(MDMA) and Its Metabolites in Plasma and Urine Byhplc-Dadandgc-MS

Journalof AnalyticalToxicology,Vol.20,October1996

Analysisof3,4-Methylenedioxymethamphetamine (MDMA) and its Metabolites in Plasma and Urine byHPLC-DADandGC-MS Hans-J6rgHelmlin, Katrin Bracher,Daniel Bourquin, David Vonlanthen, and Rudolf Brenneisen* Institute of Pharmacy, University of Bern, Baltzerstr.5, CH-3012 Bern, Switzerland Juraj Styk Psychiatrist, Birmannsgasse39, CH-4055 Basel, Switzerland

Abstract I stance. It showsdistinct psychotropic effects,which are dif- ferentfrom those ofthe structurally relatedstimulant amphet-

InEurope, the compound 3,4-methylenedioxymethamphetamine amine or hallucinogenicphenylalkylamines(e.g.,3,4-methyl- (MDMA, Ecstasy, Adam), in addition to cannabis, is the most enedioxyamphetamine[MDA]and 3,4,5-trimethoxyphenethyl- abused illicit drug at all-night "techno" parties. Methods for the amine [mescaline]) (2-6). MDMA is reported to possess determinationof MDMAandits metabolites, 4-hydroxy-3- antidepressant and anxiolyticproperties and to evokea con- methoxymethamphetamine (HMMA), 3,4-dihydroxy- trollable emotional experiencewith relaxation, peacefulfeel- methamphetamine (HHMA), 3,4-methylenedioxyamphetamine trigs,increasedempathy,and a drop in fear responsesthatare (MDA),4-hydroxy-3-methoxyamphetamine (HMA),and mostlywithout distortion of sensor3,perception and thought 3,4-dihydroxyamphetamine (HHA)in, biological fluidswere and without marked stimulation (5-10). Anovelpsychoactive established. Plasma and urine samples were collected from two substance class, which is named the "entactogens" (greco- patients in a controlled clinical study over periods of 9 and 22 h, latinic term: "produce (gert) an inner (en) touching (tact)") was respectively. MDMA and MDA were determined in plasma and recently proposed for MDMA(3,5-7.11). The mechanismof urine by reversed-phase high-performance liquid chromatography withdiodearraydetection(HPLC-DAD)aftersolid-phase action of MDMAis characterized by a high affinity at sero- extractionon cation-exchange columns.Acidicor enzymatic tonin uptake sites, whereas at norepinephrine and dopamine hydrolysis was necessary to detect HMMA, HMA, HHMA, and uptake sites of central neurons the affinity is lower (12). HHA,whichare mainly excretedas glucuronides. Gas Reviewsof the pharmacologyof MDMAhave appearedin the chromatography-mass spectrometry (GC-MS) was used for literature (13,14). confirmationSa. mpleextractionandon-discderivatizationwith The acute and chronic toxicity ofMDMAis controversial.In heptafluorobutyric anhydride (HFBAwere) performedonToxi-Lab animal experiments MDMAwas shown to exert dose- and SPEC solid-phase extraction concentrators. After administration of species-dependentneurotoxic effects on central serotonergic a single oral dose of 1.5 rog/kg body weight MDMA, peak plasma neurons in terms of degenerationofaxon terminals(12,15-22). levels of 331 ng/mL MDMA and 15 ng/mL MDA were measured However,the relevanceof these findings for humans is still after 2 h and 6.3 h, respectively. Peak concentrations of unclear (23-25). There are no reports of individualsor drug- 28.1 pg/mL MDMA in urine appeared after 21.5 h. Up to addictswhotake frequent or largedosesof MDi%&for extended 2.3 pg/ml MDA, 35.! pg/ml. HMMAa,nd2.1 pg/mL HMA were periods of time. The reason for this is that the positiveeffects measuredwithin16-21.5h. Conjugated HMMAandHHMAare of the drug seem to diminish and the negativeeffectsseemto the mainurinary metabolitesof MDMA. increasewithtime (5,8).Well-documenteddeaths relatedtothe use of MDMAare exceptionally rare and are mostly the result of underlying cardiac diseases, cardiovascular complications. Introduction and hyperthermia (26-28). A recent postmortem study on seven men (age range 20-25) who died after the consumption of MDMA("Ecstasy")or 3,4-methylenedioxyethylamphetamine ! The recreational use of 3,4-methylenedioxymethylamphet- (MDEA,"Eve")or bothshowedsignificantchangesinthe liver, amine (MDMA;Figure 1)dramaticallyincreasedin the United heart, or brain (1).Asthe possiblemechanism of damage,the , States and Europe by the mid to late 1980s. In Britain, more authors suggested hyperthermia, direct toxic effects on or- than 500,000 people are said to use the drug each week (1). gans, individual susceptibility to ring substituted am-

