Stereoselective separation of dimethenamid by cyclodextrin electrokinetic chromatography using deep eutectic solvents

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.

Contentslistsavailableat ScienceDirect

journalhomepage: www.elsevier.com/locate/chroma

María Ángeles Garcíaa,b, Sara Jiménez-Jiméneza, María Luisa Marinaa,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 Química 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

AnElectrokineticChromatography(EKC)methodwasdevelopedinthisworkenablingforthefirsttime theseparationofthefourstereoisomersoftheacetamideherbicidedimethenamid.Ascreeningof differ-entanioniccyclodextrins(CDs)revealedthattheuseofasingleCDsystemdidnotallowtheseparation

ofthefourdimethenamidstereoisomerswhiledualsystemsimprovedthechiralseparation.The combi-nationof15mM(2-carboxyethyl)-β-CD(CE-β-CD)with10mMmethyl-γ-CD(M-γ-CD)originatedthe partialseparationofdimethenamidstereoisomers.Toobtainthebaselineseparationbetweenall consec-utivepeaks,theeffectoftheadditionofionicliquidsanddeepeutecticsolventstotheCDsdualsystem wasinvestigated.WhileionicliquidsdidnotimprovethechiralseparationobtainedwiththeCDsdual system,the additionofdeepeutecticsolventsshowedgenerallybeneficialeffectsontheseparationin termsofresolution.Theinfluenceofthenatureofthedeepeutecticsolventwasstudiedandtheeffects

oftheready-madedeepeutecticsolventanditscomponentsontheseparationwerecompared.Choline chloride-D-fructose(ChCl-D-fructose) whenaddedtothe CDsdualsystemunderoptimized conditions (15mMCE-β-CD,10mMM-γ-CD,1.5 %ChCl-D-fructose (2:1)ina100mMboratebuffer(pH9.0), a separationvoltageof25 kVand atemperatureof20 ˚C)enabledseparatingthe fourstereoisomers of dimethenamidin21minwithresolutionsbetweenconsecutivepeaksof6.0,2.1and1.5.Theanalytical characteristicsofthedevelopedmethodwereevaluatedandconsideredadequatetoachievethe stereos-electiveanalysisofdimethenamid-Pincommercialagrochemicalformulations.Resultsdemonstratedthe potentialofthemethodtocontrolthequalityoftheseformulationsandtodeterminethestereoisomeric purityofdimethenamid-Pintheseproducts

© 2022TheAuthors.PublishedbyElsevierB.V ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

1 Introduction

Theuseofpesticidestoimprovefoodqualityandcropyieldsis

increasingconsiderably,duetothecontinuousgrowthoftheworld

population, thegreat demand forfood, therapid developmentof

agriculture andthe multitudeof pests,weeds andfungi that can

be found in crops[1,2] However, despite their numerous

advan-tages, pesticidesareconsidered oneofthemostdangerous

pollu-tants inthe environment, not onlyfor their toxicity, butalso for

theirmobilityandbioaccumulationcapacity

Arounda 30-40%ofthecurrentlyregisteredpesticidescontain

atleastone chiralcentre,givingriseto differentenantiomers [3]

Theseenantiomerscanbehavedifferent,showingdifferentactivity,

∗ Corresponding author

E-mail address: mluisa.marina@uah.es (M.L Marina)

toxicityandpersistence.Whenoneoftheenantiomersisthemost activeandpresentsalesserriskfortheenvironmentornon-target organisms,the use of enantiomerically pure pesticidesis recom-mendedtopreparecommercialformulations.Nevertheless,dueto economic reasons, most of the chiral pesticides are marketedas racemates[4]whichimpliesinmanycasesanunnecessaryriskfor theenvironment

[2-chloro-N-(2,4-dimethyl-3-thienyl)-N-(2-methoxy-1-methylethyl)acetamide], commonly known as dimethenamid,

is a chiral acetamide herbicide widely used on raw agricultural commodities[5].Dimethenamidconsistsof4stereoisomers(aS,1S; aR,1S;aS,1RandaR,1R)duetotwochiralelements,acarbonatom asymmetrically substituted and a chiral axis (Fig 1) [6,7] The low energyrequired forthe rotationaround the chiral axisgives 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/ )

Fig 1 Chemical structure of dimethenamid

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

and aS,1Sdiastereomers)hasdemonstrated tobe more active,so

the agrochemical formulations are marketed as S-dimethenamid

[7].In orderto achieve thequality control ofthese formulations,

the development of analytical methodologies enabling the

sep-aration 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 neverbeen reportedbefore BuserandMüller

[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 ofthese isomers inthe columnwasunknown In addition,

the partialseparationofthree isomersofdimethenamid byHPLC

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

unresolvedstereoisomersinpeak 2.The HRGCmethoddeveloped

for dimethenamidwasapplied by the sameauthors tostudythe

degradation ofthiscompoundinenvironmentalsamples showing

a low to moderate enantioselectivityin soilsandsewage sludges

while no enantioselective degradation was observed for surface

watersandinrain[8]

