Pharmacokinetic and clinical effects of two bupivacaine concentrations on axillary brachial plexus block

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REVISTA

BRASILEIRA

DE

ANESTESIOLOGIA

PublicaçãoOﬁcialdaSociedadeBrasileiradeAnestesiologia

www.sba.com.br

SCIENTIFIC

ARTICLE

Pharmacokinetic

and

clinical

effects

of

two

bupivacaine

concentrations

on

axillary

brachial

plexus

block

Leonardo

H.C.

Ferraro

a

_,

_Alexandre

_Takeda

a

_,

_Cleber

_N.

_Barreto

b

_,

_Bernadete

_Faria

b

_,

Nilson

A. Assunc

¸ão

b_,_∗

a_Universidade_Federal_de_São_Paulo_(UNIFESP),_Disciplina_de_{Anestesiologia,}_Dor_e_Terapia_Intensiva,_São_Paulo,_SP,_Brazil b_Universidade_Federal_de_São_Paulo_(UNIFESP),_Instituto_de_Ciências_Ambientais,_Químicas_e_{Farmacêuticas,}_São_Paulo,_SP,_Brazil

Received7December2016;accepted4September2017 Availableonline24November2017

KEYWORDS Bupivacaine; Brachialplexus; Pharmacokinetics; Regionalanesthesia Abstract

Introduction:Theriskofsystemicbupivacainetoxicityisapersistentproblem,whichmakes itspharmacokineticstudyfundamentalforregionalanesthesiasafety.Thereislittleevidence ofitsinﬂuenceonplasmapeakatdifferentconcentrations.Thepresentstudycomparestwo bupivacaineconcentrationstoestablishhowtheconcentrationaffectsthisdrugplasmapeak inaxillarybrachialplexusblock.Postoperativelatencyandanalgesiawerealsocompared. Methods:30 patientswere randomized.In the0.25%Group, 0.25%bupivacaine(10mL)was injectedpernerve.Inthe0.5%Group,0.5%bupivacaine(5mL)wasinjectedpernerve. Periph-eralbloodsampleswerecollectedduringtheﬁrst2haftertheblockade.Forsampleanalyses, highperformanceliquidchromatographymassspectrometrywasused.

Results:Plasmapeakoccurred45minaftertheblockade,withnodifferencebetweengroupsat theassessedtime-points.Plasmapeakwas933.97_±328.03ng.mL−1(mean_±SD)in0.25%Group and1022.79±253.81ng.mL−1in0.5%Group(p=0.414).Latencywaslowerin0.5%Groupthan in0.25%Group (10.67±3.71×17.33min±5.30,respectively,p=0.004).Nopatienthadpain withintheﬁrst4haftertheblockade.

Conclusion: Foraxillarybrachialplexusblock,therewasnodifferenceinbupivacaineplasma peak despitethe useofdifferentconcentrations with thesame local anestheticmass. The concentrationinverselyinﬂuencedlatency.

∗_{Corresponding}_author.

E-mail:nilson.assuncao@gmail.com(N.A.Assunc¸ão). https://doi.org/10.1016/j.bjane.2017.09.007

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PALAVRAS-CHAVE

Bupivacaína; Plexobraquial; Farmacocinética; Anestesiaregional

Efeitosfarmacocinéticoseclínicosdeduasconcentrac¸õesdebupivacaína nobloqueiodoplexobraquialviaaxilar

Resumo

Introdução:Oriscodeintoxicaçãosistêmicapelousodabupivacaínaéum problema persis-tenteetornaseuestudofarmacocinéticofundamentalparaasegurançadaanestesiaregional. Sãoescassasasevidênciassobreainfluênciadediferentesconcentraçõesnopicoplasmático dessefármaco.Opresenteestudocomparaduasconcentraçõesdebupivacaínapara estabele-cercomoaconcentraçãoafetaopicoplasmáticodessefármaconobloqueiodoplexobraquial viaaxilar.Tambémsecompararamlatênciaeanalgesiapós-operatória.

Métodos: Foramrandomizados 30 pacientes. NoGrupo 0,25%,injetaram-se 10 mLde bupi-vacaína0,25%pornervo.NoGrupo0,5%, injetaram-se5mLdebupivacaína0,5%pornervo. Amostrasdesangueperiféricoforamcolhidasduranteasduasprimeirashorasapósobloqueio. Para análisedas amostras,usou-se acromatograﬁa líquida de alta frequência acoplada ao espectrômetrodemassas.

