*Glyphosate Residues in Soil and Air: An Integrated Review DOI: http://dx.doi.org/10.5772/intechopen.93066*

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

[75]

**128**

**Year**

**Sample** 

**Derivatization** 

**Analytical method**

**LOD/LOQ μg/g**

**Other information**

**Reference**

**(pre- or** 

**post-column)**

**preparation** 

**(extraction/**

**cleanup)**

2008 2009 2009 2010 2011 2012 2013 2014 2014 2014

0.01 M

FMOC-CI

10% H PO3 4

0.6 M KOH

FMOC-CI

0.1

Μ

ΚΟΗ/SPE

FMOC-CI

0.2 M KOH

SWV using CFME

HPLC-ESI-MS/MS

using reversed-phase

C18

HPLC-FLD (λex = 267,

0.5 GLY

0.5 AMPA

λem =317 nm) using

reversed-phase C18

SPE-HPLC-ESI-MS/MS

0.37 GLY

Recoveries 85–126%

[61]

[78]

0.61 AMPA

0.6/2.0 ng/mL GLY

0.4/1.3 ng/mL

AMPA

(using HILIC)

SPE-HPLC-FLD (λex

=263 nm and λem

=317 nm)

25/83 μg/L GLY 0.02/0.05 GLY

0.01/0.03 AMPA

Water

1. Ca(ClO)2

2.OPA/ME

Water

2 M NH OH 4

1. CS2

UV 435 nm

1.1./3.7 μg/mL GLY

80–87%

2. Ammonical

solution of Cu(II)

CE/MS (negative with

20 GLY

Recoveries 76–121% (20/200/2000) for GLY

[74]

and 83–89% (40/400/4000) for AMPA

Pre-column conversion: 1. of glyphosate to glycine

by Ca(ClO)2; 2. followed by reaction with OPA/ME

in borate buffer (pH 9.5) to produce the fluorescent

1-(2′-hydroxyethylthio)-2-N-alkylisoindole

Recoveries 89–102%

Recoveries 79–117%

[76]

[77]

[57]

40 AMPA

0.08/0.25 mmol/L

GLY

amino capillary)

SIA-FLD (λex =270 nm,

λem =315 nm)

0.1 M KOH/CAX

cleaned up

2 M NH OH 4

TFAA/TFE

FMOC-CI

GC-NPD HPLC-FLD

0.01 GLY

0.025

Recoveries 88–94%

[72]

[55]

[73]



**Table 1.**

**131**

*Glyphosate Residues in Soil and Air: An Integrated Review*

derivative are formed suitable for GC analysis.

gradual degradation of GLY along with the presence of AMPA.

volatile derivatives for GC analysis. These derivatization approaches can be applied

GLY as a compound permits its detection (in conjunction with GC) by several detectors such as the flame photometric detector (FPD), flame ionization detector (FID), electron capture detector (ECD), nitrogen-phosphorus detector (NPD),and also the more sensitive and selective mass spectrometer detector (MSD). The quantification of GLY in the soil through NPD has reached an limit of quantification (LOQ ) equivalent of 0.02 mg/kg [93, 94]. GC analytical methods are reliable, sensitive, and selective, but the sample preparation is very time-consuming, complicated, and tedious as all ionic groups must be derivatized. In addition, they involve anhydrous conditions, extensive cleanup using solvent partitioning steps, charcoal elimination of pigments, and a large-volume anion of cation exchange.

Borjesson and Torstensson using GC-MS managed to sensitively detect GLY and AMPA in soil [48]. One point that should have been addressed was the content of humic acids a problem mentioned above as well. Extraction was tedious, involving extraction under basic conditions, adjustment of the pH to acidic, and then subjected to column purifications to achieve ligand-anion exchanges. To derivatize GLY and AMPA TFE and TFAA were used. By this way the respective ester and acetyl

Utilizing the S/N approach, they presented one of the lower LOQs of the bibliography for GLY, established at 0.006 mg/kg. The application of the method in soils collected from Swedish embankments after being treated with GLY revealed the

Bergstrom et al. investigated at laboratory level GLY and AMPA in sand and clay soils [13]. More specifically, its degradation was monitored using a GC-MS method, after derivatization with TFE and TFAA. The extraction of the soil was accomplished using an alkaline solution. Selected ion monitoring was utilized to enhance selectivity and optimize sensitivity of the method. The LOQ of the method was established at 0.01 mg/kg. The studied kinetics demonstrated that GLY had a very slow degradation rate in the clay soil. Concerning AMPA, though it is more tenacious than GLY (when derived from GLY), it degrades faster than GLY.

