Journal of Chromatography A 1673 (2022) 463114

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Stereoselective separation of dimethenamid by cyclodextrin
electrokinetic chromatography using deep eutectic solvents
María Ángeles García a,b, Sara Jiménez-Jiménez a, María Luisa Marina a,b,∗
a

Universidad de Alcalá, Departamento de Química Analítica, Química Física e Ingeniería Química, Ctra. Madrid-Barcelona Km. 33.600, Alcalá de Henares
(Madrid) 28871, Spain
b
Universidad de Alcalá, Instituto de Investigación Qmica Andrés M. del Río, Ctra. Madrid-Barcelona Km. 33.600, Alcalá de Henares (Madrid) 28871, Spain

a r t i c l e

i n f o

Article history:
Received 15 March 2022
Revised 29 April 2022
Accepted 2 May 2022
Available online 7 May 2022
Keywords:
Cyclodextrin
Chiral separation
Electrokinetic chromatography
Deep eutectic solvents

Ionic liquids

a b s t r a c t
An Electrokinetic Chromatography (EKC) method was developed in this work enabling for the first time
the separation of the four stereoisomers of the acetamide herbicide dimethenamid. A screening of different anionic cyclodextrins (CDs) revealed that the use of a single CD system did not allow the separation
of the four dimethenamid stereoisomers while dual systems improved the chiral separation. The combination of 15 mM (2-carboxyethyl)-β -CD (CE-β -CD) with 10 mM methyl-γ -CD (M-γ -CD) originated the
partial separation of dimethenamid stereoisomers. To obtain the baseline separation between all consecutive peaks, the effect of the addition of ionic liquids and deep eutectic solvents to the CDs dual system
was investigated. While ionic liquids did not improve the chiral separation obtained with the CDs dual
system, the addition of deep eutectic solvents showed generally beneficial effects on the separation in
terms of resolution. The influence of the nature of the deep eutectic solvent was studied and the effects
of the ready-made deep eutectic solvent and its components on the separation were compared. Choline
chloride-D-fructose (ChCl-D-fructose) when added to the CDs dual system under optimized conditions
(15 mM CE-β -CD, 10 mM M-γ -CD, 1.5 % ChCl-D-fructose (2:1) in a 100 mM borate buffer (pH 9.0), a
separation voltage of 25 kV and a temperature of 20 ˚C) enabled separating the four stereoisomers of
dimethenamid in 21 min with resolutions between consecutive peaks of 6.0, 2.1 and 1.5. The analytical
characteristics of the developed method were evaluated and considered adequate to achieve the stereoselective analysis of dimethenamid-P in commercial agrochemical formulations. Results demonstrated the
potential of the method to control the quality of these formulations and to determine the stereoisomeric
purity of dimethenamid-P in these products.
© 2022 The Authors. Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license
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1. Introduction
The use of pesticides to improve food quality and crop yields is
increasing considerably, due to the continuous growth of the world
population, the great demand for food, the rapid development of
agriculture and the multitude of pests, weeds and fungi that can
be found in crops [1,2]. However, despite their numerous advantages, pesticides are considered one of the most dangerous pollutants in the environment, not only for their toxicity, but also for
their mobility and bioaccumulation capacity.
Around a 30-40% of the currently registered pesticides contain
at least one chiral centre, giving rise to different enantiomers [3].

These enantiomers can behave different, showing different activity,
∗

Corresponding author.
E-mail address: (M.L. Marina).

toxicity and persistence. When one of the enantiomers is the most
active and presents a lesser risk for the environment or non-target
organisms, the use of enantiomerically pure pesticides is recommended to prepare commercial formulations. Nevertheless, due to
economic reasons, most of the chiral pesticides are marketed as
racemates [4] which implies in many cases an unnecessary risk for
the environment.
[2-chloro-N-(2,4-dimethyl-3-thienyl)-N-(2-methoxy-1methylethyl)acetamide], commonly known as dimethenamid,
is a chiral acetamide herbicide widely used on raw agricultural
commodities [5]. Dimethenamid consists of 4 stereoisomers (aS,1S;
aR,1S; aS,1R and aR,1R) due to two chiral elements, a carbon atom
asymmetrically substituted and a chiral axis (Fig. 1) [6,7]. The
low energy required for the rotation around the chiral axis gives
rise to racemization and thus, to two main isomers: aRS,1R and
aRS,1S (designed as S-dimethenamid and commercially known

/>0021-9673/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( />

M. Ángeles García, S. Jiménez-Jiménez and M.L. Marina

Journal of Chromatography A 1673 (2022) 463114

Fig. 1. Chemical structure of dimethenamid.