On the other hand, there is also an ongoing discussion about *Author to wh ...... pond ..... hould be add ..... d the therapeutic potency ofMDMA.For example,MDMAisused

_i_ Since432 1986,MDMAhas beenReproductionan internationally(photocopying) controlledof editorial contentsub-of this journalphetamines,is prohibited withoutabnormalpub}ishermetabolism,'s permission and water intoxication. ' i.

JournalofAnalytical Toxicology, Vol. 20, October 1996

in medicineasan adjunct in psychotherapy(5,7,9,29,30), 3,4-methylenedioxymethamphetamineand 6-hydroxy-3,4- where it can facilitate the therapeutic communication and methylenedioxyamphetamine have been found in rat plasma increasepatient insight and self-esteem(26), and as an and brain (39),whereas2,4,5-trihydroxymethamphetamine analgesicfor patients with terminal cancer (31). and 2,4,5-trihydroxyamphetaminehave been identifiedin rat High-performance liquid chromatography (HPLC) with elec- liver (40). trochemical (32,33), LTV(34), or diode array detection (DAD) Veryfew MDMAstudies have been performed in man. In aj (35)and gas chromatography with nitrogen-phosphorus de- postmortem urine specimen (with unknown dosage),HMMAv' tection (GC-NPD) (36) or mass spectrometry (GC-MS, was detected as the main urinary metabolite of MDMA.Other GC-MS-MS,GC-MS-MS-MS)(32,37-44) havebeen used for metabolites found were MDA_-'HMA3,,4-dihydroxymetham- thedetection ofMDMAin biologicalspecimens, phetamine (HHMA)3-hydroxy-4-methoxymethamphetamine, , Severalanimal studies (rat and mouse) have shown that and 3,4-methylenedioxyphenylacetone(38).In a singlepatient, MDMAis metabolized by N-demethylation. O-dealkylation, controlled study, unchanged MDMAwas the major urinary deaminationO, -methylation,and O-conjugationto glucuronides excretionproduct, and MDAwas the onlymetaboliteidentified and/orsulfates.Metabolitesdetectedinurine are 3,4-methylene- after the ingestion of50 mg MD.MA.Ofthe dosagegiven,72% / dioxyamphetamine(Figure 1, MDA),4-hydroxy-3-methoxy- was recoveredfrom the urine within 72 h. The MDMAplasma methamphetamine (HMMA),3-hydroxy-4-methoxymeth- levelpeakedat about 106ng/mL 2 h after administration and amphetamine, 4-hydroxy-3-methoxyamphetamine (HMA), declinedto about 5 ng/mL by24 h (43). 3-hydroxy-4-methoxyamphetamine, 4-hydroxy-3-methoxy- It was the aim of the present study to establish the analytical phenylacetone, 3,4-rnethylenedioxyphenylacetone,and 3,4- methodology for monitoring MDMAand its metabolites in dihydroxyphenylacetone (32,36.37,41,42). Most of these metabo- body fluids and to investigate the pharmacokinetic behavior of lites are also present in the blood. In addition, 6-hydroxy- MDMAin man under controlled conditions.

