Determination of Etomidate in Human Postmortem Fluids and Tissues

Robert D. JohnsonRussell J. Lewis

Civil Aerospace Medical InstituteFederal Aviation AdministrationOklahoma City, OK 73125

February 2009

Final Report

DOT/FAA/AM-09/3Office of Aerospace MedicineWashington, DC 20591

NOTICE

This document is disseminated under the sponsorship of the U.S. Department of Transportation in the interest

of information exchange. The United States Government assumes no liability for the contents thereof.

___________

This publication and all Office of Aerospace Medicine technical reports are available in full-text from the Civil Aerospace Medical Institute’s publications Web site:

www.faa.gov/library/reports/medical/oamtechreports/index.cfm

�

Technical Report Documentation Page 1. Report No. 2. Government Accession No. 3. Recipient's Catalog No.

DOT/FAA/AM-09/3 4. Title and Subtitle 5. Report Date

February 2009 Determination of etomidate in human postmortem fluids and tissues 6. Performing Organization Code

7. Author(s) 8. Performing Organization Report No. Johnson RD, Lewis RJ

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS) FAA Civil Aerospace Medical Institute P.O. Box 25082 11. Contract or Grant No. Oklahoma City, OK 73125

12. Sponsoring Agency name and Address 13. Type of Report and Period Covered Office of Aerospace Medicine Federal Aviation Administration 800 Independence Ave., S.W. Washington, DC 20591 14. Sponsoring Agency Code

15. Supplemental Notes Work was accomplished under approved task AM- B-05-TOX-204. 16. Abstract Following an aviation accident, biological specimens from the operator of the aircraft are submitted to the Federal Aviation Administration’s Civil Aerospace Medical Institute for toxicological analysis. During the course of medical treatment following an aviation accident, pilots who later died as a result of their injuries may have been administered etomidate as an intravenous anesthetic. Our laboratory has developed a sensitive method for the identification and quantitation of etomidate in the biological specimens received from these pilots. Furthermore, we have evaluated the distribution of this compound in various postmortem tissues and fluids from 3 fatal aviation accident cases. When available, 10 specimen types were analyzed for each case, including blood, urine, vitreous humor, liver, kidney, skeletal muscle, lung, spleen, heart muscle, and brain. Specimens were extracted using solid-phase base extraction and analyzed by GC/MS. Deuterated etomidate was not available as an internal standard, so to eliminate any possible matrix effects during extraction all quantitative values in specimens other than blood were determined through standard addition. Blood etomidate concentrations in these three cases ranged from 12 to 41 ng/mL. Distribution coefficients for etomidate were determined for each of the specimen types analyzed. These coefficients are expressed relative to the blood concentration in that case. To our knowledge, this is the first report presenting the distribution of etomidate in humans at therapeutic concentrations.

17. Key Words 18. Distribution Statement

Forensic Toxicology, Etomidate, Distribution, Postmortem, Aircraft Accident Investigation

Document is available to the public through the Defense Technical Information Center, Ft. Belvior, VA 22060; and the National Technical Information Service, Springfield, VA 22161

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price Unclassified Unclassified 11

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized

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CONTENTs

IntroductIon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

MaterIals and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

. Chem�cals.and.Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

. Gas.Chromatograph�c/Mass.Spectroscop�c.Cond�t�ons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

. Sample.Select�on.and.Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

. Cal�brator.and.Control.Preparat�on. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

. Sample.Preparat�on.and.Extract�on.Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

. Extract�on.Effic�ency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

results and dIscussIon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

. Method.Val�dat�on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

. Postmortem.Concentrat�ons.of.Etom�date. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

conclusIon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

references. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1

Determination of etomiDate in Human Postmortem fluiDs anD tissues

INTrOduCTION

Etom�date,. [R-(+)-ethyl-1(phenylethyl)-1-H. �m�d-azole-5-carboxylate],.�s.an.�ntravenous.anesthet�c.agent.first.approved.for.use.�n.the.Un�ted.States.�n.1983.and.marketed.under.the.trade.name.Am�date® .1.Pr�or.to.the.�ntroduct�on.of.etom�date,.the.med�cal.commun�ty.lacked.an.�deal.agent.for.the.�nduct�on.of.unconsc�ousness.be-fore.certa�n.med�cal.procedures ..Th�s.compound.causes.rap�d.hypnos�s.�n.pat�ents.and.may.be.used.repeatedly.w�thout.tolerance .1,2

Etom�date.�s.rap�dly.absorbed.follow�ng.�ntravenous.adm�n�strat�on .. Peak. plasma. concentrat�ons. typ�cally.occur.w�th�n.4.m�nutes.of.�ntroduct�on,.and.the.half-l�fe.has.been.reported.to.range.from.2.to.11.hours .3.Etom�-date. �s. extens�vely. metabol�zed. by. hydrolys�s. �n. both.the.plasma.and.�n.the.l�ver.to.an.�nact�ve.carboxyl�c.ac�d.metabol�te .1,3,4.The.chem�cal.structure.of.etom�date.can.be.seen.�n.F�gure.1 ..The.�nternal.standard,.SERTIS,.was.used.for.all.etom�date.analys�s ..SERTIS,.n-methyl-4-(4-bromophenyl)-1,2,3,4,-tetrahydro-1-napthylam�ne,.�s.a.gener�c.�nternal.standard.used.by.our.laboratory.when.no.deuterated.analog.�s.ava�lable ..SERTIS.�s.not.sold.for.human.consumpt�on,.so.there.�s.no.r�sk.of.th�s.compound.be�ng.present.�n.human.spec�mens ..

