Study of the Reaction Derivatization Glyphosate and Aminomethylphosphonic Acid (AMPA) with <i>N</i>,O-Bis(trimethylsilyl)trifluoroacetamide

doi:10.4236/ajac.2013.411077

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American Journal of Analytical Chemistry, 2013, 4, 647-652

Published Online November 2013 (http://www.scirp.org/journal/ajac)

http://dx.doi.org/10.4236/ajac.2013.411077

Open Access AJAC

Study of the Reaction Derivatization Glyphosate and

Aminomethylphosphonic Acid (AMPA) with

N,O-Bis(trimethylsilyl)trifluoroacetamide

Tereza Cristina Pimenta Gonçalves Catrinck1, Maria Clara Santana Aguiar2, Amanda Dias2,

Flaviano Oliveira Silvério2, Paulo Henrique Fidêncio1*, Gevany Paulino de Pinho2

1Department of Chemistry, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, Brazil

2Institute of Agricultural Sciences, Universidade Federal de Minas Gerais, Montes Claros, Brazil

Email: *[email protected]

Received September 15, 2013; revised October 25, 2013; accepted November 5, 2013

Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original

work is properly cited.

ABSTRACT

This work aimed to study the derivatization unprecedented of glyphosate and AMPA solutions using N,O-bis

(trimethylsilyl)trifluoroacetamide (BSTFA) combined with trimethylchlorosilane (TMCS), evaluating the composition

of the reaction medium, use of ultrasound, volume of BSTFA:pyridine and pH of the reaction medium. From this study

it was inferred that the reaction medium was composed of BSTFA:pyridine in ratio 60:100, respectively, without ultra-

sonic vibration and pH adjustment that provide optimal conditions for analysis by GC-MS. Furthermore, the methodol-

ogy used was simple and fast, and that was the most practical method commonly used.

Keywords: Glyphosate; GC-MS; Derivatization; BSTFA

1. Introduction

Chemical control of weeds was adopted in the second

half of the twentieth century leading to a significant de-

velopment in the industry of herbicides [1]. Among these

substances, glyphosate (N-phosphonomethylglycine) has

been widely used due to its excellent performance and

effective pest control [2]. This compound is presented as

a polar molecule, post-emergent, non-selective and sys-

temic action [3]. It may be degraded by two catabolic

routes (Figure 1), producing aminomethylphosphonic

acid (AMPA) as the major metabolite and sarcosine as an

intermediary in the alternative route [4].

Glyphosate (GLY) has been worldwide used in differ-

ent cultures, however, their potential toxicological risks

to human health [5] and environmental pollution [6] have

demonstrated the need to develop simple methodologies,

fast and sensitive to monitor GLY residues and their me-

tabolites in the environment [5]. Some techniques have

been used, including high performance liquid chroma-

tography (HPLC) [6,7], capillary electrophoresis (CE) [8]

and spectrophotometry in the visible region.

Among the proposed techniques, the gas chromatogra-

phy is frequently used due to their high selectivity and

sensitivity [9]. However, the low volatility of GLY and

AMPA molecules makes the determination of these ana-

lytes difficult [10], requiring the use of derivatization

techniques (pre- or post-column) [8].

The derivatization procedure consists in chemically

modifying a compound, to increase the sensitivity and/or

make it volatilizable [11]. In the analysis by gas chroma-

PNH OH

OH OH

PNH2

OH OH

NH OH

AMPA

Glyphosate

Sarcosine

bacteria or fungi

Figure 1. Degradation scheme of glyphosate with the pro-

duction of aminomethylphosphonic acid (AMPA) and sar-

cosine [4].

*Corresponding author.

T. C. P. G. CATRINCK ET AL.

648

tography, substances containing functional groups-OH

and -NH, as the GLY and AMPA may form hydrogen

bonds with each other and/or matrix components, making

their volatilization difficult [11]. Thus, some reagents

may be used to reduce the polarity of the compound re-

placing labile hydrogens by aliphatic groups [11]. A-

mong the reagents, trifluoroacetic anhydride (TFAA), tri-

fluoroethanol (TFE) [12] and N-methyl-N-tert-butyldi-

methylsilylfluoracetamide (MTBSTFA) [13] have been

used for the derivatization of the GLY.