H I

CH3 3,4- methylenedioxy- 3,4-methylenedioxyamphetamine methamphetamine MDA MDMA

1.I

c sulphate conjugates I- CH z I H_ I 4-hydroxy-3-methoxy- t-,-- I 4-hydroxy-3-methoxyamphetamine methamphetamine HMA HMMA

1.I

CH3 CH3 ,-d_ ydroxy- ' ' 3,4-dihydroxyamphetamine methamphetamine HHA HHMA

Figure 1. Metabolism of 3,4-methylenedioxymethamphetamine (MDMA). ,_ h/"_.,_

433 Journal of Analytical Toxicology, Vol. 20, October 1996

Experimental Preparationof plasmaextractsforHPLC.The solid-phase extraction (SPE) and cleanup of plasma samples were per- Standardsand chemicals formedon an automated SPEsystem (ASPEC,GilsonMedical (_+)-3,4-Methylenedioxymethamphetaminehydrochloride Electronics France S.A., Synmedic, Zurich, Switzerland). (MDMA)was purchased from Eprova (Schaffhausen,Switzer- Frozen plasma samples were warmed up to room tempera- ' land); (+)-3,4-methylenedioxyamphetamine hydrochloride ture in an ultrasonic bath and centrifuged, ifnecessary.Toan (MDA)and (+)-methamphetamine hydrochloride (MA)were aliquot of 1.2 mL plasma 100 pL of the internal standard i obtained from Sigma (Buchs,Switzerland).(±)-4-Hydroxy-3- solution (800 ng/mL MAin water) and 0.6 mL 75 mMH3P04 methoxymethamphetamine (HMMA)was synthesized by (85%)were added,sonicated for 1 min in a stoppered2.5-mL ii reaction of methylamine hydrochloride, sodium cyanoboro- vial, and centrifuged for 10min at 2000 rpm. The supernatant hydride, and 4-hydroxy-3-methoxyphenylacetonewhich, was (1.5 mL) was then applied to the AdsorbexSCX (100rng) prepared by the oxidation of 4-hydroxy-3-methoxyphenyl-2- cation-exchange SPE column (Merck,Zurich, Switzerland), nitropropene (45). (+)-4-Hydroxy-3-methoxyamphetamine which was preconditioned with 2 mL methanol, 1 mLwater, (HMA) was synthesized by the reduction of 4-hydroxy-3- and 2 mL 25mM KH2P04.After drying the column with 6 mL methoxyphenyl-2-nitropropene,which was prepared by reac- of air, plasma interferences were removed by washingwith tion of 4-hydroxy-3-methoxybenzaldehyde with nitroethane 1.5 mL of 25mM KH2P04 and 1 mL of methanol, followedby (45). (+)-3,4-Dihydroxymethamphetamine (HHMA) was syn- 1.6 mL of air. The elution was done twice with 1.25 mL of thesized by demethylation of 3,4-dimethoxymethamphetamine methanol-HCl 7.3% (97.5:2.5), followed by 1 mL of air. After with borotribromide (46). 3,4-Dimethoxymethamphetamine additionof90gL of 1MK2HPO4to a 2-mLaliquotofthe eluate, waspreparedbyreacting3,4-dimethoxyphenylacetonemethyl-, the solutionwas concentrated to about 100pL undera stream amine hydrochloride, and sodium cyanoborohydride (45). of nitrogen and reconstituted to exactly 200 pL with water. (_+)-3,4-Dihydroxyamphetamine(HHA)was synthesized by Afterfiltration through the tip of a Pasteur pipette filledwith reactionof3,4-dibenzoylbenzaldehydewith nitroethaneto yield cotton wool,25]aLofthe filtrated solutionwas usedfor HPLC. 3,4-dibenzoylphenyl-2-nitropropenefollowed, by reduction to Preparationof urineextractsforHPLC.SPEand cleanupof 3,4-dibenzoylamphetamineand debenzylationto HHA(47).All urine samples were carried out according to the procedure synthesized standards were prepared as hydrochlorides, and describedearlier (35),but modifiedfor the use on the ASPEC the structures were confirmed by MS, infrared spectroscopy system. Frozenurine sampleswere warmed up to roomtern- (IR), proton (IH-), and carbon nuclear magnetic resonance perature in an ultrasonic bath and centrifuged ifnecessary.To spectroscopy(]3C-NMR).15-Glucuronidase/sulfatasetype H-1 an aliquot of 1.4 mL urine 14 pL of the internal standard was purchased from Sigma.Allother chemicalsand reagents solution(1.24mg/mLMAin water)and 0.6 mL75mMKH2P04 were of HPLC or analytical grade and were purchased from were added. The pH was adjusted to 5 with 200 mM H3P04 Merck(Zurich, Switzerland)or Fluka(Buchs,Switzerland). when necessaryand sonicatedfor 1min in a stoppered2.5-mL vial. An aliquot (1.5 mL) of this sample was then applied tothe Sample collection and preparation AdsorbexSCX(100mg) cation-exchangeSPE column,which Plasma and urine samples. The plasma and urine speci- waspreconditionedwith2 mLmethanol, 1mLwater,and1mL mens were obtained from a female (40-year-old subject A)and 25 mM KH2PO4.After drying the column with 6 mL of air, a male patient (23-year-old subject B)treated with MDMAbya urine interferences were removed by washing with 1.5 mLof psychiatrist (J.S.,one of the authors) from The SwissAssocia- 25 mM KH2P04and 1 mL of methanol, followedby 1.6mLof tion for Psycholytic Therapy (SAPT) under theauthorizationofthe SwissFederalOffice ofPublicHealth.Bloodsamples(10-20mL 2 3 each) were obtained through an indwelling catheter at 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 170,190,220, 250, 310, 380, 450,and 530min after the oral administra- _ tion of 1.5 mg MDMA(calculated as base) E per kg body-weight. The heparinized blood sampleswerecentrifugedfor 10rain at 2000rpm;theplasmawastransferredto ,_ _ polypropylenetubes and storedat -20°C. UrinesamplesfromsubjectAwerecollected .... 0, 1.5,3.5,5.5,7.5, 10,22, and 23.75 h after 0 2 4 6 8 10 the administration of MDMA,and urine Time(min)

