Conversion of Methamphetamine to N-MethyI-Methamphetamine in ...

Journal of Analytical Toxicology, Vol. 29, January/February 2005

Conversion of Methamphetamine to N-MethyI-Methamphetamine in Formalin Solutions Padma S. Tirumalai, Diaa M. Shakleya, Peter M. Gannett, Patrick S. Callery, Tina M. Bland,

and Timothy

S. Tracy*

Departmentof Basic PharmaceuticalSciences,School of Pharmacy, West Virginia University, Morgantown, West Virginia

Abstract ] Embalming is common, and yet it can create problems for the forensic scientist if a drug has been the cause of death and if this drug is also reactive toward the embalming fluid. Previous studies have focused on the amines such as nortriptyline, desipramine, and fenfluramine. In the presence of formalin, a typical component of embalming fluid, these compounds can be rapidly converted to their methylated derivatives amitriptyline, imipramine, and N-methyl-fenfluramine, respectively. We have begun a larger project designed to determine the reactivity and reactions of a wide range of drugs with formalin and have extended it to amphetamines. We report here our results from methamphetamine, which is converted into its N-methyl derivative in the presence of formalin. The rate of conversion is dependent upon pH and formalin concentration with the greatest conversion occurring under basic conditions and the highest formalin concentration. Up to 100% conversion in 24 h was observed under certain conditions. When studied in human tissue exposed to methamphetamine and treated with formalin, again, conversion to N-methyl-methamphetamine was readily apparent as early as 30 rain after exposure to formalin. Finally, we note that the reactions of methamphetamine with formalin studied here are

probably general and should be consideredwhen performing

postmortem/postembalmingforensicanalysis.

Introduction The amphetamines and related 1-phenyl-2-aminopropanes have been known as stimulants for centuries and include such natural products as ephedrine and its congeners. More recently, man-made amphetamines have been synthesized to optimize some of the pharmacologic and physicochemical properties of these agents. Methamphetamine is one such synthetic amphetamine and is a highly addictive stimulant drug, also known in various forms as "speed", "crystal meth", "crank", or "ice". Unfortunately, the abuse of me[ham* Author to whom correspondenceand reprint requestsshould be addressed:Timothy S. Tracy, Ph.D., Departmentof Experimentaland Clinical Pharmacology,College of Pharmacy, Universily of Minnesota, 7-168 Weaver-Densford Hall, 308 Harvard St. SE, Minneapolis, MN 55455. E-mail: [email protected].

48

phetamine has reached epidemic proportions, fueled in part by the relative ease of synthesis in "bathtub chemistry" labs. According to the 2000 National Household Survey on Drug Abuse an estimated 8.8 million people (4% of the U.S. population) have tried methamphetamine at some time in their lives (1). Tolerance to methamphetamine occurs rapidly, requiring higher and higher doses to achieve effects. Thus, toxic concentrations of methamphetamine may be encountered in situations of accidental overdose or suicide-type deaths. In cases where a drug has been implicated in death, postmortem autopsy samples are usually analyzed for such evidence. However, if foul play is not suspected at death, a body is typically embalmed with a formalin-based embalming fluid. Any drug present in the body at the time of death will be exposed to formalin, a highly reactive compound that may chemically react with the drug of interest. It is not uncommon to discover evidence suggesting drug involvement in a death post-embalming, presenting a challenge for forensic analysis and interpretation. Previous studies have demonstrated that formalin can react with various drugs, including amphetamine derivatives, in a time, pH and formalin concentration dependent manner resulting in the production of new chemical entities (2-7). Because the body undergoes numerous postmortem changes with respect to pH, initially being slightly acidic but then becoming alkaline as proteins begin to breakdown (8), the pH to which the drug and formalin are exposed becomes important. Furthermore, embalmers use different concentrations of formalin-containing embalming solutions depending on the condition of the body and even higher concentrations may be used by forensic examiners in preserving tissue for potential later analysis. Based on the previous studies listed, we hypothesized that formalin would react with methamphetamine, forming another chemical entity, thus complicating forensic analysis. Based on our previous work with fenfluramine (3), it was predicted that the primary reaction of formalin and methamphetamine would result in the formation of N-methyl-methamphetamine (Figure 1). This study was undertaken to provide the forensic community with information regarding the decomposition rate of methamphetamine in the presence of formalin under various pH conditions and to identify the decomposition product(s). Furthermore, chemical synthesis of authentic standards of

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N.-CH~ ~~CH

"H

NICH3 Formalin

"CH3

. ~

3

Methamphetamlne

~

CH3

N-Methyl-Melhamphetamine

Figure 1. Predicted decomposition pathway of methamphetamine in the presence of formalin. the product could provide an alternate analyte that could be used by forensic investigators and assist in the establishment of accurate conclusions.

