Advances in the Determination of Alkylsilyl Derivatization of

Glyphosate and Aminomethylphosphonic acid by Gas

Chromatography-Mass Spectrometry

Tuğçe Arkan
Institute of Chemistry, Department of Analytical Chemistry
Eötvös Loránd University, Budapest, Hungary

Ph.D. Dissertation
Submitted to the Ph.D. School of Environmental Sciences
Eötvös Loránd University

Supervisor: Ibolya Molnár-Perl

Professor Emerita, D.Sc.
Eötvös Loránd University, Institute of Chemistry
Department of Analytical Chemistry, Environmental Sciences

Doctoral School of Environmental Sciences

Head of School: Professor Imre M. Jánosi
Environmental Chemistry Program
Head of Program: Professor Tamás Turányi

Budapest
2017
RESEARCH TOPIC AND OBJECTIVES

The constantly increasing production of glyphosate is expected to exceed 1,200 kilotons

by 2024 [1]. Due to the continuous demand and heavy application of the broad spectrum
herbicide, the compound and its metabolite accumulate in the environment, endangering the
wellbeing of the living organisms [2]. Thorough, fast and reliable analysis methods are
required for the efficient detection of the compounds in order to control the herbicide
application levels and to ensure their accumulation in the environment do not reach dangerous
levels.

Derivatization is a key step in the analysis of this highly polar herbicide and one of the
main chromatographic techniques confirmed is the gas chromatography-mass spectrometry
acquisition. However, the GC-MS studies concerning silylation techniques are lacking in the
literature. Thus, formulating a novel method in the form of GLYP's trialkylsilylated
derivatives as well as the reinvestigation of the tert.-butyldimethylsilylation prior to GC
analysis becomes imperative to ensure a complete study of the unrecognized potential of the
method.

Taking the newly published studies into consideration mass selective detection proved
to be the detection method of choice for coupling with both separation methods. In the studies
described, the identification and quantification of the compounds was done by both direct
injection and after their derivatization with FMOC prior to LC detection most commonly
[3,4]. Derivatization compositions prior to GC analysis with TFAA/TFE [5] or MTBSTFA [6]
are described in literature for acquiring the most efficient quantification analyses. Where LC-
MS techniques are well detailed and readily published in the literature, the GC-MS studies
concerning silylation techniques are lacking [6-8]. Thus, formulating a novel method in the
form of glyphosate's trialkylsilylated derivatives as well as the reinvestigation of the tert.-
butyldimethylsilylation prior to GC analysis becomes imperative to ensure a complete study
of the unrecognized potential of the method.

The aim of this dissertation is to investigate and examine the determination of GLYP
and AMPA, in regards to chromatographic techniques applied previously; to present a novel
method for the rapid and simultaneous analysis following a trimethylsilylation step with N-
Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The secondary aim is to reinvestigate
the study of alkylsilylation with N-Methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide
(MTBSTFA) and to propose an alternative and novel method in addition.

1
 The limited labeling of GLYP and AMPA serves as the basis for reconsideration. The
mass fragmentations for the characteristics ions in the literature are further studied and the full
derivatization products that were previously unrecognized are described in order to provide a
complete study of the methods. The optimum reagent-solvent composition and derivatization
conditions for ensuring the identification and quantification of full derivatization ions are
studied and the analytical performance characteristics of the optimized working strategy are
defined.

In summary, the aim of this research is to fully exhaust the triamethylsilyl- (TMS) and
tert.-butyldimethylsilyl- (TBDMS) derivatization properties of GLYP and AMPA with
MSTFA, BSTFA and MTBSTFA by means of

a) ascertaining the identification and quantification of all characteristic fragments by

extending the acquisition range of the GC-MS protocol up to the m/z values of 1000,

b) determining the optimum reagent/solvent compositions and ratios by comparing the

application of three solvents; ACN, PYR and ETAC,

c) establishing the optimum derivatization parameters and working strategy in

comparison for the TMS and TBDMS derivatives efficiency,

d) defining the analytical performance characteristics as well as complete labeling of the

fully derivatized masses as opposed to impartial data from the literature, and

e) measuring the AMPA impurities in GLYP market samples as a means to demonstrate

the practical utility of the method

MATERIALS AND METHODS

GLYP standard was purchased from Molecula Fine Chemicals (distributor: France,
Europe; manufacturer: Jiang SU Feng Shan Group, CO. LTD, China). AMPA ( 99%),
MTBSTFA (with 1% tert-Butyldimethylchlorosilane;  95%), ACN and PYR were products
of Sigma-Aldrich Ltd. (St. Louis, MO, USA), in highest purity available. The 360 g/l GLYP
containing commercial herbicide samples are GlialkaR Star (Glialka; as K salt), Medallon
Premium (Medallon; as diammonium salt) and FozátR 480 (Fozát). Glialka and Medallon are
products of Monsanto Europe, S.A., Brussels, Belgium and Syngenta, AG Suisse,

2
respectively. FozátR 480 was from Agro-Chemie Kft, Budapest, Hungary, while Total Spray
containing 7.2 g/L GLYP (isopropyl amine salt), was acquired from Sinon Corporation
(Taiwan, Republic of China; distributor: Cresco Chemical Kft, Budapest, Hungary).