Frequently,.p�lots.�nvolved.�n.av�at�on.acc�dents.surv�ve.and.are.treated.surg�cally.�n.an.effort.to.save.the�r.l�ves ..However,.these.p�lots.do.not.always.surv�ve.surgery ..If.death.occurs.follow�ng.med�cal.treatment,.postmortem.spec�mens. are. sh�pped. to. our. laboratory. for. analys�s ..

These.spec�mens.are.subsequently.found.pos�t�ve.for.the.compounds.used.by.med�cal.personnel.dur�ng.l�fe-sav�ng.procedures ..Etom�date.�s.one.such.compound ..There.are.relat�vely.few.publ�shed.reports.descr�b�ng.the.analys�s.of.etom�date,.and.only.1.of.these.publ�cat�ons.descr�bes.the.analys�s.by.GC/MS .5-9.Furthermore,.sc�ent�fic.�nforma-t�on.concern�ng.the.d�str�but�on.of.etom�date.�n.human.spec�mens.�s.not.ava�lable ..Th�s.manuscr�pt.presents.the.quant�tat�on.and.d�str�but�on.of.etom�date.�n.postmor-tem.human.spec�mens,.�nclud�ng.ur�ne,.v�treous.humor,.skeletal.muscle,. l�ver,. k�dney,. lung,. spleen,. bra�n,. and.heart.muscle ..

MaTErIals aNd METhOds

Chemicals and ReagentsAll. aqueous. solut�ons. were. prepared. us�ng. double.

de�on�zed.water. (DDW),.wh�ch.was.obta�ned.us�ng.a.M�ll�-QT

plus. Ultra-Pure. Reagent. Water. System. (M�l-

l�pore®,.Cont�nental.Water.Systems,.El.Paso,.TX) ..All.chem�cals.descr�bed.below.were.purchased.�n.the.h�ghest.poss�ble.pur�ty.and.used.w�thout.any.further.pur�ficat�on ..Etom�date.was.purchased.from.Bedford.Labs.(Bedford.Laborator�es,.Bedford,.OH).and.rece�ved.as.a.2.mg/mL.standard.�n.35%.v:v.propylene.glycol ..The.�nternal.stan-dard,.SERTIS,.was.obta�ned.from.Pfizer.(Pfizer.Inc .,.New.York,.NY) ..Methanol,.aceton�tr�le,.ammon�um.hydrox�de,.acet�c.ac�d,.ethyl.acetate,.sod�um.fluor�de,.and.potass�um.phosphate.monobas�c.were.purchased.from.F�sher.Sc�en-t�fic.(F�sher.Sc�ent�fic.Co .,.P�ttsburgh,.PA) ..The.pH.of.all.

N

N

O

O

Br

H

N

Etomidate SERTISFigure 1. Chemical structures of etomidate and SERTIS.

2

solut�ons.was.measured.us�ng.a.Corn�ng.model.430.pH.meter.(Corn�ng.L�fe.Sc�ences,.Acton,.MA).connected.to.a.Corn�ng.3-�n-1.model.pH.electrode .

Gas Chromatographic/Mass Spectroscopic ConditionsAll. analyses.were.performed.us�ng. a.bench-top. gas.