as dimethenamid-P) [7]. Among both isomers, the S form (aR,1S

and aS,1S diastereomers) has demonstrated to be more active, so
the agrochemical formulations are marketed as S-dimethenamid
[7]. In order to achieve the quality control of these formulations,
the development of analytical methodologies enabling the separation of the four stereoisomers of dimethenamid has a high
interest. Although some works reported the chiral separation of
this herbicide [6,8], the separation of the four stereoisomers of
dimethenamid has never been reported before. Buser and Müller
[6] described the partial separation (Rs 1.2) of two isomers of
dimethenamid by High Resolution Gas Chromatography (HRGC)
using a chiral OV1701-BSCD column in around 22 min. Authors
supposed that atropisomers of dimethenamid were unstable at
the column temperatures employed in the analyses. The elution
order of these isomers in the column was unknown. In addition,
the partial separation of three isomers of dimethenamid by HPLC
using a Pirkle column in around 46 min was also reported in
this work [6]. Since a 1:2:1 ratio was observed for these three
peaks, authors suggested the possibility of the elution of two
unresolved stereoisomers in peak 2. The HRGC method developed
for dimethenamid was applied by the same authors to study the
degradation of this compound in environmental samples showing
a low to moderate enantioselectivity in soils and sewage sludges
while no enantioselective degradation was observed for surface
waters and in rain [8].
Capillary Electrophoresis (CE) has demonstrated a great potential to achieve chiral separations mainly using the separation mode
Electrokinetic Chromatography (EKC) being cyclodextrins (CDs) the
most employed chiral selectors. In spite of the high discrimination power of CDs, in the case of very hydrophobic chiral compounds or with multiple chiral centres, the use of dual systems
of chiral selectors and/or the addition of other compounds to the
separation buffer is necessary in many cases to obtain stereoisomeric separations [9]. In recent years, the trend to develop environmentally friendly analytical procedures has led to the use of
new compounds that can be added to the background electrolyte
(BGE) in combination with CDs, such as deep eutectic solvents

(DES) or ionic liquids (ILs) that have received considerable attention for being chemically sustainable solvents [10,11]. ILs are salts
with weakly coordinated ions which melting points are below the
boiling point of water. They are constituted by a bulky organic

cation and an organic or inorganic anion [10] and have extensively
been studied in many fields due to their interesting physicochemical properties [10,12]. ILs can be used in CE directly as background
additives [13–16], as sole chiral selectors [9,13,17–20] or chiral ligands [9,13,21,22] when they are chiral, but their most frequent use
is based on their combination with chiral selectors including CDs
[9,10,13–16,23] generating in many cases a synergistic effect.
DES are homogeneous liquids at ambient temperature formed
by a mixture of a donor (HBD) and an acceptor (HBA) of hydrogen bond compounds (usually solids) [11,16,24–26]. The synthesis of DES, unlike ILs, is simple and environmentally friendly as
it does not involve the use of any organic solvent since the two
solid components are mixed and heated until a homogeneous liquid is obtained. Quaternary ammonium, tetraalkylammonium or
phosphonium salts are generally used as HBA components, while
carboxylic acids, amines, polyols or carbohydrates are usually used
as HBD components [16,25]. The formation of hydrogen bonds between the two components leads to a significant decrease in the
melting point of DES in comparison with their precursors. They
are non-volatile, with high thermal stability and readily dissolve
many organic and inorganic compounds. Compared to ILs, DES are
cheaper, more environmentally friendly, and easier to obtain [25].
Despite these advantages, DES have been very scarcely used in
CE and only in combination with CDs [11,16,24,25]. Thus, the presence of a DES in the CE separation medium can alter the chiral
separation based on a change in the ionic strength of the separation buffer and/or the adsorption of the DES on the capillary inner surface that could reduce or even reverse the EOF [11,24,26].
Moreover, the addition of a DES can improve the inclusion capacity of the CDs allowing more enantiomers to enter the CD cavity
and then increasing the resolution [24].
Mu et al. were the first who investigated the effect of different
DES based on choline choride (ChCl) as HBA, and urea, ethylene
glycol, propylene glycol and butylene glycol as HBD, on the chiral
separation of zopiclone, salbutamol and amlodipine using β -CD as
chiral selector [11]. Resolution was improved in the presence of the

DES for the three drugs suggesting the existence of a synergistic
effect. Deng et al. [24] studied the effect of different ChCl-based
DES (with urea, ethylene glycol, propylene glycol, lactic acid, and
glycerol as HBD) on the enatioseparation of tropicamide, homatropine, ofloxacin, atenolol and propanolol when using different
2


M. Ángeles García, S. Jiménez-Jiménez and M.L. Marina

Journal of Chromatography A 1673 (2022) 463114

β -CDs as chiral selectors in non-aqueous capillary electrophoresis
(NACE). An improvement in the chiral separation efficiency and in
the inclusion efficiency of the CD in presence of a DES was demonstrated through fluorescence measurements. Salido-Fortuna et al.
investigated the effect of different ChCl-based DES (urea, ethylene
glycol, D-glucose, D-sorbitol as HBD) on the enantioseparation of
the drug lacosamide when they were combined with different CDs.
The combination of ChCl-D-sorbitol and succinyl-β -CD allowed the
separation of lacosamide for the first time by CE. This dual system
was necessary to obtain a resolution value large enough to perform
the analysis of the enantiomeric impurity of lacosamide in a pharmaceutical formulation [25]. Finally, García-Cansino et al. studied
the effect of adding five ChCl DESs (ethyleneglycol, D-sorbitol, Dglucose, D-fructose, urea as HBD) to the separation medium containing sulfobutylated-β -CD at two pH values (3.0 and 9.0) on the
enantiomeric separation of clopidogrel [16]. In this case, there was
not a significant change in the resolution or analysis time when
adding the DES with respect to the addition of ChCl alone to the
separation buffer containing the CD at each pH value.
The aim of this work was to separate for the first time the
four stereoisomers of dimethenamid by EKC using CDs as chiral
selectors. The effect of the addition of DES and ILs to the separation medium was investigated and the variables affecting the chiral
separation were optimized. Application of the developed method

to the stereoselective analysis of dimethenamid-P-based commercial agrochemical formulations was also described.

