In this study, we developed and validated a simple, fast and sensitive LC-MS/MS method for the measurement of tetrodotoxin (TTX) in human plasma. Three HILIC-type solid phase extraction (SPE) carriers (PSA, silica, Siphila i HILIX) with different stationary phase functional groups were compared.
Trang 1journalhomepage:www.elsevier.com/locate/chroma
spectroscopy
Liang Xina,b,c,1, Yan Lianga,b,c,1, Shuangshuang Yanga,b,c,1, Fengli Jianga,b,c, Fan Yua,b,c,
Meiwei Zhanga,b,c, Wei Changd, Wei Wangd, Chen Yua,b,c, Gangyi Liua,b,c,∗, Youli Lua,b,c,∗
a Central Laboratory, Shanghai Xuhui Central Hospital/Zhongshan-Xuhui Hospital, Fudan University, Shanghai, China
b Shanghai Engineering Research Center of Phase I Clinical Research & Quality Consistency Evaluation for Drugs, Shanghai, China
c Shanghai Institute of Clinical Mass Spectrometry, Shanghai, China
d Department of Emergency Medicine, Shanghai Xuhui Central Hospital/Zhongshan - Xuhui Hospital, Fudan University, Shanghai, China
a r t i c l e i n f o
Article history:
Received 26 August 2022
Revised 11 October 2022
Accepted 12 October 2022
Available online 18 October 2022
Keywords:
Tetrodotoxin
Hydrophilic-interaction/ion-exchange
Mixed-mode solid phase extraction
LC-MS/MS
Human plasma
a b s t r a c t
Inthisstudy, wedeveloped andvalidatedasimple,fastandsensitiveLC-MS/MSmethodforthe mea-surementoftetrodotoxin(TTX)inhumanplasma.ThreeHILIC-typesolidphaseextraction(SPE)carriers (PSA,silica,SiphilaiHILIX)withdifferentstationaryphasefunctionalgroupswerecompared.TheSiphila
iHILIXSPEplatecontainingmulti-carboxylgroupswasfinallyselectedduetoobviouslybetterextraction recoveryofTTX(about80%ofrecoveryfromplasmasamples)thantheothertwoandnosignificant ma-trixeffectswereobserved,whichwasspeculatedtohavemixed-modesynergisticeffectsofhydrophilic interactionandionexchange.100μLplasmasamplewasprecipitatedrapidlywithacetonitrilecontaining 1%trichloroaceticacid,and filtrateswereloaded ontoSiphilaiHILIX 96well SPEplate.Afterwashed with95%acetonitrile,TTXwaselutedwith200μLof50%acetonitrilecontaining1%trichloroaceticacid
2μLofelutionsolutionwasdirectlyinjectedintoLC-MS/MSandthetotalruntimeonaBEHamide col-umnwas4.5min.Themethodavoidstheevaporationandultrafiltrationprocesseswhichissimpleand timesaving(<30min).TTXandinternalstandard(arginine-15N4)weremonitoredinpositivemodeusing
m/z320.3→162.2(quantificationtransitionforTTX),320.3→284.1(confirmationtransitionforTTX)and 179.2→63.0(transitionforIS),respectively.Themethodwaslinearintherangeof0.1–20ng/mLforTTX withthelowlimitofquantification(S/N >10)of0.1ng/mL;theintra-andinter-assayaccuracieswerein therangeof98.5%-99.8%(relativestandarddeviations,RSDs≤ 5.92%)and98.8–99.5%(RSDs≤ 6.23%), re-spectively.Biasesofspikinganalysiswererangedfrom-7.00%to7.43%forhealthyhumanplasmasamples (RSDs≤ 8.83%)andfrom-5.00%to3.93%forhemolytic,hightriglyceride,highcholesterolandhigh biliru-binplasmasamples(RSDs≤ 6.40%),whichprovedthegoodanti-interferencepropertyofthemethod.The resultsshowedthatthemethodissensitive,accurate,specific,reliable,andcanbeusedtomonitorthe concentrationofTTXinplasmatomeettheneedsofclinicalresearchandpoisoningscreening
© 2022TheAuthors.PublishedbyElsevierB.V ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Introduction
Tetrodotoxin (TTX) poses a serious threat to human health in