Solutions of the standards 200 mg/50 mL of GLYP and 20 mg/50 mL of AMPA,

weighed with  0.01 mg uncertainty were prepared using distilled water, and refrigerated (at 4
°C) up to three months. Dilutions from both standards were combined in vials (2 L - 20 L),
and the duplicate solutions were sonicated for 5 minutes, and dried using rotary evaporated (at
30 – 40 C). Stock solutions were prepared from the commercial market samples. The salts
were dissolved in distilled water and diluted in a 50 mL volumetric flask in accordance with
their GLYP contents. 360 g/L GLYP salt containing Glialka, Medallon and Fozát samples
were diluted by 400-fold. 7.2 g/L GLYP salt containing Total Spray sample was diluted by
25-fold.

The evaporated residues were treated with 60 - 100 L PYR (or ACN, or ETAC) and
100 - 120 L MSTFA (or BSTFA) to yield the TMS derivatives. The reagent compositions
for TBDMS derivatives study were a mixture of 60 - 100 L PYR (or ACN), and 100 - 120
L MTBSTFA (with 1% TBDMCS). The solutions were mixed at room temperature; making
up to a total of 180 - 200 L in volume. Derivatization was carried out by heating the samples
at 80 C for 30 minutes. Derivatized solutions and their corresponding blank tests were
transferred into the autosampler vial and one L was injected into the GC-MS/MS apparatus
(Varian 4000 model).

SUMMARY OF RESULTS AND NOVELTY STATEMENTS

In this study, trimethylsilylation and tert.-butyldimethylsilylation of GLYP and AMPA,

with the application of MSTFA and MTBSTFA under optimum conditions, were described.
Upon extending the acquisition up to 1000 m/z, the fully derivatized products were obtained
in the spectra while demonstrating the previously unrecognized, unique reactivity of MSTFA
and the reactivity potential of MTBSTFA. The conclusions drawn from this study are as
follows:

 In order to determine the optimum conditions, trimethylsilylation reagents BSTFA

and MSTFA were applied in different mediums, varying the solvents (ACN, ETAC and
PYR), the reagent/solvent composition ratios and time and temperature parameters of the

3
derivatization process. Under the same conditions, the optimum derivatization parameters
were determined as 30 minutes of heating at 80 C. The retention behavior and the structural
compositions of the derivatization products proved to be consistent, regardless of the
reagent/solvent and ratio compositions.

 The fully derivatized products quantitated as the AMPA.4TMS ([M+1] + m/z 400),
GLYP.4TMS ([M]+/[M+1]+ m/z 457/458) and GLYP.5TMS ([M+1]+ m/z 530) ions. The
existence of the five active sites containing GLYP species was confirmed by DFT modeling
and computation. The differences were observed in the distribution of the products where
compositions containing ACN and PYR favor the full derivatization product formations.

 The detailed fragmentation patterns prove that full derivatization with BSTFA is also
possible under optimum conditions despite the absence of studies in the literature. In the
absence of the catalyst, the strength of the donor silyl reagent, BSTFA in PYR provide an
unsatisfactory yield and even in comparison, ACN as the solvent proves to be a better
selection for an efficient derivatization step. As opposed to PYR, in studies with the addition
of and in the absence of TMCS, the MSTFA/ACN application proves higher response yield.

 Between BSTFA/ACN and MSTFA/ACN methods, the latter favors formation of the
full derivatization products, as the stronger reagent composition, as MSTFA is more volatile
than BSTFA (1%TMCS) which would cause less interference with the by-products after the
silylation process while increasing the detection limit of trace analysis.

 MTBSTFA holds a significant place in the derivatization studies concerning

glyphosate and its metabolite as the more commonly applied reagent in the literature prior to
GC-MS analysis. Under the optimum time and temperature conditions, the tert.-
butyldimethylsilylation of the compounds revealed that in addition to the characteristic ions
described in the literature, the fully derivatized products GLYP.4TBDMS and
AMPA.4TBDMS are also formed. The varied reagent compositions as MTBSTFA/ACN and
MTBSTFA/PYR proved to be equally suitable for the quantitation analysis.

 Repeatability of model measurements was characterized with the relative standard

deviation percentages (RSD %). In the study, LOQ values, expressed as the injected amounts
of species, proved to be 0.45 ng (AMPA) and 1.4 ng (GLYP) in MSTFA/ACN medium with
an average of 4.24 RSD%. In the MTBSTFA/PYR medium, LOQ values were determined as
2.36 ng/ 1μL (GLYP) and 0.036 ng/ 1μL (AMPA) with a 2.63% RSD average; and in the

4
MTBSTFA/ACN medium, 1.18 ng/ 1μL (GLYP) and 0.036 ng/ 1μL (AMPA) with a 3.30%
RSD average.

 The optimized methods were utilized for the analysis of the four commercial samples;
GlialkaR Star, FozátR, Medallon Premium and Total Spray. Following trimethylsilylation with
MSTFA/ACN, AMPA contents varied between Total Spray, 0.17 g/L (2.77 RSD%) up to
Glialka, 3.70 g/L (6.1 RSD%). The analysis of the AMPA impurities of the same set of
samples following derivatization with MTBSTFA/PYR provided values between Total Spray,
0.038 g/L (4.64 RSD%) and up to Medallon, 7.2 g/L (4.28 RSD%).

