Journal of Analytical Toxicology, Vol. 14, September/October 1990

Technical Note I Detection of D,L-, D,L-, and Illicit Amphetamine Analogs Using Diagnostic Products Corporation's Amphetamine and Methamphetamine Radioimmunoassay*

John T. Cody Air Force Testing Laboratory, Brooks AFB, Texas 78235-5000

samples for of abuse mayor may not detect an analog. I Abstract I Because of the ease of synthesis, numerous illicit analogs of am- phetamine have appeared. The structure of amphetamine can be Cross-reactivity with Diagnostic Products Corporation (DPC) altered by substitution of the ethyl amine or on the benzene ring. amphetamine and methamphetamine radioimmunoassay (RIA) These modifications may retain the properties of am- reagents was determined for amphetamine, methamphetamine, phetamine or extend them. Recentlyseveral amphetamine analogs and a number of amphetamine analogs. Concentrations from have been seen on the illicit market with increasing frequency. 100 to 100,000 ng/mL were assayed. 3,4-Methylenedioxyam- One of the most common is "esctasy" (MDMA), which remains phetamine (MDA) and 3,4-methylenedioxymethamphetmaine popular even after its placement on the schedule of controlled (MDMA) showed significant cross-reactivity for the amphetamine substances in 1988. Other amphetamine analogs that have not and methamphetamine reagents respectively. 4-Hydroxymeth- amphetamine, 3,4-methylenedioxyethylamphetamlne (MDEA), been popular for some time have reappeared in clandestine and N,N-dimethyl-MDA also showed significant cross-reactivity laboratories. An example of this is the seizure of 4-bromo- with the methamphetamine reagents, but less than MDMA. None 2,5-dimethoxyamphetamine (DaB) by the DEA in 1989. Lawen- of the other analogs showed a positive result with the forcement efforts that address the starting materials for one il- amphetamine or methamphetamine reagents at even the highest licit drug often prompt the synthesis from another starting concentration, although several did show measurable cross- material or the synthesis of another drug. To evaluate the ability reactivity. The L isomers of amphetamine and methamphet- of commercial screening methods to identify these compounds, amine showed substantially less cross-reactivity than the 0 a number of amphetamine analogs were tested with the double forms to which the respective antibody systems are targeted. antibody for amphetamine assay and the Coat-a-Count metham- phetamine assay from Diagnostic Products Corporation (DPC).

Introduction

Abuse of amphetamine, methamphetamine, and amphetamine Materials and Methods analogs is neither a new nor unique phenomenon but is currently a significant problem. Although amphetamine abuse is not Materials. 2-Methoxyamphetamine, 4-hydroxymethamphet- widespread in the U,S., methamphetamine abuse is and there amine, 2,5-dimethoxyamphetamine (DMA), DaB, 4-bromo- is growing interest and concern over "ice", the solid form of 2,5-dimethoxy-B- (BDMPEA), 3,4,5-trimethoxy- methamphetamine. The use of amphetamine analogs poses not amphetamine (TMA), 3,4-methylenedioxyamphetamine (MDA), only societal concerns but also analytical challenges. Many am- N,N-dimethyl-3,4-methylened.ioxyamphetarnine, and N-hydroxy- phetamine analogs from the illicit market of the 1960s have been 3,4-methylenedioxyamphetamine (N-OB MDA) were obtained reintroduced and new analogs have recently surfaced. New from the National Institute on Drug Abuse (NIDA). 3,4-Methyl- analogs have been synthesized and marketed for increased enedioxymethamphetamine (MDMA), 3,4-methylenedioxyethyl- potency, altered pharmacological effects, and difficulty of detec- amphetamine (MDEA), 2,5-dimethoxy-4-ethylamphetamine tion and prosecution. In some cases, synthesis of an analog (DOE), 2,5-dimethoxy-4-methylamphetamine (DOM), 3,4,5-tri- yielded a compound not under the schedule of illegal drugs and methoxyphenethylamine () and L-methamphetamine which therefore could be prepared, marketed, and used without were obtained from Alltech. D- and L-amphetamine and n-meth- interference from law enforcement agencies. To address this amphetamine were obtained from Sigma. Amphetamine and problem, the Controlled Substance Analogue Act was enacted methamphetamine RIA reagents were obtained from DPC. to prevent synthesis of new unscheduled drugs of abuse. Sample preparation. Solutions of test compounds were pre- Standard methods commonly used for screening biological pared in ethanol and stored at 4°C. These drug solutions were mixed with negative control resulting in final concentrations ranging from 100 to 100,000 ng/mL. • Presented at the annual meeting of the International Association of Forensic Toxicologists, Glasgow, Scotland, August 14-19, 1989. Amphetamine test. Radiommunoassay analysis was per-