coastalareaswheremarinefoodisthemainfoodsource[1–2]
In-cidentsoffoodpoisoningcausedbyTTXwerereportedalmost
ev-eryyear[3–7].Comparedtoothermarinetoxins,TTX intoxication
∗ Corresponding authors
E-mail addresses: gyliu@shxh-centerlab.com (G Liu), yllu@shxh-centerlab.com
(Y Lu)
1 These authors are considered as co-first authors
leadstomoreseveresymptomsandhighermortality[8].Thegrade
ofTTX poisoningdependsontheingestedamount,thetime since ingestion,andthehealthstatusbeforepoisoning[9–10].Thereisa strongcorrelationbetweenthesymptomsandthelevelsofTTXin thebloodofvictims[11]
Asa potenttoxin,TTX selectivelyblocksvoltage-gated sodium (NaV)channels,suchasNaV1.1, NaV1.2,NaV1.3,NaV1.6, andNaV1.7 [12–13],whicharewidelydistributedinthehumancentraland pe-ripheralnervoussystem[14],making itaverypromisingdrugfor the treatment ofpain [15–16].The therapeutic doses reportedin currentclinical trialswere 15–45 μg withCmax ofapproximately https://doi.org/10.1016/j.chroma.2022.463567
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/ )
Trang 2chro-matographywithfluorescencedetection(LC-FL)[22],mostofthem
are for shellfish orfish tissue samples.Dealing withhuman
bio-logical samplesismuch morechallengingbecausetheamount of
TTXintheurineandplasmaisgenerallyextremelylow.Theliquid
chromatography-tandem mass spectroscopy (LC-MS/MS) method
wasconsideredthemostpopularmethodfortheidentificationand
quantificationofTTXbecauseofitshighsensitivity,selectivity,and
specificity[23–24]
SinceTTX isahighlypolarsubstance,therewouldbepoor
re-tentionontheconventionalreverse-phasecolumnandlow
ioniza-tion efficiencyin massspectrometry [25] Hydrophilicinteraction
liquid chromatography(HILIC) could help to address theseissues
andshowedgoodretentioncapabilityandimprovedionization
ef-ficiencyinLC-MS[26–27].Whileionsuppression(onekindof
ma-trix effect) wasanother challenge in analyzing TTX in biological
samples.Ionsuppressionusuallyresultsfromthecoelutionof
non-detected interferences[28–29].Ingeneral, ionsuppressioncanbe
reduced by samplepreparation, chromatographic separation, and
MSanalysis[30–32]
The most daunting analysis bottleneck is sample preparation
Because its effectiveextractionandenrichment fromcomplicated
human biological samples are challenging [33] The most
popu-lar technique for the cleanup of TTX samples is solid phase
ex-traction (SPE), usually using cation exchange extraction [34–35]
The positively charged guanidinium group of TTX strongly
inter-actswiththesulfonicacidgroupinthestationaryphase,allowing
TTX to be strongly retained on SPE cartridges Onlystrong acids
such ashydrochloricacidcaneluteit,requiringadditionalsolvent
evaporationandresidueredissolutionsteps,whichare both
time-consuming andlabor-intensive Therefore, strongcation exchange
extraction techniques are rarelyused for the detectionof TTX at
present
Another SPEtechnique applied to TTX detectionis the use of
graphitized carboncarriers.However,theTTX recoveriesofurine,
serum, and plasma samples were 43%, 23%, and 20%,
respec-tively [36] Lowrecoveries can be attributedto toxin loss and/or
ion suppression during SPE cleanup The limits of quantification
for serum and plasma samples were 21.8 and 25.4 ng/mL,
re-spectively, which were not sensitive enough for pharmacokinetic
studies Ochi [37]also usedgraphitized carboncarriers and