A 2019 study on GLY and AMPA analysis in soil showed that still GC-FPD can function as an adequate tool for such demanding analyses [89]. This research was intrigued by the obstacles observed in derivatization in connection with the by-product interferences in high-performance liquid chromatography (HPLC) analysis (when MS/MS mode is not utilized), which lead to inferior selectivity. Consequently, the soil samples were mixed, turned to powder, and then ultrasonic extracted using water assisted by a solid-phase extraction (SPE). After derivatization, the samples were subjected to chemical analysis. A breakthrough of this process was the three-cross derivatization, and the elaborate investigation of its optimization, aided by the orthogonal experimental design. Such design is fundamental in the selection of the optimum conditions, in this case, reaction temperature, time, and ratio of the derivatizing-coupling reagents.

GC-NPD was utilized by Hu and coworkers to analyze GLY in soil, using GC-MS for verification [72]. Extraction was performed in alkaline environment, followed

not only to soil (or sediment) but to other commodities as well [91].

*DOI: http://dx.doi.org/10.5772/intechopen.93066*

*2.3.3.1 GC-MS*

*2.3.3.2 GC-FPD*

*2.3.3.3 GC-NPD*

*Main characteristics on the methods used for the determination of GLY and its main metabolite in soil matrices.*

#### *Glyphosate Residues in Soil and Air: An Integrated Review DOI: http://dx.doi.org/10.5772/intechopen.93066*

volatile derivatives for GC analysis. These derivatization approaches can be applied not only to soil (or sediment) but to other commodities as well [91].

GLY as a compound permits its detection (in conjunction with GC) by several detectors such as the flame photometric detector (FPD), flame ionization detector (FID), electron capture detector (ECD), nitrogen-phosphorus detector (NPD),and also the more sensitive and selective mass spectrometer detector (MSD). The quantification of GLY in the soil through NPD has reached an limit of quantification (LOQ ) equivalent of 0.02 mg/kg [93, 94]. GC analytical methods are reliable, sensitive, and selective, but the sample preparation is very time-consuming, complicated, and tedious as all ionic groups must be derivatized. In addition, they involve anhydrous conditions, extensive cleanup using solvent partitioning steps, charcoal elimination of pigments, and a large-volume anion of cation exchange.

#### *2.3.3.1 GC-MS*

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

**130**

**Year**

**Sample** 

**Derivatization** 

**Analytical method**

**LOD/LOQ μg/g**

**Other information**

**Reference**

**(pre- or** 

**post-column)**

1. Ca(ClO)2

SIC-FLD

0.03/0.10 GLY

Pre-column conversion: 1. of glyphosate to glycine

[90]

by Ca(ClO)2; 2. followed by reaction with OPA/ME

in borate buffer (pH 9.5) to produce the fluorescent

1-(2′-hydroxyethylthio)-2-N-alkylisoindole

*Solid-phase extraction = SPE; anion exchange = SAX; cation exchange = CAX; Flame photometric detector = FPD; nitrogen-phosphorus detector = NPD; fluorescence detector = FLD (λex = excitation;* 

*λem = emission); capillary electrophoresis = CE; immunosensor = IS; square wave voltammetry = SWV; carbon-fiber microelectrode = CFME; double template imprinted polymer film-gold nanoparticlemodified pencil graphite electrode = DIPN-GNPs-PGE; carbon dot-labeled antibodies = lgG-CDs; sequential injection chromatography = SIC; sequential injection analysis = SIA; linker-assisted* 

*enzyme-linked immunosorbent assay = L'ELISA; laser-induced fluorescence detection = LIF; Fluorenylmethylchloroformate = FMOC-Cl; 1-fluoro-2,4-dinitrobenzene = FDNB; trifluoroethanol = TFE;* 