CapillaryElectrophoresis(CE)hasdemonstratedagreat

poten-tialtoachievechiralseparationsmainlyusingtheseparationmode

ElectrokineticChromatography(EKC)beingcyclodextrins(CDs)the

most employed chiral selectors In spite of the high

discrimina-tion power of CDs, in the caseof very hydrophobic chiral

com-pounds or with multiple chiral centres, the use of dual systems

ofchiral selectorsand/or theaddition ofothercompounds tothe

separation buffer is necessary inmany casesto obtain

stereoiso-meric separations[9].In recentyears, thetrend todevelop

envi-ronmentally friendly analytical procedures has led to the use of

newcompounds that canbe addedtothe backgroundelectrolyte

(BGE) in combination with CDs, such as deep eutectic solvents

(DES) or ionicliquids (ILs)that havereceived considerable

atten-tion forbeingchemicallysustainablesolvents[10,11].ILsare salts

withweaklycoordinatedionswhichmeltingpointsarebelowthe

boiling point of water They are constituted by a bulky organic

cationandanorganicorinorganicanion[10]andhaveextensively beenstudiedinmanyfieldsduetotheirinteresting physicochemi-calproperties[10,12].ILscanbeusedinCEdirectlyasbackground additives[13–16],assolechiralselectors[9,13,17–20]orchiral lig-ands[9,13,21,22]whentheyarechiral,buttheirmostfrequentuse

isbasedon their combinationwithchiralselectorsincludingCDs [9,10,13–16,23]generatinginmanycasesasynergisticeffect DESare homogeneous liquids at ambienttemperature formed

by a mixture ofa donor (HBD)andan acceptor (HBA)of hydro-gen bond compounds (usually solids) [11,16,24–26] The synthe-sis of DES, unlike ILs, is simple and environmentally friendly as

it doesnot involve the useof anyorganic solvent since the two solidcomponentsaremixedandheateduntila homogeneous liq-uid is obtained Quaternary ammonium, tetraalkylammonium or phosphoniumsalts aregenerallyused asHBA components,while carboxylicacids,amines,polyolsorcarbohydratesareusuallyused

asHBDcomponents[16,25].Theformationofhydrogenbonds be-tween thetwo components leadstoa significant decreasein the melting point of DES in comparison with their precursors They are non-volatile, with highthermal stability and readily dissolve manyorganicandinorganiccompounds.ComparedtoILs,DESare cheaper,moreenvironmentallyfriendly,andeasiertoobtain[25] Despitetheseadvantages,DEShavebeenveryscarcelyusedin

CEandonlyincombinationwithCDs[11,16,24,25].Thus,the pres-enceof a DES in the CE separation medium can alter the chiral separationbasedona changeintheionicstrengthofthe separa-tion buffer and/or theadsorption of theDES onthe capillary in-ner surfacethat couldreduce oreven reversethe EOF [11,24,26] Moreover,theadditionofaDEScanimprovetheinclusion capac-ityofthe CDsallowing more enantiomersto enterthe CDcavity andthenincreasingtheresolution[24]

Muetal.were thefirstwhoinvestigatedtheeffectofdifferent DES basedon choline choride(ChCl) as HBA, andurea, ethylene glycol,propyleneglycol andbutyleneglycolasHBD,onthechiral separationofzopiclone,salbutamolandamlodipineusingβ-CDas chiralselector[11].Resolutionwasimprovedinthepresenceofthe DESfor thethree drugs suggestingthe existence of a synergistic effect.Deng etal [24] studied the effect ofdifferent ChCl-based DES(with urea,ethylene glycol, propylene glycol,lactic acid, and glycerol as HBD) on the enatioseparation of tropicamide, homa-tropine, ofloxacin, atenolol and propanolol when using different