tetramethylammonium-glutamic acid ([TMA][L-Glu]) and 2tetrabutylammonium-glutamic acid ([TBA]2 [L-Glu])), were synthesized by the Center for Applied Chemistry and Biotechnology
(CQAB) from the University of Alcalá following different procedures
previously reported [28]. CQAB also synthesized the ILs L-alanine
tertbutyl ester bis(trifluoromethane)sulfonamide ([L-AlaC4][NTf2 ]),
L-carnitine methyl ester bis(trifluoromethane)sulfonimide ([LCarniOMe][NTf2 ]) and L-carnitine methyl ester L-lactate ([LCarniOMe][L-lactate]) following previously optimized procedures
[29,30].
The five DES used in this work (ChCl-urea, ChCl-EtGly, ChClD-sorbitol, ChCl-D-glucose and ChCl-D-fructose) were synthesized
following the literature [25,27]. In brief, ChCl-urea and ChCl-EtGly
were synthesized both in the molar ratio 1:2 by mixing the appropriate amount of ChCl with the appropriate amount of urea and
of EtGly, respectively. Both mixtures were stirred for 30 min in a
water bath at 70-80 °C. ChCl-D-glucose and ChCl-D-fructose were
prepared both in the molar ratio 2:1, while ChCl-D-sorbitol was
prepared in the molar ratio 1:1. These last three DES were stirred
in a water bath at 70-80 °C for 4 h. All DES were stored at room
temperature in the dark before dilution in the buffer.
Dimethenamid and dimethenamid-P were from Sigma-Aldrich.
The commercial agrochemical formulations (M1 and M2) were
kindly donated by BASF Española, S.L (Barcelona, Spain). According
to the labelled data, M1 and M2 contained 720 g L−1 and 212.5 g
L−1 of dimethenamid-P, respectively.

2. Materials and methods

2.2. Preparation of solutions and samples

2.1. Chemicals, reagents, DESs, ILs and samples

In order to prepare the borate buffer solution, first, the appropriate amount of boric acid was dissolved in Milli-Q water to

reach a concentration of 100 mM. Then, the pH was adjusted with
sodium hydroxide 1M to the desired value (pH 9.0) before completing the volume with Milli-Q water. Background electrolyte solutions (BGEs) were prepared by dissolving the appropriate amount
of the different CDs, DES or ILs in the borate buffer solution.
Stock standard solutions of racemic dimethenamid and
dimethenamid-P, and stock solutions of agrochemical formulations (M1 and M2) (20 0 0 mg L−1 ) were prepared in methanol
and stored at -20°C. Working solutions containing racemic
dimethenamid and/or dimethenamid-P or agrochemical formulations (M1 and M2) were prepared from the stock solutions by
appropriate dilution in Milli-Q water. Disposable nylon 0.45 μm
pore size filters purchased from Scharlau were used to filter all
solutions before their injection in the CE system.
Reagents, DES, ILs and standards were weighed on an OHAUS
Adventurer Analytical Balance (Nänikon, Switzerland). A pH-meter
model 744 from Metrohm (Herisau, Switzerland) was employed to
adjust the pH of the borate buffer solutions. An ultrasonic bath
B200 from Branson Ultrasonic Corporation (Danbury, USA) was
used to degas all the solutions.

Boric acid, sodium hydroxide, choline chloride (ChCl), guanidine hydrochloride, betaine, ethylene glycol (EtGly), D-sorbitol, Dglucose and D-fructose were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Methanol, urea and orthophosphoric acid were
obtained from Scharlau (Barcelona, Spain).
Carboxymethyl-α -CD (CM-α -CD, DS ∼ 3.5), carboxymethyl-γ CD (CM-γ -CD, DS ∼ 3.5), succinyl-β -CD (Succ-β -CD, DS ∼ 3.4),
succinyl-γ -CD (Succ-γ -CD, DS ∼ 3.5), (2-carboxyethyl)-β -CD (CEβ -CD, DS ∼ 3.5), (2-carboxyethyl)-γ -CD (CE-γ -CD, DS ∼ 3.5), phosphated β -CD (Ph-β -CD, DS ∼ 4), phosphated γ -CD (Ph-γ -CD, DS
∼ 3.5), sulfated α -CD (S-α -CD, DS ∼ 12), sulfated γ -CD (S-γ -CD,
DS ∼ 10), sulfobutylated β -CD (SB-β -CD, DS ∼ 6.3), γ -CD, methylγ -CD (M-γ -CD, DS ∼ 12), (2-hydroxypropyl)-γ -CD (HP-γ -CD, DS
∼ 4.5), heptakis(2,3,6-tri-O-methyl)-β -CD (TM-β -CD), acetyl-β -CD
(Ac-β -CD, DS ∼ 7) and acetyl-γ -CD (Ac-γ -CD, DS ∼ 7) were
from Cyclolab (Budapest, Hungary). Carboxymethyl-β -CD (CM-β CD, DS ∼ 3), sulfated β -CD (S-β -CD, DS ∼ 18), α -CD, methylβ -CD (M-β -CD) and heptakis(2,6-di-O-methyl)-β -CD (DM-β -CD)
were from Sigma-Aldrich. β -CD and (2-hydroxypropyl)-β -CD (HPβ -CD, DS ∼ 0.6) were purchased from Fluka (Buchs, Switzerland).
Sulfobutylether-β -CD (captisol) was from Cydex Pharmaceuticals
(Lawrence, Kansas). Water used to prepare solutions was purified