opti-mized themethod by ion-pair solid-phaseextraction The
differ-encewasthattheuseofion-pairsolventinsampleloading
condi-tionimprovetheretentionofTTXongraphitizedcarbon.Whilethe
methodwasonlyappliedtothedeterminationoftoxinsinbivalve
extracts,therewasnodatatoproveitsrecoveryandmatrixeffect
inhumanbiologicalsamples
Hydrophilic interaction liquidchromatography-solid phase
ex-traction (HILIC-SPE) can be a thirdoption dueto thehydrophilic
nature ofTTX.However, fewattempts havebeenmadetoextract
TTX from plasma using HILIC-SPE technology One reason is the
limitedimpurity removalcapabilityofasingleHILIC-SPE
Consid-eringthis, doubleSPEusingC18-HILIChadbeenusedto improve
theefficiencyofremovingionsuppression[38]
In thisstudy, we attempted to use three HILIC-type SPE
car-riers for samplepreparationto comparetheir extraction
recover-ies and influence on matrix effects A simple and fast SPE
pro-tocol based on hydrophilic-interaction/ion-exchange mixed-mode
1.1 Reagents and materials
TTX(99.5%)waspurchasedfromStandardpharmCo.,Ltd.(New York, USA).Arginine-15N4 (90.4%) waspurchased from TLC Phar-maceutical Standards Ltd (Ontario, Canada) Voglibose(98%) was purchasedfromAltaScientific.Ltd (Tianjin,China).SiphilaiHILIX
96wellplates(30mg/2mL)wereobtainedfromSiphila Technolo-gies Co., Ltd (Tianjin, China) PSA 96 well plates (30 mg/2 mL) and silica 96 well plates (30 mg/2 mL) were obtained from Tu-pLabsCo.,Ltd (Tianjin,China).The linksforthethree SPEplates were shown in the supplementary materials Protein precipita-tion plates (2 mL/well) were obtained from TupLabs Co., Ltd (Tianjin, China).Trichloroaceticacid(TCA, ≥99%)wassuppliedby Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) HPLC-grade acetonitrile (ACN) and methanol were supplied by Merck KGaA (Darmstadt, Germany) HPLC-grade formic acid (FA, 99%) andammoniumformate(98%)weresuppliedbyACS(Wilmington, USA) Water was purified using Simplicity system (Merck KGaA, Darmstadt, Germany) Blank human plasma was obtained from healthydonorswhosignedtheInformedConsentForms
ThestandardstocksolutionsofTTX(100μg/mL)wereprepared
indeionizedwaterandstoredat−80 °Cuntilfurther use Work-ingstandardsolutionswerepreparedbyserialdilutionofstandard stocksolutionsusingdeionizedwaterat5differentpoints(1,2,10,
50,200ng/mL)beforetheywereaddedtohumanblankplasmato create calibration curves withranges from 0.1to 20 ng/mL The same waywas used to make quality control samples (QCs) at 4 differentconcentrationlevels(0.1,0.5,2,10ng/mL)
1.2 Sample pretreatment
Onehundred microlitersofhumanplasmasample(calibration curves, QCs or real samples) and 10μL arginine-15N4 (internal standard,IS,100 ng/mL) workingsolution weremixedand trans-ferredto96wellproteinprecipitationplate,then700μLof 1%TCA-ACNwasaddedforproteinprecipitation.Afterfilteringwitha pos-itivepressuredevice,allfiltratesweretransferredtoSiphilaiHILIX
96 well SPE plate, which was previously activated with 1 mL of methanolandequilibratedwith1mLACN.Washoncewith200μL
of95%ACNandelutewith200μLof1%TCA-50%ACN.2μLof elu-tionsolutionwasdirectlyinjectedintoLC-MS/MS
1.3 LC-MS/MS analysis
The LC-MS/MS system consisted of a triple quadrupole mass spectrometer 6500 system (SCIEX, USA) and an ExionLC system (SCIEX,USA).Datawasacquiredandstatisticallycalculatedby An-alyst1.6.3(SCIEX,USA)