β-CDs aschiralselectorsinnon-aqueouscapillaryelectrophoresis

(NACE).Animprovementinthechiralseparationefficiencyandin

theinclusionefficiencyoftheCDinpresenceofaDESwas

demon-strated through fluorescence measurements Salido-Fortuna et al

investigatedtheeffectofdifferentChCl-basedDES(urea,ethylene

glycol, D-glucose,D-sorbitol asHBD) onthe enantioseparation of

thedruglacosamidewhentheywerecombinedwithdifferentCDs

ThecombinationofChCl-D-sorbitolandsuccinyl-β-CDallowedthe

separationoflacosamideforthefirsttimebyCE.Thisdualsystem

wasnecessarytoobtainaresolutionvaluelargeenoughtoperform

theanalysisoftheenantiomericimpurityoflacosamideina

phar-maceutical formulation[25].Finally, García-Cansino etal.studied

the effectofaddingfive ChClDESs (ethyleneglycol,D-sorbitol,

D-glucose, D-fructose,urea asHBD) totheseparation medium

con-tainingsulfobutylated-β-CDattwopHvalues(3.0and9.0)onthe

enantiomericseparationofclopidogrel[16].Inthiscase,therewas

not a significant change in the resolution oranalysis time when

addingthe DESwithrespecttothe additionofChCl alonetothe

separationbuffercontainingtheCDateachpHvalue

The aim of this work was to separate for the first time the

four stereoisomers of dimethenamid by EKC using CDs as chiral

selectors Theeffectoftheaddition ofDESandILstothe

separa-tionmediumwasinvestigatedandthevariablesaffectingthechiral

separation were optimized Application of the developedmethod

to thestereoselective analysisofdimethenamid-P-based

commer-cialagrochemicalformulationswasalsodescribed

2 Materials and methods

2.1 Chemicals, reagents, DESs, ILs and samples

Boric acid, sodium hydroxide, choline chloride (ChCl),

guani-dine hydrochloride,betaine,ethyleneglycol (EtGly),D-sorbitol,

D-glucose and D-fructose were purchased from Sigma-Aldrich (St

Louis, MO, USA) Methanol, urea and orthophosphoric acid were

obtainedfromScharlau(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

(CE-β-CD,DS∼ 3.5),(2-carboxyethyl)-γ-CD(CE-γ-CD,DS∼ 3.5),

phos-phated β-CD(Ph-β-CD, DS∼ 4),phosphatedγ-CD(Ph-γ-CD, DS

∼ 3.5), sulfatedα-CD(S-α-CD, DS ∼ 12), sulfatedγ-CD(S-γ-CD,

DS∼ 10),sulfobutylatedβ-CD(SB-β-CD,DS∼ 6.3),γ-CD,

methyl-γ-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,

methyl-β-CD (M-β-CD) and heptakis(2,6-di-O-methyl)-β-CD (DM-β-CD)

were fromSigma-Aldrich.β-CDand(2-hydroxypropyl)-β-CD

(HP-β-CD, DS∼ 0.6) werepurchasedfromFluka(Buchs,Switzerland)

Sulfobutylether-β-CD (captisol) was from Cydex Pharmaceuticals

(Lawrence,Kansas) Waterusedto preparesolutions waspurified

throughaMilli-QsystemfromMillipore(Bedford,MA,USA)

Two of the sixteen ILs used in this work,

2-hydroxyethyl-trimethylammonium L-lactate ([HETMAm][L-lactate]) and

1-ethyl-3-methylimidazolium L-lactate ([EMIm][L-lactate])

([TBA][L-Arg]), tetramethylammonium-arginine

([TMA][L-Arg]), tetrabutylammonium-aspartic acid ([TBA][L-Asp]),

tetrabutylammonium-lysine([TBA][L-Lys]),

tetramethylammonium-lysine ([TMA][L-Lys]), tetrabutylammonium-isoleucine

([TBA][L-Ile]), tetramethylammonium-isoleucine

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

tetramethylammonium-glutamic acid ([TMA][L-Glu]) and 2-tetrabutylammonium-glutamic acid ([TBA]2[L-Glu])), were syn-thesized by the Center for Applied Chemistry and Biotechnology (CQAB)fromtheUniversityofAlcalá followingdifferentprocedures previously reported[28].CQABalso synthesizedtheILs L-alanine tertbutylester bis(trifluoromethane)sulfonamide([L-AlaC4][NTf2]), L-carnitine methyl ester bis(trifluoromethane)sulfonimide ([L-CarniOMe][NTf2]) and L-carnitine methyl ester L-lactate ([L-CarniOMe][L-lactate]) following previously optimized procedures [29,30]

The five DES used in this work (ChCl-urea, ChCl-EtGly, ChCl-D-sorbitol, ChCl-D-glucoseandChCl-D-fructose)were synthesized followingtheliterature[25,27].Inbrief,ChCl-ureaandChCl-EtGly weresynthesizedbothinthemolarratio1:2bymixingthe appro-priate amount of ChCl withthe appropriate amountof urea and

ofEtGly,respectively Bothmixtureswere stirredfor30minin a waterbathat70-80°C.ChCl-D-glucoseandChCl-D-fructosewere prepared both in the molar ratio 2:1, while ChCl-D-sorbitol was preparedinthemolarratio1:1.Theselast threeDESwerestirred

inawaterbathat70-80°C for4h AllDESwerestoredatroom temperatureinthedarkbeforedilutioninthebuffer