*N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide = MTBSTFA; 2,2,3,3,4,4,4-heptafluoro-1-butanol = HFB; trifluoroacetic anhydride = TFAA; heptafluorobutyric anhydride = HFBA; isopropyl* 

*chloroformate = iso-PCF; o-phthalaldehyde/2-mercaptoethanol = OPA/ME; 4-toluenesulfonyl chloride = TsCl.*

*Main characteristics on the methods used for the determination of GLY and its main metabolite in soil matrices.*

**Table 1.**

2.OPA/ME

**preparation** 

**(extraction/**

**cleanup)**

2019

Borjesson and Torstensson using GC-MS managed to sensitively detect GLY and AMPA in soil [48]. One point that should have been addressed was the content of humic acids a problem mentioned above as well. Extraction was tedious, involving extraction under basic conditions, adjustment of the pH to acidic, and then subjected to column purifications to achieve ligand-anion exchanges. To derivatize GLY and AMPA TFE and TFAA were used. By this way the respective ester and acetyl derivative are formed suitable for GC analysis.

Utilizing the S/N approach, they presented one of the lower LOQs of the bibliography for GLY, established at 0.006 mg/kg. The application of the method in soils collected from Swedish embankments after being treated with GLY revealed the gradual degradation of GLY along with the presence of AMPA.

Bergstrom et al. investigated at laboratory level GLY and AMPA in sand and clay soils [13]. More specifically, its degradation was monitored using a GC-MS method, after derivatization with TFE and TFAA. The extraction of the soil was accomplished using an alkaline solution. Selected ion monitoring was utilized to enhance selectivity and optimize sensitivity of the method. The LOQ of the method was established at 0.01 mg/kg. The studied kinetics demonstrated that GLY had a very slow degradation rate in the clay soil. Concerning AMPA, though it is more tenacious than GLY (when derived from GLY), it degrades faster than GLY.

#### *2.3.3.2 GC-FPD*

A 2019 study on GLY and AMPA analysis in soil showed that still GC-FPD can function as an adequate tool for such demanding analyses [89]. This research was intrigued by the obstacles observed in derivatization in connection with the by-product interferences in high-performance liquid chromatography (HPLC) analysis (when MS/MS mode is not utilized), which lead to inferior selectivity. Consequently, the soil samples were mixed, turned to powder, and then ultrasonic extracted using water assisted by a solid-phase extraction (SPE). After derivatization, the samples were subjected to chemical analysis. A breakthrough of this process was the three-cross derivatization, and the elaborate investigation of its optimization, aided by the orthogonal experimental design. Such design is fundamental in the selection of the optimum conditions, in this case, reaction temperature, time, and ratio of the derivatizing-coupling reagents.

#### *2.3.3.3 GC-NPD*

GC-NPD was utilized by Hu and coworkers to analyze GLY in soil, using GC-MS for verification [72]. Extraction was performed in alkaline environment, followed

by acidification in the dry extract. The authors stated that NH4OH was the most adequate extractant due to lesser extracted interferences than other alkaline agents, though in other works other agents are selected [48]. Derivatization was accomplished by the use of TFE and TFAA, followed by a liquid extraction using methylene chloride. The method verified the degradation of GLY in soil in apple orchards.

#### *2.3.4 Liquid chromatography and derivatization*

### *2.3.4.1 Fundamentals*

The availability of derivatization techniques compatible with an aqueous extract or sample and the chromatographic separation makes LC a more attractive precolumn derivatization [91]. Derivatization approach is used to produce fluorescent derivatives and to enhance their retention in hydrophobic stationary phases prior to detection by fluorescence detection (FLD), UV detection, electrochemical detection (ECD), or tandem mass spectrometry (MS/MS). In post-column procedures, the most known reactions are ninhydrin derivatization accompanied by UV detection and fluorogenic labeling with o-phthalaldehyde (OPA) in mercaptoethanol or N,N-dimethyl-2-mercaptoethylamine after oxidation of glyphosate to glycine.