TTXandISweremonitoredusingmultiplereactionmonitoring (MRM)inpositivemode withthetransitionsofm/z 320.3→162.2 (quantification transition for TTX), 320.3→284.1 (confirmation transitionforTTX)and179.2→63.0(transitionforIS),respectively Ionsourceparameterswerealsooptimized:550°Csource temper-ature,60psiionsourcegas1,70psiionsourcegas2,30psicurtain gas,4000Vionsprayvoltage,120/43Vdeclusteringpotential,and 49/27eVcollisionenergy
2
Trang 3Fig 1 Effects of ammonium formate (a) and formic acid (b) concentration in mobile phase for TTX
Chromatographic separation was performed on an ACQUITY
UPLC BEH Amide column (50×2.1 mm, 1.7 μm, Waters, USA) at
room temperature The mobile phase consisted of (A) deionized
waterand(B)ACN,bothcontaining10μmol/Lammoniumformate
and 0.01% FA The following gradient program was used [mobile
phaseB]:85–55%at0–2min;held55%at2–3min;55–85%at3–
3.01min;held85%at3.01–4.5minwithaflowrateof0.5mL/min
1.4 Method validation
The lower limit of quantification (LLOQ), linearity,
inter/intra-batch accuracy and precision, matrix effect, extraction recovery,
specificity,andinterferenceoftheestablishedmethodwere
inves-tigated accordingto the U.S foodand drugadministration(FDA)
bioanalyticalmethodvalidationguidelines[39]
2 Results and discussion
2.1 Development of chromatography and mass spectrometry method
TTX is difficult toretain on reversed-phase columns [26], and
mostrecentreportsusedanamidecolumnwithalengthof10cm
toimprovetheretentionof10TTXanalogs[37].Here,anACQUITY
UPLCBEHC18Amide column(50×2.1mm,1.7μm,Waters,USA)
wasusedtoseparate TTXfromitsinterferences,whichperformed
better compared with an ACQUITY UPLC BEH C18 HILIC column
(50×2.1 mm, 1.7μm, Waters,USA) The retention times forTTX
andISontheamidecolumnwere2.41minand2.31min,
respec-tively,withatotalanalysistimeof4.5min
The pHvalue andthe concentration ofammonium formate in
the mobile phase were considered to have significant effects on
retentiontimeandpeakarea[37].Therefore,differentFAand
am-moniumformateconcentrationsweretestedunderthesame
sepa-rationconditions.Itwasobservedthattheretentiontimeandpeak
areaofTTXincreasedwiththedecreaseofFA(from0.05%to0.01%,
v:v)(seeFig.1a)andammoniumformateminimizedtheretention
timeshiftbetweenpuresolventandmatrixsamplesbutdecreased
the peakarea ofTTX (Fig.1b).In thisstudy,10μmol per literof
ammonium formateand0.01%FA(v:v)were usedinbothmobile
phasestoacquirebettersensitivityandmakethepeaksmore
sta-ble.Thevariationof56consecutiveinjectionspreparedbyplasma
sampleswastested(seeFig.S1)andthemeanretentiontimewas
2.42±0.1min(2.37–2.47min),whichindicatedthatthe
chromato-graphicseparationconditionsperformedwellwithexcellent
repro-ducibility MS/MS parameters for TTX and IS were optimized by
directinfusionofindividualstandardsolutions.Thequantifierand
confirmationproductionforTTXwerem/z162.2and284.1,
respec-tively, although the mostabundant product ionwas observed as
[M+H –H2O]+ion(m/z302.2).Thelatterhashigherbaselineand
strongerinterferenceinplasmasamples
2.2 Selection and optimization of SPE
In the beginning, MCX and WCX cation exchange SPE plates (Waters,USA)weretestedtocleanuptheplasmasamplesand pu-rify TTX Itwas found that theWCX plate can hardly retain TTX duringloading,andtheMCXplatecanhardlyeluteTTXusing or-ganic solventcontainingmoderate acids likefomic acid oracetic acid.Strongacidlikehydrochloricacidwasn’ttried onMCXplate duetoits damagetoliquidchromatographyandmass spectrome-try,evenifitcanbedriedup,poorpeakshapesandseverematrix effectmightnotbeavoided