through a Milli-Q system from Millipore (Bedford, MA, USA).
Two of the sixteen ILs used in this work, 2-hydroxyethyltrimethylammonium
L-lactate
([HETMAm][L-lactate])
and
1-ethyl-3-methylimidazolium
L-lactate
([EMIm][L-lactate])
were purchased from Sigma-Aldrich. Amino acid-based
ILs
used
in
this
work
(tetrabutylammonium-arginine
([TBA][L-Arg]),
tetramethylammonium-arginine
([TMA][LArg]),
tetrabutylammonium-aspartic
acid
([TBA][L-Asp]),
tetramethyl-ammonium-aspartic
acid
([TMA][L-Asp]),
tetrabutylammonium-lysine ([TBA][L-Lys]), tetramethylammoniumlysine
([TMA][L-Lys]),
tetrabutylammonium-isoleucine
([TBA][L-Ile]),
tetramethylammonium-isoleucine
([TMA][LIle]),

tetrabutylammonium-glutamic
acid
([TBA][L-Glu]),

2.3. CE analysis
An Agilent 7100 CE system from Agilent Technologies (Waldbronn, Germany) was used to carry out the electrophoretic experiments. Detection was performed with a diode array detector
(DAD) set at a wavelength of 205 nm with a bandwidth of 30
nm (reference off). The HP 3D CE ChemStation software from Agilent Technologies was used to control the electrophoretic system.
Separations were carried out using uncoated fused-silica capillary
provided by Polymicro Technologies (Phoenix, AZ, USA). Injections
were made applying a pressure of 50 mbar for 5 s.
Before its first use, the new capillary was conditioned (applying 1 bar) with 1 M sodium hydroxide for 30 min, Milli-Q water
for 15 min, followed by 60 min with buffer solution and finally
3


M. Ángeles García, S. Jiménez-Jiménez and M.L. Marina

Journal of Chromatography A 1673 (2022) 463114

mM M-γ -CD. ILs in which the cationic counterpart was an alkylammonium chain and the anionic part was an amino acid were
employed together with other ILs in which the anionic part was
L-lactate or NTf2 being the cationic part an amino acid derivative
or EMIm or HETMAm. Table S1 shows the analysis times and resolutions between consecutive peaks obtained when these ionic liquids were added to the separation medium. ILs in which the anionic counterpart was an amino acid made possible the separation
in four peaks except when L-Asp and L-Ile were the amino acids
or when [TBA]2 [L-Glu] was employed. In fact, the addition of the
ILs [TBA][L-Arg], [TMA][L-Arg], [TBA][L-Lys], [TMA][L-Lys], [TBA][LGlu], [TMA][L-Glu] gave rise to four peaks although their baseline separation was not observed. Although the addition of TBA
ILs enabled the stereoselective separation in shorter analysis times
than TMA ILs, resolutions obtained with TMA ILs were slightly
higher for the separation of the three last peaks. Regarding the ILs

[LCarniOMe][L-Lact], [EMIm][L-Lactate] and [HETMAm][L-Lactate],
their addition to the dual CDs system also originated four peaks.
However, any of the ILs investigated in this work allowed obtaining
the baseline separation of the four stereoisomers since resolutions
between consecutive peaks obtained when adding ILs were equal
or lower than 3.6/0.9/0.6. In addition, these resolution values did
not improve in any case those obtained with the CDs dual system
without ILs (3.8/0.9/0.6).

with the corresponding BGE for 10 min. Every working day, at the
beginning, the capillary was pre-washed (applying 1 bar) with 0.1
M sodium hydroxide for 10 min, Milli-Q water for 5 min, buffer
solution for 20 min and with the corresponding BGE for 10 min.
After each run, with the aim of ensuring repeatability between injections, the capillary was rinsed 4 min with 0.1 M sodium hydroxide, 2 min with Milli-Q water, 2 min with buffer solution and
3 min with BGE.
2.4. Data treatment
Migration time values, resolution values between consecutive
peaks (Rs) and peak area values were obtained using the HP 3D CE
ChemStation software. In order to compensate the differences in
the electrophoretic conditions and to obtain better reproducibility of data, corrected peak areas (Ac) were used for data treatment. For the analysis of experimental data, development of statistical tests and the composition of graphs and figures, the programs used were Excel Microsoft, Origin Pro 8, Statgraphics Centurion XVII software and ChemDraw 20.0.
3. Results and discussion
3.1. Development of a chiral analytical methodology by CD-EKC for
the separation of the four stereoisomers of dimethenamid

3.1.3. Effect of the addition of DES to the dual system
Since the use of dual CDs systems or the addition of ILs to
the separation medium did not suppose the baseline separation of
dimethenamid stereoisomers although four peaks were obtained,
the effect of the addition of DES to the dual CDs system was studied.
Preliminary experiments were achieved to select the most adequate HBA part of the DES to be assayed. With this aim, the addition of three different HBAs (betaine, guanidine hydrochloride and