321 Journal of Analytical Toxicology, Vol. 14, September/October 1990

formed with the double antibody for amphetamine from DPC tested in this study were consistent with this statement, 4-meth- essentially as described by the manufacturer (1,2). For the am- oxyamphetamine was not tested and may be predicted to have phetamine test, 25 JLLof sample were placed into a 12- x 75-mm higher cross-reactivity than methoxyamphetamines tested in this tube, to which was added 200 JLLof IlSI amphetamine and study. Structures of compounds that showed substantial cross- 100 JLLof amphetamine antiserum. The racks were shaken to reactivity are shown in Figure 1. Results from compounds that thoroughly mix the components. After incubation at room did not demonstrate significant measurable cross-reactivity to temperature for an hour, 1.0 mL of the cold precipitating solu- the amphetamine reagents are found in Table III. tion was added to each tube and the racks were again shaken. MDMA demonstrated significant cross-reactivity to the The racks were then centrifuged for 15min at 3,000 g, the super- methamphetamine reagents. The methylenedioxy group substan- natant was decanted, and the precipitate counted on a Micro- tially enhanced the binding of the MDMA, giving a result . medic 10/600 gamma counter. equivalent to 1,000 ng/mL of D-methamphetamine at a con- Methamphetamine test. The methamphetamine procedure centration between 200 and 300 ng/mL. This high degree of with the Coat-a-Count methamphetamine reagents was similar cross-reactivity becomes even more significant when one con- to that of the amphetamine assay except it involved antibody- siders that the MDMA in this study was the racemic mixture coated tubes rather than a double antibody system, thus and racemic methamphetamine is only 67% cross-reactive when eliminating addition of antibody solutions and centrifugation. compared to D-methamphetamine at 1,000 ng/mL. MDEA and 25 JLLof sample was placed into a 12- x 75-mm metham- 4-hydroxymethamphetamine both gave positive results at con- phetamine antibody-coated polypropylene tube, to which was centrations of 2,000-3,000 ng/mL. The degree of cross-reactivity added 1.0 mL of 12SImethamphetamine solution. The racks were demonstrated with N,N-dimethyl-MDA would require approx- shaken and then incubated at room temperature for one hour. imately 25 JLg/mL to produce a positive result (assuming a The tubes were then thoroughly decanted to remove all liquid. 1,000-ng/mL cutoff). Results of the MDMA, MDEA, 4-hy- The tubes were counted on a Micromedic 10/600 gamma counter. droxymethamphetamine, and N,N-dimethyl-MDA testing are Positive and negative control solutions (provided by the shown in Table IV. For the methamphetamine reagents, an ethyl manufacturer) were used to compare the cross-reactivity of the group on the amine nitrogen impeded but did not eliminate compounds tested to amphetamine or methamphetamine. Stan- binding and the hydroxy group at the 4 position also did not dard curves at 0, 500, 1,000, and 1,500 ng/mL analyte were eliminate binding. Addition of a second methyl group to the analyzed with each batch of analogs tested. Each rack of 20 tubes amine nitrogen substantially decreased binding as demonstrated also contained duplicate standards at the 0- and l,OOO-ng/mL by the N,N-dimethyl-MDA data. Methoxy groups at various levels for evaluation of assay drift or decanting irregularities. positions on the ring also significantly decreased binding. Results Concentrations of the various amphetamine analogs were an- of the other compounds tested with the methamphetamine alyzed in quadruplicate. Average CPM determinations of repli- reagents are shown in Table V. cates were used to determine percent cross-reactivity. The data show the L-isomers of amphetamine and metham- Data analysis. Quantitations were based on comparison of phetamine to be significantly less reactive than the D-isomers. the mean CPM of four replicates for each compound at each These results have important implications in the interpretation concentration against a log-logit curve derived from five of data. In some countries, including the U.S., Vicks Inhalers replicates of standards at 0,500, 1,000, and 1,500 ng/mL of contain 50 mg of L-methamphetamine (t-desoxyephedrine). n-arnphetamine for the amphetamine assay and D-metham- With the advent of RIA reagents that are targeted specifically phetamine for the methamphetamine assay. Any assay rack that to n-methamphetamine, the potential for a positive result from showed values for the 0- or l,OOO-ng/mL standards that were not within acceptable limits was repeated. Table I. Typical Precision Data for OPC Amphetamine and Methamphetamine Reagents Results and Discussion Coefficient of variation (0/0) Concentration Amphetamine Methamphetamine Behavior of the assay reagents for both amphetamine and (ng/mL) reagents reagents methamphetamine was excellent. Precision data for both assays showed coefficients of variation (CV) less than 30/0 for all con- 0 1.97 1.87 centrations (Table I). The coated-tube assay required a slightly 500 1.44 2.16 1,000 2.80 2.22 different decanting technique, but the procedure was quickly 1,500 1.98 2.97 mastered by personnel experienced with double antibody assays. Substantial cross-reactivity was seen with MDA in the amphetamine assay. Results of the MDA analysis with the am- Table II. CrOSS-Reactivity of 3,4-Methylenedioxyam- phetamine reagents are shown in Table II. The value of 1,000 phetamine (MOA) with OPC Amphetamine Reagents ng/mL was given specific significance in this study because it Concentration (ng/mL) is defined as the positive cutoff level by the Department of Cross- Health and Human Services guidelines (3) and the Department Actual Measured Reactivity (0/1) of Defense and recommended by the manufacturer (1,2). Eval- uation of results from the amphetamine reagents revealed that 100 173 173 200 314 157 compounds with a substitution on the amine nitrogen showed 300 445 148 substantially reduced cross-reactivity. The methylenedioxy 400 616 154 group slightly enhanced binding with the antibody, but the 500 754 151 presence of one or more methoxy groups on the ring substantially 1,000 1,377 138 10,000 10,837 108 decreased binding to the antibody. Although all compounds