samplesfrom subject Bwere collected0, 1, Figure 2. HPLC profile of a plasma extract; subject B, 120 min after an oral dose of 1.5 rog/kg 3,4-

16,and 21.5h after theadministrationof ng/mL);2, methamphetamine(MA,internalstandard);3, 3,4-methylenedioxymethamphetamine MDMA.Thesampleswerestoredat -20°C. (MDMA,331.3ng/mL).

I ,_ 1.25434 ,1.5,3.75,4.2,5,5.5,6.4,8.4,10.5,12.5, methylenedioxymethamphetamine(MDMA).Peaks:1,3,4-methylenedioxyamphetamine(MDA,3.9 1996 JournaloiAnalyticalToxicology,Vol.20,October1996 base air.Theelutionwas donetwicewith 1 mLmethanol-HCl 7.3% Acidichydrolysis.HCl(O.4mL;37%)wasaddedto2mLof per- (97.5:2.5)followed, by 1mL of air.Afterthe additionof 68 pL urine with 20 pL internal standard solution, and the mixture tical 1MK2HPO4to a 1.5-mLaliquotof the eluate, the solution was was incubatedfor 15min at 120°Cand l0s Pa pressure. After :nd). concentratedto about 100pLunder a stream ofnitrogen and cooling to room temperature, the mixture was centrifuged era- reconstituted to exactly 150 pL with water. After filtration (2000rpm for 5 min), and 1.5mLof the supernatant solution b an throughthe tip of a Pasteur pipette filledwith cotton wool, was appliedto the BakerbondC-18(300mg)SPEcolumn (P.H. ,lard 3tJLof the filtrated solution was used for HPLC. Stehelin &Co., Basel,Switzerland). The column was precon- J)04 Enzymatichydrolysis.For the enzymatic hydrolysisof the ditionedwith 3 mL methanol and 6 mL water.Urine interfer- -mL conjugates0.6, mL75mMKH2PO 4, containing10000units/mL ences and the excessivehydrochloric acid were removed by _tant urineof 13-glucuronidase/sulfatase(fromHelixpomatia,type washing with 6 mLwater.Afterdrying the column with 6 mL rog) H-1)wasaddedto 1.4mLofurine with 141_Linternal standard of air, the elution was done with 1.5 mL of methanol- nd), solution.The mixture was then incubated for 16 h at 37°C. 75mM KH2PO4(95:5) followedby 5 mL of air. The methanol _ter, Aftercentrifugation, 1.5 mL of the supernatant solution was was evaporated to 50 pL under a stream of nitrogen and mL extractedas described previously,and a 3-pL aliquot of the reconstituted to 1.75mL with 50 mM KH2PO 4. Analiquot of vith filtratedextractwasusedfor HPLC. 1.5mLwasextractedon the AdsorbexSCX(100mg) cation- .tby exchangeSPEcolumnasdescribedprevi- Lof ,0o A ously. After the addition of 68 pL of 1M tfter K2HPO4,theeluatewasconcentratedto _ate, 200 pL, and a 3-pL aliquot of the filtrated eam extractwasusedforHPLC. PreparationofurineextractsforGC-MS. _ith_ter' <_Eso s Extractionandcleanupofurinespecimens p of On-DiscDerivatizationTM (ODD)with hep-