chromatograph/mass. spectrometer. (GC/MS),. wh�ch.cons�sted.of.a.Hewlett.Packard.(HP).6890.ser�es.GC,.�nterfaced. w�th. a. HP. 5973. quadrupole. MS. (Ag�lent.Technolog�es.Inc .,.Santa.Clara,.CA) ..The.MS.was.tuned.on.a.da�ly.bas�s.us�ng.perfluorotr�butylam�ne ..The.electron.mult�pl�er.voltage.was.set.at.106.eV.above.the.tune.value ..Chromatograph�c.separat�on.was.ach�eved.us�ng.a.Var�an.FactorFour.crossl�nked.100%.methyl.s�loxane.cap�llary.column.12.m.x.0 .2.mm.� .d .,.0 .33.µm.film.th�ckness.(Var-�an.Co .,.Harbor.C�ty,.CA .) ..Hel�um.was.employed.as.the.carr�er.gas.and.used.at.a.flow.rate.of.1 .0.mL/m�n ..The.GC/MS.transfer.l�ne.and.source.temperatures.were.set.to.280°C.and.250°C,.respect�vely ..An.HP.6890.autosam-pler.was.used.to.�nject.1.µL.of.extract.�nto.the.GC/MS ..The. GC. was. equ�pped. w�th. a. spl�t/spl�tless. �nject�on.port.operated.at.250°C.�n.the.spl�tless.mode.w�th.the.purge.t�me.of.0 .5.m�n ..The.oven.temperature.profile.was.establ�shed.as.follows:.70°C.–.290°C.at.30°C/m�n.and.a.final.hold.t�me.of.2 .67.m�n,.result�ng.�n.a.total.run.t�me.of.10.m�n ..In�t�ally,.neat.standards.of.each.compound.(1.µL.of.a.100.ng/µL.solut�on).were.�njected.�nd�v�dually.and.analyzed.us�ng.the.full.scan.mode.of.the.GC/MS,.wh�ch.scanned.from.50.to.600.AMU ..The.full.scan.mass.spectra.from.these.2.compounds.can.be.seen.�n.F�gures.2.and.3 ..Quant�tat�on.and.qual�fier.�ons.for.each.analyte.were.then.selected.based.on.both.abundance.and.mass-to-charge.rat�o.(m/z) ..To.�ncrease.reproduc�b�l�ty.and.reduce.�nterference,.h�gh-mass.�ons.were.selected.when.poss�ble ..The.�ons.chosen.for.each.respect�ve.analyte.can.be.seen.�n.Table.1 ..Upon.select�on.of.un�que.�ons,.the.MS.was.run.�n.selected.�on.mon�tor�ng.(SIM).mode.w�th.a.dwell.t�me.of.30.msec.for.each.recorded.�on .

Sample Selection and StorageA.search.of.the.CAMI.database.�dent�fied.3.etom�date-

pos�t�ve.fatal�t�es.from.separate.c�v�l.av�at�on.acc�dents.from.the.prev�ous.3.years.that.had.a.major�ty.of.the.des�red.b�olog�cal.t�ssues.and.flu�ds.(blood,.ur�ne,.v�treous.humor,.l�ver,.k�dney,.muscle,.lung,.spleen,.heart,.and.bra�n).ava�l-able.for.analys�s ..In.all.cases,.blood.was.stored.at.-20°C.�n.tubes.conta�n�ng.1 .00%.(w/v).sod�um.fluor�de/potass�um.oxalate.unt�l.analys�s ..All.other.spec�mens.were.stored.at.-20°C,.w�th.no.added.preservat�ve,.unt�l.analys�s ..

Calibrator and Control PreparationA.cal�brat�on.curve.for.etom�date.was.prepared.by.se-

r�al.d�lut�on,.ut�l�z�ng.bov�ne.whole.blood.as.the.d�luent ..

Controls.were.also.prepared.us�ng.bov�ne.whole.blood.as.the.d�luent ..Cal�brators.were.prepared.from.1.stock.stan-dard.solut�on,.wh�le.controls.were.prepared.from.a.second.separate.stock.solut�on.w�th.a.d�fferent.manufacturer’s.lot.number ..A.cal�brat�on.curve.was.prepared.at.concentra-t�ons.rang�ng.from.0 .39.–.800.ng/mL ..A.m�n�mum.of.7.cal�brators.were.used.to.construct.the.cal�brat�on.curve ..Controls.were.prepared.at.concentrat�ons.of.80.and.320.ng/mL.and.extracted,.w�th.each.batch.of.unknowns.to.ver�fy.the.accuracy.of.the.cal�brat�on.curve ..The.�nternal.standard.solut�on.was.prepared.at.a.concentrat�on.of.400.ng/mL.�n.DDW.by.d�lut�on.from.the.stock.standard.of.th�s.compound .

Quant�tat�on.of.blood.spec�mens.was.ach�eved.v�a.an.�nternal.standard.cal�brat�on.procedure ..Response.rat�os.for.etom�date.were.determ�ned.for.every.blood.sample.analyzed ..The.response.rat�o.was.calculated.by.d�v�d�ng.the.area.of.the.analyte.peak.by.the.area.of.the.�nternal.standard.peak ..Cal�brat�on.curves.were.der�ved.by.plott�ng.a.l�near.regress�on.of.the.analyte/�nternal.standard.response.rat�o.versus.the.analyte.concentrat�on.for.each.respect�ve.cal�brator ..Standard.add�t�on.was.used.to.determ�ne.the.concentrat�on.of.etom�date.�n.every.spec�men.type.other.than.blood ..Th�s.was.necessary.due.to.the.poss�ble.ma-tr�x.effects.assoc�ated.w�th.the.extract�on.of.an.�nternal.standard.that.�s.not.a.deuterated.analog.of.the.compound.of.�nterest ..The.standard.add�t�on.procedure.used.�n.th�s.work.has.been.descr�bed.extens�vely.elsewhere .10.Br�efly,.a.spec�men.was.d�v�ded.�n.half,.1.half.was.sp�ked.w�th.a.prec�sely.known.amount.of.etom�date,.the.other.half.was.not.altered ..Internal.standard.was.added.to.each.tube.and.the.2.halves.were.analyzed ..After.analys�s,.the.rat�o.of.the.etom�date.concentrat�on.�n.the.unsp�ked.sample.compared.to.the.sp�ked.sample.allowed.us.to.der�ve.the.concentrat�on.�n.the.or�g�nal.spec�men .

