A technique combining trifluoroacetyl derivatization and gas chromatography-mass spectrometry to distinguish methamphetamine and its 4-substituted analogs

Full text of DOI:10.1002/jms.1851

JMS Letter
Received: 15 July 2010
Accepted: 20 September 2010
Published online in Wiley Online Library: 21 October 2010
(wileyonlinelibrary.com) DOI 10.1002/jms.1851
JMS Letter
Dear Sir,
(6 ml) was added to make the solution alkaline. The aqueous layer was
extractedwithdichloromethane(5 ml×2),andthecombinedorganiclayer
was washed with brine (5 ml), dried over anhydrous sodium sulfate and
concentrated. Ethereal hydrochloric acid (1 ml) was added to the residue.
The solution was allowed to stand at 5 ◦C until precipitation occurred.
The precipitate was then filtered, and the residue was washed with pre-
chilled diethyl ether (5 ml) and air dried to afford 4-FMA hydrochloride
(129 mg, 63%).
A technique combining trifluoroacetyl derivatization and gas chro-
matography–mass spectrometry to distinguish methamphetamine
and its 4-substituted analogs
The abuse of drugs has become a serious problem throughout the
world. One synthetic psychotropic drug that has been extensively
abused is methamphetamine (MA). Although MA is controlled around
the world, many newly emerging designer drugs are modified from
MA. For example, the 4-substituted psychoactive analogs of MA, 4-
methoxymethamphetamine (4-MMA) and 4-fluoromethamphetamine (4-
FMA), have recently emerged on the illicit drug market.[1–3] In addition to
4-FMA, other 4-halogenated analogs include 4-chloromethamphetamine
(4-CMA), 4-bromomethamphetamine (4-BMA) and 4-iodomethamphet-
amine (4-IMA).
So far, there have been no reports of the abuse of 4-CMA, 4-BMA,
4-IMA or 4-nitromethamphetamine (4-NMA). However, their abuse is
expected since Ledgard[4] has reported the psychotomimetic properties
of 4-CMA, 4-BMA and 4-NMA along with their synthetic processes.
Furthermore,like4-iodo-2,5-dimethoxy-phenethylamine(2C-I)and4-iodo-
2,5-dimethoxy-amphetamine (DOI), which have been widely abused as
designer drugs,[5–7] 4-IMA also has a 4-iodinated aromatic part. Because
of their psychotomimetic effects, it is expected that novel 4-substituted
analogs of MA will be encountered in the future.
4-CMA: The same procedure as that used to obtain 4-FMA hydrochloride
was carried out on 4-chlorophenylacetone (168 mg, 1 mmol), which was
used as the starting material to obtain 4-CMA hydrochloride (86 mg, 39%).
4-BMA: The same procedure as that used to obtain 4-FMA hydrochloride
was carried out on 4-bromophenylacetone (213 mg, 1 mmol), which was
used as the starting material to obtain 4-BMA hydrochloride (96 mg, 36%).
4-IMA: 4-Iodophenylacetone was prepared by refluxing 4-iodo-
phenylacetic acid (524 mg, 2 mmol) and acetic anhydride (1 ml) with
pyridine (1 ml).[12] The same procedure as that used to obtain 4-FMA
hydrochloride was carried out on 4-iodophenylacetone, which was used
as the starting material to obtain 4-IMA hydrochloride (106 mg, 17%).
4-NMA: The same procedure as that used to obtain 4-FMA hydrochloride
wascarriedouton4-nitrophenylacetone(179 mg,1 mmol),whichwasused
as the starting material to obtain 4-NMA hydrochloride (107 mg, 47%).
The synthesized compounds (4-FMA, 4-CMA, 4-BMA, 4-IMA and
4-NMA) were identified by MALDI-TOF/MS and NMR. The spectral data
are listed in Table S1 (Supporting Information). The MALDI-TOF/MS data
wereobtainedusinganAppliedBiosystemsVoyagerRPmassspectrometer
in the reflector mode with 2,5-dihydroxybenzoic acid (DHB) as the matrix.
The 1H and 13C-NMR spectra were obtained using a Varian Unity-300
spectrometer.
The GC–MS analysis of MA and six 4-substituted MAs was performed as
follows. The instrument, a Shimadzu 17-A gas chromatograph equipped
with a QP-5050A mass spectrometer (Kyoto, Japan), was operated under
the following conditions: ionization mode set to electron ionization (EI);
an ionization energy of 70 eV; helium as the carrier gas; a flow rate of
1.9 ml/min; a DB5-MS capillary column (Agilent, Santa Clara, CA, USA, 30 m
× 0.25 mm i.d., 0.25 µm film thickness); an injector temperature of 200 ◦C;
the injection mode set to the splitless mode; an oven temperature of 60 ◦C
(2-min hold) followed by an increase to 250 ◦C (3-min hold) at a rate of
20 ◦C/min; and a scan range of m/z 35–375.
The TFA derivatives of the analytes were prepared by adding 100 µl
of trifluoroacetic anhydride to 100 µl of standard solutions of MA and six
