American Journal of Analytical Chemistry, 2013, 4, 647-652
Published Online November 2013 (http://www.scirp.org/journal/ajac)
Open Access AJAC
Study of the Reaction Derivatization Glyphosate and
Aminomethylphosphonic Acid (AMPA) with
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
Received September 15, 2013; revised October 25, 2013; accepted November 5, 2013
Copyright © 2013 Tereza Cristina Pimenta Gonçalves Catrinck et al. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.
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
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 . Among these
substances, glyphosate (N-phosphonomethylglycine) has
been widely used due to its excellent performance and
effective pest control . This compound is presented as
a polar molecule, post-emergent, non-selective and sys-
temic action . 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 .
Glyphosate (GLY) has been worldwide used in differ-
ent cultures, however, their potential toxicological risks
to human health  and environmental pollution  have
demonstrated the need to develop simple methodologies,
fast and sensitive to monitor GLY residues and their me-
tabolites in the environment . Some techniques have
been used, including high performance liquid chroma-
tography (HPLC) [6,7], capillary electrophoresis (CE) 
and spectrophotometry in the visible region.
Among the proposed techniques, the gas chromatogra-
phy is frequently used due to their high selectivity and
sensitivity . However, the low volatility of GLY and
AMPA molecules makes the determination of these ana-
lytes difficult , requiring the use of derivatization
techniques (pre- or post-column) .
The derivatization procedure consists in chemically
modifying a compound, to increase the sensitivity and/or
make it volatilizable . In the analysis by gas chroma-
bacteria or fungi
Figure 1. Degradation scheme of glyphosate with the pro-
duction of aminomethylphosphonic acid (AMPA) and sar-
T. C. P. G. CATRINCK ET AL.
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 . Thus, some reagents
may be used to reduce the polarity of the compound re-
placing labile hydrogens by aliphatic groups . A-
mong the reagents, trifluoroacetic anhydride (TFAA), tri-
fluoroethanol (TFE)  and N-methyl-N-tert-butyldi-
methylsilylfluoracetamide (MTBSTFA)  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).
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).
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
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.
Composition of the reaction medium (v/v) Acetonitrile/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
TMS O P
TMS O P
TMS O SiCH
TMS O P
TMS O PCH
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]+,
These ions were also proposed by Ngim and collabora-
tors (2011)  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
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 . Thus, the combina-
tion of pyridine and BSTFA was selected for the next
6912 15 18 21 24
6912 15 18 21 24
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.
3.2.2. Ultrasonic Vibration
The ultrasonic waves create, increase and implode steam
cavities and gases in a liquid, promoting activation in
chemical reactions . This process generates heat en-
ergy sufficient to favor homolytic cleavage of the com-
pounds present . 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  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 .
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.
6912 15 18 21 24
Ultras onic vib ration
Without u ltras onic vib ratio n
Figure 4. Part of the chromatogram of GLY e AMPA solu-
tion 1 mg·L−1 with and without ultrasonic vibration.
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 .
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
. 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.
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).
The authors thank the Conselho Nacional de Desenvol-
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
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