ChCl which structures are shown in Fig. S2) at a concentration of
0.5% w/v to the dual CDs system selected in this work, was studied
(Fig. S3). When betaine was added, only three peaks were observed
in an analysis time of 7.8 min. With guanidine hydrochloride and
ChCl four peaks were obtained. Although analysis time was shorter
when using guanidine hydrochloride than when using ChCl (10
min versus 12 min), resolution values between consecutive peaks
were lower when using guanidine hydrochloride (Rs 3.6, 1.0 and
0.6) than when using ChCl (Rs 4.1, 1.2 and 0.7). Therefore, ChCl was
chosen. Next, the influence of the percentage of ChCl added to the
separation medium was evaluated (0.2, 0.5 and 1 % w/v). Fig. S4
shows that by increasing the added concentration of ChCl up to 1%
w/v, higher analysis times (13.8 min) were obtained, but also better resolution values between consecutive peaks (Rs 4.4, 1.5 and
1.0). However, repeatability between analyses got worse and peak
efficiency was lower than at a 0.5 % ChCl. On the other hand, when
the percentage of ChCl added was 0.2% w/v, analysis times and resolutions between consecutive peaks decreased (10 min; Rs 3.6, 0.9
and 0.6). As a result, next experiments were carried out using a
concentration of ChCl of 0.5% w/v as a compromise between analysis time and resolutions.
After selecting the most suitable HBA, in this case ChCl at a percentage of 0.5 %, the effect of the addition to the dual CDs system
of five different DES (three chiral: ChCl-D-fructose, ChCl-D-glucose
and ChCl-D-sorbitol and two achiral: ChCl-urea and ChCl-EtGly,
which structures are shown in Fig. S2), maintaining a percentage
of 0.5% ChCl, was investigated (Fig. S5). It was observed that chiral DES gave rise to higher resolution values between consecutive
peaks than achiral DES, being ChCl-D-fructose the chiral DES producing the best resolution values between consecutive peaks (Rs
4.9, 1.4 and 0.9). The improvement observed in the chiral separation of dimethenamid under these conditions could be due to an

3.1.1. Effect of the nature of the cyclodextrin
Since dimethenamid is a neutral compound, 14 negatively
charged (at pH 9) CDs were evaluated (CM-α -CD, CM-γ -CD, Succβ -CD, CE-β -CD, CE-γ -CD, Ph-β -CD, Ph-γ -CD, S-α -CD, S-γ -CD, SBβ -CD at a concentration of 10 mM and S-β -CD, Succ-γ -CD, CMβ -CD and sulfobutylether-β -CD (captisol) at a concentration of 2%
w/v). A 100 mM borate buffer, using a separation voltage of 20

kV, a temperature of 20°C, an uncoated fused-silica capillary 50
μm id × 50 cm (58.5 cm to the detector) and an injection of 50
mbar x 5 s were employed. Among all the CDs tested, only five
of them (Ph-γ -CD, Captisol, SB-β -CD, Ph-β -CD and CE-β -CD) interacted stereoselectively with the analyte, resulting in a certain
stereomeric discriminating power and two peaks (Fig. S1). However, CE-β -CD gave rise to the highest resolution value (0.8 in less
than 8 min) and, in addition, it originated the unfolding of the
second peak obtained with a less peak broadening. Thus, CE-β -CD
was selected as the chiral selector.
The effect of combining CE-β -CD with other negatively charged
or neutral CDs under the above-described initial experimental conditions, was investigated. The dual systems formed by 10 mM CEβ -CD + 10 mM HP-γ -CD; 10 mM CE-β -CD + 2% S-β -CD; 10 mM
CE-β -CD + 10 mM M-γ -CD; 10 mM CE-β -CD + 10 mM CM-β -CD;
and 10 mM CE-β -CD + 10 mM S-γ -CD gave rise to the best results
(three peaks were obtained for all of them in less than 10 min).
However, none of these combinations gave rise to the 4 peaks corresponding to the stereoisomers of dimethenamid. Then, the concentration of CE-β -CD was increased to 15 mM in those dual systems in which the last peak was broaden (CE-β -CD + M-γ -CD and
CE-β -CD + S-β -CD). The dual system composed of 15 mM CE-β CD and 10 mM M-γ -CD was chosen since it enabled the partial
separation of the four stereoisomers of dimethenamid (resolutions
between consecutive peaks 3.8, 0.9, 0.6 in 8.9 min) (Fig. 2A). This
last CDs combination was selected in order to study the effect of
the addition of ILs and DES on the separation with the objective to
improve the resolution among stereoisomers.
3.1.2. Effect of the addition of ionic liquids to the dual CDs system
In order to improve the stereoselective separation obtained for
dimethenamid, 16 different ILs were added at a concentration of
10 mM to the dual system formed by 15 mM CE-β -CD and 10
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M. Ángeles García, S. Jiménez-Jiménez and M.L. Marina

Journal of Chromatography A 1673 (2022) 463114


Fig. 2. Electropherograms corresponding to the separation of dimethenamid stereoisomers when using the dual system 15 mM CE-β -CD + 10 mM M-γ -CD (A) alone and
combined with (B) a 0.585 % of D-fructose, (C) a 0.915 % of ChCl, (D) a 0.585 % of D-fructose + a 0.915 % of ChCl and (E) a 1.5 % of ChCl-D-fructose (2:1) DES. Experimental
conditions: BGE in 100 mM borate buffer (pH 9.0); uncoated fused-silica capillary 50 μm id × 60 cm (68.5 cm to the detector); injection by pressure 50 mbar × 5 s;
temperature 20 °C; applied voltage 25 kV; λ 205 ± 30 nm (reference off) and [racemic dimethenamid]: 100 mg L−1 .

