'" Hallucinogenic Derivatives Using .I,' Thin-Lager Chromatography

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;t Differentiation of Amphetamine and Its ,fiajor '" Hallucinogenic Derivatives Using .i,' Thin-Lager Chromatography

· Barbara A. O'Brien, Judith M. Bonicamp*, and Donald W. Jones Analytical Syslems, 23162 La Cadena Drive, Laguna Hills, California 92653

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(GC), aud various researchers have reported methods to Abstract facilitate and improve the GC detection (13,15-18). Even with these improvements, GC is complicated, time-consuming, and Psychotroplc, ring-substituted amphetamine derivatives expensive, requiring highly trained personnel to interpret the can be differentiated from each other and from over-the- results and maintain proper instrument function. Alternative counter drugs using a sequential TLC detection techni- procedures such as immunoassay techniques do not pro',ide a qua. The improved detection is accomplished by distinct single, quick screen for many specific drugs, but instead, usually differences in color through four detection stages, identify certain drug categories. Reported in the tables are Rf values in two solvent We report here an improved TLC technique for differen- systems, the color characteristics through the four detection stages and in two confirmatory reagents, and the tiating several amphetamines, which, by virtue of the detection minimum detectible concentrations in urine of 19 am- methods used, greatly reduces the need for confirmation. When phetamine derivatives, confirmation is necessary, the number of possible interfering ', substances is minimal. Because of the five-parameter specifici- ty (coloc reactions with four reagents and Rr) the method of thin-layer chromatography for amphetamines has advantages over other analytical techniques in vogue. Introduction

Due to their psychotropic properties, various ring-substituted amphetamine derivatives have gained popularity as recreational Experimental drugs (1-3). Included in a list of illicitly marketed compounds compiled by Taylor et al. (4) are several of these psychotropic Applied Sciences (State College, Pennsylvania) supplied amphetamines, among them 2,5-dimethoxy-4-methylam- 4-methoxyamphetamine, 2,5-dimethoxyamphetamine, TMA, phetamine (DOM or STP) (2,5), 3,4,5-trimethoxyamphetamine 2,4,5-trimethoxyamphetamine, 2,4,6-trimethoxyamphetamine, (TN1A) (6), 3-methoxy-4,5-methylenedioxyamphetamine DOM, 2,5-dimethoxy-4-ethylamphetamine (DOE), and (MMDA) (7), and 3,4-methylenedioxyamphetamine (MDA)(I). MMDA. Methylenedioxyamphetamine was supplied by U.S. Ratcliffindicated the occurrence of 4-methoxyamphetamine and Pharmacopeial Convention, Inc. (Rockville, Maryland). 2,5-dimethoxyamphetamine in street drugs, alleged to be MDA Mescaline (3,4,5-trimethoxyphenethylamine) was obtained from (8). Other amphetamine derivatives appearing recently in street Sigma Chemical Co. (St. Louis, Missouri). Except for MD*I drugs are N-methylG,4-methylenedioxyamphetamine (MDM) and MDE, which were synthesized, all other drugs x_ere put- (9,10) and N-ethyl-3,4-methylenedioxyamphetamine (MDE or chased from the manufacturers. XTC) (9,10,11). Methanol and ethyl acetate were spectrophotometric quali- Repeated mentions of this class of drugs in the literature in- ty. Other chemicals were reagent grade. dicate a need for a reliable differentiation technique to assist Mandelin's reagent was prepared by heating 200 mg of am- both clinicians and toxicologists, monium metavanadate with 250 mL of concentrated sulfuric Several v,orkers have detected amphetamine derivatives by acid until solution of the salt was complete. thin-layer chromatography (TLC); however, due to limitations To prepare modified Dragendorff's reagent (iodinated), 200 of the methods used, results were considered presumptive until mg of bismuth subnitrate, 5 mL of water and 10 mL of glacial confirmed by other techniques (5,13-16). Usually TLC anatysis acetic acid were heated until the bismuth subnit'rate dissolved of these substances is accompanied by gas chromatography (Solution A). In a separate container, 5 g of potassium iodide and 2 g of iodine were dissolved in 100 mL of distilled water (Solution B). Solution A was added to Solution B and the corn- · Pressenl address 22906 Via G ut Laguna Nmgue CaMo nia 92677 bination was diluted with water to 250 mL.