'lure?ECLC. o _ _ 4 _ L andtafluorobutyricbyusingthe preparationTOXI-LABanhydride®ofSPECHFBAwereVCderivativesMP1performed(15mg)by em- ' microcolumns (Toxi-Lab,Irvine, CA;Spec- ,.To '°°1 tronex AG, Basel, Switzerland) and fol- lard B lowingtheinstructionsofthemanufacturer

.,PO4 methamphetamine.Afterderivatization, 200 pL water was added to the hexane- mLPO4 _ s fortheextractionofamphetamineand rich 3 water phase was discarded,and 200 pL of mE 4% NH4OH was added. The mixture was air, 4 shaken again.A 1-pLaliquot of the hexane Lof _ j k_ layerwasusedforGC-MSanalysis.The Lof o enzymaticandacidichydrolysisweredone asdescribedforHPLCanalysis. O HPLCanalysis The HPLC system consisted of a Hewlett- J Packard(HP)(Waldbronn,Germany)

1040M DAD and an HP HPLC3DChem- station (software version A.02.00). The 4 separationofplasmaand urine samples __,4__3_ ) _ was performed isocratically at 40°C on a 150090x 4.6-mmuahomtointernal diameter column rph th _ _ _ _ _b , with a 20 x 4.0-mm internal diameter precolumn packed with 3-pm Spherisorb Time(rain) ODS-1.The mobilephasewasacetoni- Figure3. HPLCprofilesof an urine extract before(A)and afterenzymatic (B)or acidic (C)hydrolysis; trile-water (96:904,v/v)containing 5.0 mL subject B,21.5 h after an oral dose of 1.5 rog/kg3,4-methylenedioxymethamphetamine.Peak iden- (8.5 g) orthophosphoric acid (85%) and tification:1,3,4-dihydroxymethamphetamine{HHMA);2, 4-hydroxy-3-methoxyamphetamine(HMA, 0.28 mL (0.22g) hexylamineper 1000mL. 2.1pg/mL,afteracidichydrolysis);3,4-hydroxy-3-methoxymethamphetamine(HMMA,31.3pg/mL); Theflowratewas1 mL/min.Thequantita- 4,3,4-methylenedioxyamphetamine(MDA)(1.6pg/mL);5, methamphetamine(MA,internalstandard); tion of MDMA,MDA,HMMA, and HMA 6,3,4-methylenedioxymethamphetamine(MDMA,18.1 pg/m[), before and after enzymatic or acidic

435 r ...... ,,,_,-,,i...... _',,_, ilirl...... mi il"_...... i _ ...... _ ...... 11',4'i'[{]11

Journal of Analytical loxicology, Vol. 20, October 1996

hydrolysiswas performed at 200 nm by measuring the peak 280°C(held 14min)at 15°C/min.Heliumwasused asthe_. areas and using the internal standard method, rier gas at a flowrate of 0.7 mL/min. The MSwas operatedin the scan and the SIMmode. The scan range was m/z33-780, GC-MS analysis and the scan-rate was set at 0.57 scans/sec.The characteristic The GC-MSsystem consisted of a HP 5990 GCwith a HP ions were m/z 389, 254, 210, 162, 135 (MDMA-HFBA);375, 5970 mass selective detector (MSD).A 1-pL aliquot of the 240, 162, 135 (MDA-HFBA);587, 360, 254, 210 (HMMA-di. derivatized urine extracts was separated on a J&WScientific HFBA);573, 360, 240, 163 (HMA-di-HFBA);769, 542,515, (HSP Friedlid Co., K6niz, Switzerland) DB-5bonded-phase 254,210 (HHMA-tri-HFBA);and 755,542,515,240,210(HHA. capillary column (20 m x 0.18-mm i.d.; 0.40-1_mfilm thick- tri-HFBA). ness) directly inserted into the ion source. The injector and transfer line temperatures were 260 and 280°C,respectively. The oven temperature was programmed from 70°C (held 1 min) to 200°Cat 10°C/minand from 200°C (held 1min) to Results and Discussion