Fig. 3. Electropherograms showing the chiral separation of dimethenamid stereoisomers when different percentages of the DES ChCl-D-fructose were added to the dual
system of CE-β -CD and M-γ -CD. Experimental conditions: 15 mM CE-β -CD + 10 mM M-γ -CD in 100 mM borate buffer (pH 9.0); uncoated fused-silica capillary 50 μm
id × 50 cm (58.5 cm to the detector); injection by pressure 50 mbar × 5 s; applied voltage 20 kV; temperature 20 °C; λ 205 ± 30 nm (reference off) and [racemic
dimethenamid]: 100 mg L−1 .

enhancement in the inclusion ability of the CD for the analyte in
the presence of the DES as well as a squeezing effect of DES on the
CD-enantiomers interactions, as previously proposed by Deng et al.
[24].

sis time and the resolution between consecutive peaks increased
with the percentage of the DES up to a value of 1.5% w/v of ChClD-fructose (20.2 min and resolutions of 5.6, 1.8, and 1.3), slightly
decreasing when this percentage increased to a 1.7% w/v (19.5 min
and resolutions of 5.5, 1.7 and 1.2). Then, a 1.5% w/v of ChCl-Dfructose was selected as the optimum value. The increase in the
analysis time observed can be justified taking into account the increase in the viscosity caused by the DES and the change in the
EOF due to a modification of the capillary wall by the DES [24,26]
(in both cases, the enantiomers and the CDs would have more time

3.1.4. Optimization of the dual system CE-β -CD + M-γ -CD in
presence of ChCl-D-fructose
The influence of the percentage of ChCl-D-fructose (0.8, 1.2, 1.5
and 1.7% w/v) added to the CDs dual system, on the chiral separation of dimethenamid was studied. As Fig. 3 shows, the analy5



M. Ángeles García, S. Jiménez-Jiménez and M.L. Marina

Journal of Chromatography A 1673 (2022) 463114

Fig. 4. Electropherograms obtained for (A) a dimethenamid standard solution (containing racemic dimethenamid at a concentration of 100 mg L−1 ), (B) a dimethenamidbased agrochemical commercial formulation solution, M1 (containing dimethenamid-P at a concentration of 50 mg L−1 ) and (C) a dimethenamid-based agrochemical commercial formulation solution, M2 (containing dimethenamid-P at a concentration of 50 mg L−1 ), under the optimized conditions (25 kV; uncoated fused-silica capillary 50
μm id × 60 cm (68.5 cm to the detector); other conditions as in Fig. 3). ∗ Peaks of dimethenamid-P.

to interact between them). These reasons together with the abovementioned effect of the DES on the inclusion ability of the CD and
on the CD-enantiomers interactions [24] could explain the increase
observed in the enantiomeric resolution.
The effect of the temperature was also studied (15, 20 and 25
°C). Results obtained are shown in Fig. S6. It can be observed that
when a temperature of 25 °C was used, the shape of the first peak
deteriorated. In addition, analysis time decreased (16.8 min), what
resulted in a decrease in the resolution values between consecutive
peaks (Rs 3.5, 1.5 and 1.0). For a temperature of 15 °C, the analysis
time increased (24.2 min) and resolution values got worse except
for the two first-migrating peaks (Rs 6.0, 1.5 and 1.1). Therefore, a
temperature of 20 °C was considered the most adequate to achieve
the chiral separation.
In order to improve the resolution values between peaks 3 and
4, the effective length of the capillary was increased from 50 cm
to 60 cm (maintaining the same electric field, thus, applying a
voltage of 20 and 23.4 kV, respectively). As it can be observed in
Fig. S7, the analysis time increased from 20.2 min for 50 cm to
24.0 min for 60 cm. However, the resolution values remarkably
improved and the baseline resolution of the 4 stereoisomers of
dimethenamid was obtained (Rs 6.5, 2.1 and 1.5).
Finally, in order to decrease the analysis time, a separation voltage of 25 kV was applied instead of 23.4 kV which enabled to
decrease the analysis time (around 3 min) without decreasing the

resolution values between the second, third and fourth peaks while
the resolution for the two first peaks slightly decreased (6.0, 2.1
and 1.5).
Fig. 4A shows the first stereoselective separation of
dimethenamid showing the separation of the four stereoisomers with resolution values between consecutive peaks ranging
from 1.5 to 6.0. This separation was not possible when the DES or
its components were present in the separation buffer under the
same optimized experimental conditions but without the addition
of the two CDs employed. In fact, no peaks were observed under
these experimental conditions in absence of the CDs (results