Dimethenamidanddimethenamid-Pwere fromSigma-Aldrich The commercial agrochemical formulations (M1 and M2) were kindlydonatedbyBASFEspañola,S.L(Barcelona,Spain).According

tothelabelleddata,M1andM2contained720 gL−1 and212.5 g

L−1ofdimethenamid-P,respectively

2.2 Preparation of solutions and samples

In order to prepare the borate buffer solution, first, the ap-propriate amountof boricacidwasdissolved in Milli-Qwater to reachaconcentrationof100mM.Then,thepHwasadjustedwith sodium hydroxide 1M to the desiredvalue (pH 9.0)before com-pletingthevolumewithMilli-Qwater.Backgroundelectrolyte so-lutions(BGEs)werepreparedbydissolvingtheappropriateamount

ofthedifferentCDs,DESorILsintheboratebuffersolution Stock standard solutions of racemic dimethenamid and dimethenamid-P, and stock solutions of agrochemical formula-tions (M1 and M2) (2000 mg L−1) were prepared in methanol and stored at -20°C Working solutions containing racemic dimethenamid and/or dimethenamid-P or agrochemical formu-lations (M1 andM2) were prepared from the stocksolutions by appropriate dilutionin Milli-Q water Disposable nylon 0.45 μm pore size filters purchased from Scharlau were used to filter all solutionsbeforetheirinjectionintheCEsystem

Reagents, DES, ILsand standardswere weighed on an OHAUS AdventurerAnalyticalBalance(Nänikon,Switzerland).ApH-meter model744fromMetrohm(Herisau,Switzerland)wasemployedto adjust the pH of the borate buffer solutions An ultrasonic bath B200 from Branson Ultrasonic Corporation (Danbury, USA) was usedtodegasallthesolutions

2.3 CE analysis

An Agilent 7100 CE system from AgilentTechnologies (Wald-bronn, Germany) was used to carry out the electrophoretic ex-periments Detection was performedwith a diode array detector (DAD) set at a wavelength of 205 nm with a bandwidth of 30

nm (referenceoff) The HP3DCEChemStationsoftware from Agi-lentTechnologieswasused tocontrol theelectrophoreticsystem Separationswere carriedout usinguncoatedfused-silicacapillary providedbyPolymicro Technologies(Phoenix,AZ, USA).Injections weremadeapplyingapressureof50mbarfor5s

Before its firstuse,the newcapillarywas conditioned (apply-ing 1bar) with1M sodium hydroxidefor30 min,Milli-Qwater for 15 min, followed by 60 min with buffer solution andfinally

beginning,thecapillarywaspre-washed(applying1bar)with0.1

M sodium hydroxide for10 min, Milli-Q waterfor 5min, buffer

solution for20 minandwiththe corresponding BGE for10 min

Aftereachrun,withtheaimofensuringrepeatabilitybetween

in-jections, the capillary wasrinsed 4 min with0.1 M sodium

hy-droxide,2minwithMilli-Qwater,2minwithbuffersolutionand

3minwithBGE

2.4 Data treatment

Migration time values, resolution values between consecutive

peaks(Rs)andpeakareavalueswereobtainedusingtheHP3DCE

ChemStation software In orderto compensate the differencesin

the electrophoretic conditions and to obtain better

reproducibil-ity of data, corrected peak areas (Ac) were used for data

treat-ment Forthe analysisof experimental data,developmentof

sta-tistical testsand thecomposition of graphs andfigures, the

pro-gramsusedwereExcelMicrosoft,OriginPro8,Statgraphics

Centu-rionXVIIsoftwareandChemDraw20.0

3 Results and discussion

3.1 Development of a chiral analytical methodology by CD-EKC for

the separation of the four stereoisomers of dimethenamid

3.1.1 Effect of the nature of the cyclodextrin

Since dimethenamid is a neutral compound, 14 negatively

charged(atpH9)CDswere evaluated(CM-α-CD, CM-γ-CD,

Succ-β-CD, CE-β-CD,CE-γ-CD,Ph-β-CD,Ph-γ-CD,S-α-CD,S-γ-CD,

SB-β-CD ata concentration of 10 mM andS-β-CD, Succ-γ-CD,

CM-β-CDandsulfobutylether-β-CD(captisol) ataconcentrationof2%

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× 50cm (58.5cm tothe detector)andan injectionof 50

mbar x5 were employed Among all the CDs tested, only five

of them(Ph-γ-CD, Captisol, SB-β-CD, Ph-β-CDandCE-β-CD)

in-teracted stereoselectively with the analyte, resulting in a certain

stereomeric discriminating power and two peaks (Fig S1)

How-ever,CE-β-CDgaverisetothehighestresolutionvalue(0.8inless

than 8 min) and, in addition, it originated the unfolding of the

second peakobtainedwithalesspeakbroadening.Thus,CE-β-CD

wasselectedasthechiralselector

TheeffectofcombiningCE-β-CDwithothernegativelycharged

orneutralCDsundertheabove-describedinitialexperimental

con-ditions,wasinvestigated.Thedualsystemsformedby10mM

CE-β-CD+ 10mMHP-γ-CD;10mM CE-β-CD+ 2%S-β-CD;10mM

CE-β-CD+10mMM-γ-CD;10mMCE-β-CD+10mMCM-β-CD;

and10mMCE-β-CD+10mMS-γ-CDgaverisetothebestresults

(three peakswere obtainedfor all ofthem in lessthan 10min)