Experimental General Methamphetamine was purchased from the Sigma Chemical Co. (St. Louis, MO). High-performance liquid chromatography (HPLC)-grade methanol and ammonium acetate were purchased from Fisher Scientific Co. (Pittsburgh, PA). All other chemicals were of the highest quality available and obtained from standard commercial sources. 1H nuclear magnetic resonance (NMR)data were collected on a Gemini 300 MHz NMR (Varian, Palo Alto, CA) in CDCI3,at room temperature, and UV spectral data were collected on a Hitachi U-2000 spectrometer (Hitachi, Schaumburg, IL). Reactions of methamphetamine with formalin A stock solution of methamphetamine (1 mg/mL) was made by dissolving the compound in water. Reactions were initiated individually by adding 20 IJL of the stock solution to 980 1JL of the appropriate reaction mixture to obtain a final methamphetamine concentration of 20 IJg/mL. The reaction mixtures consisted of 5, 10, or 20% formalin in water (no pH adjustment, ~ pH 3.5), in 10raM K2HPO4(pH 7) or in ]0raM K2HPO4 (pH 9.5). Each reaction condition was conducted in triplicate. Control reactions consisted of 20 IJg/mL methamphetamine in water, in 10mM K2HPO4(pH 7) and in 10raM K2HPO4 (pH 9.5) analyzed in a fashion analogous to the formalin reactions. Samples were analyzed by HPLC for products of decomposition immediately upon initiation of the reaction (day 0) and on days 1, 7, and 30.

Reaction of methamphetamine HCI and formalin in human liver in vitro Methamphetamine HCI (2 ~g) in water (2 I~L)was injected into the approximated center of pieces of human liver (100-150 rag). The liver pieces in centrifuge tubes were covered with 20% (v/v) formalin in water and vortex mixed briefly to give a final volume of 200 ~L. Control mixtures consisted of water in place of formalin or absence of methamphetamine HCI. The mixtures were held at room temp for 0, 0.5, 1, 2, and 24 h and 7 days. The samples were homogenized in a PotterElvejhem apparatus and sonicated for 5 min and then centrifuged at 13,000 rpm in a desk-top centrifuge for 5 min. Supernatant aliquots (10 ~L) were added to 500 ~L of 0.1% formic acid in acetonitrile for mass spectral analysis.

HPLCconditions Methamphetamine and the products of degradation were separated on a reversed-phase column (YMC-PackPro C]s, 4.6 • 150 mm) attached to a Waters Alliance 2690XE chromatographic system. The mobile phase consisting of 50raM ammonium acetate (pH 4.5)/methanol (72:28, v/v) was pumped through the column at 1.0 mL/min. Samples were directly injected onto the HPLC system and ultraviolet absorption of methamphetamine and reaction products was monitored at 251 nm. The retention times of methamphetamine and the decomposition product, N-methyl-methamphetamine,were 5.2 and 4.5 min, respectively. The correlation coefficients of standard curves for methamphetamine and N-methyl-methamphetamine (1, 2.5, 5, 10, 17.5, and 25 I~g/mLfor both analytes) were greater than 0.99 for all sample sets. Mass spectral analysis for identification of N-methyl-methamphetaminewas conducted on a Micromass ZMD mass spectrometer (MS). Chromatography conditions were identical to those described previously. Samples were analyzed in positive ion electrospray mode with the following conditions: capillary, 3.6 kV; sample cone, 33 V; and extraction cone, 4.0 V. The source block temperature was set at 100~ and desolvation temperature set at 350~ The desolvation gas flow and cone gas flow were 402 and 183 L/h, respectively.

