu and soy sauce), breakfast cereals (corn flakes and pops), durum wheat, pastry and snacks (crisps, etc.), bread, flour and baking mixtures, and beverages (beer, wine, milk, fruit juices, and mineral water) for a total of 243 samples [86]. The authors applied a well-tested analytical method based on solvent extraction with methanol and LC-MS/MS analysis for determining GLYP and AMPA (LODs 0.2–0.4 and 0.5–1 μg kg<sup>−</sup><sup>1</sup>

<13.9%). Further, in this paper, the authors assessed a dietary risk of each food for a child of 15 kg body weight and for an adult of 60 kg body weight. The first findings of this work were that the GLYP maximum residue levels did not exceed more

for animal products). So, the scores reported by authors for the risk assessment highlighted a low exposure only for the pulses (5% of the acceptable daily intake, ADI, and acute reference dose, ARfD), whereas in all the other cases, honey samples included, the exposure to GLYP is less than 1% of the ADI/ARfD, meaning there is no any human health issue in all samples. Further, the authors, simulating a daily ingestion of the different investigated foods, estimated the probable GLYP content in urine. They found levels in agreement with those found by other authors in German [3, 17] and Swiss [87] populations, whereas some differences could be

A pilot study for monitoring GLYP and AMPA in 32 honey samples was set up by Pareja et al. based on IC coupled to a Q-Orbitrap accurate high-resolution mass spectrometry [88]. It is still confirmed that the use of IC simplifies the polar pesticide determination, whereas the use of an orbitrap detector allows to reach

) and recoveries ranging between 80 and 110% with RSDs <20% in the

Still in 2019, a Canadian group developed an easy method for analyzing GLYP,

FMOC-Cl in acetonitrile solution and online SPE(C18)-LC-MS/MS analysis [89] and the use of isotopically labeled internal standards (as just evidenced previously). In particular, for all the investigated compounds, the authors obtained accuracies ranging between 95.2 and 105.3% (intraday precision 1.6–7.2%) and LOQ 1 μg kg<sup>−</sup><sup>1</sup>

By this method, 200 honey samples were analyzed: GLYP was found in 196 samples

no risks for the consumers. Further, the authors performed a survey between their data with others from worldwide studies (the United States, Estonia, Switzerland,

A 2020 paper evaluated the exposure risk of bees and humans to GLYP and AMPA residues in three different bee matrices, that is, beebread, wax, and paired samples of wax/honey collected from 379 Belgian apiaries using an analytical method based on clean-up on SPE-C18 followed by derivatization step with FMOC-Cl and

for the other pesticides), and correlation coefficients >0.990 for all

; recoveries 92–103 and 92–115%; RSDs <9.5 and

for plant products and 0.05 mg kg<sup>−</sup><sup>1</sup>

for AMPA), less than the allowed EU MRL

levels based on both the derivatization with

with a 95th percentile of 14.2 μg kg<sup>−</sup><sup>1</sup>

(and

, respec-

.

, evidencing

**108**

0.010 mg kg<sup>−</sup><sup>1</sup>

tively; LOQs 0.5–1 and 1–2.5 μg kg<sup>−</sup><sup>1</sup>

a GLYP LOQ of 5 μg kg<sup>−</sup><sup>1</sup>

linearity range of 5–500 μg kg<sup>−</sup><sup>1</sup>

AMPA, and GLUF at low μg kg<sup>−</sup><sup>1</sup>

at maximum level of 49.8 μg kg<sup>−</sup><sup>1</sup>

some just cited in this review) [85, 86, 90–92].

(50 μg kg<sup>−</sup><sup>1</sup>

than the legally tolerated ones (0.1 mg kg<sup>−</sup><sup>1</sup>

expected in AMPA concentration comparison [17].

(20 μg kg<sup>−</sup><sup>1</sup>

.

compounds.