However,noneofthesecombinationsgaverisetothe4peaks

cor-responding tothestereoisomersofdimethenamid.Then,the

con-centrationofCE-β-CDwasincreasedto15mMinthosedual

sys-temsinwhichthelastpeakwasbroaden(CE-β-CD+M-γ-CDand

CE-β-CD+ S-β-CD).The dualsystemcomposed of15mM CE-β

-CD and 10 mM M-γ-CD waschosen since it enabledthe partial

separationofthefourstereoisomersofdimethenamid(resolutions

betweenconsecutivepeaks3.8,0.9, 0.6in8.9min)(Fig.2A).This

last CDs combinationwasselected inordertostudythe effectof

theadditionofILsandDESontheseparationwiththeobjectiveto

improvetheresolutionamongstereoisomers

3.1.2 Effect of the addition of ionic liquids to the dual CDs system

Inordertoimprovethestereoselectiveseparationobtainedfor

dimethenamid, 16 differentILs were added ata concentration of

10 mM to the dual system formed by 15 mM CE-β-CD and 10

mM M-γ-CD ILsin whichthe cationic counterpart wasan alky-lammoniumchain andthe anionic part wasan amino acidwere employed together with other ILs in which the anionic partwas L-lactateorNTf2 beingthecationic partanamino acidderivative

orEMImorHETMAm.TableS1showstheanalysistimesand reso-lutionsbetweenconsecutivepeaksobtainedwhentheseionic liq-uids wereadded tothe separationmedium ILsin whichthe an-ioniccounterpartwasanaminoacidmadepossibletheseparation

infourpeaks exceptwhen L-AspandL-Ilewere the aminoacids

orwhen[TBA]2[L-Glu]wasemployed.In fact,theaddition ofthe ILs[TBA][L-Arg],[TMA][L-Arg],[TBA][L-Lys],[TMA][L-Lys], [TBA][L-Glu], [TMA][L-Glu] gave rise to four peaks although their base-line separation wasnot observed Although the addition of TBA ILsenabledthestereoselectiveseparationinshorteranalysistimes than TMA ILs, resolutions obtained with TMA ILs were slightly higherfortheseparationofthethreelastpeaks.RegardingtheILs [LCarniOMe][L-Lact], [EMIm][L-Lactate] and [HETMAm][L-Lactate], their addition tothe dualCDs systemalso originatedfourpeaks However,anyoftheILsinvestigatedinthisworkallowedobtaining thebaselineseparationofthefourstereoisomerssinceresolutions betweenconsecutivepeaksobtainedwhen addingILswere equal

orlower than 3.6/0.9/0.6.In addition,theseresolution valuesdid notimproveinanycasethoseobtainedwiththeCDsdualsystem withoutILs(3.8/0.9/0.6)

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 theseparationmediumdidnotsupposethebaselineseparationof dimethenamid stereoisomers although four peaks were obtained, theeffectoftheadditionofDEStothedualCDssystemwas stud-ied

Preliminaryexperimentswereachievedtoselectthemost ade-quateHBApartoftheDEStobeassayed.Withthisaim,the addi-tionofthreedifferentHBAs(betaine,guanidinehydrochlorideand ChClwhichstructures are showninFig.S2)ata concentrationof 0.5%w/vtothedualCDssystemselectedinthiswork,wasstudied (Fig.S3).Whenbetainewasadded,onlythreepeakswereobserved

inan analysistimeof7.8min.Withguanidinehydrochlorideand ChClfourpeakswereobtained.Althoughanalysistimewasshorter when using guanidine hydrochloride than when using ChCl (10 minversus 12min), resolutionvaluesbetweenconsecutivepeaks were lower when using guanidine hydrochloride (Rs 3.6, 1.0and 0.6)thanwhenusingChCl(Rs4.1,1.2and0.7).Therefore,ChClwas chosen.Next,theinfluenceofthepercentageofChCladdedtothe separationmedium wasevaluated (0.2,0.5 and1% w/v) Fig S4 showsthatbyincreasingtheaddedconcentrationofChClupto1% w/v,higheranalysistimes(13.8 min)wereobtained,butalso bet-ter resolution values between consecutivepeaks (Rs 4.4, 1.5and 1.0).However,repeatabilitybetweenanalyses gotworse andpeak efficiencywaslowerthanata0.5%ChCl.Ontheotherhand,when thepercentageofChCladdedwas0.2%w/v,analysistimesand res-olutionsbetweenconsecutivepeaksdecreased(10min;Rs3.6,0.9 and0.6) As a result, next experiments were carried out using a concentrationofChClof0.5%w/vasacompromise between anal-ysistimeandresolutions

