Isomeric amphetamines — A problem for urinalysis?

153

Forensic Science International, 50 (1991) 153 - 165 Elsevier Scientific Publishers Ireland Ltd.

ISOMERIC

AMPHETAMINES

FREDERICK

-

A PROBLEM

FOR URINALYSIS?

P. SMITHat* and DAVID A. KIDWELLb

aGraduate Program in Forensic Science, Department of Criminal Justice, University of Alabama at Birmingham, Birmingham, AL 35294 and bChemistry Division, Naval Research Laboratory, Washington, DC 20375-5000 (U.S.A.)

(Received December 4th, 1990) (Accepted April 18th, 1991)

Summary Alkyl amphetamine isomers (amphetamine, l-phenyl-2-butylamine (PBA), methamphetamine, Nmethyl-PBA, N,N-dimethylamphetamine, N-ethylamphetamine, N-ethyl-PBA and N,N-diethylamphetamine) were purchased or synthesized and tested by immunoassay and GClMS for their detectability in urine. Some cross reactivity was observed with PBA, N-methyl-PBA N-ethylamphetamine, and N-ethyl-PBA when analyzed using a series of commercial amphetamine and methamphetamine immunoassays. Chromatographic co-elution problems were observed for the underivatized isomeric group N,N-dimethylamphetamine, N-ethylamphetamine, and N-methyl-PBA under GClMS conditions used; and their GClMS spectra were quite similar. Of the potential derivatives, pentafluoropropionyl (PFP) anhydride and heptafluorobutyryl (HFB) anhydride provided adequate separation and easily distinguishable spectra using the electron-impact GClMS conditions specified. Key words:

Urinalysis;

Immunoassay;

Gas chromatography/mass

spectrometry;

Amphetamine

analogues

Introduction

The U.S. Department of Defense maintains a strong drug abuse deterrence program, partially through the use of urinalysis. In this urinalysis program, a wide variety of drugs of abuse are detected using a two-step procedure: screening and confirmation. Immunological tests (usually radioimmunoassay-RIA) screen urine samples while gas chromatography/mass spectrometry (GC/MS) is used for confirmation. Immunoassays employ highly selective antibodies as the means to recognize specified concentrations of drugs. An individual abusing similar drugs (such as so-called ‘designer’ drugs) that exhibit little or no cross reactivity with immunoassays would evade detection by the initial screening test. When this result is ‘negative’, the urine sample is discarded; no further tests are performed. Hence, a user of designer drugs would not be deterred by the urinalysis program. *To whom all correspondence

should be addressed.

0379.0738/91/$03.50

@ 1991 Elsevier Scientific Publishers Printed and Published in Ireland

Ireland Ltd.

154

m

Amphetamine

I

Abused

d-Methonphetunine

m

Drugs

kMethanphetamine

Pheny Ipropono

I Amine

Cold

Preporot

ions

; Ephedrine Pseudcephedr ine (The rvsne depends upon the sterochemistry at the starred carbons)

Phentermine

Fig. 1. Structures

of abused amphetamines

Prescript Anorexic

ion

and relatively innocuous amphetamine

analogues.

Because of the way antibodies for amphetamine-class immunoassays are prepared, they can recognize amphetamine and several other amphetamine-like analogues. Many cold preparations contain materials that are sufficiently similar ephedrine, phenylpropanolamine, lto amphetamine (i.e. pseudoephedrine, methamphetamine, and phentermine, see Fig. 1) that urine from users may screen positive for amphetamines and be held for confirmation by GUMS. GUMS, being a more specific test, can easily distinguish cold preparations from amphetamines (with the exception of Z-methamphetamine, in which case chiral derivatives must be prepared to allow the separation of optical isomers). However, GUMS is time consuming and expensive. To lower the cost of a urinalysis program a second, more-specific immunoassay is often performed. This immunoassay detects d-methamphetamine and excludes most cold preparations [l]. By using a two-test screening, the workload for GUMS confirmations is reduced. In the 198Os, access to many of the precursors used in clandestine drug laboratories manufacturing amphetamines became more restricted by law enforcement officials. As a result, readily available materials were used with alternative chemical routes to synthesize amphetamine-like compounds. One synthesis resulted in the production of NJ-dimethylamphetamine (a potent CNS stimulant [2-41) in place of methamphetamine [5]. The presence of this compound in the illicit marketplace is substantial [6,7]. When purchased by law en-