In turn, three differentHILIC-type SPE plates were attempted
to separate TTX from the plasma matrix PSA plate containing polyamidematerial,silica platecontainingsilica gelmaterial,and SiphilaiHILIXplatecontainingmulti-carboxylmaterialwere com-pared The flow charts of comparisonand optimizationfor three differentHILIC-SPEwereshowninFig.2.Alltheloading,washing, andelutionsampleswere collectedandanalyzedto calculatethe lossorrecoveryofTTX
HILIC-typeSPEusually requires highconcentrationsoforganic solvents as loading solutions and water is used as a strong elu-ent [40] In this study, pure ACN and ACN containing 1%TCA, 5%TCA, and 2%FA were investigated respectively as loading solu-tions, which also acted as protein precipitating agents, denatur-ingplasmaproteinsandreleasingTTX.100μLofstandardsolution (prepared bydeionized water)andplasmasamples were precipi-tated with700μL ofloading solutions in 96-well protein precip-itation platesrespectively andthen all filtrates were loaded onto three different SPE plates, which were previously activated with methanolandequilibratedwithACN.TheresultsshowedTTXwas moststronglyretainedontheSiphilaiHILIXSPEplateduring sam-ple loading ACNwas the optimalloading solvent and facilitated theretentionofTTXonSPEplates,followedby2%FA-ACN, 1%TCA-ACN, and5%TCA-ACN(Fig.3) Theloss ofTTX inplasmasamples were lessthan that instandard solution samples duringloading, whichmightbeduetothepoorbufferingcapacityofstandard so-lutionsamplescomparedwithplasma
TofurtherinvestigatethelossandrecoveryofTTXduring wash-ingandelutionsteps,200μLof95%ACNwasusedforwashing fol-lowed by 200μL of 1%TCA-50%ACNfor elution In the 2%FA-ACN andACN loadinggroups, due to thelow recoveriesafter elution, another200μLof5%TCA-ACNwasusedforfurtherelutionto im-provetherecovery.TheresultswereshowninFig.4
Different retention and elution capabilities of TTX were ob-served on the same SPEplate underdifferent loading conditions duringwashingandelution.SiphilaiHILIX wasthemostdistinct andoptimalone(Fig.4a) Inthe1%TCA-ACNloadinggroup, wash-ingwith95%ACNresultedinalossof18%TTX,whileelutionwith 1% TCA-50% ACN resulted in the recovery of 80% TTX inplasma samples While during the same loading and washing steps, the corresponding standard solution samples would lose even more, withless than 30% TTX recovered by elution Inthe 5%TCA-ACN loading group, both the loading and washing steps resulted in
Trang 4Fig 2 The flow charts of comparison and optimization for three different HILIC-SPE
moreloss ofTTX,about60%forplasmasamplesandeven higher
for standard solution samples (>80%) In the 2%FA-ACN loading
group, no significant TTX loss wasobserved for plasmasamples
afterwashingwith95%ACN,andtherecovery wasabout22%after
elution with 1%TCA-ACN Comparedto the two TCA-ACN loading
groups, thetotal recoveryofTTX inplasmasampleswasreduced
throughouttheSPE,so anadditional5%TCA-ACNelutionwas
per-formed to look for the lost TTX and the total recovery was
in-creasedto60%aftertheadditionalelution.Thiswasquitedifferent
from the total recovery of standard solution samples which was
closeto100%.ThetotalrecoveryofTTXinplasmasampleswas