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".:- Journal of Analytical Toxicology, Vol. 14, September/October 1990 use of an over-the-counter inhaler must be considered. Because reactivity with the reagents used in this study (1,2), but the of the low cross-reactivity of the t-isomer, a substantial amount potential must not be ignored. Because the illicit analogs have of L-methamphetamine would be required to cause a positive a high abuse potential, it is important to be aware of their cross- result. Though this fact diminishes the probability of a positive reactivity. In addition, when the use of an amphetamine analog result from a Vicks inhaler, a disadvantage of this low cross- is suspected, it is important to evalute negative RIA results with reactivity is that illicit methamphetamine synthesized from regard to these cross-reactivity data. phenyl-2-propanone will be less cross-reactive (670/0for the D,L mixture) than that synthesized from , thus decreasing the ability to detect the use of the drug. In addition, though L-methamphetamine has a legitimate use, it can still be abused. Conclusions Because of the potential for a common over-the-counter medica- tion to be identified as methamphetamine, it is imperative to The reagents are fairly specific for the n-isomer of amphet- identify the isomer present in the sample. This can easily be ac- amine and methamphetamine, which is generally considered a complished with the use of a chiral column or assymetrical positive factor. Cross-reactivity of over-the-counter medications derivatizing reagent in gas chromatography. to the amphetamine reagents is quite low and very few positives The data reported in this study are limited to the radioim- would be expected to result from even fairly heavy use. The munoassay results from the DPC reagents. Other studies of methamphetamine reagents have higher cross-reactivity and MDA, MDMA, and MDEA have been reported elsewhere for more samples will be positive due to the presence of over-the- Syva EMIT (4,5) and Abbott TDx (4) and for MDA and counter medications. This can be a detrimental aspect of the MDMA using the Abbott TOx system (6). One of these reports reagents, unless the cross-reactivity to the illicit analogs is im- also tested the Roche Abuscreen reagents with MOA, MDMA, portant, in which case the higher cross-reactivity is beneficial. and MDEA (4). Another study, using a group of analogs iden- Of the amphetamine analogs tested, only MDA showed tical to those in the present study, reported the cross-reactivity substantial cross-reactivity with the OPC amphetamine reagents. with Roche Abuscreen amphetamine reagents (7). The cross-reactivity was slightly greater than that of am- Immunoassay positive results for amphetamine and metham- phetamine are not absolutely selective for the drug and can possibly be caused by a number of other , some Table III. Cross-Reactivity of Other Amphetamine of which are found in over-the-counter medications. Most over- Analogs with OPC Amphetamine Reagents the-counter medications have a reasonably low degree of cross- Concentration (ng/mL) Compound 1,000 10,000 50,000 100,000