155

& d-Amphetonine

d, l-N-Ethyl-l-phenyl-2-butylomine

d-Dimethylanphetamine

d,

d-Diethylcmphetcrnine

d, l-l-Phenyl-2-butylanine

I-N-Methyl-1-phenyl-Z-buty lonine

Fig. 2. Amphetamine analogues chosen for study, including two isomeric groups: (1) methamphetamine and 1-phenyl-2-butylamine and (2) dimethylamphetamine, ethylamphetamine, and Nmethyl-1-phenyl-2-butylamine.

forcement agents, this drug was purveyed a3 methamphetamine; hence, users do not know they are taking N&V-dimethylamphetamine [6,7]. Shown in Fig. 2 are the CNS stimulant analogues chosen for this study, in[ 11,121, and a series cluding N-ethylamphetamine [&lo], phenyl-2-butylamine of other amphetamine analogues [13-201. Most exhibit CNS stimulant properties. For example, in humans NJ-dimethylamphetamine has been reported to cause the same degree of CNS stimulation at 10 times the amphetamine dose [4]. Due to stricter concern for the ethical use of human subjects in new drug testing, intensified after the abuse of human subjects prior to 1945, and because amphetamine isomers studied here have been proposed for medicinal or food product use after 1947 [21], the body of literature describing the physiological effects of these analogues in animals and humans [8-191 precedes this interval and little modern data are available. This report discusses the synthesis and characterization of a series of amphetamine analogues. To determine the detectability of these compounds in urine, they were analyzed using the major commercially available immunoassays: Abbott TD X8 Abused Drug Assays AmphetaminelMethamphetamine (TD,@ -Class and TD,@ -Specific), Syva EMIT da@ System (EMIT@ -Class), and Roche Diagnostic Systems AbuscreerP radioimmunoassays (RIA) for amphetamine and methamphetamine. GUMS confirmation of these materials in urine was adapted from existing methodology [5].

156

Materials and Methods Syntheses The amphetamine analogues chosen for synthesis are shown in Fig. 2. They were prepared by two well-documented syntheses [2,5,22] involving either the phenylalkyl ketone (which provides a product mixture of d- and Z-isomers) or the N-alkylation of d-amphetamine (which produces the d-isomer product only). d,ZIV-ethylamphetamine, d,l-iv-ethyl-1-phenyl-2-butylamine, d,l-Wmethyl-l-phenyl2-butylamine, d,Z-l-phenyl-2-butylamine were synthesized by the first method, using platinum oxide catalysis hydrogenation of the imine intermediate. d-N,iVdimethylamphetamine, d-NJ-diethylamphetamine were prepared from d-amphetamine by alkylation with either methyl iodide or diethyl sulfate. (The mass spectrum of the PFP derivative of commercially purchased phentermine was obtained using the derivatization and GUMS methods described below.) Gas chromatography/mass spectrometry Drugs were extracted from 10 ml of urine containing 50 ng drug/ml, derivatized, and analyzed by GUMS. Temperature programming and selective ion monitoring modes were utilized to separate and detect small quantities of amphetamine analogues. Aqueous solutions of the salts of the amphetamine isomers were prepared by adding the appropriate volume of solvent to approximately 10 mg of drug substance. A working stock solution of standards was prepared by adding 5000 ng of each standard to q.s. 10.0 ml water. An aliquot (LO-ml) of the resulting solution (500 ng/ml) was added to 9.00 ml urine so that the resulting concentration was 50 ng/ml. To 10.0 ml urine was added 2.0 ml sodium hydroxide (1.0 M) and 1.0 ml chloroform/isopropanol (25:l) in a 15-ml cone-shaped, screw-cap glass tube. The tube was capped and rocked for 15 min. After centrifugation at 1000 rev./min for 5 min, the chloroform layer was placed in a cone-bottom reaction vial and evaporated carefully to near dryness so that the remaining volume was approximately 20 ~1. Pyridine (5 ~1) and PFP anhydride (5 ~1) were added and vortexed. After incubation at room temperature for 5 min, the reaction was quenched with 50 ~1 water followed by 50 ~1 of 1.0 M sodium hydroxide. After vortexing, the reaction tubes were centrifuged for 2 min at 1000 rev./min. An aliquot of 1 ~1 was injected for GUMS analysis. Samples were analyzed using a Hewlett Packard Model 5890A series MSD GUMS. The gas chromatographic injector was operated in the splitless mode and a DB-5 capillary column (30 m, 0.25 mm i.d.) at an injection temperature of 250°C. The oven temperature began at 100°C and ramped to 300°C at ZOWmin, where the temperature was held constant. The analysis was terminated at 12 min. Acquisition parameters for the selective ion monitoring file were, first, to monitor ions 72, 86, 91, 118, and 190 until 5 min after injection. Next, ions 86, 91, 100, 160, 190, 204, 218, and 232 were monitored. The dwell time on all ions was 100 ms.

