;,,,,.,alo/ChromatograS93p(199_a_hy, 7-94 /k_)__¢_._ Elsevier Science Publishers B.V., Amsterdam CHROMSYMP. 2429 Determination of psychotropic phenylalkylamine derivatives in biological matrices by high-performance liquid chromatography with photodiode-array detection Hans-J6rg Helmlin and Rudolf Brenneisen* Instituteof Pharmacy, University of Berne, Baltzerstrasse 5, CH~3012 Berne (Switzerland) ABSTRACT Several procedures using high-performance liquid chromatography with photodiode-array detection have been developed to create phytochemical and toxicological profiles of phenylalkylamine derivatives in biological samples (e.g. plant materials and urine). Mesca- line-containing cactus samples were extracted with basic methanol, using methoxamine as internal standard; the extraction and clean- up of urine samples were performed on cation-exchange solid-phase extraction columns. The extracts were separated on a 3-#m ODS column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was performed a_ 198 or 205 nm; peak identity and homogeneity were ascertained by on-line scanning of the UV spectra from 190 to 300 nm. The detection limit of phenylalkylamine derivatives in urine and cactus material was 0.026-0.056 #g/ml and 0.04 t_g/mg, respectively. Following a single oral dose of 1.7 mg/kg methylenedioxymethylamphetamine (MDMA) the concentrations found in urine ranged from 1.48 to 5.05 #gml MDMA and 0.07-0.90 #g/mi methylenedioxyamphetamine (a metabolite of MDMA). The mescaline content of the cactus Trichocereus ?achanoi varied between 1.09 and 23.75 #g/mg. INTRODUCTION South America, especially in the Andean regions and belongs, together with Lophophora williamsii Many natural and synthetic phenylalkylamine (Leto. ex Salm-Dyck) Coult. [4], to the mescaline- derivatives such as mescaline, amphetamine, meth- containing cactus species which are commercially ylamphetamine and 4-bromo-2,5-dimethoxyphen- available without legal restrictions in Switzerland ethylamine (see Table I) are known for their stim- and other European countries. ulant and/or hallucinogenic properties. Some very Considering the potential of abuse of phenyl- active ring-substituted amphetamines such as 3,4- alkylamine derivatives and mescaline-containing methylenedioxyamphetamine (MDA), 3,4-methyle- cactus species, it was the aim of this work to devel- nedioxymethylamphetamine (MDMA) and 3,4- op a selective, specific and sensitive analytical pro- methylenedioxyethylamphetamine (MDE) have cedureusinghigh-performanceliquidchromatogra- now appeared as "designer drugs" on the illicit pry with photodiode-array detection (HPLC- market, produced by clandestine laboratories [1]. DAD). This method should allow not only the iden- Owing to their high potential of abuse, most of tification of such compounds, but also the acquisi- these popular recreational substances are now in- tion ofphytochemical and pharmacokinetic profiles ternationally controlled. Despite this, MDMA in complex biological samples to estimate their toxi- I"Ecstasy", "XTC", "Adam") is used more fre- cological or therapeutic potency. The efficiency of quently as a controversial adjunct in psychotherapy HPLC-DAD in drug analysis, analytical toxicol- [2,3].Trichocereus pachanoi Britt. et Rose ("San Pe- ogy, forensic chemistry and phytochemistry of psy- dro") grows in subtropical and temperate areas of chotropic drugs has been shown previously [5-18]. 0021-9673/92/$05.00 (O, 1992 Elsevier Science Publishers B.V. All rights reserved 88 H.-J. HELMLIN, R. BRENNEISEN "[_ TABLE I & -_._'_ STRUCTURES OF PHENYLALKYLAMINE DERIVATIVES I s,",'_ Structure Peak no. Compound [[__ NHa I Amphetamine t 2 3,4-Methylenedioxyamphetamine (MDA) ._/_ ! L_[[t'_YA_ N _ 3 Methylamphetamine '3 s 4 4 4-Methoxyamphetamine '" H.,,CO' v [_"'_ NHa 5 Phentermine ) [J_ [ 6 3,4-Methylenedioxymethamphetamine (MDMA) °X..o/ ! CH30 i__ _,__ A. T NHa 7 5-Methoxy-3,4-methylenedioxyamphetamine (MMDA) ' H3COi_NH2 8 3,4,5-Trimethoxyamphetamine H3CO' T t OCH3 H I ' 9 3,4-Methylenedioxyethylamphetamine(MDE) ! HaCO j_NHa 10 2,5-Dimethoxyamphetamine '_ ["'_ /_"' 'OCH3 11 4-Bromo-2,5-dimethoxyphenylethylamine (DOBP,2-CB) HaCOj._/-_jNH2 Br" '"'cf -OCH3 H3CO j_NHa 12 2,5-Dimethoxy-4-methylamphetamine (DOM,STP) /' _ 'OCH_ · (Continued on p. 89) 4 LENNEISEN HPLC OF PHENYLALKYLAMINE DERIVATIVES 89 TABLE ] (continued) Structure PeakNo. Compound I H3C_ [_NH2 13 4-Bromo-2,5-dimethoxyamphetamine (DOB) '14 2,5-Dimethoxy-4-ethylamphetamine (DOET) V _ 'OCH3 15 Mescaline H3CO i_ ''._N H2 H_C0' T I., OH OCH3'1" H3CO _NH2I,_,II 16 Methoxamine v 'OCH3 } EXPERIMENTAL through a membrane filter (regenerated cellulose, 0.45 #m, Schleicher and Schuell) and degassed by Instrumentation sonication and during use with a constant flow of The HPLC-DAD system consisted of a Hewlett- helium. Methanol was used for washing the col- 0 