AfterselectingthemostsuitableHBA,inthiscaseChClata per-centageof0.5%,theeffectoftheadditiontothedualCDssystem

offivedifferentDES(threechiral:ChCl-D-fructose,ChCl-D-glucose and ChCl-D-sorbitol and two achiral: ChCl-urea and ChCl-EtGly, whichstructures are shownin Fig. S2 ), maintaining apercentage

of0.5%ChCl, wasinvestigated(Fig.S5) It wasobserved that chi-ralDESgaveriseto higherresolution valuesbetweenconsecutive peaksthanachiralDES, beingChCl-D-fructose thechiralDES pro-ducing the best resolution valuesbetween consecutivepeaks (Rs 4.9, 1.4and0.9).The improvementobserved inthechiral separa-tionof dimethenamidundertheseconditionscould be duetoan

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; λ205 ± 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; λ205 ± 30 nm (reference off) and [racemic dimethenamid]: 100 mg L −1

enhancement inthe inclusionabilityofthe CDfortheanalyte in

thepresenceoftheDESaswellasasqueezingeffectofDESonthe

CD-enantiomersinteractions,aspreviouslyproposedbyDengetal

[24]

3.1.4 Optimization of the dual system CE-β-CD+M-γ-CD in

presence of ChCl-D-fructose

TheinfluenceofthepercentageofChCl-D-fructose(0.8,1.2,1.5

and1.7% w/v) addedto theCDs dualsystem, on thechiral

sepa-ration of dimethenamid wasstudied As Fig.3 shows, the

analy-sis time andthe resolution betweenconsecutive peaksincreased withthepercentageoftheDESuptoavalue of1.5%w/vof ChCl-D-fructose(20.2 minandresolutions of5.6, 1.8, and1.3),slightly decreasingwhenthispercentageincreasedtoa1.7%w/v(19.5min andresolutions of5.5, 1.7and 1.2).Then, a 1.5% w/v of ChCl-D-fructose was selectedas the optimum value The increase in the analysistimeobservedcanbejustifiedtakingintoaccountthe in-creasein theviscosity caused by theDES andthe changein the EOFduetoamodificationofthecapillarywallbytheDES[24,26] (inbothcases,theenantiomersandtheCDswouldhavemoretime

Fig 4 Electropherograms obtained for (A) a dimethenamid standard solution (containing racemic dimethenamid at a concentration of 100 mg L −1 ), (B) a dimethenamid- based agrochemical commercial formulation solution, M1 (containing dimethenamid-P at a concentration of 50 mg L −1 ) and (C) a dimethenamid-based agrochemical com- mercial 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

tointeractbetweenthem).Thesereasonstogetherwiththe

above-mentionedeffectoftheDESontheinclusionabilityoftheCDand

ontheCD-enantiomersinteractions[24]couldexplaintheincrease

observedintheenantiomericresolution

The effectofthetemperaturewasalsostudied(15, 20and25

°C).ResultsobtainedareshowninFig.S6.Itcanbeobservedthat

whenatemperatureof25°Cwasused,theshapeofthefirstpeak

deteriorated.Inaddition,analysistime decreased(16.8min),what

resultedinadecreaseintheresolutionvaluesbetweenconsecutive

peaks(Rs3.5,1.5and1.0).Foratemperatureof15°C,theanalysis

time increased(24.2min)andresolutionvaluesgot worse except

forthetwofirst-migratingpeaks(Rs6.0,1.5and1.1).Therefore,a

temperatureof20°Cwasconsideredthemostadequatetoachieve

thechiralseparation

Inordertoimprovetheresolutionvaluesbetweenpeaks3and

4, theeffectivelength ofthecapillarywasincreasedfrom50 cm

to 60 cm (maintaining the same electric field, thus, applying a

voltage of20and23.4kV, respectively).As itcan beobserved in

Fig S7, the analysis time increased from 20.2 min for50 cm to

24.0 min for 60 cm However, the resolution values remarkably

improved and the baseline resolution of the 4 stereoisomers of

dimethenamidwasobtained(Rs6.5,2.1and1.5)

Finally,inordertodecreasetheanalysistime,aseparation

volt-age of 25 kV was applied instead of 23.4 kV which enabled to

decreasetheanalysistime(around3min) withoutdecreasingthe

resolutionvaluesbetweenthesecond,thirdandfourthpeakswhile

the resolution for thetwo first peaksslightly decreased (6.0, 2.1

and1.5)

Fig 4A shows the first stereoselective separation of

dimethenamid showing the separation of the four

stereoiso-mers with resolution values between consecutive peaks ranging

from1.5to6.0.ThisseparationwasnotpossiblewhentheDESor

its components were presentin the separation buffer under the

sameoptimizedexperimentalconditionsbutwithouttheaddition

ofthe twoCDs employed.In fact,nopeaks wereobserved 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 improvetheseparationobtainedwiththedualCDssystem