157

Immunoassays

Negative control urine collected from laboratory personnel was added to the free base equivalent of the hydrochloride or sulfate salts of the amphetamine analogues to obtain 1000 ng drug/ml. Aliquots of 3.0 ml were sent to the following manufacturers of commercial immunoassays: Roche Diagnostic Systems, Abbott Diagnostics Company, and Syva Company.* Stock solutions of the free base equivalent of the hydrochloride or sulfate salts of the amphetamine analogues (1 mglml) were prepared by adding the appropriate volume of water (approximately 10 ml) to a vial containing approximately 10 mg of the free base equivalent of the hydrochloride or sulfate salts, weighed to 0.1 mg. A working solution containing 10,000 nglml was prepared by adding 0.2 ml of the stock solution to 19.8 ml negative control urine. Samples tested (1000 nglml) were prepared by adding 0.3 ml of the working solution to 2.7 ml negative control urine and sent by overnight delivery to three manufacturers of immunoassay products. Results and Discussion Immunoassays

As shown in Table 1, commercial immunoassays failed to detect amphetamine analogues more often than not. Results were expressed as d-amphetamine (columns 1, 3, and 5) or d-methamphetamine (columns 2 and 4) equivalents. For example (column l), d-methamphetamine reacted with the Abuscreene RIA for Amphetamine to the extent that 1000 ng d-methamphetaminelml caused a result comparable to 20 ng d-amphetamine/ml, equivalent to 2% cross reactivity (or relative reactivity). Except for d-ethylamphetamine and c&l-PAB, none of the other amphetamine analogues showed more than 8% relative reactivity. If one assumes that only the d-isomer of d, Z-PAB reacts significantly with this immunoassay, its 43% cross reactivity compared with d-amphetamine could be as high as 86% for the d-isomer alone. Of the six analogues, only one (d,Z-ethylamphetamine) was recognized by all the immunoassays at greater than 10% cross reactivity. At the other extreme, none of the immunoassays recognized d-diethylamphetamine at more than 6% cross reactivity. In a comparison of immunoassays, Abbott TD,@ -Class showed the broadest selectivity. Five of the six analogues cross reacted to some extent; ddiethylamphetamine showed poor, single-digit cross reactivity with this test. The next highest number of compounds (three of five) was recognized with the Abbott TD,e -Specific. The single, most cross reactive example (261%) was observed in the Syva EMIT@ -Class with d,Lethylamphetamine, although this assay did not detect other analogues. The lowest overall recognition of amphetamine compounds other than d-amphetamine and d-methamphetamine was seen in both of the Abuscreen immunoassays. Conversely, Abuscreen showed the highest ‘SYVACompany,900 Arastradero Road, Palo Alto, CA 94303; Abbott Diagnostics Division, Dept. IL 60064; and Roche Diagnostic Systems, 11 Franklin Avenue,

9’& Bldg. -4P20, Abbott Park, Belleville, NJ 07109, U.S.A.

TABLE

1

PERCENT

CROSS REACTIVITY Roche Amph. RIA

m

(95)

OF AMPHETAMINE Roche Meth.RIA

6

ANALOGUES

Abbott TDx-Class

(88)

AT 1000 ng/ml URINE

Abbott TDx-Specific

(124)

Syva EMIT-Class

60

d-&h&nine

(137)

(125)

78

13

5

28

63

13

11

251

1

6

109

4

8

14

1

6

5

2 (g6)