Sample Preparation and Extraction ProcedurePostmortem.spec�mens,.cal�brators,.and.controls.were.

extracted.�n.the.follow�ng.manner ..T�ssue.spec�mens.were.homogen�zed.us�ng.an.Omn�.post-mounted.homogen�zer.(Omn�.Int .,.Mar�etta,.GA) ..The.generator.used.w�th.th�s.homogen�zer.had.a.d�ameter.of.30.mm.and.rotated.at.22,000.rpm ..T�ssues.were.homogen�zed.follow�ng.a.1:2.d�lut�on.w�th.1 .00%.NaF.�n.DDW ..Three.mL.al�quots.of.postmortem.flu�ds,.cal�brators,.controls,.and.3 .00.g.al�quots. of. each. t�ssue.homogenate. (1 .0. g. t�ssue). and.each.standard.add�t�on.sample.were.transferred.to.�nd�-v�dual.16.x.150.mm.screw-top.tubes ..To.each.spec�men,.cal�brator,.and.control,.1 .00.mL.of.the.�nternal.standard.(400.ng).was.added ..Samples.were.vortexed.br�efly.and.allowed.to.stand.at.room.temperature.for.10.m�n ..N�ne.mL.�ce-cold.aceton�tr�le.was.added.to.each.sample ..The.m�xture.was.then.placed.on.a.rotary.m�x�ng.wheel.and.

3

1

Table 1. Ions utilized for the quantitation of fluoxetine etomidate.

Compound Ions utilized for quantitation (m/z)*

Etomidate 105, 244, 199 SERTIS 286, 284, 314

* Ions in bold used for quantitation, other ions used as qualifiers.

40 60 80 100 120 140 160 180 200 220 240 2600

500000

1000000

1500000

2000000

2500000

3000000

3500000

m/z-->

Abundance 105

244

77

95

199

51 17211567 143128 15540 183 215 229 258

Figure 2. Full scan mass spectra of etomidate.

40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 0100000

300000

500000

700000

900000

1100000

1300000

1500000

1700000

m/z-->

Abundance 286

205 159 272

178 129 104

116 191 314 144 258 897742 63 235

Figure 3. Full scan mass spectra of SERTIS.

4

m�xed.at.15.rpm.for.15.m�n ..Centr�fugat�on.at.820×g.for.5.m�n.removed.cellular.debr�s.and.prote�ns ..Follow-�ng. centr�fugat�on,. the. supernatant. was. transferred. to.clean.16.x.125.mm.culture.tubes.and.evaporated.�n.a.TurboVap.Concentrat�on.Workstat�on.at.40°C.(Cal�per.L�fe.Sc�ences,.Hopk�nton,.MA).under.a.stream.of.dry.n�trogen.to.a.volume.of.approx�mately.1.mL ..Follow�ng.aceton�tr�le.evaporat�on,.4 .00.mL.0 .10.M.phosphate.buf-fer,.pH.6 .00,.was.added.to.each.sample ..The.extracts.were.transferred.to.Bond.Elute.Cert�fy®.sol�d-phase.extract�on.(SPE).columns.obta�ned.from.Var�an.(Var�an.Co .,.Harbor.C�ty,.CA .),.wh�ch.had.been.pre-cond�t�oned.w�th.2 .00.mL.methanol,.followed.by.2 .00.mL.0 .10.M.phosphate.buffer,.pH.6 .00 ..Care.was.taken.not.to.dry.the.column.pr�or. to.add�ng.the.extract ..Column.flow.rates.of.1-2.mL/m�n.were.ma�nta�ned.�n.each.SPE.step.us�ng.a.Var-�an.24.port.Cerex.SPE.processor.(Var�an.Co .,.Harbor.C�ty,.CA .).w�th.a.n�trogen.pressure.of.3.ps� ..Once.each.sample.had.passed. through. �ts. respect�ve. column,. the.columns.were.washed.w�th.1 .00.mL.of.1 .00.M.acet�c.ac�d.and.then.dr�ed.completely.w�th.25.ps�.n�trogen.for.5.m�n ..The.columns.were.aga�n.washed.w�th.6 .00.mL ..Follow�ng.the.methanol.wash,.the.columns.were.aga�n.dr�ed.completely.w�th.25.ps�.n�trogen.for.5.m�n ..The.analytes.were.eluted.off.the.columns.w�th.3 .00.mL.of.2%.ammon�um.hydrox�de.�n.ethyl.acetate,.wh�ch.was.prepared.fresh.da�ly ..Eluents.were.evaporated.to.dryness.�n.a.TurboVap.set.at.40°C.under.a.stream.of.dry.n�trogen ..Once.dry,.the.contents.of.each.tube.were.reconst�tuted.�n.50.µL.of.ethyl.acetate.and.transferred.to.GC/MS.v�als.for.analys�s .