Resultados: Opicoplasmáticoocorreu45 minutosapósobloqueio,semdiferença entreos gruposnostemposavaliados.Opicoplasmático(média±DP)foi933,97±328,03ng.mL−1no Grupo0,25%e1.022,79_±253,81ng.mL−1noGrupo0,5%(p=0,414).OGrupo0,5%apresentou menorlatênciacomrelaçãoaoGrupo0,25%(10,67±3,71×17,33min±5,30;respectivamente; p=0,004).Nenhumpacienteapresentoudornasprimeirasquatrohorasapósobloqueio. Conclusão:Paraobloqueiodoplexobraquialviaaxilar,nãofoidetectadadiferençanopico plasmáticodebupivacaínaapesardousodediferentesconcentrações,comamesmamassade anestésicolocal.Aconcentraçãoinfluenciouinversamentealatência.

Introduction

Thesuccess ofregionalanesthesia(RA)is directly related to the evolution of knowledge and the development of localanesthetics(LA).Despiteeffortstoincreaseitssafety, a persistent problem in clinical practice is local anes-theticsystemictoxicity(LAST).1,2_The _rapid_increase _in_LA

plasmalevelsleadstodevastatingneurologicalandcardiac complications;LAsystemictoxicityaccountsfor one-third of deaths or brain damage during regional anesthesia.3,4

Regarding RAtype, peripheral nerveblock mayrequire a greaterLAvolume,withahigherriskofLASTcomparedto epiduralblock.1,5,6

Theincreaseduseof ultrasoundinclinical practicehas reducedtheoccurrenceofcomplicationsrelatedto periph-eral block, mainly due to the reduction of inadvertent vascular puncture.7,8 _Moreover, _in _order _to _improve _the

safetyof regional anesthesia, anesthesiasocieties around theworldrecommendusingtheminimumdoserequiredand stipulate the maximum LA dose to be used in peripheral blocks.9 _However,_there_is_no_consensus_among_the

differ-entsocietiesaboutthemaximumrecommendeddoseofLA, whichrevealsagapinunderstandingthepharmacokinetics ofthesedrugs.9_Furthermore,_there_are_several_factors_that

mayinﬂuenceLAplasmapeaksuchastheinfusionsite vas-cularizationandthetissuebindingability.9

Surprisingly, data on the effect of different LA con-centrations on plasma levels are scarce and conﬂicting so far. However, understanding the pharmacokinetics

of these chemical compounds is essential to prevent complications.10---12

Thisstudywasperformedtoevaluatethisrelationship, particularlyregardingaxillary brachialplexus block.Thus, thepresent study providesananalysisof the pharmacoki-netic proﬁles of twobupivacaine concentrations obtained after axillary brachial plexus block. Despite the different concentrationsused,thetotalmassoflocalanestheticwas maintainedtoevaluatetheeffectofthesedifferent concen-trationsonthisdrugplasmapeak.Asasecondaryobjective, latencyandpostoperativeanalgesiawereevaluatedinboth groups.

Material

and

method

Aprospectiveandrandomizedclinicaltrialwasperformed attheoperatingroomoftheHandandUpperLimbSurgery DepartmentataquaternaryUniversityHospital.The anes-thetic procedures were initiated in January 2014, after approvalbytheEthicsandInstitutionalResearchCommittee approval number 288,461. Inclusion criteria were: candi-dates for elective distal forearm and hand surgery, with brachialplexusblockindicationforanesthesiaand analge-sia,agedbetween18and65years,withphysicalstatus(ASA Ior II)according totheAmericanSociety of Anesthesiolo-gists,bodymassindex(BMI)lessthan35kgm−2,andasigned writteninformedconsent.Exclusioncriteriawere:cognitive impairment,infection at thepuncture site,coagulopathy, historyofbupivacaineallergy.

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Clinicalmethodology

Aftermeetingtheinclusioncriteria,patientswere random-ized into groups to receive either 0.25% bupivacaine or 0.5%bupivacaine,accordingtocomputer-generatedrandom numbers,whichwereconsideredasblocksoftencases.