Although GLY and its derivatives show high sensitivity in LC determination, a laborious cleanup procedure such as ion-exchange column chromatography is required which may result in some sample loss and lower reproducibility, or many laboratories do not have the facilities required for this type of pre- or post-column fluorogenic labeling. The use of either hydrophilic/weak exchange or reversedphase/weak exchange mixed-mode chromatography without any derivatization, followed by diverse detection techniques including tandem mass spectrometry detection, is gaining interest [77, 95]. HPLC methods are highly sensitive with fluorogenic labeling, but they lack specificity and usually require a laborious cleanup procedure such as ion-exchange column chromatography, which may result in some sample loss and lower reproducibility.

#### *2.3.4.2 Pre-column procedures*

#### *2.3.4.2.1 FMOC derivatization*

Pre-column procedures are a good alternative to post-column ones, and this has gradually come to play an important role in the analysis of glyphosate. The easier, less demanding and more current popular method to analyze these compounds is derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl) followed by HPLC with FLD or MS/MS. A factorial experimental design was applied by a Chilean group in a critical analysis of this derivatization reaction [78]. The design was studied in aqueous soil extracts, unveiling the proper equilibrium between agents for the successful completion of the reaction. For example, excess of FMOC-Cl is required since there are also other active centers (amine-hydroxy groups) with which FMOC-Cl can react. Isotherm data verified the broad applicability of this method.

Back in the 1990s, Sancho et al. established a method for the analysis of GLY in soil samples that involved a pre-column derivatization step with FMOC-Cl and subsequent estimation by coupled-column liquid chromatography with fluorescence detection (LC–LC/FLD) [68]. However, for the determination of glyphosate in soils based on FMOC derivatization analytics, an extraction procedure including an SPE cleanup step has been used in many studies and considered more efficient [37]. In particular, Todorovic et al. extracted soils using sodium tetraborate. Once

**133**

*Glyphosate Residues in Soil and Air: An Integrated Review*

based on the analytical results on three different types of soils.

again, a group devoted substantial time in the extraction of GLY and AMPA from soil due to the complex sorption and desorption in soil which is also pH dependent. The sodium tetraborate performed better in terms of chromatographic efficiency than KOH extraction (more matrix interferences, more humic substances, etc.). The authors after derivatizing GLY and AMPA with FMOC-Cl cleaned up-enriched the extract with a polymeric SPE cartridge. Overall, the method was fit for purpose

Botero-Coy et al. have established a method based on LC–MS/MS, which was successfully applied to soil samples from Colombia and Argentina [77]. This work was an improvement of the previous work in the same domain [49]. In that work, the soil samples were extracted with potassium hydroxide solution and purified with SPE Oasis HLB cartridges. A pre-column derivatization step was also required in this method for which 9-fluorenylmethylchloroformate (FMOC-Cl) was used and the purification method using SPE cartridges was troublesome and expensive. Despite these difficulties, the analysis was conducted in Spanish soils with success. But, when soil samples from the mentioned countries were analyzed by the specific protocol, their high organic content proved an obstacle in the analysis. For this reason the authors introduced a dilution step of the extract assisted by pH adjustment to 9, before the SPE step. For SPE the polymeric reversed-phase Oasis HLB cartridges proved better in retaining-releasing the FMOC derivative than Oasis

Internal standard's use compensated possible downsides during sample preparation and corrected matrix effects. An additional tool in this work was the use of high-resolution mass spectrometry exploiting the time-of-flight technology. By this way additional interferences that would affect the analysis were further elucidated using the accurate mass full-acquisition data. It is noteworthy that the authors investigated MS ions used in the MS/MS mode. Interestingly, MRM transition containing the m/z 179 was problematic since it is related to FMOC and lacks specificity. In our work (see below), the specific ion was monitored only in AMPA transition, solving this issue [88]. Overall, the analysis verified the presence of GLY

Another work in the field of GLY analysis in soil/sludge using FMOC-Cl as a derivatizing agent was presented by Sun and coworkers [96]. In this context, an optimized sample preparation protocol was developed, applying extraction with sodium phosphate and trisodium citrate solutions (aqueous) and a purification step using hexane in acidified soil. The rationale behind the use of trisodium citrate was to counteract the effect of other metal ion complexing agents (such as Mg2+, Ca2+, etc.), in which GLY binds. The method was validated in three types of soils (and sludge samples) verifying that it was fit for purpose. The demonstrated LOQ was