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JourrciJ of Arlaiehcal Tox;colo'_y Vo I 6. Ma_, j,.ne 1...4c_;_,

Gallic acid and chromouopic acid reagents ,,','crc prepared b> quid/liquid extraction tubes, v.hich contain a mixture of 2.5 heating 2.5 [: gallic acid or 0.5 g chromolropio acid ill 250 mL mi. el organic soh'cnts (methylene chloride and dich!oroc:han,, h of concenlratcd sulfuric acid until solution *,as complete, pre-mca',urcd, buf[ercd salts and a phaqe marking d.,.e. 'Fhz Symhese., fei MDM and MDE ha,.e been included duc to tube,, x_cre mixed by inversion for I minute, and then cemn:u.g the difficuh.', in obtainmg these standards from commercial ed. ] lie organic extracts ,.'.ere remo,.ed and concentrated by heal sources, and c,.aporation, onto small discs (3.5 mm) of glass microfibcr ' 'Fhe N-methyl derivative ofMDA (MDM) was prepared by media impregnated with silica gel (21/. 'ILC plates , reacting 5 mg of free base MDA *,'ith 0.5 mL of methyl iodide. (ToM Grams _ , Analytical SystemsJ made of the same ,iiica [2el- The solution ,,_as boiled for I0 minutes and allowed to cool to impregnated material ,.,,'ereinoculated by inserting die dried sam- room temperature. The N-eth','l derivative (NIl)E) was prepared pie disc into one of the two center holes located near the low ct t similarly using ethyl iodide (191. The products are the secon- edge of the chromatograms. Two solvem systems _ere used tor dar)' amine: (.'slDM or MDE), the tertiary amines (N,N- separation: System I. ethyl acetate:methanol:*,ater:NH,OH dimethyl-M DA or N,N-diethyl-MDA), and thc quaternary am- (95:3.5:1.5 by volume plus 7.5 i_L of concemrated ammonium monium salts. The amines were isolated from the salts by Ii- hydroxide per mL of sol',ent); System Il, acetone plus 51& of quid/liquid extraction at pH 9.0 (Text-Tubes e , Analytical concentrated ammonium hydroxide per milliliter of solvent. i Systems, Laguna Hills, California), followed by separation on Chromatograms were developed for 10 cm. The solvent *,as the TLC systems described below. The identities of MDM and removed from the chromatograms by heating them on a hotplate i MDE from these syntheses were verified by comparing their col- at 70°C for about 30 seconds. or reactions and Rr values with those of known standards (20). The main detection system for broad screening consisted of Methanolic solutions of all drugs (l t_g/mL) were used to spike dipping the chromatograms into several reagents sequentially, urine (drug free, except for caffeine). Aliquots of spiked urine and observing the results at four stages (211. At each stage, the were extracted at pH 9.0; 5 mL of urine were added to the Ii- chromatograms were removed from the reagents and viewed

Table I. Rf Values and Detection Characteristics of Amphetamine and the Major Substituted Derivatives

Ri DetectionCharacteristics"*

Drug I* Il'* Stage I Stage Il Stage I[I StageIV t Amphetamine ,37 .70 yellow--brown paleolive blue brown 2,5-dimethoxyamphetamine .32 .69 yellowishgreen brightgreen_ deeporange brown t (DMA) orange-yellow 2.5-dimethoxy-4-ethyt- .32 .65 greenishyelIow yellow dull blue brown amphetaminet (DOE) 2,5-dimethoxy-4-methyl- .32 .65 greenishyellow yellow dull blue brown arnphetaminet(DOM,STP) N-ethyl-3,4-methytene- .37 .35 - blue-green light olive nog brown dioxyamphetamine (MDE) Methamphetamine .23 .20 yellow--brown paleolive blue brown , 4-methoxyamphetamine .35 .68 blue-purple fades dull brown 2-methoxy-4,5-methylene- .32 .65 greenishyetlow greenishyellow faint or neg. brown dioxyamphetaminef (MMDA) N-methyl-3,4-methylene- .23 .20 _ blue-green light olive neg. brown di0xyamphetamine(MDM) 3,4-methylenedioxy- .37 .70 - blue-green grey-tan brightblue brown amphetamine{MDA) Phendimetrazinet; .66 65 neg., slowyellow neg., neg.. strongbrown or yellow--green or palegreen or bJue Phenrnetrazine:l: .39 .50 neg,, slowyellow neg., neg., strongbrown or yellow--green or palegreen or blue Phentermine .38 .40 yellow--brown paleolive blue brown Phenylpropanolamine(PPA) .32 .90 yellow--green palegreen blue brown Pseudoephedrine .15 1.0 yellow--green palegreen btue brown 2,4.54rimethoxyamphetamine.20 .65 yellow fades neg. brown 2,4,6-trimethoxyamphetamine.20 .65 rose . pink-tan faint or nog brown 3,4.5-trimethoxy- .20 .65 yellow fades dull blue brown amphetamine(TMA) 3.4,5-trimethoxyphen- .13 .60 orange fades dull green brown ethylamine(mescaline) _.,.