Extraction EfficiencyThe.method.used. for. the.determ�nat�on.of. analyte.

recovery.has.prev�ously.been.reported .11.Br�efly.descr�bed,.2.groups.of.controls,.X.and.Y,.prepared.us�ng.negat�ve.whole.blood,.were.extracted.�n.the.same.manner.as.de-scr�bed.above ..Group.X.was.sp�ked.w�th.a.prec�sely.known.concentrat�on.of.etom�date.pr�or.to.SPE.extract�on,.wh�le.group.Y.was.sp�ked.w�th.the.same.prec�sely.known.con-centrat�on.of.etom�date.follow�ng.SPE.extract�on ..Upon.analys�s,.the.average.response.factor.obta�ned.from.group.X.was.d�v�ded.by.the.average.response.factor.obta�ned.from.group.Y.to.y�eld.the.percent.recovery.value.(100.*.(X/Y).=.%.recovery).for.etom�date .

rEsulTs aNd dIsCussION

Method ValidationThe.procedure.descr�bed.here�n,.wh�ch.ut�l�zes.SPE.

and.GC/MS.for.the.detect�on.of.etom�date,.�s.rap�d,.re-produc�ble,.and.sens�t�ve ..Analyte.peaks.were.completely.resolved,.and.each.prov�ded.quant�tat�on.�ons.w�th.un�que.

m/z,.so.no.�nterference.was.observed ..Retent�on.t�mes.for.etom�date.and.SERTIS.were.typ�cally.4 .75.and.6 .10.m�n,.respect�vely ..No.analyte.suffered.�nterference.from.endogenous/exogenous. matr�x. components .. Standard.add�t�on.was.used.for.spec�men.types.other.than.blood.wh�le.us�ng.a.blood.cal�brat�on.curve,.thereby.el�m�nat�ng.any.matr�x.effect.concerns .

The.full-scan.mass.spectra.of.etom�date.and.SERTIS.prov�ded.numerous.h�gh.mass.�ons;.these.spectra.can.be.seen.�n.F�gures.2-3 ..Quant�tat�on.and.qual�fier.�ons.for.each.compound.are.shown.�n.Table.1 ..Acceptab�l�ty.cr�ter�a.employed.for.analyte.�dent�ficat�on.and.quant�tat�on.were.as.follows:.1).�on.rat�os.for.a.g�ven.analyte,.measured.as.the.peak.area.of.a.qual�fier.�on.d�v�ded.by.the.peak.area.of. the.quant�tat�on. �on,.were. requ�red. to.be.w�th�n.±.20%.of.the.average.of.the.�on.rat�os.for.cal�brators.used.to.construct.the.cal�brat�on.curve;.2).each.�on.mon�tored.was.requ�red.to.have.a.m�n�mum.s�gnal-to-no�se.rat�o.(S/N).of.10;.and.3).the.analyte.was.requ�red.to.have.a.retent�on.t�me.w�th�n.±.2 .00%.of.the.average.retent�on.t�me. for. cal�brators. used. to. construct. the. cal�brat�on.curve ..Analytes.not.meet�ng.these.cr�ter�a.were.reported.as.e�ther.negat�ve.or.�nconclus�ve .

The.l�near.dynam�c.range.(LDR),.l�m�t.of.detect�on.(LOD),.and.l�m�t.of.quant�tat�on.(LOQ).for.etom�date.were.determ�ned.us�ng.whole.blood.as.the.matr�x ...The.LDR. was. determ�ned. to. be. 0 .78. –. 400. ng/mL ..The.correlat�on.coeffic�ent.for.the.cal�brat�on.curve.used.to.evaluate.the.LDR.was.0 .998.when.employ�ng.a.we�ght-�ng.factor.of.1/X ..The.�nject�on.of.an.ethyl.acetate.blank.follow�ng.the.400.ng/mL.cal�brator.showed.no.carryover.contam�nat�on ..Subsequently,.ethyl.acetate.blanks.were.ut�l�zed.between.each.postmortem.spec�men.throughout.the.sample.sequence.to.ver�fy.that.no.sample-to-sample.contam�nat�on.had.occurred ..The.LOD.was.defined.as.the.lowest.etom�date.concentrat�on.detectable.that.met.the.above-d�scussed.�dent�ficat�on.cr�ter�a ..The.LOQ.was.defined.as.the.lowest.etom�date.concentrat�on.detectable.that.not.only.met.all.�dent�ficat�on.cr�ter�a.d�scussed.above.but.also.had.an.exper�mentally.determ�ned.concentra-t�on.w�th�n.±.20%.of.�ts.prepared.value ...The.LOD.for.etom�date.was.determ�ned.to.be.0 .39.ng/mL,.wh�le.the.LOQ.was.determ�ned.to.be.0 .78.ng/mL .