The N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)

combined with trimethylchlorosilane (TMCS) is prefer-

entially employed to promote trimetylsylation of alcohols,

amines, carboxylic acids, among others. Being an alter-

native for the analysis of low volatile compounds by gas

chromatography, the combination of these compounds

favors the replacement of the amine and phosphonate

groups which may be found in the structures of GLY and

AMPA. TMCS acts as a catalyst, increasing the strength

of the donor silyl (BSTFA) and assuring greater effi-

ciency for the reaction. However, few reports have been

found in the literature regarding the derivatization of

GLY and AMPA using this combination of reagents.

With this study, we sought to develop and optimize a

derivatization technique as from the silylation of GLY

and AMPA using a combination of BSTFA and TMCS

and analysis by gas chromatography, and detection by

mass spectrometry (GC-MS).

2. Experimental

2.1. Reagents

Standard stock solutions of GLY (99.2% m/m) and

AMPA (99.0% m/m) obtains of Sigma-Aldrich (St. Louis,

MO, EUA) were prepared in deionized water with con-

centration of 500 mg·L−1 and stored at 4˚C. Working

solutions were prepared from stock solutions at the con-

centrations of 15 e 50 mg·L−1 in the same solvent.

As solvents used Pyridine (99.8% v/v) and N,O-bis(tri-

methylsilyl)trifluoroacetamide (BSTFA) with 1% TMCS,

both obtained from Sigma-Aldrich (St. Louis, MO, USA).

Sodium hydroxide with a purity greater than 97.0% m/m

(Dynamic, Brazil) and hydrochloric acid (37.0% v/v)

acquired from Vetec (Rio de Janeiro, Brazil).

2.2. Instrumentation

For chromatographic analysis was used Agilent Tech-

nologies gas chromatograph (GC 7890A) coupled to a

mass spectrometer (MS 5975). Was used a capillary

column DB-5 MS (Agilent Technologies, stationary

phase 5% phenyl and 95% methylpolysiloxane, 30 m ×

0.25 mm d.i. × 0.25 µm film thickness). Helium

(99.9999%) was used as carrier gas at a rate of 3.0

mL· mi n −1. The injector was maintained at 280˚C. The

system initially at 100˚C increased the temperature at a

rate of 8˚C·min −1 to 300˚C. The sample volume intro-

duced was 1µL in injection mode without flow divider,

splitless, using an injector Combi PAL. The mass spec-

trometer was operated in electron ionization at 70 eV,

and a quadrupole mass analyzer, operated in selective ion

monitoring (SIM) (m/z 232, 312 e 340 for GLY and m/z

102, 298 e 312 for AMPA). The interface was kept at

300˚C and the ion source to 280˚C.

2.3. Sample Preparation

The optimized parameters of the reaction derivatization

of GLY and AMPA are described afterwards. Samples of

10.6 µL of GLY and AMPA standard solution at 15

mg· L −1 respectively, were transferred for a derivatization

vial (0.3 mL) and heated to dryness (60˚C). Then, was

added 60 µL of pyridine and, after five minutes, 100 µL

of the reagent derivatizing (BSTFA + TMCS 1%). The

mixture was heated at 60˚C for 30 minutes, previously

the analysis by GC-MS.

2.4. Optimized Parameters

To optimize the derivatization reaction, the following

parameters were evaluated: the composition of the reac-

tion medium, homogenization, volume of BSTFA:Pyri-

dine and pH of the reaction medium according to Table

The pH of the reaction medium was adjusted using

concentrated hydrochloric acid and solution of sodium

hydroxide with pH 10 and 13 (0.1 and 1.0 mol·L−1 re-

spectively). The pH values were obtained in the pH me-

ter micro processed of Quimis (São Paulo, Brazil). The

best conditions were determined based on the mass spec-

tra obtained for the studied compounds.

3. Results and Discussion

3.1. Structural Characterization of AMPA and

GLY Derivatized

Representative mass spectra with the major ions pro-

posed for GLY and AMPA derivatized may be observed

in Figure 2. The identification of derivatives of these

compounds was performed by interpretation of their

mass spectra with respect to their molecular mass and

expected elution order.