not shown). These results show the crucial role of the chiral
selectors employed (CE-β -CD + M-γ -CD) in the separation of
dimethenamid stereoisomers and also the fact that the addition
of the ready-made DES or the mixture of its components can
improve the separation obtained with the dual CDs system.
3.1.5. Effect of the addition of the individual components of
ChCl-D-fructose to the CDs dual system
To investigate the effect of the eutectic mixture on the chiral
separation of dimethenamid with respect to that of the individual components of the DES, both ChCl and D-fructose were added,
individually and together, to the dual CDs system selected in this
work and the results obtained were compared with those corresponding to the addition of the ready-made DES. The components
of the DES were added at the same percentages (w/v) as those employed in the synthesis of the DES. While components of the DES
were individually dissolved to the aqueous separation buffer, the
DES was synthesized previously by mixing the components in solid
state to obtain an eutectic mixture at a given temperature, which
is subsequently added to the separation buffer once synthesized.
Results obtained are shown in Fig. 2B–E. As it can be observed, the
addition of D-fructose at a percentage of 0.585% w/v to the 15 mM
CE-β -CD + 10 mM M-γ -CD system originated similar resolution

values and analysis times as when using the dual system alone.
When ChCl was added at a percentage of 0.915% w/v to the dual
CDs system, a considerable improvement in the resolution values
for dimethenamid stereoisomers was observed (resolution values
between consecutive peaks increased from 3.8, 0.9 and 0.6 to 5.2,
1.8 and 1.1) although in a longer analysis time (15.2 min), probably due to a modification in the capillary wall by the ChCl (25).
Similarly, when both DES components were simultaneously added
(ChCl 0.915% + D-fructose 0.585%) to the dual CDs system, better resolution values between consecutive peaks were achieved (Rs
values of 5.9, 2.0 and 1.3) in 20.2 min. However, the baseline separation of all dimethenamid stereoisomers (Rs values of 6.0, 2.1 and
1.5) was only obtained when the ready-made DES was added to
6


M. Ángeles García, S. Jiménez-Jiménez and M.L. Marina

Journal of Chromatography A 1673 (2022) 463114

Table 1
Analytical characteristics of the developed chiral method.
First-migrating stereoisomer
External standard calibration method a
Range
1-50 mg L−1
Slope ± t · Sslope
0.045 ± 0.001
Intercept ± t · Sintercept
0.03 ± 0.03
R2
0.998
Standard additions calibration method for M1 b

Range
0-35 mg L−1
Slope ± t · Sslope
0.046 ± 0.002
R2
0.994
Accuracy
p-value of ANOVA
0.0649
Recovery (%) c
101 ± 4
Standard additions calibration method for M2 d
Range
0-35 mg L−1
Slope ± t · Sslope
0.046 ± 0.002
R2
0.996
Accuracy
p-value of ANOVA
0.0873
Recovery (%) e
104 ± 5
Precision
Instrumental repeatability f
t, RSD (%)
1.1
Ac, RSD (%)
2.4
Method repeatability g

t, RSD (%)
1.6
Ac, RSD (%)
2.9
Intermediate precision h
t, RSD (%)
1.9
Ac, RSD (%)
4.1
LOD i
0.5
LOQ j
1.8

Second-migrating stereoisomer

Third-migrating stereoisomer

Fourth-migrating stereoisomer

1-50 mg L−1
0.043 ± 0.002
0.06 ± 0.06
0.992

1-50 mg L−1
0.037 ± 0.001
0.03 ± 0.04
0.995


1-50 mg L−1
0.036 ± 0.001
0.04 ± 0.04
0.995

0-35 mg L−1
0.044 ± 0.002
0.996

0-35 mg L−1
0.039 ± 0.002
0.991

0-35 mg L−1
0.038 ± 0.002
0.991

0.3150
101 ± 4

0.0528
103 ± 4

0.1704
99 ± 3

0-35 mg L−1
0.043 ± 0.002
0.996


0-35 mg L−1
0.037 ± 0.003
0.991

0-35 mg L−1
0.038 ± 0.002
0.993

0.7783
103 ± 6

0.9511
98 ± 2

0.0603
104 ± 4

1.1
2.6

1.1
2.5

1.1
2.5

1.8
3.0

1.9

3.2

1.9
2.9

2.1
3.9
0.6
1.8

2.1
4.3
0.6
2.0

2.2
3.7
0.6
2.0

Ac : corrected area.
a
Eight standard solutions at different concentration levels injected in triplicate.
b
Addition of eight known amounts of racemic dimethenamid standard solution to commercial formulation sample containing a constant concentration of
dimethenamid-P.
c
Accuracy was evaluated as the mean recovery obtained from six samples solutions (n=6) of commercial formulation containing 30 mg L−1 of dimethenamid-P
(as labelled amount) spiked with 60 mg L−1 of racemic dimethenamid standard solution.
d

Addition of six known amounts of racemic dimethenamid standard solution to commercial formulation sample containing a constant concentration of
dimethenamid-P.
e
Accuracy was evaluated as the mean recovery obtained from six samples solutions (n=6) of commercial formulation containing 30 mg L−1 of dimethenamid-P
(as labelled amount) spiked with 60 mg L−1 of racemic dimethenamid standard solution.
f
Instrumental repeatability was calculated from six consecutive injections of racemic dimethenamid standard solution (100 mg L−1 ).
g
Method repeatability was determined by using the value obtained for three replicates of racemic dimethenamid standard solutions injected in triplicate on
the same day (100 mg L−1 ).
h
Intermediate precision was calculated by using the value obtained for three replicates (injected in triplicate during three consecutive days) of racemic
dimethenamid standard solutions (100 mg L−1 ).
i
LOD obtained experimentally for a S/N = 3.
j
LOQ obtained experimentally for a S/N = 10.