HPLCanalysis A The same SPE procedure, which canbe automated for serial analysis,hasbeenused 3.sE+s forthepreparationofplasmaandurine samples for HPLC analysis. As shown b)' Figures 2 and 3, most of the endogenous '_ 2.ot+s matrix is eliminated bythe highlyselective : cation-exchangecolumn, whereasabout98 < and99%ofMDMAisrecoveredfromplasma

__ 3 andMDMAurinewas, respectively.determined usingThe recoverytwo plasmaof 5.oE+4__ . _ ___AA. __ L_J[ samples spiked with 49 and 493 ng/mL MDMA and two urine samples spiked with B 1800and 8800 ng/mL MDMA.The extrac- 2.oE+s tion efficiencyfor the MDMAmetabolites (MDA,HMMAHMA), was determinedtobe 7 goodto excellent.Urinesampleswere [ spiked with 400 ng and 800 ng/mLMDA, I.OE+S [ 725 and 7250 ng/mL HMMAand, 800and 4000ng/mLHMA.The recoverieswere100,

z.o£+4 3 respectively.Methamphetamine(MA)was _J._ _ _ _ , , chosen90, andas68%the forinternalMDAstandard,HMMAforand, plasmaHMA, L and urine. Amphetamine was too polar and (3 interfered with the biological matrix. For the determination of the conjugated 4.5E+4 mono- and dihydroxylated MDMAmeta_ lites (HMMAHMA, HHMA, ,etc.)enzymatic

,.sE+4 SPE.Thehydrolysiswithhydrochloricacid was faster (completed within 1 h), more

< Ii 3s thanefficientwith(see[3-glucuronidaseFigure3)and/sulfatase.lessexpensive 5.oE+3 The chromatographic systemdescribed _,,J_ __ l oracidichydrolysiswasnecessarypriorto here was originally developedforthe _d.o'' ' i_'.o'' ' i22o'' ' i3'.o'' ' i4'.o'' ' is_o'' ' i6'.o'' i7'.o ' analysis of methadone in pharmaceutical ]Sine(rain) preparations (48).Subsequently,itwasrou- tinely used in our laboratory for the anal)_is Figure4. GC-MSfullscanprofileofa derivatizedurineextractbefore(A)andafterenzymatic(B)and of psychotropic substances in biological acidic(C)hydrolysis;subjectB,21.5hafteranoraldoseof1.5rog/kg3,4-methylenedioxymetham-matrix (e.g., determination of cathinone phetamine(MDMA).Peakidentification:1,methamphetamine(MAinternal, standard);2,3,4-dihy- and its metabolitesinplasma[49]andurine

amphetamine(HMA);5, 3,4-dihydroxymethamphetamine(HHMA)6,; 4-hydroxy-3- [35]).TheselectivityofthisuniversalHPLC methoxymethamphetamine(HMMA);7,3,4-methylenedioxymethamphetamine(MDMA). methodcan easilybe optimizedfor basic

,_ 436droxyamphetamine(HHA); 3, 3,4-methylenedioxyamphetamine(MDA); 4, 4-hydroxy-3-methoxy- [50] aswell as for MDMA and MDA in urine Journalof AnalyticalToxicologyVol., 20,October1996