3.1.5 Effect of the addition of the individual components of ChCl-D-fructose to the CDs dual system

Toinvestigate the effectof the eutecticmixture on the chiral separation of dimethenamid with respect to that of the individ-ualcomponentsoftheDES,bothChClandD-fructosewereadded, individually andtogether,to thedualCDs systemselectedinthis work andthe results obtained were compared withthose corre-spondingtotheadditionoftheready-madeDES.Thecomponents

oftheDESwereaddedatthesamepercentages(w/v)asthose em-ployedinthesynthesisoftheDES.While componentsoftheDES were individually dissolved tothe aqueous separation buffer, the DESwassynthesizedpreviouslybymixingthecomponentsinsolid stateto obtainan eutecticmixtureata giventemperature, which

is subsequentlyadded to the separation buffer oncesynthesized ResultsobtainedareshowninFig.2B–E.Asitcanbeobserved,the additionofD-fructoseatapercentageof0.585%w/vtothe15mM CE-β-CD + 10 mM M-γ-CDsystem originated similar resolution values andanalysis times as when using the dual system alone WhenChCl wasaddedata percentageof0.915%w/vto thedual CDs system,a considerableimprovement inthe resolutionvalues for dimethenamid stereoisomers wasobserved (resolution values betweenconsecutivepeaksincreasedfrom3.8, 0.9and0.6to5.2, 1.8and 1.1) although ina longeranalysis time (15.2 min), prob-ablydueto amodification inthecapillarywall by theChCl(25) Similarly,whenbothDEScomponentswere simultaneouslyadded (ChCl 0.915% + D-fructose 0.585%) to the dual CDs system, bet-terresolutionvaluesbetweenconsecutivepeakswereachieved(Rs valuesof5.9,2.0and1.3)in20.2min.However,thebaseline sepa-rationofalldimethenamidstereoisomers(Rsvaluesof6.0,2.1and 1.5) wasonly obtained when the ready-made DESwas added to

Table 1

Analytical characteristics of the developed chiral method

First-migrating stereoisomer Second-migrating stereoisomer Third-migrating stereoisomer Fourth-migrating stereoisomer External standard calibration method a

Standard additions calibration method for M1 b

Accuracy

Standard additions calibration method for M2 d

Accuracy

Precision

Instrumental repeatability f

Method repeatability g

Intermediate precision h

A c : 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

thedualsystemataconcentrationof1.5%.Inlasttwocaseswhere

the two components of the DES and the ready-made DES were

added to theseparation medium,the above-mentioned effects of

the addition of theDES on thechiral separation (higher analysis

times andresolutions)were againobserved.From the results

ob-tained,theready-made DESwasselectedasthebestconditionto

reachthebaselineseparationofalldimethenamidstereoisomersin

21.2min

3.2 Analytical characteristics of the developed method

Thedevelopedmethodwasappliedtothestereoselective

anal-ysis of dimethenamid-P (aRS,1’S isomers) commercial

agrochem-ical formulations With this aim, the analytical characteristics of

themethodwereevaluatedintermsoflinearity,selectivity,

preci-sion,accuracy,limitsofdetection(LODs)andlimitsofquantitation

(LOQs)).TheresultsobtainedaregroupedinTable1

Linearity was determined using eight standard solutions

con-tainingracemicdimethenamidatconcentrationsfrom4to200mg

L−1 Corrected peakareas (Ac)for eachof thefourstereoisomers

wererepresentedasafunctionoftheir concentrationsinmgL−1 LinearitywasadequatesinceR2 valueswere≥ 0.992forthefour stereoisomers andconfidence intervalsfor theintercept included

de zerovalue whileconfidenceintervalsfortheslope didnot in-cludethezerovalue(inbothcasesfora95%confidencelevel) Thet-testwasemployedtostudythepresenceofmatrix inter-ferencesby comparisonofthe confidenceintervalsfortheslopes corresponding to the external standard and the standard addi-tions calibration methods forthe commercial formulations Since the confidence intervals for the slopes of the calibration meth-ods overlapped andp-values were ≥ 0.05 fora 95 % confidence levelforallcases,therewerenomatrixinterferences(seeTable1) Therefore,theexternalcalibrationmethodcouldbeusedto quan-titatethecontentofdimethenamid-Pintheagrochemical formula-tions.Fig.4BandCshowthat nointerferenceswereobservedfor anyofthecommercialagrochemicalformulationsanalyzed,which demonstratedan adequateselectivityofthe developed methodol-ogy