The 0.25%bupivacaineconcentrationwasusedbecause itisthelowesteffectiveconcentrationforaxillarybrachial plexusblock.13_The_0.5%_{concentration}_was_used_because_it

isthehighestbupivacaineconcentrationusedinourservice. Forgreateraccuracyofinformation,thepatientreceived explanationtothedifferencesbetweentactileandpainful sensation. Routine monitoring for surgical procedure with electrocardioscopy, sphygmomanometer, andpulse oxime-trywasperformed.Venousaccesswasobtainedintheupper limbcontralateraltothesurgicalprocedure,exclusivelyfor bloodsamplecollection.

The axillary brachialplexus blockguided byultrasound (SSeries,FujifilmSonosite,Seattle,USA)wasperformedby a single experienced anesthetist, withthe patient in the horizontaldorsaldecubitus position,aiming at standardiz-ingtheblockadetime,definedastheintervalbetweenthe needleinsertionandtheendofLAinjection.Thus,thistime wouldnotinfluencethelatencyofgroups.Skinasepsiswas madewitha chlorhexidine-alcoholsolution.Afterbrachial plexusnervevisualization,LAwasinjectedintoeachnerve identifiedin thispathway--- namely,radial,ulnar,medial, andmusculocutaneous.Forthe0.25%Group,10mLof0.25% bupivacaine(CristáliaProdutosQuímicos,SãoPaulo,Brazil) wereinjectedintoeachnerve,totaling40mLperpatient. Forthe0.5%Group,5mLof0.5%bupivacaine(Cristália Pro-dutosQuímicos,SãoPaulo,Brazil)wereinjectedintoeach nerve,totaling20mLperpatient.

An anesthesiologist, who was not present during the injectionandwasblindedtotheconcentrationandvolume ofanesthetics used,evaluatedthe nerveblocksaccording tomotorfunction,thermalsensitivity,andpainsensitivity.

In ordertoevaluatemotor function,the modiﬁed Bro-mage scale wasused (Table 1).14,15 _The _assessed _muscles

were:fingerflexors(mediannerve),fingerextensors(radial nerve),firstfingeradductor(ulnarnerve),andbiceps (mus-culocutaneousnerve).

Thermalsensationassessmentwasmadewithgauzeand alcohol, testing the sensitivity of the dermatomes inner-vatedbytheulnar,median, radial,andmusculocutaneous nerves.

Theassessmentofpainsensationattheupperlimbwas performedwiththepinpricktest(23Gneedle),testingthe sensitivity of theulnar, median, radial,and musculocuta-neousdermatomesnerves.

Thisassessmenthappenedeveryﬁveminutesupto30min aftertheblockade.Inthisperiod,ifsurgicalanesthesiahad notbeenachieved,asupplementalinjectionof ultrasound-guidedbupivacainewasperformed,distaltotheaxilla,and thepatientwasexcludedfromtheprotocol.

Surgicalanesthesiawasdeﬁnedasamotorscalelessthan orequaltoBromagetwo,withnosensationofcoldand pin-prickintheassesseddermatomesandalsoiftherewasno needforanestheticsupplementationduringtheprocedure. The end of the LA solution injection was considered as timezero (T0)to evaluate the blockade success rate.

Latencywasdeﬁnedasthetimeintervalbetween T0and thetimeatwhichthesurgicalanesthesiawasachieved.

Duringsurgicalprocedure,patientsreceivedmidazolam (0.05mg.kg−1)for sedation.After surgical procedure, the patient was admitted to the post-anesthesia care unit, whereheremainedforfourhoursinordertoassesstheneed foranalgesicsupplementation.

Laboratorymethodology

Venousbloodsampleswerecollectedbeforetheblockade, every15minutes(min)duringtheﬁrsthourandevery30min inthe second hourafter the blockade, throughan exclu-sivecannula.Initially, 5mLwere collectedandscavenged toavoid any contaminationfrom the previous collection. Subsequently, another 5mL were collected and stored in twoEDTAtubes.EDTA tubeswerecentrifugedat 3500×g

for10mintoobtainbloodplasma.Theobtainedplasmawas storedin cryogenictubesinafreezeratatemperatureof −80◦_C_until_analysis.

AHighPerformanceLiquidChromatography---HPLC (Shi-madzu,Kyoto,Japan)wasusedfortheanalysis,coupledto themassspectrometer(BrukerAmazon,USA)with electro-sprayionizationsource16_and_sequential_mass_spectrometry

system(MS/MS).