measured after 380 and 150 min, respectively. Significant The HMMApeak concentration may even exceedthat ofthe amounts of MDMAwere found 8 h after administration, parent compound MDMA.The metabolic scheme ofMDMAis Figure 6 represents the urine excretion profiles of subjects summarized in Figure 1.Our results confirm the findingsofan Aand B afteradministration of 1.5mg/kg-bodyweightMDMA. uncontrolled study wherein one urine specimenfromafatally MDMAconcentrations ranged from 13.09 to 28.14 pg/mL in injured motorcyclistwasanalyzedbyGC-MS(38).Althoughno the urine ofsubject Aand from 0 to 18.12pg/mL in the urine quantitationwasperformed,it wassuggestedthat HMMAisthe ofsubjectBwith peakvaluesafter5 and 21.5h, respectively·Up major metabolite and HHMA,HMA,and MDAare formedas to24.58gg/mL HMMA(subjectA)and 35·08pg/mL(subjectB) further metabolites. could be detected between 1.5and 16 h. The MDAurine con- The present study has been followed by a trial with six centrations varied from 0.11 to 2.30 pg/mL (subject A) and patients and slight modificationsof the protocol (longercol- from 0 to 1.58 pg/mL (subject B) with maximum levels lection periods)·The full pharmacokineticand statisticaldata occurring after 5 and 21.5 h. Concentrations of0-1.20 pg/mL evaluationis currently in progress. of HMA, the other minor MDMAmetabolite, were measured for subject Aand 0-2.05 pg/mLwere measured for subject B.The highestconcentrationsappearedbetween5.5and 21.5 h. When comparing the urine profiles before and after hydrolysis Conclusion 1 (Figure 3), it is obvious that the metabolites with an open methylenedioxy ring (HMMA,HMA,HHMA)are excreted HPLCin combination with automated SPE is the method 1 mainly as conjugates (glucuronides and/or sulfates). These of choice for the pharmacokinetic profiling of MDMAand urine data reflect the interindividual differences in the excre- its conjugated and unconjugated metabolites in plasmaand tion pattern and that HMMA(as conjugate), and not MDAas urine. GC-MSwith SPEC-ODDsamplepreparation is recom- found earlier (43), is the major urinary metabolite of MDMA. mended as the confirmation method. The controlled clinical _: trial with orally administered MDMAhas shown that the main metabolicpathwaysof 30000 SubjectA MDMAin manarecleavageofthemethyl- 2soo0 enedioxybridge, demethylation,andconju- 12 --E" gationwith HMMAand HHMAasmajoruri- 2o0o0 narymetabolites.

O,- t4

15OO0 Acknowledgment 15

c- Thisworkwaspartlysupportedbygrants 16. _o0 from the Swiss Federal Office of Public - _ Health (Foundationof NarcoticsResearchl. 0 o s lo is 20 17. Time (h)

40000 Subject B References 18. 3soon 1. C.M. Milroy, J.C.Clark, and A.R.W.Fortes: Pathology of deaths associatedwith "e_- _' 30000 stasy" and "eve" misuse.J. Clin.Pathol.49: 149-53 (1996). ,- lsoo0 2. A.T. ShuIgin. The background and chemist", 19. .o of MDMA. J. Psychoactive Drugs 18: 20000 ,- 291-304 (1986). c, 3. D.E.Nichols.Differencesbetweenthemech. § _sooo anism of action of MDMA, MBDB,andIht

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438 .__. Figure6. Urine profiles of subjectsA and 13after an oral doseof 1.5 mg/kg 3,4-methylene- [:ds., NIDA Res.Monograph 94, 1989.P; 1996 Journal of Analytical Toxicology, Vol. 20, October 1996 f the 5. L. Hermle,M. Spitzer,D. Borchardt,K-A. Kovar,and E.Gouzoulis. 22. M.I. Colado andA.R. Green.A studyof the mechanismof MDMA IA is Psychological effects of MDE in normal subjects. Neuropsy- ('Ecstasy')-induced neurotoxicity of 5-HT neurones using )fan chopharmacol.8:171-76 (1993). chlormethiazole, dizociJpine and other protective compounds. taJJy 6. ativeK.M. behaviouralHegadoren,andM.T.neurochemicaiMartin-lverson,studiesand G.B.with Baker.a psychomotorCompar- 23. L.H.Br.J.Price,PharmacG.A. oRicaurte,l.111' 131-36J.H. Krystal,(1994).and G.R. Heninger. Neuro- h no stimulant, an hallucinogen and 3,4-methylenedioxy analogues of endocrine and mood responses to intravenous L-tryptophan in the amphetamine. Psychopharmacol118'. 295-304 (1995). 3,4-methylenedioxymethamphetamine (MDMA) users. 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