sig-nificantlylower intheACNloadinggroup,nomatter inthe
load-ingandwashingsolutions,ortheelutedandtwice-elutedsamples,
whiletherecovery ofstandardsolutionsampleswasnearly 100%
In the 2%FA-ACN and ACNloading groups, the lower recovery of
TTX fromplasmasamplescomparedtostandardsolutionsamples
maybeduetotheincompletereleaseofTTXfromplasmaproteins
upon precipitation of thesetwo loading solutions Similarresults
were observed whenusingPSAandsilica plates(Fig.4b and4c)
Strong acids such asTCA inthe precipitatingagent maybe
nec-essaryforthereleaseofTTX fromplasmaproteins.ExcessiveTCA
resulted in weak retentionof TTX onSPE, whichmay be related
to the decreased polarity of theTTX-TCA conjugates inthe
pres-enceof ionpairssolvents, thus reducing theretentionof TTXon
HILIC-typeSPEplates
TheresultsshowedthattheretentioncapacityamongSiphilai HILIX,PSA,andsilicaSPEwerequitedifferent.ComparedtoSiphila
iHILIX,PSAandsilicahadweakerretentionofTTX underalmost all loadingconditions.It wasspeculatedthat both SiphilaiHILIX and silica may have two interaction modes: hydrophilic interac-tionandionexchange.SomeSiOH-containingsilica materialshad beenshowntohavecation-exchangeproperties[41].Whileinour study,wefoundthattheionexchangeeffectofsilicaSPEmightbe veryweak whichcouldbe easilyinhibitedby TCAorFA In addi-tiontohydrophilicinteraction,SiphilaiHILIXmayalsoretainpolar basiccompounds through cation-exchangeinteraction which was similarorevenstrongertothat ofweak cation-exchangematerial with carboxylic acid moieties [42] It seemed that PSA had only
a purehydrophilic effecton TTX,andretention wastheweakest Among the three HILIC materials, Siphilai HILIX wasconsidered
tohavebettersynergisticeffectofhydrophilicinteractionandion exchange,whichwassuitablefortheretentionofTTX
After optimizing the loading conditions,the washing solvents wereselected among95%ACN, 98%ACN, andACN No significant differencewasobserved,so95%ACNwaschosenbecauseithadthe strongestcleaningcapability.Theelutionsolventremained 1%TCA-50%ACN,which had no obviousion suppression orenhancement effects on TTX Since stable isotope labeled TTX is very expen-sive and difficult to obtain, we used the reported voglibose and theavailable arginine-15N4 ascandidateinternal standardsinthis
Trang 5Fig 3 The loss of TTX under different sample loading conditions on three HILIC-SPE plates
Table 1
The major validation parameters for TTX and IS in human plasma
Spiked conc (ng/mL)
TTX Intra-assay ( n = 6) Inter-assay ( n = 6 × 3) Extraction recovery ( n = 6) IS-normalized MF ( n = 6) Mean accuracy (%) RSD (%) Mean accuracy (%) RSD (%) Mean (%) RSD (%) Mean (%) RSD (%)
Extraction recovery ( n = 6) Extraction recovery ( n = 6)
study,bothofwhichshowedsimilarchromatographicbehaviorto
TTXontheamidecolumn
The performance ofthe two candidateinternal standards was
further compared inplasmamatrix Post-columninjection
exper-iment wasusedto investigatethe differentmatrixeffects ofTTX
and the two candidate internal standards in pure solvent and
plasma samples It was found that TTX wasbarely enhanced or
inhibited by plasma matrix,arginine-15N4 was slightlyenhanced,
whilevoglibose wasseverelyenhanced (Fig.5).Bycomparingthe
recoveries, itcan be foundthat both arginine-15N4 andvoglibose
had similarextraction recoveriesas TTX.Therefore, arginine-15N4