Mass spectrum for GLY (Figure 2(a)) after substitu-

tion by groups TMS ((CH3)3Si) showed fragmentation

profile containing the main íons m/z 73 [(CH3)3Si]+, 147

[(CH3)3SiOSi(CH3)2]+, 232 [(CH3)3SiOCOCH2N

((CH3)3Si)CH2]+, 298 [((CH3)3SiO)2PO(CH3)3Si], 312

[(CH3)3SiOPOO((CH3)3Si)CH2NH(Si(CH3)2]+ and 340

[((CH3)3SiO)2POCH2N((CH3)3Si)CH2]+. The peak in m/z

Open Access AJAC

T. C. P. G. CATRINCK ET AL. 649

Table 1. Variables evaluated in optimizing the derivatiza-

tion reaction of GLY and AMPA.

Variables Levels

Composition of the reaction medium (v/v) Acetonitrile/BSTFA;

Pyridine/BSTFA

Ultrasonic vibration (min) 0 e 2

Volume of BSTFA:Pyridine (µL) 20:200; 60:100

pH of the reaction medium 1, 6 and 13

[ ]

50000

100000

150000

200000

250000

300000

350000

102

108

116

122

131

137

145

151

160

166

176

182

192

199

210

221

227

239

256

281

287

302

315

330

Abundance

312

298

102

NTMS

TMS O P

OTMS

TMS

M15

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

106

117

128

139

151

161

172

183

195

207

218

229

242

254

266

278

290

302

314

327

344

367

384

399

419

443

462

m/z

Abundance

232

340

312

298

147

TMS

TMS O P

OTMS

TMS

TMS O SiCH

TMS

OTMS

TMS O P

OCH

TMS

TMS O PCH

TMS

Si(CH

)

[

[ ]

457

327

[M]

[ ]

(a)

(b)

Figure 2. Mass spectrum showing the principal fragments

of GLY (a) and AMPA (b) after derivatization with BSTFA.

73 represents the formed ion by trimethylsilane group.

The peaks in m/z 147, 232, 298, 312 and 340 are cleav-

age and rearrangement products of the structure of GLY

after the derivatization and electron impact at 70 eV.

Among the major ions obtained for GLY, peaks at m/z

232, 312 and 340 were selected for selective ion moni-

toring (SIM), having greater abundance.

For AMPA derivatized, there is the following frag-

ments common ionic m/z 73 [(CH3)3Si]+, 102

[(CH3)3SiNHCH2]+, 298 [((CH3)3SiO)2PO(CH3)3Si]+,

312 [(CH3)3SiOPOO((CH3)3Si)CH2NH(Si(CH3)2]+.

These ions were also proposed by Ngim and collabora-

tors (2011) [14] to the optimize procedure for character-

izing impurities in AMPA using the analyte in the solid

state and analyzes by GC-MS.

3.2. Development and Optimization of the

Silylation Procedure

3.2.1. Composition of the Reaction Medium

To favor the derivatization reaction with BSTFA one

base was added to the medium. In this study, was used

acetonitrile and pyridine, the latter is most often selected

for derivatization reactions for analysis by GC [15,16].

The chromatograms obtained from the use of basic re-

agent: BSTFA in the proportion 20:200 µL may be ob-

server in Figure 3.

In chromatograms was not observed sign of studied

compounds. However this is the first measured parameter

in the optimizating method allowing observing that the

use of acetonitrile favored for derivatization of some

impurities presents in the medium mainly represented by

the compounds between 12 e 20 min (Figure 3). Al-

ready the use of pyridine gave a chromatogram with few-

er interferences. These results differ from those found for

amino acid analyzes that the use of acetonitrile favored

for derivatization of the analyte [17]. Thus, the combina-

tion of pyridine and BSTFA was selected for the next

experiments.

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

6912 15 18 21 24

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

6912 15 18 21 24

Acetonitrile:BSTFA

Pyridine:BSTFA

Time (minutes)

Abundance Abundance

(a)

(b)

Figure 3. Part of the chromatogram of GLY e AMPA solu-

tion 1 mg·L−1 obtained by employing basic reagent: BSTFA

in proportion 20:200 μL (acetonitrile (a) e pyridina (b)).