'System I. ethyl acetate:melha_ol:water (95:3 5:!.5) plus 7.5 _L of concentrated ammomum hydroxide per mL of solvent. · 'Syslem II, acelone plus S uL of concentrated ammonium hydroxide per mL of solvenl ' ' 'Please reler to texl Ior description of Slages I-IV, tNo differentiation in the main detection system Chromotropic acid et gallic acid reagents will dffferenliate MMDA from DOM and DOE :tOharacterlsbcs al Stages Mil are dependent on concenlration and formaldehyde exposure

144 _. still wel. F'ollowing a two-minute exposure to for- t_t diltcrcntiaGon :.ullong isomers of TMA. [he N-methyl and L Jchyde vapors. Ibc chromatogram:, wc_e dipped sloà ly in- N-edtyl deriva!ives o1' MI)A (M DM and NIl)IL) were distin?uish- il landelin'sreagent. Thechromatograms were then viewed ecl from each other and trom MI)A by Rf in each solvent !i : about 20 seconds to one minute (Stage Il. Next they ,acre system, and by their lack of fluorescence in Stage I11. :t ,ed quickly into and out of à'ater (Stage 11). At Stage II, Deri,.atives that differ from amphetamine only irt side-chain !i ' -rs appeared imrnediately for some drugs; others appear modifications (Figure 1), migrate to differem positions in each i: ;/. ) seconds. ] he chromatograms were vier,ed subsequent- sol',ent sy,,Icnt. This distJ_ction ',',as reinforced b5 nua_ces of nderlongwave(366 nm) ultravioletlight(Stage111).At this color in Stages I and II. e many of the drugs showed an immediate absorbance or Though the distinction between MMDA, DOM and DOE is ,rcscence. At Stage IV, the chromatograms ,,','ere diPPed in- poor with the main detection system, MMDA can be differen- nodi'fied Dragendorff's reagent. Various shades of brown dated from the others using gallic or chromotropic acid reagents. c visible immediately. Both reagents form chromophores with formaldehyde, which or confirming the presence of a methylenedioxy group, w'e is liberated when the methylenedioxy group decomposes in the d either gallic acid or chromotropic acid reagents (22,23). sulfuric acid present in the tào reagents (23). Water then causes omatograms ',','ere dipped into one or the other reagent, then a vivid color development, because the heal of solution of edippedonceinto water.A positivetestis the immediate marion of a bright green chromophore à'ith gallic acid, or ale lavender chromophore with chromotropic acid. Table II. Detection Limits in Urine of Amphetamine and tuman urine specimens were ana!yzed for some amines: am- the Major Substituted Derivatives :tamine, methamphetamine, phentermine, phenylpropanol- :ine, MDA, phenmetrazine and ephedrine. Urine specimens Detection Limit, _glmL ntaining the more exotic amphetamine derivatives ,,,,'ere available for testing. In place of these, w-etested spiked urine. Stages Gallic Chromo- least five spiked specimens at each amphetamine concen- Drug I-IV Acid tropicAcid _tion (0.5, 1.0, 1.5, 2.0 p.g/mL) v,,rere extracted in order to Amphetamine 0.5 -- -- termine the detection limits. The color patterns at the 2,5-dimeth0xyamphetamine 1.0 -- -- nimum detectible concentrations were then evaluated. 2,5-dimeth0xy-4-ethylamphetamine 2.0 -- -- 2,5-dimethoxy-4-methylamphetamine 20 -- -- Methamphetamine 0.5 -- -- 4-methoxyam0hetamine 0.5 -- -- Phendimetrazine 1.0' -- -- ! Results and Discussion ._ Phenmetrazine 1.0' -- -- Phentermine 30 -- --