Afterobtainingthe precursorion, afragmentfromthe precursor ion dissociation was obtained by the collision-induced dissociation process. HPLC---MS/MS data were monitored for internal standard (tryptophan) and bupiva-caine standard. Positive mode analyzes were performed in the MS/MS mode with selection of molecular ions at 289m/z⇒140.1m/z, bupivacaine. The methodology was validatedin accordance withthe Foodand Drug Adminis-trationinternationalrecommendations.17

Statisticalanalysis

Theaimofthisstudywastoevaluatethedifferenceinthe maximum plasma levelsachieved after ultrasound-guided axillarybrachialplexusblockwithtwodifferent concentra-tionsofbupivacaineandmaintenanceoftheinfusedmass. Tocalculate the sample sizeneeded torevealthis differ-ence,the followingassumptions wereconsidered:(1) null hypothesis:H0:P025max=P05max;alternative hypothe-sisHa:P025max>P05max,whereP025max=peakplasma achieved in the 0.25% Group and P 0.5%=peak plasma achievedinthe0.5%Group;(2)testforcomparisonoftwo independentsamplemeanswithaknownstandarddeviation (Student’st-test);(3)signiﬁcancelevelof5%(˛=0.05);(4) Samplepowerof80%(1−ˇ=0.80).

Consideringthedifferenceofabout40%reportedinthe literatureforintramuscularinjection,thesamplesizewas calculatedas14patientspergroup.Consideringalossrate of approximately 10% during collection and analysis, the samplenumberwasincreasedto32patients. Allanalyzes wereperformedusingSPSS(v18.0)andMinitab(v16).

Shapiro---Wilk test was used to verify if the analyzed data were normally distributed. For quantitative varia-bles with normal distribution, the Student’s t-test was used. ANOVA was used to assess the variance between

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Table1 Motorandsensorytest.14,15

Motortest Localsensitivetest

Median Fingerﬂexion Thenareminence

Radial Wristextension Backofthehand Ulnar Abductionofthe5thﬁnger Hypothenareminence Musculocutaneous Elbowﬂexion Forearmlateralregion

Score Deﬁnition

4 Fullmusclestrengthinrelevantmusclegroups 3 Reducedstrength,butabletomoveagainstresistance 2 Abilitytomoveagainstgravity,butnotagainstresistance 1 Discretemovements(trembling)ofmusclegroups

0 Lackofmovement

ModiﬁedBromageScale.14,15

quantitativevariables. The level of signiﬁcance was 5% (˛<0.05) andthe tests witha descriptivelevel below 5% (p<0.05)wereconsideredsigniﬁcant.18

Results

Atotal of 35 patients wereselected for the study.Three patients were not included for not meeting the inclusion criteriawhile 32patientswererandomized. Therewasno difﬁcultyinvisualizingtheaxillarybrachialplexusvia ultra-sound.Twopatients,onefromeachgroup,wereexcluded from the protocol due to blockade failure; 30 patients completedtheprotocoluptosampleanalysis(Fig.1). Demo-graphicdataofpatientsareshowninTable2.

Blockade time was 192±28s for the 0.5% Group and 204±32s forthe0.25% Group(p=0.462);it didnot inﬂu-encethelatencytimemeasurement.However,theblockade latencyofthe0.5%Groupwasstatisticallylowerthanthat ofthe0.25%Group(Table3).Inaddition,nodifferencewas seeninthedurationofsurgicalprocedures(Table3).

Inallpatientsinwhomsurgicalblockadewasachieved, thesurgicalprocedureswereuneventful.Duringthe block-ade,noinadvertent intravascular puncturewasobserved, which could impair the outcome evaluation. In addition, nomild or severe symptoms of LAsystemic toxicitywere observed (e.g., tinnitus, perioral tingling, or seizures). Regardingpostoperativeanalgesia,nopatientreportedpain uptofourhoursaftertheblockade.Allpatientswere dis-chargedonthesamedayoftheprocedureandtherewasno caseofhospitalreadmission.

Regardingthepharmacokineticstudy,theuseddosesof bupivacaine,inmg.kg−1,weresimilarbetweengroupsand did not interfere with the results achieved(0.25% Group ---1.33±0.19;0.5%Group---1.32±0.19,p=0.826).Plasma concentrationsofbupivacaineafteraxillarybrachialplexus blockfor 0.5%and0.25%groups areshown inTable4and

Fig.2.Therewasnodifferencebetweenplasmaanesthetic concentrationsatanytime.Thepeakplasmaforbothgroups occurred 45min after the blockade. The mean maximum concentrationofbupivacainewas933.97±328.03ng.mL−1 for 0.25% Group and 1022.79±253.81ng.mL−1 for 0.5% Group.