The.extract�on.effic�ency.for.etom�date.was.determ�ned.at.two.d�fferent.concentrat�ons ..The.recovery.of.etom�-date.at.50.ng/mL.was.determ�ned.to.be.79%,.wh�le.the.recovery.at.400.ng/mL.was.91% ..Recovery.values.above.75%.are.except�onal.when.cons�der�ng.the.s�mpl�c�ty.of.the.extract�on.and.the.use.of.whole.blood.as.the.matr�x.for.these.exper�ments .

Intra-day.and.�nter-day.accuracy.and.prec�s�on.were.exam�ned. for. th�s. extract�on.procedure ..Accuracy.was.measured.as.the.relat�ve.error.between.the.exper�mentally.

5

determ�ned.and.prepared.concentrat�ons.of.a.whole-blood.control ..Prec�s�on.was.measured.as.the.relat�ve.standard.dev�at�on.(RSD).of.the.exper�mentally.determ�ned.con-centrat�ons.of.a.group.of.whole.blood.controls ..Pools.of.controls.were.created.at.80.ng/mL.and.320.ng/mL.�n.volumes.large.enough.to.be.used.for.the.ent�re.accuracy.and.prec�s�on.�nvest�gat�on ..These.controls.were.stored.at.4°C.for.the.durat�on.of.th�s.study ..For.the.�ntra-day.accuracy.and.prec�s�on.exper�ment,.a.cal�brat�on.curve.was. extracted,. along.w�th.5. repl�cates. of. each. control.concentrat�on ..As.shown.�n.Table.2,.etom�date.at.both.concentrat�ons.y�elded.relat�ve.errors.w�th�n.5%.of.the.target.concentrat�on ..Furthermore,.the.RSD.assoc�ated.w�th.th�s.exper�ment.was.found.to.be.2%.at.each.con-centrat�on ..These. results. demonstrate. the. except�onal.accuracy.and.prec�s�on.of.th�s.method .

Inter-day.accuracy.and.prec�s�on.were.evaluated.by.ex-tract�ng.5.repl�cates.of.each.of.the.2.control.concentrat�ons.on.Days.2,.4,.and.7 ..The.quant�tat�ve.values.determ�ned.on.each.of.these.days.were.der�ved.from.the.cal�brat�on.curves.or�g�nally.prepared.on.Day.1 ..The.results.obta�ned.after.storage.of.each.control.lot.at.4°C.for.2,.4,.and.7.days.can.be.seen.�n.Table.2 ..Etom�date.concentrat�ons.determ�ned.over.th�s.7-day.per�od.showed.no.s�gn�ficant.d�fference. from. those. obta�ned. on. Day. 1 ..The. RSDs.for.etom�date.were.less.than.5%.on.Days.2,.4,.and.7 ...Even.though.no.apparent.decrease.�n.concentrat�on.was.observed.over.the.7-day.storage.per�od.at.4°C,.as.a.good.laboratory.pract�ce,.we.recommend.prompt.tox�colog�cal.analys�s.once.a.postmortem.spec�men.has.been.thawed.and.prepar�ng.a.new.cal�brat�on.curve.at.the.beg�nn�ng.of.each.new.analys�s .

Postmortem Concentrations of EtomidateBlood.concentrat�ons.found.�n.these.3.cases.ranged.