This last matrix is really important considering the large use of beverages in the daily dietary intake. Beverages such as water, beer, milk, and fruit juices are under strict attention by the different national authorities. For instance, in 2019, a study by Cook of the CalPIRG Education Fund (available at https://uspirg.org/sites/pirg/ files/reports/WEB\_CAP\_Glyphosate-pesticide-beer-and-wine\_REPORT\_022619. pdf?\_ga=2.33097086.1581849178.1551185850-857148262.1551185850) reported that 19 of wine (5) and beer (14) brands contained GLYP at levels ranging between 4.8 and 51.4 ppb. Several papers have been published in recent years, some of which have already been mentioned in this review [49, 58, 71, 73, 86].

The first interesting paper by Hao et al. describes a method for analyzing GLYP, AMPA, and GLUF in drinking water, surface water, and groundwater samples [96], that is, a LC-MS/MS method with reversed-phase and weak anion-exchange mixedmode Acclaim® WAX-1 column. Good analytical parameters were obtained: LODs of 1.5, 3.9, and 1.7 μg L<sup>−</sup><sup>1</sup> for GLYP, AMPA, and GLUF, respectively; LOQs of 4.5, 11.6, and 5.3 μg L<sup>−</sup><sup>1</sup> ; and recoveries between 62 and 102%. The main aspect is the analysis by direct injection of aqueous samples without derivatization or clean-up procedures with the risk of artifacts.

In 2015, a Chinese group developed a procedure for analyzing GLYP and GLUF in tea samples by means of FMOC-Cl derivatization and UPLC–MS/MS analysis [97]. The method shows good linearity (*r* > 0.990) in the range of 0.003– 0.1 mg L<sup>−</sup><sup>1</sup> , LODs of 0.03 mg kg<sup>−</sup><sup>1</sup> for both compounds, and recoveries between 81.4 and 99.1% with RSDs <2.3%.

Two papers published in 2015 reported the GLYP, AMPA, and GLUF determination in milk and milk-based products. Ehling and Reddy carried out a derivatization with FMOC-Cl followed by means of LC-MS/MS in different nutritional milk matrices such as cow's milk, human breast milk, soy milk, and whole milk powder [98]. This study is important because the reported analytical method does not require any analytical treatment such as clean-up, evaporation, or concentration; so, the possible artifact formation is drastically reduced. Further, the importance of the use of a triple-quadrupole mass spectrometry is still confirmed in terms of selectivity and fragment analysis. This occurrence gives good analytical parameters: *R*2 > 0.99 in the entire investigated linearity range (5–500 ng mL<sup>−</sup><sup>1</sup> ); recoveries between 91.1 and 115.2%; LODs of 0.012 and 0.01 μg g<sup>−</sup><sup>1</sup> for GLY and AMPA, respectively; LOQ of 0.05 μg g<sup>−</sup><sup>1</sup> for both; high intra-day (<4.0 and <7.7% for GLYP and GLUF, respectively) and inter-day (<8.4 and <3.8, respectively) precision. The second paper investigates the direct injection of milk extract after deproteination and SPE on Oasis cartridge [99]: the LC–MS/MS analysis under the negative ion-spray ionization mode allowed to reach low method detection limits (MDLs), that is, 0.3, 1.4, and 0.4 ng mL<sup>−</sup><sup>1</sup> for GLYP, AMPA, and GLUF, respectively, and low method quantification limits (MQLs), 1, 4, and 1 ng mL<sup>−</sup><sup>1</sup> , respectively, with recoveries ranging between 81 and 107% and RSDs 2.04–8.36%. A LC-MS/MS method (6 min chromatographic run) was successfully applied to a sample of fortified milk with a very low herbicides concentration (0.025 μg mL<sup>−</sup><sup>1</sup> ). Further, the use of negative mode ion spray offers high sensitivity and selectivity. According to the study's

authors (and these authors agree), this methodology could be competitive with the enzyme-linked immunosorbent assay (ELISA) method.