Table2 Demographicsofpatients.

0.25%Group 0.5%Group p n 15 15 Age(years) 36.0(10.3) 35.7(12.1) 0.93a BMI(kgm−2) 25.7(3.9) 26.2(3.2) 0.7a Sex M/F 10/5 11/4 ASA I/II 7/8 6/9

BMI,BodyMassIndex.

a _Student’s_t-test,_age_and_BMI_presented_as_mean_(SD).

The differentconcentrations of LAused didnotaffect howfastthepeakplasmawasreached(Table4andFig.2).

Discussion

LA plasma concentration depends on the dose adminis-tered,rateofsystemicabsorption,tissuedistribution,and drug clearance. The decrease in LA systemic absorption increasesitssafetymargininclinicalpractice.LAdoseisthe variablemostmanipulatedbythephysicianduringclinical practice.Thedoctorcansetthetotaldose tobe adminis-teredandwhetherthedrugisgiveninamoreconcentrated ormoredilutedsolution.Thepresentstudyaimwasverify therelationshipbetweentheLAconcentrationanditspeak plasma. Despite the difference in bupivacaine concentra-tionbetween groups, thepresent study outcomesshowed thatthemaximumplasmalevelachievedwassimilar.

These results are similar to those of a previous study that failed to ﬁnd a difference in the maximum level of LA in epidural anesthesia when the mass was main-tainedconstant.12 _Thus,_this_result_increases_the_evidence

that it is the total dose of LA that regulates the drug pharmacokinetics.19,20

Onepossibleexplanationwouldbethatwhenusingthe higher concentration drug, the surface area available for absorption is smaller, which would result in a systemic absorptionequaltothatofthelowerconcentrationdrugbut withalargersurfaceareaavailableforabsorption.Itmay

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16 0.5% Group 16 0.25% Group 15 patients Collected Samples 15 patients Collected Samples 15 patients Collected Samples 15 patients Collected Samples 1 excluded due to block failure 1 excluded due to block failure 32 Randomized 35 Selected

3 did not meet inclusion criteria

Figure1 Clinicalﬂowchartofthestudy.

Table3 Blockadelatencyandsurgicaltimepergroup.

Techniquevariables 0.25%Group

(n=15)

0.5%Group (n=15)

p

Latency(min) 0.004

Mean(standarddeviation) 17.3(5.3) 10.7(3.7)

Median 20 10

Durationofsurgery(min)

Mean(standarddeviation) 88.6(49.8) 83.9(44.9) 0.41

Median 70 70

Student’st-test.

Table4 Comparisonofplasmabupivacaineconcentrationateachtimepointafterblockade. Time.min−1 Bupi0.25%(ng.mL−1) Mean(SD)a 95%CIa _Bupi_0.5%_(ng.mL−1₎ Mean(SD)a 95%CIa _p 15 447.07(162.37) 364.91---529.24 525.22(149.66) 449.49---600.96 0.181 30 791.76(251.32) 664.58---918.95 826.59(232.27) 709.05---944.14 0.696 45 933.97(328.03) 767.97---1099.98 1022.79(253.81) 894.35---1151.24 0.414 60 631.37(214.45) 521.83---740.9 709.98(191) 613.32---806.6 0.301 90 614.85(307.71) 459.13---770.58 615.18(196.33) 515.83---714.54 0.997 120 410.95(117.09) 351.69---470.2 470.14(122.8) 408---532.29 0.187

a _SD,_standard_deviation;_CI,_conﬁdence_interval;_Bupi,_bupivacaine. Student’st-test.

besuggestedthatthesurfaceareaavailableforabsorption playsanimportantroleintheplasmapeakofLA,neutralizes the effect of the differencein concentrations. This ﬁnd-ingisdemonstratedbythesamepeakplasmaconcentration andbythesamepatternofplasmaconcentrationovertime in both groups, despite different concentrations. In addi-tion,apossibledifferenceinosmolaritybetweensolutions couldresultindifferentpeakplasmalevelsbetweengroups. The osmolarity of the solutions used in this study were

293mOsmoL.L−1for0.25%bupivacaineand239mOsmoL.L−1 for 0.5% bupivacaine. However, the osmolarity difference betweengroupsdidnotinﬂuencetheplasmapeak.