from.12.to.41.ng/mL ..Therapeut�c.levels.of.etom�date.�n.plasma.range.from.220.to.410.ng/mL .3.The.publ�shed.plasma/whole.blood.rat�o.�s.0 .62 ...Therefore,.therapeut�c.blood.concentrat�ons.for.etom�date.�n.whole.blood.range.from.354.to.661.ng/mL ..Blood.concentrat�ons.�n.these.cases.were.all.found.to.be.below.therapeut�c.levels ..The.concentrat�on.of.etom�date.�n.each.postmortem.spec�-men.from.these.3.cases.can.be.seen.�n.Table.3 ..W�th.a.volume.of.d�str�but�on.of.2-7.L/kg,1.etom�date.was.ex-pected.to.be.found.at.h�gher.concentrat�ons.�n.the.t�ssue.spec�mens.analyzed ..As.can.be.seen.�n.Table.3,.th�s.was.the.case ..It.appears.that.etom�date.�s.rap�dly.d�str�buted.follow�ng. adm�n�strat�on .. It. has. also. been. found. that.th�s.compound.�s.extremely. l�p�d.soluble .12.Therefore,.bra�n.concentrat�ons.are.expected.to.be.relat�vely.h�gh ..The.follow�ng.mean.concentrat�ons.of.etom�date.were.found:.blood.25.ng/mL,.ur�ne.9.ng/mL,.v�treous.humor.3.ng/mL,.l�ver.128.ng/g,.lung.40.ng/g,.k�dney.150.ng/g,.spleen.119.ng/g,.muscle.61.ng/g,.bra�n.128.ng/g,.and.heart.119.ng/g ..The.�nd�v�dual.concentrat�ons.from.each.case.can.be.seen.�n.Table.3 ..The.d�str�but�on.coeffic�ents.for.etom�date,.expressed.as. spec�men/blood.rat�os,.are.summar�zed. �n. Table. 4 .. The. d�str�but�on. coeffic�ents.for.etom�date.were.determ�ned.to.be:.ur�ne.0 .4.±.0 .1,.v�treous.humor.0 .17.±.0 .08,.l�ver.4.±.1,.lung.1 .6.±.0 .5,.k�dney.5.±.1,.spleen.4.±.1,.muscle.3.±.1,.bra�n.5.±.1,.and.heart.6.±.2 ..As.can.be.seen.�n.Table.3,.etom�date.d�str�but�on.coeffic�ents.for.ur�ne,.b�le,.v�treous.humor,.muscle,.k�dney,.lung,.spleen,.bra�n,.l�ver,.and.heart.had.coeffic�ent.of.var�at�on.(CV).values.between.20.and.47% ..It.�s.w�dely.accepted.that.bas�c.drugs.w�th.large.volumes.of.d�str�but�on.can.undergo.postmortem.red�str�but�on ..Th�s.red�str�but�on.may.account.for.some.of.the.larger.CV.values.observed .

2

Table 2. Intra-day accuracy and precision for repeated determinations over 7 days.*

Etomidate

Day 1 Target Conc. 80 320

Mean SD 79 2 333 8 Relative Error -1% +4%

R.S.D. 2% 2%

Day 2

Target Conc. 80 320

Mean SD 73 2 315 7 Relative Error -9% -2%

R.S.D. 3% 2%

Day 4

Target Conc. 80 320

Mean SD 80 1 334 4 Relative Error 0% +4%

R.S.D. 1% 1%

Day 7

Target Conc. 80 320

Mean SD 82 2 335 7 Relative Error +3% +5%

R.S.D. 2% 2%

*n=5 at each concentration for each day; controls were run on days 1, 2, 4, and 7.

6

There.are.no.w�dely.accepted.cr�ter�a.for.what.con-st�tutes.a.rel�able.d�str�but�on.coeffic�ent;.however,.w�th.certa�n.drugs.�t.may.be.poss�ble,.w�th.caut�on,.to.use.a.t�ssue.or.flu�d.d�str�but�on.coeffic�ent.to.crudely.est�mate.a.blood.concentrat�on.�n.cases.where.blood.�s.not.ava�l-able,.�f.the.d�str�but�on.coeffic�ent.has.a.CV.of.<.25% ..Our. laboratory. rece�ves. blood. �n. only. approx�mately.70%.of.the.cases.that.we.analyze ..Therefore,.the.results.obta�ned.from.the.l�m�ted.number.of.cases.(n=3).�n.th�s.study.suggest.that.etom�date.concentrat�ons.found.�n.l�ver,.k�dney,.bra�n,.and.spleen.could.be.used.w�th.caut�on.to.est�mate.blood.etom�date.concentrat�ons.that.are.sl�ghtly.below.therapeut�c.levels ..We.acknowledge.that.a.study.�nvolv�ng.a.greater.number.of.samples.from.a.larger.pool.of.cases.needs.to.be.completed.to.more.defin�t�vely.ver�fy.these.results ..However,.based.on.these.find�ngs.one.could.caut�ously.est�mate.a.range.for.postmortem.etom�date.concentrat�ons.�n.blood .

Table 3. Etomidate concentrations obtained from 3 pilot fatalities.*

Case Blood Urine VH Liver Lung Kidney Spleen Muscle Brain Heart

1 12 6 3 29 12 48 32 33 49 83

2 21 — 2 92 46 152 79 87 132 167

3 41 11 — 262 63 251 246 63 202 108

* All concentrations shown in units of ng/mL or ng/g—Specimen type not available for analysis

Table 4. Postmortem etomidate-positive specimen distribution coefficients.