Open Access AJAC

T. C. P. G. CATRINCK ET AL.

650

3.2.2. Ultrasonic Vibration

The ultrasonic waves create, increase and implode steam

cavities and gases in a liquid, promoting activation in

chemical reactions [18]. This process generates heat en-

ergy sufficient to favor homolytic cleavage of the com-

pounds present [18]. To evaluate this parameter was used

ultrasonic bath and two minutes as ultrasonic vibration

time as shown in Figure 4.

The chromatograms showed that the ultrasonic vibra-

tions have not favored in the derivatization of the analyte.

This is because during the cavitation process, few radi-

calar species may be formed [18] interfering negatively

in the derivatization process. However, this result differs

from that found for derivatization with BSTFA of car-

boxylic acid wherein the homogenization and ultrasonic

favored by 14% in the chromatographic response [19].

3.2.3. Volume of BSTFA:Pyridine

The relation between the basic reagent volume (Pyridine)

and derivatizing reagent (BSTFA) was also evaluated

and the results can be verified in Figure 5.

500000

1000000

1500000

2000000

2500000

3000000

3500000

6912 15 18 21 24

Série1

Série2

Ultras onic vib ration

Without u ltras onic vib ratio n

Time

(

minutes

)

Abundance

Figure 4. Part of the chromatogram of GLY e AMPA solu-

tion 1 mg·L−1 with and without ultrasonic vibration.

200000

400000

600000

800000

60:200

60:100

Time (minutes)

AMPA

Abundance

Pyridine:BSTFA

20:200

50000

100000

150000

200000

250000

13 14

60:200

60:100

Time (minutes)

GLY

Abundance

Pyridine:BSTFA

20:200

(b)

(a)

Figure 5. Part of the chromatogram of AMPA solution (a)

and GLY (b) 1 mg·L−1 using differents volums of basic re-

agente (Pyridine) and derivatizing reagent (BSTFA) in

proportions 20:200 and 60:100 μL.

Lower volumes of pyridine (20 μL) were not sufficient

to basify the medium and favor the reaction of derivati-

zation (Figure 5). It is observed that the derivatization

reaction was promoted only when using proportion pyri-

dine:BSTFA 60:100 μL. Under these conditions the

AMPA eluted at 8.2 min and GLY 13.4 min. This ratio

has been used for derivatization of plant extracts [20].

3.2.4. pH of the Reaction Medium

The GLY has secondary chemical equilibrium having its

structure changed various forms in a certain medium pH

[21]. In this work, pH 1.00 was used to ensure complete

protonation of the molecule and pH 13.0 promoting

complete desprotonation of the same.

The use of an acidic medium did not favor the deriva-

vitization of the analytes and no signal was observed

corresponding to the compounds obtained in the chro-

matograms. By using basic medium, occurred derivitiza-

tion of GLY, however, the signal obtained in 13.4 min.

showed lower intensity and in this condition the deriviti-

zation of AMPA was not favored (Figure 6). Thus, the

step of adjusting the pH of the reaction medium was not

inserted in the optimized methodology.

4. Conclusion

The optimization technique of derivitization of GLY and

AMPA resulted in a rapid and simple method for the

analysis of these compounds by GC-MS. It was observed

that the process of derivitization occurred more favorably

when using pyridine: BSTFA in proportions 60:100 μL,

respectively, without the need to add steps to ultrasonic

vibration or adjust the pH of the reaction medium (pH 6).

5. Acknowledgements

The authors thank the Conselho Nacional de Desenvol-

200000

400000

600000

800000

Time

(

minutes

)

Absorbance

AMPA

50000

100000

150000

200000

250000

13 1

pH 13

sem ajust

Time

(

minutes

)

Absorbance

GLY

pH 6

pH 13

(b)

(a)

Figure 6. Part of the chromatogram of AMPA solution (a)

and GLY (b) 1 mg·L−1 obtained by employing basic medium

(pH 13) and no pH adjustment (pH 6).

Open Access AJAC

T. C. P. G. CATRINCK ET AL. 651

vimento Científico e Tecnológico (CNPq) and Fundação

de Amparo à Pesquisa do Estado de Minas Gerais

(FAPEMIG) for their financial supports. At Universidade

Federal de Minas Gerais for infrastructure available.

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