Synthesisof N-methyl-methamphetamine (1-phenyl-2-dimethylaminopropane) Synthesis of N-methy]-methamphetamine was based on a previous synthetic method established in our laboratory (3). To a stirred suspension of 1-phenyl-2-methylaminopropane hydrochloride (methamphetamine hydrochloride) (100 rag, 0.34 mmol) in 3.0 mL acetonitrile, was added formalin (37%, 0.4 mL), sodium cyanoborohydride (40 rag, 0.64 retool, 6 eq), and sufficient glacial acetic acid to adjust the pH to 6.0. The pH of the reaction mixture was checked every 30 rain, and glacial acetic acid added as necessary to maintain the pH at 6.0. After a constant pH was obtained, the mixture was stirred overnight, quenched by the addition of 3.0 mL of water and sufficient solid sodium hydroxide to render the reaction mixture strongly basic (pH 12-14). It was then extracted with pentane (4 • 6 mL), the combined organic extracts dried (MgSO4), filtered and the pentane removed by distillation to yield 1-phenyt-2-dimethylaminopropane (73 mg, 80%) as a colorless oil. 1H NMR data were collected on a Gemini 300 MHz NMR (Varian, Palo Alto, CA) in CDC13,at room temperature: 1H NMR (CDCI3) 5 (ppm) 0.88 (3H, d, J -- 6.6 Hz), 2.75 (1H, m), 2.94 (2H, dd, J -- 4, 12.6 Hz), 7.1-7.24 (5H, m); UV (ethanol) ~ nm (log a) 260 (64), 230 (131). Spectra are reported as chemical shift in parts per million relative to tetramethylsilane along with proton integration, their multiplicity, and coupling constants.

Data analysis Data were calculated as the mean plus or minus standard deviation of the percent of methamphetamine remaining and the N-methyl-methamphetamine formed under each set of reaction conditions and at each time point.

49

Journal of Analytical Toxicology, Vol. 29, January/February 2005 Results

In the presence of formalin, methamphetamine decomposed over the course of the 30-day period in a time pH and formalin concentration dependent manner (Figures 2--4). The decomposition of methamphetamine to the predicted product, Nmethyl-methamphetamine,was observed as early as day one at both neutral and basic pH, with the most significant changes observed at basic pH and high formalin concentration. On day one, ~ 90% of the methamphetamine had decomposed in 20% formalin at pH 9.5 (Figure 2). Concentrations of N-methylmethamphetamine produced under the pH 9.5 conditions (Figure 2) were roughly inversely proportional to the measured methamphetamine concentration. Substantial decomposition of methamphetamine also occurred at pH 7.0 (Figure 3). Again, the rate of decomposition increased with increasing formalin concentration and with time. N-Methyl-methamphetamine was also generated at pH 7.0, though to a lesser degree than observed under alkaline conditions, and was approximately inversely proportional to the methamphetamine concentration measured. Conversely,no substantial changes in methamphetamine concentration were observed under pH 3.5 (pH not adjusted) conditions regardless of the time of exposure or formalin concentration suggesting stability of methamphetamine under acidic formalin conditions (Figure 4). Methamphetamine in the control reactions remained stable at all pH conditions during the entire course of the 30-day period. The production of N-methyl-methamphetamine from methamphetamine was authenticated using both HPLC with UV detection and HPLC-MS. A peak was observed at 5.3 min that eluted at the same time as authentic standard. In the case of the mass spectral analysis, this peak resulting from the pH 9.5 F o r m a l i n

~120 ]

solution

reaction mixture had a mass-to-charge ratio of 164 (M + H+), again directly corresponding to that of authentic standard. In order to determine if formalin methylates methamphetamine when the methamphetamine is in liver tissue, human liver samples were injected with an aqueous solution of methamphetamine hydrochloride. Formalin (20% aqueous 120 -

pH 7.0 Formalin solution

8 loo-

~

80-

=~ 60-

~ 40-

..= 20-

0

10

5 ---O----~----e----O--+ +

15 Days

20

25

30

35

Methamphetaminecontrol (no formalin) Metharnphetamine(5%formalin) Methamphetamine(10%formalin) Methamphetamine(20%formalin) N-Methyl-methamphetamine(no formalin) N-Methyl-methamphetamine(5% formalin) N-Methyl-methamphetamine(10%formalin) N-Methyl-methamphetamine(20%formalin)

Figure 3. Decomposition of methamphetamine and formation of N-methylmethamphetamine over 30 days in the presence of various concentrations of formalin (5, 10, or 20%) at pH 7.