/he characteristics of the amphetamines detected are listed PseudoephedrinePhenylpropanolamine 33.00 ------'e l, as are the R,-values with Systems I and Il. Table 11 2,4.5-trimeth0xyamphetamine 20 -- -- t d_,.,xtes the detection limits of each drug, which was defined 2,4.6-trimeth0xyamphetamiae 1.0 -- -- the lowest concentration of drug in urine that would give 3,4,5-trimeth0xyamphetamine 1.0 -- -- _aracteristic reactions in all stages of detection. Exceptions are 3.4,5-trimeth0xyphenelhylamine 1.0 -- -- N-ethyl-3,4-methylenedioxyamphetamine 1.5 025 0.25 aced in the table. Very little problem ',','asencountered due to 2-methoxy-4,5-methylenedioxy- 1.5 0.25 0.25 terference from other drugs. Table 111 indicates the color araphetamine _aracteristics and Rfvalues in System I of some other drugs that 3,4-methylenedi0xyamphetamine 1.0 025 0.25 e commonly used or abused. N-methyl-3,4_melhylenedi0xy- 1.5 0.25 025 The colors observed for amphetamine and the major ring_ amphetamine

_bstituted derivatives, 4-methoxyamphetamine, M DA, DMA · Stage Iv pray. 15 _9/mL Ls necessary to detect al Singe I. _d TMA, were strikingly different. There was even a degree

fable Ill. R r Values and Detection Characteristics of Other Commonly Used Drugs

DetectionCharacteristics"

Drug R,I* StageI StageII StageIII StageIV I Acetaminophen .73 blanch honey neg., or dull red brown ,< Caffeine .60 neg. neg neg. slategrey j Cocaine .83 neg nco neg. rose-brown 1 Codeine .22 darkblue blue--straw absorbsor green brown t Imipramine .50 neg. brightblue blue-green brown j Meprobamate .82 neg neg neg. yellow-lan i Methadone 67 blue fades neg.or faint greell brown Methaqualone 89 neg. neg neg. rose-brown Morphine .13 grey-purple straw absorbs brown Nicotine .43 neg neg neg. rose-brown

,em I. ethyl acetale melhanol wa_e¢ (953515) plus 75 _L of concentrated ton*urn hyGfox_de per mL of solvent ' · ,_ase refer {o lexl eof a descrmpl*on of Stages IIV

145 Jour:]:'!', o _ f',na z' _'_ 'r_;, £,.9!o_/ V,9', 6 L_,w/_J_r',. ",.,:_

...... sulfuric acid ill '.',arm cncouraoes further decomposilion of Ibc /_- /SH 2 rncth',lcncdioxy lzrotJp. :' _ '1' hin-lavcr chroruatoe,raphv is a convcnicm screenin_ method H ..... _ 3 but has not previously lent itself K) more than presumptive, idcn- ) amPhoti'al'n° OMA tificafion of most of these sub,,tancc.,. 'iht unique m.-'ti,od discussed here improxe_ the. Icxcl al confidence in diffe,remia-

CH30"_X'x,/_'""-,r/NH2 CHaO_'_TNH2 lion of amphelaminc derkatixcs; the improxcment req,, on

2'-0C H3 CH3_X_3v /_OC Oh { distinct differences m color characteristics, xd'fich increa-,e the CH3CH2 specificiLv of thc method and decrease thc need for numerous solvent systems. DOE DOM, STP

/_NH_CH2C H 3

MDE methamphetamine We thank Dr. Alexander T. Shulgin for gifts of MDM and MDE.

/0_ NH2

CH30'_'_ / c,, "ao.a.. oct, 4-methoxyamphet amine MMDA References

1. P.N. Thiessen and D.A. Cook. The properties of

/O_NH-CH3 /O_ NH2 3,4-methylenedioxyamphetamine (MDA). I. A review of the H2CXo_ CH 3 H2Cxxo.--'"'-__' CX3 literature. Clin. Toxicol. 6:45-52 1973). 2. S.H. Snyder, L. Faillace, and L. Hotlister. 2,5' MDM MDA Dimethoxy-4-methylamphetamine (STP): A new hallucinogemc drug. Science. 158:669-72 (1967).

o"_ o"_ 3. S.N.BasicPradhanAspects.andC V.S.N.Mosby,DuttaSt.(Ed.).Louis,DrugMissoAbuuri, se197, C7,linicpp al30-a45.nd ,

i_NCH3 4. B.L. Taylor, J.I. Maurer, and J.R. Tinklenberg. Management of 3 a "bad trips" in an evolving drug scene. JAMA. 213:422-25 (1970) , 5. K. Genest and D.VV Hughes. Chromatographic methods for the phendimetrazme phenmelrazine identification of the new hallucinogen, 4-methyl - ' 2,5-dimethoxy-o-methylphenethylamine, and related drugs. Analyst. 93:485-89 (t968).