Urine/

Blood

VH/

Blood

Liver/

Blood

Lung/

Blood

Kidney/

Blood

Spleen/

Blood

Muscle/

Blood

Brain/

Blood

Heart/

Blood

n 2 2 3 3 3 3 3 3 3

Mean 0.4 0.17 4 1.6 5 4 3 5 6

s.d. 0.1 0.08 1 0.5 1 1 1 1 2

CV 25 47 25 31 20 25 33 20 33

CONClusION

In.th�s.study,.our.laboratory.developed.and.val�dated.a.novel.method.for.the.extract�on.and.analys�s.of.etom�date.�n.human.b�olog�cal.spec�mens ..The.results.obta�ned.from.these.cases.suggest.that.etom�date.�s.read�ly.absorbed.by.all. t�ssues.and.flu�ds. �n. the.body ..Although.etom�date.d�str�but�on.var�ed.among.�nd�v�duals,.there.were.several.spec�men.types.that.showed.relat�vely.cons�stent.d�str�bu-t�on.coeffic�ents ..L�ver,.k�dney,.bra�n,.and.spleen.each.had.a.CV.of.less.than.25%.(n=3) ..The.relat�vely.small.CV.assoc�ated.w�th. these.d�str�but�on. coeffic�ents. suggests.that.these.spec�mens.may.be.used.w�th.extreme.caut�on.to.obta�n.an.approx�mate.blood.concentrat�on.for.etom�date.when.blood.�s.not.ava�lable.for.analys�s .

7

rEfErENCEs

1 .. G�ese,. J .L ..and.Stanley,.T .H ..Etom�date:.A.New.Intravenous.Anesthet�c.Induct�on.Agent ..Pharma-cotherapy,.3:.251-8.(1983) .

2 .. Doen�cke,.A ..Etomidate, a New Hypnotic Agent for Intravenous Application.,. 1. ed .. (Spr�nger-Verlag,.Berl�n,.1977) .

3 .. Moffat,. A .C .,. Osselton,. M .D .,. and.W�ddop,. B ..eds ..Clarke’s Analysis of Drugs and Poisons in Phar-maceuticals, Body Fluids, and Postmortem Materials,.3.ed ..(Pharmaceut�cal.Press,.London,.UK,.2004) .

4 .. Van. Hamme,. M .J .,. Ghone�m,. M .M .,. and. Am-bre,. J .J .. Pharmacok�net�cs. of. Etom�date,. a. New.Intravenous.Anesthet�c ..Anesthesiology,.49:.274-7.(1978) .

5 .. de.Boer,.A .G .,.Smeekens,.J .B .,.and.Bre�mer,.D .D ..Assay. of. Etom�date. �n. Plasma. by. Cap�llary. Gas.Chromatography.w�th.N�trogen-Select�ve.Detec-t�on ..J Chromatogr,.162:.591-5.(1979) .

6 .. Deng,.X ..and.S�mpson,.V .J ..Gas.Chromatograph�c--Mass.Spectrometr�c.Determ�nat�on.of.Etom�date.�n.Mouse.Bra�n ..J Pharmacol Toxicol Methods,.43:.73-7.(2000) .

7 .. Ell�s,.E .O ..and.Beck,.P .R ..Determ�nat�on.of.Etom�-date.�n.Human.Plasma.by.H�gh-Performance.L�q-u�d.Chromatography ..J Chromatogr,.232:.207-11.(1982) .

8 .. Har�ng,.C .M .,.D�jkhu�s,.I .C .,.and.van.D�jk,.B ..A ..Rap�d. Method. of. Determ�n�ng. Serum. Levels. of.Etom�date.by.Gas.Chromatography.W�th.the.A�d.of.a.N�trogen.Detector ..Acta Anaesthesiol Belg,.31:.107-12.(1980) .

9 .. Le. Mo�ng,. J .P .. and. Levron,. J .C .. H�gh-Perfor-mance.L�qu�d.Chromatograph�c.Determ�nat�on.of.Etom�date.�n.Plasma ..J Chromatogr,.529:.217-22.(1990) .

10 .. S�ek,.T .J .. and.Dunn,.W .A ..Documentat�on.of. a.Doxylam�ne. Overdose. Death:. Quant�tat�on. by.Standard.Add�t�on.and.Use.of.Three.Instrumental.Techn�ques ..J Forensic Sci,.38:.713-20.(1993) .

11 .. Johnson,.R .D .,.Lew�s,.R .J .,.Canf�eld,.D .V .,.et.al ..Accurate.Ass�gnment.of.Ethanol.Or�g�n.�n.Postmor-tem.Ur�ne:.L�qu�d.Chromatograph�c-Mass.Spectro-metr�c.Determ�nat�on.of.Seroton�n.Metabol�tes ..J Chromatogr B Analyt Technol Biomed Life Sci,.805:.223-34.(2004) .

12 .. Heykants,.J .J .,.Meuldermans,.W .E .,.M�ch�els,.L .J .,.et.al ..D�str�but�on,.Metabol�sm.and.Excret�on.of.Etom�date,.a.Short-Act�ng.Hypnot�c.Drug,.�n.the.Rat ..Comparat�ve.Study.of.(R)-(+)-(--)-Etom�date ..Arch Int Pharmacodyn Ther,.216:.113-29.(1975) .