wasfinallyselectedasISinthisstudy(seeTable1)
2.3 Method validation
After determining the optimization of SPE conditions,
valida-tionwasattemptedtoinvestigatethepracticabilityofthepresent
method The method displayed excellent linearity of TTX with
1/x asweighting factor,and R value was higherthan 0.999 over
a 200-fold linear dynamic range (0.1–20 ng/mL) with the LLOQ (signal-to-noiseratio,S/N >10)ofabout0.1ng/mL.Moreover, spike-recoverystudiesassessed forfourdifferentQCs(at0.1,0.5, 2,and
10ng/mL,n =6) atthreeconsecutivedays.The intra- and inter-assay accuracies (%) were 98.5%−99.8% and98.8%−99.5%, respec-tively, calculated by comparing measured concentrations to the-oretically spiked concentrations.The intra- and inter-assay preci-sioninvestigatedintermsofrelativestandarddeviations(RSDs%), were≤ 5.92%and≤ 6.23%,respectively.Theresultswere summa-rizedinTable1,whichshowedacceptable accuracyandprecision forplasmaTTX inthe investigatedconcentration range The vali-dationofthemethodselectionprovedthatsampleswereprepared wellbySiphilaiHILIX.Therepresentativechromatogramsofblank samplesand LLOQ samplesatconcentration of0.1ng/mL of TTX and10ng/mLofISwereshowninFig.6.Noobviousinterferences coelutingwiththeTTXandtheISwasobservedandthebaselines
ofnoisewereacceptable.TheLLOQwasabout0.3ng/mLfor con-firmationtransition (m/z 320.3→284.1) accordingto thecriterion
ofS/N >10,indicatingthatthemethodinthisstudywouldbe
Trang 6suit-Fig 4 The recoveries of TTX in each step on three HILIC-SPE plates under different sample loading conditions a Siphila i HILIX SPE, b Silica, c PSA
6
Trang 7Fig 5 Plasma matrix effect of a TTX, b arginine- 15 N 4 and c voglibose
Fig 6 The representative chromatograms of blank, LLOQ (0.1 ng/mL) of a TTX and b IS (10 ng/mL)
Trang 8Plasma5 −1.43 5.90 −7.00 4.07
hemolytic plasma −3.53 4.28 −2.50 3.86
high triglyceride plasma 0.90 4.80 −4.67 2.92
high cholesterol plasma −3.67 3.10 −2.50 6.40
high bilirubin plasma −5.00 4.95 3.93 2.37
able forthe identification andquantification of TTX at poisoned
concentrationlevels
Theextractionrecoverywasassessedbycomparingthepeak
ar-easofspikedplasmasampleswiththoseofpost-extractionspiked
samples at three QC levels The quantitative matrix factors were
evaluated by comparing the peak areasof post-extraction spiked
samplesfromsixsourcesattwoQClevelswiththoseofneat
stan-dard solutions.The methoddemonstratedexcellent extraction
re-coveryforTTX,rangingfrom80.8%to81.5%atthreedifferent
con-centrations(RSDs≤ 6.78%).ThemeanISnormalizedmatrixfactor
of samples from sixsources at two levels were 0.979 and 0.968
respectively,whichindicatednosignificantionsuppressionor
en-hancementexistedbytheoptimizedmethod(Table1).Thelost
ex-tractionrecoveryofTTXwasmainlyinthewashingstep,asshown
inFig.4a,whichwasspeculatedtobeimprovedbyincreasingthe
amountofSPEpackedmaterialorreducingthewashingvolume
Good specificity means that the analytical method can
differ-entiate between thetarget analyteand interferingsubstances for
samples from different sources and the bias and precision were
acceptable Ten blank human plasma samples including samples
fromsixhealthyvolunteersandpooledblankhumanplasma
sam-ples mixingwithhigh concentrationsofhemoglobin,triglyceride,
cholesterol, andbilirubinwerepreparedandinvestigatedby