0.x

d-Methanphetmine

u8

l8

126

d.l-I!-Methyl-1-pheql-2-butylonine

43

1

d,l-1-Phil-Z-butylonine

45

261

& d,l-Ethylaipktaniw 6

d.l-N-Ethyl-Fptql-2-butylmiw

cl----x dOinMiylonph&nine

&

N

d-Diethylnphetmine

’ 1

0

159

degree of specificity for the drugs intended to be detected, compared with Abbott TD,@ -Class, Abbott TD, @-Specific, and Syva EMIT@ -Class. Accuracy in detecting d-amphetamine was highest with Abuscreen RIA for Amphetamine@ (95%), lowest with Syva EMIT@ -Class (60%) and intermediate with both Abbott TD,@ assays (88% and 124%). The low EMIT@ value may have resulted from the manufacturer’s use of a d,Z-amphetamine calibration. Similarly, accuracy in detecting d-methamphetamine was highest (96%) with reporting of dAbuscreen RIA for Methamphetamine@ . Most divergent methamphetamine concentration (137%) was observed with Abbott TD,@ -Class test, followed by Syva EMIT@ -Class (126%) and Abbott TD,@ Specific (125%). The metabolism of most amphetamine analogues has not been studied extensively because they have not been introduced as pharmaceuticals. Most of the members of the amphetamine-class drugs that have been studied are excreted in urine partially as the unmetabolized parent drug [5,8] and partially as the nitrogen-dealkylated metabolite [23]. Given its structural similarity to methamphetamine, NJ-dimethylamphetamine would be expected to be metabolized, in part, to amphetamine and methamphetamine [5,8]. If this metabolism is indeed general for all these materials, some of these amphetamine analogues would appear in urine as detectable metabolites (amphetamine and/or methamphetamine). GUMS would detect these metabolites in sufficient concentration rather than the parent drug, unless the instrumentation was operated to probe for the parent drug. TABLE 2 RETENTION PHETAMINE

TIMES IN MINUTES ANALOGUES

Compound

Amphetamine Methamphetamine 1-Phenyl-2-butylamine N-Ethylamphetamine N,N-Dimethylamphetamine N-Methylphenyl-2butylamine N-Ethylphenyl-2butylamine N,N-Diethylamphetamine

OF

DERIVATIZED

AND

UNDERIVATIZED

No derivative

N-alkyl derivatives TFA

PFP

HFB

PFBzoyl

PFBzyI

3.70 4.12 4.32

4.90 5.30 5.47

4.71 5.44 5.27

4.93 5.68 5.48

8.60 9.09 8.68

7.32 7.72 *

4.43

5.88

5.64

5.89

8.85

8.01

4.54

-

4.66

5.99

5.83

6.02

*

8.10

4.99

6.36

6.19

6.35

*

8.41

5.33

-

-

-

*Unreactive under derivatizing conditions. -Tertiary amine, no derivatizable functionality.

_

AM-

160

I00

a)

\

90 80

72

fW=163

80

60 100

b) 90

\

I00

120

140

72

80 70 3 ‘u, 5 +g F ._

60 50 40

ax Fw=‘63 -L

91 72

Fig. 3. Electron impact mass spectrum of (a) d,l-N-methyl-1-phenyl-2-butylamine, dimethylamphetamine and (c) d,l-ethylamphetamine.

(b) d-

161

Gas chromatography/mass

spectrometry

Co-elution problems were observed for the underivatized isomeric group N,Nand N-methyl-1-phenyl-2-butyldimethylamphetamine, N-ethylamphetamine, amine using the final chromatographic conditions described. This is illustrated in Table 2, which shows that the retention times of the individual isomers varied from each other by as little as 0.11 min and 0.12 min when conditions were optimized. The problem is compounded by the similarity of the electron impact mass spectra of these compounds (Fig. 3a-c). Beyond this, other columns and conditions tested resulted in co-elution of derivatized isomers. Laboratories analyzing urine specimens for amphetamines may wish to conduct independent confirmation studies with this group of amphetamine analogues.* Unless properly separated during GC/MS analysis certain isomeric (and potentially uncontrolled) amphetamines could be misidentified as controlled substances. For example, methamphetamine was not separated from its structural isomer, 1-phenyl-2-butylamine unless a derivative was employed. Since GC/MS techniques used for amphetamine urinalysis usually employ derivatives, a greater chance for false identification exists with solid dosage analysis techniques that

3.0E5

Fig. 4. Total ion chromatogram

.-E B z 2

for the designer amphetamines.