CH, CH, CH, 3,4·Methylenedioxymeth- 0 87 244 NT .I .I I amphetamine (MDMA) 0.00% 0.87% 0.49% H-C-NH, H-C-NH-CH, H-C-NH-CH, 3,4-Methylenedioxyethyl- 0 49 NT 203 I I I amphetamine (MDEA) 0.00% 0.49% 0.20% 2-Methoxyamphetamine 41 88 NT 664 4.10% 0.88% 0.66% 2,5-Dimethoxyamphetamine 42 207 NT NT 4.20% 2.07% 6 6 ¢ N-Hydroxy-3,4-methylene- 15 112 NT 372 o dioxyamphetamine 1.50% 1.12% 0.37% H (N-OH-MDA) 4-Hydroxymethamphet- 0 0 NT 128 A B c amine 0.00% 0.00% 0.13% 2,5-Dimethoxy-4-methyl- 0 0 72 NT amphetamine (DOM) 0.00% 0.00% 0.14% 3,4,5- Trimethoxyphenethyl- 0 0 33 NT amine (Mescaline) 0.00% 0.00% 0.07% 2,5-0imethoxy-4-ethyl- 0 0 NT 54 amphetamine (DOE) 0.00% 0.00% 0.05% 4-Bromo-2,5-dimethoxy- 0 0 NT 27 B-Phenethylamine 0.00% 0.00% 0.03% (BDMPEA) 4-Bromo-2,5-dimethoxy- 0 0 NT 46 amphetamine (DOB) 0.00% 0.00% 0.05% N,N-Dimethyl-3 ,4-methyl- 0 0 NT 75 enedioxyamphetamine 0.00% 0.00% 0.08% 3,4,5- Trimethoxyam phet- 0 0 NT 40 amine (TMA) 0.00% 0.00% 0.04% Figure 1. Structures of compounds with high cross-reactivity to RIA l-Amphetamine 92 836 NT 7,557 reagents: (A) amphetamine, (B) methamphetamine, (e) 4-hydroxy- 9.20% 8.36% 7.56% methamphetamine, (D) 3,4-methylenedioxyamphetamine (MDA), (E) D-Methamphetamine 0 6 NT 140 3,4-methylenedioxymethamphetamine (MDMA), and (F) N,N-dimethyl- 0.00% 0.06% 0.14% 3,4-methylenedioxyamphetamine. •Assymetric carbon (All structures drawn in the D configuration). l-Methamphetamine 0 48 NT 137 0.00% 0.48% 0.14%