were represented as a function of their concentrations in mg L−1 .
Linearity was adequate since R2 values were ≥ 0.992 for the four
stereoisomers and confidence intervals for the intercept included
de zero value while confidence intervals for the slope did not include the zero value (in both cases for a 95 % confidence level).
The t-test was employed to study the presence of matrix interferences by comparison of the confidence intervals for the slopes
corresponding to the external standard and the standard additions calibration methods for the commercial formulations. Since
the confidence intervals for the slopes of the calibration methods overlapped and p-values were ≥ 0.05 for a 95 % confidence
level for all cases, there were no matrix interferences (see Table 1).
Therefore, the external calibration method could be used to quantitate the content of dimethenamid-P in the agrochemical formulations. Fig. 4B and C show that no interferences were observed for
any of the commercial agrochemical formulations analyzed, which
demonstrated an adequate selectivity of the developed methodology.
Accuracy of the method was evaluated as the recovery values

(%) obtained for the four stereoisomers of dimethenamid when the

the dual system at a concentration of 1.5%. In last two cases where
the two components of the DES and the ready-made DES were
added to the separation medium, the above-mentioned effects of
the addition of the DES on the chiral separation (higher analysis
times and resolutions) were again observed. From the results obtained, the ready-made DES was selected as the best condition to
reach the baseline separation of all dimethenamid stereoisomers in
21.2 min.
3.2. Analytical characteristics of the developed method
The developed method was applied to the stereoselective analysis of dimethenamid-P (aRS,1’S isomers) commercial agrochemical formulations. With this aim, the analytical characteristics of
the method were evaluated in terms of linearity, selectivity, precision, accuracy, limits of detection (LODs) and limits of quantitation
(LOQs)). The results obtained are grouped in Table 1.
Linearity was determined using eight standard solutions containing racemic dimethenamid at concentrations from 4 to 200 mg
L−1 . Corrected peak areas (Ac) for each of the four stereoisomers
7


M. Ángeles García, S. Jiménez-Jiménez and M.L. Marina

Journal of Chromatography A 1673 (2022) 463114

agrochemical commercial formulation solutions containing 30 mg
L−1 of dimethenamid-P were spiked with 60 mg L−1 of racemic
dimethenamid standard solution. Good recovery values were obtained, since the 100 % value was included in all cases in the confidence interval (Table 1).
Precision was evaluated considering the RSD values obtained for
migration times and corrected peak areas for the instrumental and
method repeatability and for the intermediate precision. As shown
in Table 1, adequate RSD values were obtained in all cases (between 1.1 and 2.2 % for migration times and between 2.4 and 4.3
% for corrected peak areas).

Finally, LODs and LOQs were experimentally determined as the
minimum concentration yielding an S/N ratio of 3 and 10 times,
respectively. LODs ranged from 0.5 to 0.6 mg L−1 and LOQs from
1.8 to 2.0 mg L−1 for all stereoisomers.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.
CRediT authorship contribution statement
María Ángeles García: Conceptualization, Methodology, Visualization, Data curation, Resources, Supervision, Writing – original
draft, Writing – review & editing, Project administration, Funding acquisition. Sara Jiménez-Jiménez: Investigation, Formal analysis, Validation, Data curation, Visualization, Writing – original
draft. María Luisa Marina: Conceptualization, Methodology, Visualization, Data curation, Resources, Supervision, Writing – original
draft, Writing – review & editing, Project administration, Funding
acquisition.

3.3. Quantitation of dimethenamid in commercial agrochemical
formulations

Acknowledgements
Once demonstrated the suitability of the method, it was applied
to the quantitative analysis of dimethenamid-P in two commercial agrochemical formulations (M1 and M2) by injecting a diluted
sample of these formulations containing each, dimethenamid-P at
a concentration of approximately 50 mg L−1 according to their labels.
Contents of dimethenamid-P of 721 ± 5 and 211 ± 4 g L−1 were
obtained for M1 and M2, respectively. These values were in agreement with the label claim of the two agrochemical formulations
(recovery percentages of 100 ± 1 and 99 ± 2 % with respect to
the labelled amounts), which pointed out the applicability of the
method for herbicide analysis in real samples such as agrochemical products. Indeed, the great potential of the developed method
to control the stereoisomeric purity of dimethenamid-P formulations was demonstrated (aRS,1’R isomers were not detected).


Authors thank financial support from the Spanish Ministry of
Science and Innovation for research project PID2019-104913GB-I00,
and the University of Alcalá for research project CCG20/CC-023.
S.J.J. thanks the Spanish Ministry of Science, Innovation and Universities for her FPU pre-doctoral contract (FPU18/00787). Authors
thank C. Huertas, S. Bernardo-Bermejo and G. Fernández-Pérez for
technical assistance.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.chroma.2022.463114.
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4. Conclusions
The four stereoisomers of dimethenamid were separated for
the first time in this work. CD-EKC with a dual system of two
CDs (CE-β -CD and M-γ -CD) enabled the partial separation of the
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good performance for the quantitation of dimethenamid-P in commercial agrochemical formulations. Results obtained indicated that
no statistically significant differences were found between the total concentration determined for the analyzed formulations and
their labelled contents showing the great potential of the method
for herbicide analysis in real samples such as commercial agrochemical products. Moreover, the applicability of the method to
control the quality and stereoisomeric purity of dimethenamid-Pbased agrochemical formulations was demonstrated (steroisomeric

impurities were not detected for the agrochemical products analyzed).
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9