A pre-column derivatization was applied by Druart and coworkers, embracing glufosinate also in their portfolio [60]. A detailed study was conducted on the parameters governing the extraction of the analytes from the matrix. Accelerated solvent extraction, ultrasonic extraction, and magnetic stirring agitation were tested to achieve optimum conditions. In the end agitation was selected. The group also optimized derivatization by selecting water as the solvent of the reaction, though the previous study showed that an equivalent mixture of H2O:ACN would compromise the solubility of both GLY and FMOC-Cl reagents [97]. In the same study, it was demonstrated that a C18 column of 30 cm superseded other columns

In addition our group has developed a methodology for GLY and AMPA detection in topsoils originating from Greece [88]. The sample preparation was envisaged by previous works (one of our group) [49, 98]. The LC–MS/MS method developed

tested, even a respective NH2 column broadly used for such separations.

*DOI: http://dx.doi.org/10.5772/intechopen.93066*

MAX used with good results.

and AMPA in the majority of samples.

determined at 0.04 mg/kg.

#### *Glyphosate Residues in Soil and Air: An Integrated Review DOI: http://dx.doi.org/10.5772/intechopen.93066*

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

*2.3.4 Liquid chromatography and derivatization*

sample loss and lower reproducibility.

*2.3.4.2 Pre-column procedures*

*2.3.4.2.1 FMOC derivatization*

*2.3.4.1 Fundamentals*

by acidification in the dry extract. The authors stated that NH4OH was the most adequate extractant due to lesser extracted interferences than other alkaline agents, though in other works other agents are selected [48]. Derivatization was accomplished by the use of TFE and TFAA, followed by a liquid extraction using methylene chloride. The method verified the degradation of GLY in soil in apple orchards.

The availability of derivatization techniques compatible with an aqueous extract

Pre-column procedures are a good alternative to post-column ones, and this has gradually come to play an important role in the analysis of glyphosate. The easier, less demanding and more current popular method to analyze these compounds is derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl) followed by HPLC with FLD or MS/MS. A factorial experimental design was applied by a Chilean group in a critical analysis of this derivatization reaction [78]. The design was studied in aqueous soil extracts, unveiling the proper equilibrium between agents for the successful completion of the reaction. For example, excess of FMOC-Cl is required since there are also other active centers (amine-hydroxy groups) with which FMOC-Cl can react. Isotherm data verified the broad applicability of this

Back in the 1990s, Sancho et al. established a method for the analysis of GLY in soil samples that involved a pre-column derivatization step with FMOC-Cl and subsequent estimation by coupled-column liquid chromatography with fluorescence detection (LC–LC/FLD) [68]. However, for the determination of glyphosate in soils based on FMOC derivatization analytics, an extraction procedure including an SPE cleanup step has been used in many studies and considered more efficient [37]. In particular, Todorovic et al. extracted soils using sodium tetraborate. Once

or sample and the chromatographic separation makes LC a more attractive precolumn derivatization [91]. Derivatization approach is used to produce fluorescent derivatives and to enhance their retention in hydrophobic stationary phases prior to detection by fluorescence detection (FLD), UV detection, electrochemical detection (ECD), or tandem mass spectrometry (MS/MS). In post-column procedures, the most known reactions are ninhydrin derivatization accompanied by UV detection and fluorogenic labeling with o-phthalaldehyde (OPA) in mercaptoethanol or N,N-dimethyl-2-mercaptoethylamine after oxidation of glyphosate to glycine. Although GLY and its derivatives show high sensitivity in LC determination, a laborious cleanup procedure such as ion-exchange column chromatography is required which may result in some sample loss and lower reproducibility, or many laboratories do not have the facilities required for this type of pre- or post-column fluorogenic labeling. The use of either hydrophilic/weak exchange or reversedphase/weak exchange mixed-mode chromatography without any derivatization, followed by diverse detection techniques including tandem mass spectrometry detection, is gaining interest [77, 95]. HPLC methods are highly sensitive with fluorogenic labeling, but they lack specificity and usually require a laborious cleanup procedure such as ion-exchange column chromatography, which may result in some

**132**

method.

again, a group devoted substantial time in the extraction of GLY and AMPA from soil due to the complex sorption and desorption in soil which is also pH dependent. The sodium tetraborate performed better in terms of chromatographic efficiency than KOH extraction (more matrix interferences, more humic substances, etc.). The authors after derivatizing GLY and AMPA with FMOC-Cl cleaned up-enriched the extract with a polymeric SPE cartridge. Overall, the method was fit for purpose based on the analytical results on three different types of soils.