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Table IV. Cross-Reactivity of MOMA, MOEA, Table V. Cross-Reactivity of Other Amphetamine 4-Hydroxymethamphetamine and N,N-Oimethyl- Analogs with OPC Methamphetamine Reagents 3,4-methylenedioxyamphetamine with OPC Concentration (ng/mL) Methamphetamine Reagents Compound 1,000 10,000 50,000 100,000 Concentration (ng/mL) cress- Reactivity 2-Methoxyamphetamine NT 0 NT 13 Compound Actual Measured (Ofu) 0.00% 0.01% 3,4-Methylenedioxymeth- 100 570 570.00 2,5-Dimethoxyamphetamine NT NT NT 0 amphetamine (MDMA) 200 818 409.00 0.00% 300 1125 375.00 3,4-Methylenedioxyamphet- 7 155 NT 1749 400 1472 368.00 amine (MDA) 0.70% 1.55% 1.75% 361.00 500 1805 N-Hydroxy-3,4-methylene- 0 68 NT 42 1,000 3127 312.70 dioxyamphetamine 0.00% 0.68% 0.04% 4-Hydroxymethamphetamine 1,000 582 58.20 (N-OH-MDA) 2,000 815 40.75 2,5-Dimethoxy-4-methyl- 0 13 45 NT 3,000 1155 38.50 amphetamine (DOM) 0.000/0 0.13% 0.09% 4,000 1454 36.35 5,000 1748 34.96 3,4 ,5-Trimethoxyphenethyl- 0 6 19 NT 10,000 3108 31.08 amine (Mescaline) 0.00% 0.06% 0.04% 3,4-Methylenedioxyethyl- 1,000 425 42.50 2,5-Dimethoxy-4-ethyl- NT 11 NT 48 amphetamine (MDEA) 2,000 821 41.05 amphetamine (DOE) 0.11% 0.05% 3,000 1077 35.90 4-Bromo-2,5-dimethoxy NT NT NT 0 4,000 1289 32.23 8-phenethylamine 0.00% 5,000 1485 29.70 (BDMPEA) 10,000 2673 26.73 4-Bromo-2,5-dimethoxy- NT 0 NT 12 N,N-Dimethyl-3, 4-methylene- 1,000 209 20.90 amphetamine (DOB) 0.00% 0.01% dioxyamphetamine 10,000 670 6.70 3,4,5- Trimethoxyamphet- NT NT NT 0 25,000 1054 4.22 amine (TMA) 0.00% so ,000 1647 3.29 100,000 2323 2.32 D-Amphetamine 25 42 NT 278 2.50% 0.42% 0.28% L-Amphetamine 19 11 NT 89 phetamine itself and any RIA positive urine that does not show 1.90% 0.11% 0.09% the presence of amphetamine should be evaluated for MDA_ L-Methamphetamine 66 408 NT 4121 The methamphetamine reagents showed substantial cross- 6.60% 4.08% 4.12% reactivity with MDMA, MDEA, 4-hydroxymethamphetarriine, and N,N-dimethyl-MDA. MDMA was easily detected and the MDEA would likely identify users for a reasonable length of Dr. V. Spiehler of Diagnostic Products Corporation for pro- time. The cross-reactivity of the N,N-dimethyl-MDA was such viding the RIA reagents used in this study. Thanks also to Mr. that a positive result would only be seen for a relatively short E. Hubster, Ms. E. Alvarado, Ms. K. Turner, Ms. L. Renteria, period of time, because of the high concentration required to Mr. M. Vargas, and Mr. J_ Davis of the Radioimmunoassay yield a positive result. Branch for assistance in processing samples, Quality Control Other analogs tested did not show positive results with either personnel for assistance in preparation of the standard solu- the amphetamine or methamphetamine reagents (at a cutoff of tions, Ms. R. Galvan for excellent administrative assistance, and 1,000 ng/mL) even at concentrations of 100 {tg/mL. In cases Dr. R. Schwarzhoff and Dr. E. Cone for valuable discussions. of suspected use of amphetamine analogs other than those that showed significant cross-reactivity with one of the two reagent systems, it must be realized that one would not expect a positive RIA result. N-hydroxy-MDA, which has been sold on the il- References licit market under the guise of MDMA did not show signifi- cant cross-reactivity with either assay procedure. 1. Amphetamine Double Antibody package insert, Diagnostic Products Corporation, Los Angeles, July 1988. Because of the lack of substantial cross-reactivity to many 2. Coat-A-Count Methamphetamine package insert Diagnostic Products of the amphetamine analogs studied, use of this testing metho- Corporation, Los Angeles November 1988. dology is limited. Use of both reagent systems increases the 3. Department of Health and Human Services. Mandatory Guidelines for capability of identifying amphetamine, methamphetamine, and Federal Workplace Drug Testing Programs, Federal Register 53(69): their analogs in urine. Alternative methods should be in- 11970-89 (April 11, 1988). 4. W. Ruangyuttikarn and D.E. Moody. Comparison of three commercial vestigated for detecting those analogs that did not demonstrate amphetamine immunoassays for detection of methamphetamine, significant cross-reactivity _Because of the increased use of these methylenedioxyamphetamine, methylenedioxymethamphetamine, and compounds, it is important to have the ability to detect these melhylenectioxyelhylamphetamine. J. Anal. Toxicol. 12: 229-33 (1988). drugs, particularly for cases in which their use is suspected. 5. W.L. Hearn, G. Hime, and W. Andolla. Recognizing ecstasy: Adam and Eve, the MDA derivatives-analytical profiles. Presented at the 16th annual meeting of the Society of Forensic Toxicologists, Reno, 1986. 6. J.M. Ramos, R.L. Fitzgerald, and A. Poklis. MDMA and MDA cross- reactivity observed with Abbott TDx amphetamine/methamphetamine Acknowledgments reagents. Clin. Chern. 34(5): 991 (1988). 7. J.T. Cody. Cross-reactivity of amphetamine analogs with Roche Abu- screen radioimmunoassay reagents. J. Anal. Toxicol. 14: 50-53 (1990). The author would like to express special thanks to NIDA for Manuscript received April 13, 1990; several of the amphetamine analogs used in this study and to revision received June 15, 1990.

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