NH2 properties of 3,4,5-trimethoxyamphetamine. Nature. (London)

189:1011-12 (1961). CH3 OJ' )2 7. A.T. Shutgin. 3-Methoxy-4,5-methylenedioxyamphetamine, a new PPA phentermine psychotomimetic agent. Nature. (London) 201:1120-21 0954). 8. B.E. Ratcliff. MDA. C/in. Tox. 7:409-11 (1974). OH 9. Analysis Anonymous _ Itemized Street Dru_ Test Results.

pp 1-10, NHCH3 CH30_ _.. A /'tH 2 PharmChem Laboratories, Menlo Park, California, August. 1980, 3 CH30 3 10. Analysis Anonymous _ Itemized Street Drug Test Results PharmChem Laboratories, Menlo Park, California, January, 1981,

pseudoephedr Ina 2,4 S-t rimetholty- pp 1-4. i amphetamine 11. Analysis Anonymous _ Itemized Street Drug Test Results. PharmChem Laboratories, Menlo Park, California, July. 1980,

CCH 3 pp 1-1 1.

PharmChem Laboratories, Menlo Park, California, September.

CH30 CH NH3 2 CH30./0'._.,y_y_ .J_'NH CH3 2 12. Analysis1980, pp An1-11ony. mous ? Itemized Street Drug Test Results.

2,4,6-trimethoxy-- TMA CCH3 13. R.C. Baselt. Analytical Procedures for Therapeutic Monitoring and Emergency Toxicology. Biomedical Publications, Davis, amphetamine California, 1980, pp 42-43. 14. G.F. Ph_ips and J. Gardiner. The chromatographic identification of psychotropic drugs. J. Pharm. Pharmacol. 21:703-807

cH3°_ NH2 15. K.(196B9).ailey, D. Legault, and D. Yarner. Spectroscopic and CH30' T chromatographic identification of dimethoxyamphetamines. J. °c_43 Assoc. Off. Anal. Chem. 57:70-78 (1974). mescaline 16. K. Bailey, P. Gagne, P. Legault, and R Pike. Spectroscopic and

Figure 1. Structures of amphetamine and the major substituted derivatives Assoc. Off. Anal. Chem. 60:642-53 (1977). 17. A.W. Wu. Rapid analysis of the central nervous system t chromatographic identification of dimethylamphetamines. J. 146 i Jouma_ of /_.aalyt_cz¢ iox_co;oc_y, 'vo_ u, F,,;aF,J_J_w Jub_

,! stimulants, amphetamfnas, via gas chromatography-mass spec- (3.4-methylened_oxyamphetamine). J. Pharm. Sci. 69:192-95 trometry: Rapid acylation in the presence ol a mercury catalyst. (1980). Clin. Toxicol. 8:225-32 (1975) 21. J. Michaud and D.W. Jones Thin-layer chromatography for ' 18. K Bailey. IH. Beckstead. D. Legault, and D. Verner Identifica- broad-spectrum drug detection. Am. Lab. 12:104-07 (1980) I tion of 2-, 3-, and 4-methylamphetamines. J. Assoc. Off. Anal 22. M.A. Shaw and N.W Peel Thin-layer chromatography of Chern 57: t 134-43 0974). 3.4-methylenedio×yamphetamme, 3,4-methylenedioxymeth- 19. H. Had and R.D. Schuetz. Organic Chemistry,'A Short Course. amphetamine and other phenethylamine derivatives J. Houghton Mifflin, Boston. Massachusetts, 1972, pp 274-75. Chrornatogr. 104:201-04 (197'5). 20. U. Braun. A.T. Shulgin, and G. Braun, Centrally active N- 23. P.W. Lure and P. Lebish. Identification of peyote via major non- substituted analogs of 3,4-methylenedioxyphenyhsopropylamine phenolic peyote alkaloids. J. Forens. Sci. 14:63-69 (1974).

Manuscript received November 25. 1981; revision received April 9, 1982.

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