spik-ing analysis In detail, working standard solutions of TTX were
spikedtotheabove10blankplasmasamplestomakeQCsatLLOQ
andHigh concentration levels(0.1, 10ng/mL) TheQCs were
pre-treatedandanalyzed.ThebiasesandRSDswerelisted inTable2
The biases oftarget value were ranged from−7.00%to 7.43% for
healthy humanplasmasamples(RSDs ≤ 8.83%)andfrom−5.00%
to3.93%forhemolytic,hightriglyceride,highcholesterolandhigh
bilirubinplasmasamples (RSDs≤ 6.40%),whichproved thegood
anti-interferencepropertyofthemethod
Saxitoxin and its approximately 60 natural analogues, which
are also potent andspecific voltage-gated sodium channel
block-ersandshare thesame bindingsiteasthat of TTX[43,44], also
cause paralysis and even death inextreme cases In our
supple-mentalstudy,gonyautoxin5(GTX5)waspurchasedforinterference
test sinceits retentiontime wasclosest toTTX onamide column
[45,37].Underthe optimizedLCandSPEconditionsdescribedin
this study, the retention time of GTX5 was2.53 min which was
closetoTTX(seeFig.S2).Nointerferenceswereobservedbetween
theTTXandGTX5MRMtransitions.Therefore,webelievethat
sax-itoxin and its analogues may not interfere the detection of TTX
Besides, in a preliminary study, the total recovery of GTX5 from
plasma samples wasabout 70% according to the peak area ratio
of spiked plasmasamples and neat standard solutions which
in-dicated that the describedmethod in our studymay be suitable
for quantitation of these compounds after optimization.In
addi-tion, the analogs of TTX like 4,9-anhydro TTX, 4–epi TTX andso
on,usuallyhavesimilarchemicalstructuresandlowertoxic
equiv-3 Conclusions
An LC-MS/MS method had been developed and validated for thequantitationofTTXinhumanplasma.Thismethodused multi-carboxylgroupSPEmaterialsanddidnotrequireevaporationand ultrafiltrationsteps Comparedwithprevious reports,the method was highly sensitive, simple, and rapid in sample pre-treatment andhadshortchromatographictime.Themethodpresentedinthis papershowedwidelinearity,lowLLOQ, goodprecisionand accu-racy,andhighrecoverycharacteristicswhichwell satisfiedthe re-quirementsofpharmacokineticstudyandclinicaldiagnosisofTTX
Credit authorship contribution statement Liang Xin:Conceptualization,Investigation,Methodology, Writ-ing OriginalDraft.Yan Liang:DataCuration,Investigation, Writ-ing Original Draft Shuangshuang Yang: Methodology, Data Curation, Visualization Fengli Jiang: Investigation, Methodology
Fan Yu: Investigation, Validation Meiwei Zhang: Data curation, Methodology Wei Chang: Funding acquisition Wei Wang: Data curation, Validation Chen Yu: Supervision Gangyi Liu: Formal analysis,Project administration,Writing Review&Editing.Youli Lu: Funding acquisition, Formal analysis, Project administration, Writing Review&Editing
Declaration of Competing Interest
Theauthorsdeclarethattheyhavenoknowncompeting finan-cialinterestsorpersonalrelationshipsthatcouldhaveappearedto influencetheworkreportedinthispaper
Data Availability
Datawillbemadeavailableonrequest
Acknowledgments
ThisresearchwasfundedbytheInnovationFundofScienceand Technology Commission of Shanghai Municipality (20Y11900800) andtheGeneralProjectofMedicalResearchFundofXuhuiDistrict, Shanghai(SHXH202121)
Supplementary materials
Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.chroma.2022.463567
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