Accuracy ofthe method wasevaluated asthe recovery values (%)obtainedforthefourstereoisomersofdimethenamidwhenthe

agrochemicalcommercial formulationsolutionscontaining30 mg

L−1 of dimethenamid-P were spiked with 60 mgL−1 of racemic

dimethenamid standard solution Good recovery values were

ob-tained,sincethe100%valuewasincludedinallcasesinthe

con-fidenceinterval(Table1)

PrecisionwasevaluatedconsideringtheRSDvaluesobtainedfor

migrationtimesandcorrectedpeakareasfortheinstrumentaland

methodrepeatabilityandfortheintermediateprecision.Asshown

in Table 1, adequate RSD values were obtained in all cases

(be-tween 1.1and2.2%formigrationtimesandbetween2.4and4.3

%forcorrectedpeakareas)

Finally,LODsandLOQswereexperimentallydeterminedasthe

minimum concentration yielding an S/N ratioof 3 and10 times,

respectively LODsrangedfrom0.5to 0.6mgL−1 andLOQsfrom

1.8to2.0mgL−1 forallstereoisomers

3.3 Quantitation of dimethenamid in commercial agrochemical

formulations

Oncedemonstratedthesuitabilityofthemethod,itwasapplied

to the quantitative analysis of dimethenamid-P in two

commer-cialagrochemicalformulations(M1andM2)byinjectingadiluted

sampleoftheseformulationscontainingeach, dimethenamid-Pat

aconcentration ofapproximately50mgL−1accordingtotheir

la-bels

Contentsofdimethenamid-Pof721± 5and211± 4gL−1were

obtainedforM1andM2,respectively.Thesevalueswerein

agree-ment with the labelclaim of the two agrochemicalformulations

(recovery percentages of 100 ± 1 and 99± 2 % withrespect to

the labelledamounts), which pointedout theapplicability ofthe

methodforherbicideanalysisinrealsamplessuch as

agrochemi-cal products.Indeed,thegreatpotential ofthedevelopedmethod

to control the stereoisomericpurity of dimethenamid-P

formula-tionswasdemonstrated(aRS,1’Risomerswerenotdetected)

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-CDandM-γ-CD) enabledthepartialseparationofthe

fourdimethenamidstereoisomersalthoughthebaselineseparation

wasonlypossibleundertheseconditionsforthetwo first-eluting

isomers The effect of the addition of different ILs and DES to

the dual CDs systemwas investigated.While the addition ofthe

ILs studied did not improve the resolution between consecutive

peaks when addedto the CDsdual system, the additionof most

of the DES had a beneficial effect on the chiral separation The

DESChCl-D-fructosemadepossiblethe baselineseparationofthe

fourstereoisomers.Undertheoptimizedconditions(15mMCE-β

-CD+10mMM-γ-CDin100mMboratebuffer(pH9.0)with1.5%

ChCl-D-fructose(2:1),aseparationvoltageof25kVanda

temper-atureof20˚C),theseparationwasachievedin21.2minwith

res-olutionvaluesbetweenconsecutivepeaks of6.0,2.1 and1.5 The

evaluationoftheanalyticalcharacteristicsofthemethodshoweda

goodperformanceforthequantitationofdimethenamid-Pin

com-mercialagrochemicalformulations.Resultsobtainedindicatedthat

no statisticallysignificant differenceswerefoundbetweenthe

to-tal concentration determined for the analyzed formulations and

their labelledcontentsshowingthegreatpotential ofthemethod

for herbicide analysis in real samples such as commercial

agro-chemical products Moreover, the applicability of the method to

control thequality andstereoisomericpurity of

dimethenamid-P-basedagrochemicalformulationswasdemonstrated(steroisomeric

impurities were not detected forthe agrochemical products

ana-lyzed)

Declaration of Competing Interest

Theauthorsdeclarethattheyhavenoknowncompeting finan-cialinterestsorpersonalrelationshipsthatcouldhaveappearedto influencetheworkreportedinthispaper

CRediT authorship contribution statement María Ángeles García: Conceptualization,Methodology, Visual-ization, Data curation, Resources, Supervision, Writing – original draft, Writing – review & editing, Project administration, Fund-ingacquisition. Sara Jiménez-Jiménez: Investigation,Formal anal-ysis, Validation, Data curation, Visualization, Writing – original draft. María Luisa Marina: Conceptualization, Methodology, Visu-alization,Data curation,Resources,Supervision,Writing– original draft, Writing – review&editing, Projectadministration, Funding acquisition

Acknowledgements

Authors thank financial support from the Spanish Ministry of ScienceandInnovationforresearchprojectPID2019-104913GB-I00, and the University of Alcalá for research project CCG20/CC-023 S.J.J thanks theSpanish Ministry ofScience, Innovation and Uni-versitiesforherFPUpre-doctoralcontract (FPU18/00787).Authors thankC.Huertas,S.Bernardo-BermejoandG.Fernández-Pérezfor technicalassistance

Supplementary materials

Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.chroma.2022.463114

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