In a previous study,Cohen etal. demonstrated that a portionofthehigherconcentrationsolutionscould precip-itate when injected into a tissue pH>6.9. Solutions with lowerconcentrationsdidnotshowthisprecipitation.10 _The

solutionsusedinthepresentstudyhadthesamepH. How-ever,onelimitationofthestudywasnottoassesswhether

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1450 1250 1050 850 650 450 250 15 30 Bupi 0.25% (ng.mL–1₎ _{Bupi 0.5% (ng.mL}–1₎ 45 60 Time/min Concentration (ng.mL –1) 90 120

Figure2 Distributionofmeanandstandarddeviationofbupivacaineplasmaconcentrationpergroupatdifferenttimepoints afterblockade.

thisprecipitationalsooccurredwithbupivacaineathigher concentrationintheaxillarypathway,whichwoulddecrease thedrugavailabilityforabsorption.

In addition, another interesting result of the present study was that blockade latency was lower for the 0.5% Group than for the 0.25% bupivacaine Group. A possible explanation for this result is that when the surface area availableforexchangeislimited,LAconcentrationbecomes themaindeterminantofthelatencyperiod.Apreviousstudy hasdetermined that theminimum LAvolume required to involvethenervesinaxillarybrachialplexusblockisabout 3.5mL.21 _In_the _present_study,_the _lowest_infusion _volume

was 5mL per nerve. Therefore, the volume injected per nervewas greater than the minimum volumerequired to involvetheentirenervestructureinbothgroups.Thus,the volumeused wassufﬁcient to encompass the entire neu-ral exchangesurface, with nodifference between groups regardingthisfactor.Inthiscase,theconcentration prob-ably becomes the most important factor related to the transmembranepassingthroughratewhenthesurfacearea isthesame.Thiswouldexplaintheshorterlatencyperiod inthe0.5%bupivacainegroup.

Motorandsensoryblockadedurationcouldnotbe eval-uatedduetotheoutpatientnatureoftheservice.Despite thisdifferenceinblockadelatency,postoperativeanalgesia uptofourhourswasthesamebetweengroups,showingthat theuseofdifferentconcentrationsdidnotalteranalgesia duringthisperiod.

Regarding LAST symptoms, a previous study demon-strated that following intravenous bupivacaine infusion, patientshadmild toxicitysymptoms(i.e., mouthtingling, dizziness,andtinnitus)withplasmaconcentrationsranging from 1000 to 2000ng.mL−1. Infusion was done at a rate of 10mg.min−1 up toa maximum dose of 150mg or until theﬁrst symptomsappeared.22 _Although _the _plasma _peak

ofbupivacaineseen insomepatients inthepresent study alsoreachedvaluesbetween1000and2000ng.mL−1inboth groups,thiseffectoccurredafter45minofperineural infu-sionandnopatienthadLASTsymptoms.Theseresultsare similar to that of previous studies on the

pharmacokine-ticsofbupivacaineduringaxillarybrachialplexusblock,23,24

whichfoundplasmapeaklevelswithinthatintervalwithout anyofthepatients showingsymptomsof LAtoxicity.23_The

rateatwhichthemaximumplasmalevelisreached proba-blyalters thesystemiceffectsoftoxicity.Inaddition,the timerequiredtoreachtheplasmapeakconcentrationinthe presentstudywassimilartothatreportedbyotherauthors whodescribed thepharmacokinetic profileof bupivacaine duringaxillarybrachialplexusblock.Thisfindingemphasizes thatan episodeofLASTismorelikelytooccurduringthe firsthouraftertheblockade,regardlessoftheconcentration used.Asallpatientsweresedatedduringthesurgical proce-dure,anyawarenessofmildsymptomsofLASTwaslimited.

Conclusion

Theaimofthisstudywastoincreaseknowledgeaboutthe LApharmacokineticstoincreasethesafetyofRA.Thisstudy shows thatthereis nodifferenceinpeakplasmalevelsof bupivacaine, despite the use of different concentrations and volumes, since the LA mass was constant. However, concentrationplayedanimportantroleindeterminingthis blockade latencyperiod,inversely inﬂuencingthelatency period.

Funding

Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

WethankthefundingbyFAPESP(grantn/2012/02514-9)and theBrazilianAgencyforInnovationtosupportthe acquisi-tionofLC-MS/MS.

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