 Compared to the published data, in papers providing the LOQ values for GLYP
[27,28,30,31], the proposed methods yield improved quantitation limits following
derivatization with both TMS and TBDMS silylation agents.

 The various of studies on simultaneous acylation and esterification technique proves

that the analysis efficiency of GLYP and AMPA samples are greatly improved, providing
LOQ as low as 0.09 μg/L (TFAA/TFE) as opposed to the quantitation limit experienced as 36
μg/L (MTBSTFA/PYR and MTBSTFA/ACN).

Between the GC-MS and LC-MS methods, LC-based studies have been thoroughly
conducted and regarded as the preferred method due to the sensitivity of the simultaneous
quantitation of the samples, providing LOQ as low as 0.005 μg/L (FMOC-Cl) in samples.

5
RESEARCH PUBLICATIONS RELATED TO DISSERTATION

T. Arkan, I. Molnár-Perl, Review: The role of derivatization technique in the analysis of

glyphosate and aminomethylphosphonic acid by chromatography, Microchem. J. 121
(2015) 99-106.

T. Arkan, A. Csámpai, I. Molnár-Perl, Alkylsilyl derivatization of glyphosate and

aminomethylphosphonic acid followed by gas chromatography-mass spectrometry,
Microchem. J. 125 (2016) 219-223.

Poster Presentation, Application of a novel derivatization technique for glyphosate and

aminomethylphosphonic acid analysis by gas chromatography-mass spectrometry;
Presentation (2016) Eger, Hungary.

T. Arkan, I. Molnár-Perl, Advances in the alkylsilyl derivatization of glyphosate and

aminomethylphosphonic acid: A critical comeback to the N-tert.-butyldimethylsilyl-N-
methyltrifluoroacetamide reagent, Microchem. J. 132 (2017) 262-267.

SELECTED BIBLIOGRAPHY

[1] https://www.gminsights.com/industry-analysis/glyphosate-market, accessed on 2 march

2017, report ID: GMI231.

[2] R.L. Rose, E. Hodgson, R.M. Roe, Pesticides: Toxicology, H. Marquardt (Ed.) Academic
Press (1999) California, USA. ISBN: 0-12-473270-4.

[3] T. Poiger, I.J. Buerge, A. Bächli, M.D. Müller, M.E. Balmer, Occurrence of the herbicide
glyphosate and its metabolite AMPA in surface waters in Switzerland determined with
on-line solid phase extraction LC-MS/MS, Environ. Sci. Pollut. Res. 24 (2017) 1588-
1596.

[4] L. Lupi, K.S.B. Miglioranza, V.C. Aparicio, D. Marino, F. Bedmar, D.A.Wunderlin,

Occurrence of glyphosate and AMPA in an agricultural watershed from the southeastern
region of Argentina, Sci. Total Environ. 536 (2015) 687–694.

[5] A. Royer, S. Beguin, J.C. Tabet, S. Hulot, M.A. Reding, P.Y. Communal, Determination
of glyphosate and aminomethylphosphonic acid residues in water by gas
chromatography with tandem mass spectrometry after exchange ion resin purification

6
 and derivatization. Application on vegetable matrixes, Anal. Chem. 72 (2000) 3826-
3832.

[6] Y. Hori, M. Fujisawa, K. Shimada, Y. Hirose, Determination of the herbicide glyphosate

and its metabolite in biological specimens by gas chromatography-mass spectrometry.
A case of poisoning by roundup herbicide, J. Anal. Toxicol. 27 (2003) 162-166.

[7] N. Tsunoda, Simultaneous determination of the herbicides glyphosate, glufosinate and

bialaphos and their metabolites by capillary gas chromatography-ion-trap mass
spectrometry, J. Chromatogr. A 637 (1993) 167-173.

[8] T. Saito, H. Aoki, A. Namera, H. Oikawa, S. Miyazaki, A. Nakamoto, S. Inokuchi, Mix-

mode TiO-C18 monolith spin column extraction and GC-MS for the simultaneous assay
of organophosphorus compounds and glufosinate, and glyphosate in Human Serum and
Urine, Analyt. Sci. 27 (2011) 999-1005.

[9] K.K. Ngim, J. Green, J. Cuzzi, M. Ocampo, Z. Gu, Optimized derivatization procedure for
characterizing (aminomethyl)phosphonic acid impurities by GC-MS, J. Chromatogr.
Sci. 49 (2011) 8-14.

[10] T.C.P.G. Catrinck, M. Aguiar, A. Dias, F. Silverio, P. Fidencio, G. de Pinho, Study of

the reaction derivatization glyphosate and aminomethylphosphonic acid (AMPA) with
N,O-bis(trimethylsilyl)trifluoroacetamide, Am. J. Anal. Chem. 4 (2013) 647-652.