Packard1090Mliquidchromatograph(Hewlett- umn. Packard, Waldbronn, Germany), an HP 1090L au- The separation of urine samples containing tosampler, an HP 1040M photodiode~array detec- MDMA and MDMA metabolites was performed _IMDA) tor, an HP 79994A Chemstation (software version isocratically at 40°C on a 125 x 4.0 I.D. column 1.05),an HP 7470A x/y plotter and an HP 2225A packed with 3-$tm Spherisorb ODS-1. The mobile Thinkjet printer, phase was acetonitrile-water (72:928, v/v; 57:943, w/w), containing 5.0 ml (8.5 g) orthophosphoric Chromatographic conditions acid (85%) and 0.28 ml (0.22 g) hexylamine per The separation of fourteen phenylalkylamine de- 1000 ml. The flow-rate was 0.8 mi/min. rivatives was performed at 40°C on a 125 x 4.0 mm The separation of the cactus samples was per- I.D. column packed with 3-tim Spherisorb ODS-1 formed isocratically at 25°C on a 150 x 4.6 mm (Phase Separations), filled by Stagroma (Wallisel- I.D. column with a 20 x 4.0 mm I.D. precolumn, len, Switzerland). The solvent gradient was devel- packed with 3-ttm Spherisorb ODS-1. The mobile oped by using the CARTAGO (computer assisted phase was acetonitrile-water (108:892, v/v), con- retention time prediction and gradient optimiza- taining 5.0 ml (8.5 g) orthophosphoric acid (85%) tion) software; details of this procedure are publish- and 0.28 mi (0.22 g) hexylamine per 1000 ml. The ed elsewhere [19,20]. Solvent A was water contain- flow-rate was I ml/min. ing 5.0 ml (8.5 g) orthophosphoric acid (85%) and BP,2-CB) 0.28 ml (0.22 g) hexylamine per 1000 mi; solvent B Chemicals and reagents was acetonitrile containing 100 mi water, 5.0 ml (8.5 Amphetamine sulphate was obtained from Sieg- g) orthophosphoric acid (85%) and 0.28 mi (0.22 g) fried (Zofingen, Switzerland) and methylampheta- _TP) hexylamineper 1000 ml. The gradient profile was as mine hydrochloride from Dr. Grogg Chemie follows: 0_10.6 min, 5.5% B in A (isocratic); 10.6- (Berne, Switzerland). Mescaline hydrochloride was 21.6 min, 5.5-39% B in A (linear gradient). The supplied by Laboratoires Plan (Geneva, Switzer- !inuedonp.89) flow-rate was 0.8 mi/min. The eluent was filtered land), methoxamine hydrochloride and MDA were 90 H.-J. HELMLIN, R. BRENNEISEN 'l provided by Sigma (St. Louis, MO, USA). MDMA, x 0.5 mi of methanol-hydrochloric acid (7.3%; ,._ MDE, 5-methoxy-3,4-methylenedioxyampheta- 97.5:2.5) at a flow-rate of about 0.5 ml/min. Ali- mine (MMDA), 4-methoxyamphetamine, 2,5-di- quots of 10 #1 of the defined volume of the eluates methoxyamphetamine, 2,5-dimethoxy-4-methylam- were injected into the HPLC-DAD system for the 5 phetamine (DOM,STP), 2,5-dimethoxy-4-ethylam- determination of MDMA and MDA. For !ow phetamine (DOET), 3,4,5-trimethoxyampheta- MDMA and MDA levels a concentration stepmay mine, 4-bromo-2,5-dimethoxyamphetamine (DOB) be necessary. An aliquot of 1.5 ml ofthe eluates was and phentermine were donated by the Division of added to 68 #1 of I M KeHPO4, concentrated to ":; Narcotic Drugs, United Nations (Vienna, Austria). about 100 #1 under a stream of nitrogen and recon- 4-Bromo-2,5-dimethoxyphenethylamine (DOBP,2- stituted to 150.0 #1 with methanol-water (50:50). _ CB) was a gift of the Swiss Association for Psycho- The resulting solution, with a pH of about 4-5, was lyric Therapy. All other chemicals and reagents then sonicated for 2 min and filtered through the tip ';7, were of HPLC or analytical-reagent grade and were of a Pasteur pipette filled with cotton wool. Aliqu- , purchased from Merck (Darmstadt, Germany) or ots of 10/al were used for determination by HPLC- Fluka(Buchs,Switzerland). DAD. The cactus samples were cut in half, lyophilized ._ Urine and cactus samples and stored in a desiccator under vacuum and pro- The urine samples were obtained from patients tected from light until used for analysis. A repre- treated with MDMA by psychiatrists of the Swiss sentative sample of the cactus specimen was pulver- Association for Psycholytic Therapy. Urine sam- ized with a grinder. An accurately weighed amount pies were collected approximately 6 h after the ad- of the powdered sample (about l0 mg) was then ministration of 1.7 mg/kg MDMA. The specimens washed four times with 1 mi of diethyl ether by so- of Trichocereus pachanoi Britt. et Rose and Lo- nication (5 min) and filtration through a 0.2-#m re- phophora diffusa (Croizat) Bravo (Cactaceae) were generated cellulose filter (Spartan 13/30, Schleicher bought at flower shops and shopping centres in & Schuell). The defatted sample was extracted four Switzerland, or obtained from private collections, times with 0.5 ml of methanol-ammonia (33%; 99:1), containing 150.0 #g/ml methoxamine hydro- ., Sample preparation chloride as internal standard (I.S.), by sonication (5 The extraction and clean-up of urine samples min) and filtration through a 0.2-#m regenerated , (real, spiked, blank) were carried out on Adsorbex cellulose filter.