No p H a d j u s t m e n t of tormalin, pH 3.5

120 1 8 loo80-

~" 6 0 -

!~" 60 4

40-

40 ~9 2 .=

//1\

"~ 20:=.=.

\

o0

5

10

15

20

25

30

35

w

0

5

Days

m.~3---8--

Methamphetaminecontrol (no formalin) Methamphetamine(5% formalin) Methamphetamine(10% formalin) Methamphetamine(20% formalin) N-Methyl-methamphetamine(no formalin) N-Methyl-methamphetamine(5% formalin) N-Methyl-methamphetamine(10% formalin) N-Methyl-methamphetamine(20%formalin)

Figure 2. Decomposition of methamphetamine and formation of N-methylmethamphetamine over 30 days in the presence of various concentrations of formalin (5, 10, or 20%) at pH 9.5.

50

-- ~ - --~'----B---'~--9 r

10

15 Days

20

25

30

35

Methamphetaminecontrol (no formalin) Methamphetamine(5% formalin) Methamphetamine(10%formalin) Methamphetamine(20%formalin) N-Methyl-methamphetamine(no formalin) N-Methyl-methamphetamine(5% formalin) NoMethyl-methamphetamine(10%formalin) NoMethyl-methamphetamine(20%formalin)

Figure 4. Decomposition of methamphetamine over 30 days in the presence of various concentrations of formalin (5, 10, or 20%) with no pH adjustment of the formalin solution (pH ~3.5).


solution) was added to the liver samples and the mixtures were allowed to stand at room temperature for up to a week. The measured pH of these tissues was pH 5.8. As early as 30 min, JVmethyl-methamphetaminewas detected by MS. Figures 5 and 6 show the multistage mass spectra of the products isolated from the liver samples containing methamphetamine hydrochloride that were treated with formalin at room temperature for seven days. Figure 5 (panels A and B) shows the electrospray mass spectrum of standard N-methyl-methamphetamine as well as the spectrum obtained from the sample of human liver tissue containing methamphetamine and reacted with formalin solution (i.e., production of N-methylmethamphetamine). Figure 6 (panels A and B) are the second stage ESI-MS2 spectra of the further fragmentation of ion m/z 164. Panels C and D in Figure 6 show the third stage ESI-MS3 spectra. The mass spectral evidence supports the conclusion that formalin penetrates liver tissue and reacts there with methamphetamine hydrochloride.

Discussion Methamphetamine is a potent central nervous system stimulant which is a popular recreational drug and in some parts of the United States has surpassed cocaine as the stimulant drug of choice. It is the inherent adverse effects of this drug of abuse which has led to its involvement in toxicological and forensic cases. In the majority of such cases, analysis of potential drugs present in the body is performed at autopsy. However, in some cases, the suspicion of foul play or overdose may ~e,

100 -

not occur until after embalming. In these situations, post-embalming drug analysis is necessary. Previous work in our laboratory (2,3,9) has demonstrated that formalin, the primary component of embalming fluid, can react with and decompose a number of drugs of forensic interest, including amphetamine analogues. Thus, studies were undertaken to assess the decomposition and reaction products of methamphetamine in the presence of varying concentrations of formalin and at various pH values. Our findings demonstrate that methamphetamine readily decomposes under basic and neutral conditions in the presence of formalin and the principal reaction product is.N-methyl-methamphetamine. The preparation of N-methyl-methamphetamine was required here for analysis of mixtures of methamphetamine and formalin. It was prepared from methamphetamine and formalin with sodium cyanoborohydride as the reducing agent. Good yields of this product were realized (80%), and it was easily purified. The analytical data (MS, ultraviolet spectrum, and NMR spectrum) are all entirely consistent with the proposed structure. The decomposition of.N-methyl-methamphetamine in the presence of formalin likely proceeds by the Eschweiler-Clarke reaction (10), which is the reductive methylation of either primary or secondary amines. The reaction mechanism has two main steps, imine formation between the amine and formalin 111

A

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