Botero-Coy et al. have established a method based on LC–MS/MS, which was successfully applied to soil samples from Colombia and Argentina [77]. This work was an improvement of the previous work in the same domain [49]. In that work, the soil samples were extracted with potassium hydroxide solution and purified with SPE Oasis HLB cartridges. A pre-column derivatization step was also required in this method for which 9-fluorenylmethylchloroformate (FMOC-Cl) was used and the purification method using SPE cartridges was troublesome and expensive. Despite these difficulties, the analysis was conducted in Spanish soils with success. But, when soil samples from the mentioned countries were analyzed by the specific protocol, their high organic content proved an obstacle in the analysis. For this reason the authors introduced a dilution step of the extract assisted by pH adjustment to 9, before the SPE step. For SPE the polymeric reversed-phase Oasis HLB cartridges proved better in retaining-releasing the FMOC derivative than Oasis MAX used with good results.

Internal standard's use compensated possible downsides during sample preparation and corrected matrix effects. An additional tool in this work was the use of high-resolution mass spectrometry exploiting the time-of-flight technology. By this way additional interferences that would affect the analysis were further elucidated using the accurate mass full-acquisition data. It is noteworthy that the authors investigated MS ions used in the MS/MS mode. Interestingly, MRM transition containing the m/z 179 was problematic since it is related to FMOC and lacks specificity. In our work (see below), the specific ion was monitored only in AMPA transition, solving this issue [88]. Overall, the analysis verified the presence of GLY and AMPA in the majority of samples.

Another work in the field of GLY analysis in soil/sludge using FMOC-Cl as a derivatizing agent was presented by Sun and coworkers [96]. In this context, an optimized sample preparation protocol was developed, applying extraction with sodium phosphate and trisodium citrate solutions (aqueous) and a purification step using hexane in acidified soil. The rationale behind the use of trisodium citrate was to counteract the effect of other metal ion complexing agents (such as Mg2+, Ca2+, etc.), in which GLY binds. The method was validated in three types of soils (and sludge samples) verifying that it was fit for purpose. The demonstrated LOQ was determined at 0.04 mg/kg.

A pre-column derivatization was applied by Druart and coworkers, embracing glufosinate also in their portfolio [60]. A detailed study was conducted on the parameters governing the extraction of the analytes from the matrix. Accelerated solvent extraction, ultrasonic extraction, and magnetic stirring agitation were tested to achieve optimum conditions. In the end agitation was selected. The group also optimized derivatization by selecting water as the solvent of the reaction, though the previous study showed that an equivalent mixture of H2O:ACN would compromise the solubility of both GLY and FMOC-Cl reagents [97]. In the same study, it was demonstrated that a C18 column of 30 cm superseded other columns tested, even a respective NH2 column broadly used for such separations.

In addition our group has developed a methodology for GLY and AMPA detection in topsoils originating from Greece [88]. The sample preparation was envisaged by previous works (one of our group) [49, 98]. The LC–MS/MS method developed

was adequate for the analysis of both active substances, showing that GLY and AMPA were detected in 37 and 45%, respectively, of the samples investigated. A breakthrough of this work was the association of the results with the land use utilizing geographical information system (GIS) databases.

GLY in soil is studied for registering not only its residual prevalence (including AMPA's) but also its degradation dynamics. With this in view, Zhang et al. investigated its dynamics using an HPLC-FD method, utilizing FMOC derivatization [3]. Results of this study showed that the degradation is dependent on the physicochemical parameters of the soil, exemplified by the pH. The behavior of GLY and AMPA was investigated in compost-amended soils by Erban and colleagues [86]. Soil depth was disclosed as a key factor on the concentrations detected. GLY and AMPA though showed a different behavior when moisture and saturated hydraulic conductivity are considered. More specifically, GLY was affected principally by moisture, whereas AMPA was impacted by this conductivity.
