Study of the Reaction Derivatization Glyphosate and Aminomethylphosphonic Acid ( AMPA ) with N , OBis ( trimethylsilyl ) trifluoroacetamide

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 ultrasonic vibration and pH adjustment that provide optimal conditions for analysis by GC-MS. Furthermore, the methodology used was simple and fast, and that was the most practical method commonly used.


Introduction
Chemical control of weeds was adopted in the second half of the twentieth century leading to a significant development 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 systemic 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 different 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 metabolites in the environment [5].Some techniques have been used, including high performance liquid chromatography (HPLC) [6,7], capillary electrophoresis (CE) [8] and spectrophotometry in the visible region.
Among the proposed techniques, the gas chromatography 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 analytes 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- 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 replacing labile hydrogens by aliphatic groups [11].Among the reagents, trifluoroacetic anhydride (TFAA), trifluoroethanol (TFE) [12] and N-methyl-N-tert-butyldimethylsilylfluoracetamide (MTBSTFA) [13] have been used for the derivatization of the GLY.
The N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) combined with trimethylchlorosilane (TMCS) is preferentially employed to promote trimetylsylation of alcohols, amines, carboxylic acids, among others.Being an alternative 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 efficiency 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).

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 concentration of 500 mg•L −1 and stored at 4˚C.Working solutions were prepared from stock solutions at the concentrations of 15 e 50 mg•L −1 in the same solvent.

Instrumentation
For chromatographic analysis was used Agilent Technologies 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•min −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 introduced was 1µL in injection mode without flow divider, splitless, using an injector Combi PAL.The mass spectrometer 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.

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.

Optimized Parameters
To optimize the derivatization reaction, the following parameters were evaluated: the composition of the reaction medium, homogenization, volume of BSTFA:Pyridine and pH of the reaction medium according to Table 1.
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 respectively).The pH values were obtained in the pH meter micro processed of Quimis (São Paulo, Brazil).The best conditions were determined based on the mass spectra obtained for the studied compounds.

Structural Characterization of AMPA and GLY Derivatized
Representative mass spectra with the major ions proposed 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   73 represents the formed ion by trimethylsilane group.The peaks in m/z 147, 232, 298, 312 and 340 are cleavage 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 monitoring (SIM), having greater abundance.For AMPA derivatized, there is the following fragments common ionic m/z 73 [(CH 3 ) 3 Si]+, 102 [(CH 3 ) 3 SiNHCH 2 ]+, 298 [((CH3) 3 SiO)2PO(CH 3 ) 3 Si]+, 312 [(CH 3 ) 3 SiOPOO((CH 3 ) 3 Si)CH 2 NH(Si(CH 3 ) 2 ]+.These ions were also proposed by Ngim and collaborators (2011) [14] to the optimize procedure for character-izing impurities in AMPA using the analyte in the solid state and analyzes by GC-MS.

Development and Optimization of the
Silylation Procedure

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 reagent: BSTFA in the proportion 20:200 µL may be observer 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).Already the use of pyridine gave a chromatogram with fewer 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 combination of pyridine and BSTFA was selected for the next experiments.

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 energy sufficient to favor homolytic cleavage of the compounds 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 vibrations have not favored in the derivatization of the analyte.This is because during the cavitation process, few radicalar species may be formed [18] interfering negatively in the derivatization process.However, this result differs from that found for derivatization with BSTFA of carboxylic acid wherein the homogenization and ultrasonic favored by 14% in the chromatographic response [19].

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. Lower volumes of pyridine (20 μL) were not sufficient to basify the medium and favor the reaction of derivatization (Figure 5).It is observed that the derivatization reaction was promoted only when using proportion pyridine: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].

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 derivavitization of the analytes and no signal was observed corresponding to the compounds obtained in the chromatograms.By using basic medium, occurred derivitization of GLY, however, the signal obtained in 13.4 min.showed lower intensity and in this condition the derivitization 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.

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).

Acknowledgements
The authors thank the Conselho Nacional de Desenvol- 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.

Figure 2 .
Figure 2. Mass spectrum showing the principal fragments of GLY (a) and AMPA (b) after derivatization with BSTFA.

Figure 4 .Figure 5 .
Figure 4. Part of the chromatogram of GLY e AMPA solution 1 mg•L −1 with and without ultrasonic vibration.

Figure 6 .
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).