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.
Journal of Chromatography A 1673 (2022) 463114 Contents lists available at ScienceDirect 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 May 2022 Available online 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 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 (http://creativecommons.org/licenses/by-nc-nd/4.0/) 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: mluisa.marina@uah.es (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 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 https://doi.org/10.1016/j.chroma.2022.463114 0021-9673/© 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) M Ángeles García, S Jiménez-Jiménez and M.L Marina Journal of Chromatography A 1673 (2022) 463114 Fig 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 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 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 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 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 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 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 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 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 s Before its first use, the new capillary was conditioned (applying bar) with M sodium hydroxide for 30 min, Milli-Q water for 15 min, followed by 60 with buffer solution and finally 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 Every working day, at the beginning, the capillary was pre-washed (applying bar) with 0.1 M sodium hydroxide for 10 min, Milli-Q water for min, buffer solution for 20 and with the corresponding BGE for 10 After each run, with the aim of ensuring repeatability between injections, the capillary was rinsed with 0.1 M sodium hydroxide, with Milli-Q water, with buffer solution and 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 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 With guanidine hydrochloride and ChCl four peaks were obtained Although analysis time was shorter when using guanidine hydrochloride than when using ChCl (10 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 % 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 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 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 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 M Ángeles García, S Jiménez-Jiménez and M.L Marina Journal of Chromatography A 1673 (2022) 463114 Fig 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 × s; temperature 20 °C; applied voltage 25 kV; λ 205 ± 30 nm (reference off) and [racemic dimethenamid]: 100 mg L−1 Fig 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 × 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 and resolutions of 5.6, 1.8, and 1.3), slightly decreasing when this percentage increased to a 1.7% w/v (19.5 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 shows, the analy5 M Ángeles García, S Jiménez-Jiménez and M.L Marina Journal of Chromatography A 1673 (2022) 463114 Fig 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 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 for 50 cm to 24.0 for 60 cm However, the resolution values remarkably improved and the baseline resolution of the 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 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 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 M Ángeles García, S Jiménez-Jiménez and M.L Marina Journal of Chromatography A 1673 (2022) 463114 Table 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 ± 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 ± 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 ± 0.0528 103 ± 0.1704 99 ± 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 ± 0.9511 98 ± 0.0603 104 ± 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 = 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 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 Linearity was determined using eight standard solutions containing racemic dimethenamid at concentrations from to 200 mg L−1 Corrected peak areas (Ac) for each of the four stereoisomers 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 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 ± and 211 ± 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 ± and 99 ± % 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 References [1] D.B Carrão, I.S Perovani, N.C.P de Albuquerque, A.R Moraes de Oliveira, Enantioseparation of pesticides: a critical review, TrAC Trends Anal Chem 122 (2020) 115719, doi:10.1016/j.trac.2019.115719 [2] P Zhao, S Lei, M Xing, S Xiong, X Guo, Simultaneous enantioselective determination of six pesticides in aqueous environmental samples by chiral liquid chromatography with tandem mass spectrometry, J Sep Sci 41 (2018) 1287– 1297, doi:10.1002/jssc.201701259 [3] J Ye, M Zhao, L Niu, W Liu, Enantioselective environmental toxicology of chiral pesticides, Chem Res Toxicol 28 (2015) 325–328, doi:10.1021/tx500481n [4] C Draghici, E Chirila, M Sica, Enantioselectivity of 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373–384, doi:10.10 02/jssc.20170 0848 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 four dimethenamid stereoisomers although the baseline separation was only possible under these conditions for the two first-eluting isomers The effect of the addition of different ILs and DES to the dual CDs system was investigated While the addition of the ILs studied did not improve the resolution between consecutive peaks when added to the CDs dual system, the addition of most of the DES had a beneficial effect on the chiral separation The DES ChCl-D-fructose made possible the baseline separation of the four stereoisomers Under the optimized conditions (15 mM CE-β CD + 10 mM M-γ -CD in 100 mM borate buffer (pH 9.0) with 1.5 % ChCl-D-fructose (2:1), a separation voltage of 25 kV and a temperature of 20 ˚C), the separation was achieved in 21.2 with 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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... 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