Steinborn et al. reported of a survey on the GLYP content in 114 breast milk samples collected in Bavaria and Lower Saxony, Germany, by comparing the data obtained by LC-MS/MS and GC-MS/MS analyses [100]. The two analyses required (a) an ultrafiltration and chromatography on an anion exchange column for LC–MS/MS and (b) a clean-up step on a cation exchange column and derivatization with trifluoroacetic acid anhydride (TFAA) and heptafluorobutanol (HFB) for the GC–MS/MS. The authors deeply investigated the difference between the chromatograms obtained with the two methods, especially for evaluating parameters such as precision, accuracy, LOD, and LOQ. Basically, GC–MS/MS allowed to reach instrumental detection limit (IDL) lower than that found in LC–MS/MS (0.02 vs. 0.5 ng mL<sup>−</sup><sup>1</sup> ), but they detected an interference on a GLYP peak, which they did not manage to identify (all reagents, ultrapure water, all components were tested). Therefore, they fixed the LOQ at 1 ng mL<sup>−</sup><sup>1</sup> , the same concentration determined by LC–MS/MS (whose LOD is 0.5 ng mL<sup>−</sup><sup>1</sup> ). The recoveries ranged between 83 and 128% with RSD < 17% for LC–MS/MS and between 71 and 102% with RSD < 13% for GC–MS/MS. Resuming, the GC–MS/MS is powerful at lower concentrations, but it simultaneously gives more bias than LC–MS/MS; both methods manage to investigate concentration above 1 ng mL<sup>−</sup><sup>1</sup> with high precision and accuracy.

Two papers investigated the GLYP and AMPA content in human milk and urine samples. In the first, a high-throughput LC–MS/MS method using stable isotope labeled internal standard and clean-up with methylene chloride allowed to reach very low LODs (0.92 and 1.2 for GLYP and AMPA in human milk samples and 0.023 and 0.033 μg mL<sup>−</sup><sup>1</sup> in human urine samples) and LOQs (10 μg mL<sup>−</sup><sup>1</sup> for both in breast human milk samples and 0.1 μg mL<sup>−</sup><sup>1</sup> in human urine samples), high recoveries (GLYP ranging between 92 and 107% in both matrices, AMPA between 89 and 107%) with low RSDs (<7.4 and <11.6% in human milk and urine samples, respectively) [101]. The authors also studied the matrix stability over a storage in 5°C (refrigerator) and at –20°C (freezer): in the first case, the recoveries were acceptable also after 24 hours, whereas in the second case, they were good also after 3 months. On the other hand, the second paper investigated the presence of GLYP and AMPA in milk (41 samples) and urine (40 samples) from healthy lactating women from Russia and the United States [102]. The authors used the same analytical procedure as reported above (i.e., LC-MS/MS, the use of stable isotope labeled internal standard and two fragments, such as precursor and product ion transitions, for the quantification) for the analysis, that is, the same analytical parameters. The results showed GLYP and AMPA in milk samples at levels below the LODs, whereas at low concentrations (<LOD and 1.93 μg mL<sup>−</sup><sup>1</sup> and <LOD and 1.33 μg mL<sup>−</sup><sup>1</sup> , respectively, in urine samples). The authors extrapolated the maximum intake of milk containing 1 μg mL<sup>−</sup><sup>1</sup> of GLYP for a 5-kg infant: their conclusions were that the expected levels should be 12,000 times lower than the health concern.

The presence of MRLs for GLYP in barley, wheat, rye, and hops is regulated by EU Regulation (EC) No. 396/2005 (i.e., 20, 10, and 0.1 mg kg<sup>−</sup><sup>1</sup> ) [37, 38]. These are the raw agricultural commodities for beer beverage. Jansons et al. (2018) analyzed 100 beer samples from 24 different producers and distributors in Latvia with LC– MS/MS method (*R*<sup>2</sup> > 0.999 in the range of 0.2–25 μg kg<sup>−</sup><sup>1</sup> ; LOD 0.2 μg kg<sup>−</sup><sup>1</sup> ; LOQ 0.5 μg kg<sup>−</sup><sup>1</sup> ; RSD < 4.1%) [103]. Among the numerous samples analyzed, 8 samples showed levels below the LOD and 9 samples below the LOQ, whereas 80 samples reported a GLYP concentration below 15 μg kg<sup>−</sup><sup>1</sup> and 1 sample reached a GLYP content of 150 μg kg<sup>−</sup><sup>1</sup> . The authors pointed out the attention on beer brands of "undisclosed" origin, that is, no country production reported on the labeling (it sounds strange to the authors of this review considering the restrictions on food