*Limited quantities of amphetamine analogues are available from Dr. David A. Kidwell, Code 6177, Chemistry Division, Naval Research Laboratory, Washington, DC 20375 by submitting a formal request and license documentation.

162

perform GUMS without derivatization. Numerous derivatives were studied, as shown in Table 2. Several of the derivatives provided adequate separation by GUMS. The derivative chosen from those studied was PFP anhydride, due to its short reaction time; moderate retention times in the GUMS system employed, and acceptable chromatographic properties. A typical ion chromatogram for the mixture of derivatized and unreactive designer amphetamines at 50 ng/ml is shown in Fig. 4. The derivative used for this

91

70 x *e z? 60 2 -

50

10 0 60

80

100

120

40

160

180

200

220

160

180

200

220

10E

b)

90

3-2

80 70 x *% P 60 2

50

2 .-

40

z

30

\ 132 119 /

lGs 20 10 0

ddL 100

120

140

Mass/Charge Fig. 5. Electron impact mass spectra of pentafluoropropanyl derivatives of (a) d,l-ethylamphetamine and (b) d,l-N-methyl-1-phenyl-2-butylamine.

163

/ 204

‘9

I

160 \

/

204 I

B0 70 x .=5

60

.,g -

50

0, .z

40

‘; u =

30 20

30

60

I00

120

140

160

180

20

I00 ‘91 c)

30 60

/ 204 z .-

40

-z

30

z

65

I,’ 7 176

20

\

60

S0

I00

120

I40

160

I80

200

Mass/Charge Fig. 6. Electron impact mass spectra of pentatluoropropanyl (b) phentermine, and (c) Q-l-phenyl-2-butylamine.

derivatives of (a) d-methamphetamine,

164

chromatogram is PFP anhydride. Variation in the baseline intensity is due to different ion packets being monitored at different retention times. Poor peak shape of dimethylamphetamine and diethylamphetamine is caused by the chromatography of underivatized tertiary amines. Mass spectra resulting from the GClMS analysis of the PFP derivatives of amphetamine analogues reveal easily discernible differences between isomers. For example, the PFP derivatives of N-ethylamphetamine (Fig. 5a) and Nmethylbutyl-2-amine (Fig. 5b) are distinctive. Similarly, the mass spectra for the PFP derivatives of the isomeric pair methamphetamine and phenyl-2-butylamine are quite different, as shown in Figs. 6a,b. Conclusions If the illicit use of amphetamine analogues continues to grow, it may be important to ensure their detection. These results illustrate the potential for undetected abuse using commercial immunoassays for amphetamines, even when GC/MS confirmation is utilized. Immunoassays studied do not guarantee a detection capability. Secondly, GC/MS data for one analogue may resemble another, illustrating the possibility of misidentification. Due to variations in derivatives, instruments, and instrumental conditions, it would seem logical for laboratories to conduct independent tests rather than rely exclusively on the results of this study. Future development of an immunoassay capable of detecting amphetamine analogues and their metabolites would facilitate mass screening of urine samples. Acknowledgement The authors wish to thank Syva company, Abbott Diagnostics Company, and Roche Diagnostic Systems for performing immunoassays on drug-fortified urine specimens and the U.S. Navy Military Personnel Command for providing funding. References 1 2

3 4 5 6

Abuscreene RIA for Methamphetamine (High Specificity) product insert, Roche Diagnostics Systems, Nutley, NJ 0’7110-1199. R.A. Glennon, Synthesis and evaluation of amphetamine analogues. In M. Klein, F. Sapienza, H. McClain, Jr. and I. Khan (eds.), Clandestinely Produced Drugs, Analogues, and Precursors, US Department of Justice, Drug Enforcement Administration, Washington, 1989, pp. 39-68. L.S. Harris, The stimulants and hallucinogens under consideration: a brief overview of their chemistry and pharmacology. Drug Alcohol Depend., 17 (1986) 107-118. W.L. Woolverton, A review of the effects of repeated administration of selected phenethylamines. Drug Alcohol Depend., 17 (1986) 143-156. R.L. Foltz, A.F. Fentiman and R.B. Foltz, GCYMS Assays for Abused Drugs in Body Fluids, NIDA Research Monograph Series 32, US Govt. Print. Off., 1980, pp. 150-178. ‘Rules and Regulations’, Fed. Reg., 55, (Friday, February 2, 1990) 3586-3588.

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542-544.