**111**

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

labeling in the EU, but we reported the authors' considerations), which could have higher GLYP content than the locally produced beer. Further, they also compared beers by malt type (barley or combined/other), color (light or dark), packaging (canned or bottled), the presence of precipitate (precipitate or no precipitate), filtration (filtered or not filtered), and pasteurization (pasteurized or not pasteur-

Over these papers, it should be underlined two other paper dealing the GLYP determination in river water and soil samples. This particular occurrence regards the analytical protocol used by authors. In the first paper, Kudzin et al. developed a procedure based on derivatization with TEA-trifluoroacetic anhydride (TFAA) trimethyl orthoacetate reagent and analyses by GC-CI(or EI)/MS (LOD 2.5–

5.0 pmol) and GC-flame ionization detection (GC-FID; LOD 30–80 pmol, recovery 97%) [104]. In the second paper, Hu et al. investigated the performance of a method based on GC with nitrogen-phosphorus detector (GC-NPD): they estimated a LOD of 9 × 10<sup>−</sup>12 g and a LOQ of 0.01 mg kg–1 in samples, recoveries between 84.4 and 94.0%, and RSDs between 8.1 and 13.7% [105]. These two papers deserve to be mentioned for having introduced the possibility to analyze GLYP by two very easy,

This long *excursus* wanted to cover the novel or advanced methodologies based on chromatographic analysis reported in the literature. The GLYP determination in foods is a really important issue, even if the different international agencies still do not totally agree on the human health concern. The importance of a continuous monitoring of such compound (and its main metabolite, AMPA), and GLUF as well, is well known by scientists and politics worldwide due to its large use in agriculture. The suggestion is to continuously develop new methods, more accurate and sensitive, based on GC-MS/MS or LC-MS/MS analysis but also routine method

In any case, the fear for the future is that the refinement of analytical methods increasingly leads to alarmist attitudes based on the discovery of very low quantities of GLYP, which is possible for a very wide range of products, even extremely toxic,

The authors would like to thank Giuseppe Ianiri for his help in the database

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

**3. Conclusion**

**Acknowledgements**

revision and data analysis.

**Conflict of interest**

ized), finding no significant differences in these cases.

cheap, and worldwide available detectors such as FID and NPD.

based on inexpensive or use-friendly detectors (FID, FPD, or NPD).

without forgetting that in nature the zero residue does not exist.

The authors declare no conflict of interest.

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

labeling in the EU, but we reported the authors' considerations), which could have higher GLYP content than the locally produced beer. Further, they also compared beers by malt type (barley or combined/other), color (light or dark), packaging (canned or bottled), the presence of precipitate (precipitate or no precipitate), filtration (filtered or not filtered), and pasteurization (pasteurized or not pasteurized), finding no significant differences in these cases.

Over these papers, it should be underlined two other paper dealing the GLYP determination in river water and soil samples. This particular occurrence regards the analytical protocol used by authors. In the first paper, Kudzin et al. developed a procedure based on derivatization with TEA-trifluoroacetic anhydride (TFAA) trimethyl orthoacetate reagent and analyses by GC-CI(or EI)/MS (LOD 2.5– 5.0 pmol) and GC-flame ionization detection (GC-FID; LOD 30–80 pmol, recovery 97%) [104]. In the second paper, Hu et al. investigated the performance of a method based on GC with nitrogen-phosphorus detector (GC-NPD): they estimated a LOD of 9 × 10<sup>−</sup>12 g and a LOQ of 0.01 mg kg–1 in samples, recoveries between 84.4 and 94.0%, and RSDs between 8.1 and 13.7% [105]. These two papers deserve to be mentioned for having introduced the possibility to analyze GLYP by two very easy, cheap, and worldwide available detectors such as FID and NPD.