4-substitutedMAspreparedinethylacetateatfivedifferentconcentrations,
after which the mixture was reacted at 65 ◦C for 10 min. The reaction
mixture was carefully evaporated and dried under a gentle nitrogen
stream and then reconstituted in 100 µl of ethyl acetate. Subsequently,
1 µl of the aliquots were automatically injected into the GC–MS system. A
quantitative analysis was performed with N-butylbenzylamine (15 µg/ml)
as the internal standard. Linearity was examined at 1, 3, 10, 30 and
100 µg/ml for each compound using calibration curves based on the
peak-area ratios in the total ion chromatograms.
To examine the impact of the urine matrix, blank urine samples
spiked with MA and six 4-substituted MAs were extracted as follows.
Fifty microliters of N-butylbenzylamine (150 µg/ml), the internal standard,
was added to 0.5 ml of urine. The mixture was alkalinized with 5% sodium
carbonateandextractedthreetimeswith0.5 mlofisopropanol/chloroform
(1 : 3).Theorganiclayerobtainedwasevaporatedtodrynessunderastream
of nitrogen, and the residue was reconstituted in 100 µl of ethyl acetate.
Then, the same procedure as that used to obtain the TFA derivatives of
standard solutions was carried out. Subsequently, 1 µl of the aliquots was
In order to control the abuse of such MA drugs, new methods of
identifying and discriminating them must continually be developed. In
Japan, 4-MMA and 4-FMA have been controlled as designated substances
by the Pharmaceutical Affairs Law since April 2007 and January 2009,
respectively. However, the other 4-substituted analogs of MA are not
currently classified as illegal drugs. Therefore, there is a pressing need for
methods to clearly distinguish each drug.
This paper reports
a successful method that combines trifluo-
roacetyl(TFA) derivatization and gas chromatography–mass spectrometry
(GC–MS). Based on previous studies in which TFA derivatization improved
the differentiation of phenethylamine-type drugs,[1,2,5,8–10] we examined
the effects of TFA derivatization of MA and its 4-substituted analogs on the
peak shapes of their GC chromatograms and the fragmentation patterns
of their mass spectra. TFA derivatization resulted in a significant improve-
ment in the peak shapes of the chromatograms and the properties of the
mass spectra. This in turn enabled the clear differentiation of MA and six of
its 4-substituted analogs.
MA was purchased from Dainippon Pharmaceutical Co., Ltd (Osaka,
Japan). 4-MMA was obtained from Sigma–Aldrich Corp. (St Louis,
MO, USA). 4-Chlorophenylacetone, 4-bromophenylacetone and sodium
cyanoborohydride were purchased from Kanto Chemical Co., Inc. (Tokyo,
Japan).4-Fluorophenylacetone,4-nitrophenylacetone,4-iodophenylacetic
acid, 40% methylamine methanol solution and acetic anhydride were
purchased from Wako Pure Chemicals Industries, Ltd (Osaka, Japan).
Trifluoroacetic anhydride was obtained from Nacalai Tesque, Inc. (Kyoto,
Japan). Other common chemicals were purchased from commercial
sources and used without further purification.
Five 4-substituted analogs of MA (Fig. 1) were synthesized. They were
obtained by the reductive amination of 4-substituted phenylacetones in
methanolwith sodium cyanoborohydride.[11] The synthesized compounds
were ascertained by matrix-assisted laser desorption/ionization time-
of-flight mass spectrometry (MALDI-TOF/MS) and nuclear magnetic
resonance spectroscopy (NMR).
4-FMA: A mixture of 4-fluorophenylacetone (152 mg, 1 mmol), 40%
methylamine methanol solution (120 µl, 1.5 mmol), acetic acid (120 µl,
2 mmol) and sodium cyanoborohydride (95 mg, 1.5 mmol) was stirred
overnight at room temperature in methanol (3 ml). After the reaction,
methanol was evaporated in vacuo, and 2 M hydrochloric acid solution
(3 ml) was added to the residue. The aqueous solution was stirred for
0.5 h at room temperature, after which 1 M aqueous sodium hydroxide
∗
Correspondence to: M. Taniguchi , Forensic Science Laboratory, Shiga Police
Headquarters, 1-34-3, Karasaki, Otsu, Shiga 520-0106, Japan.
E-mail: tanimasa@belle.shiga-med.ac.jp
c
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.

JMS Letter
Figure 1. Structures of five 4-substituted analogs of MA synthesized in this
study: 4-fluoromethamphetamine (4-FMA), 4-chloromethamphetamine
(4-CMA), 4-bromomethamphetamine (4-BMA), 4-iodomethamphetamine
(4-IMA) and 4-nitromethamphetamine (4-NMA).
Table 1. Retention indices of MA and six 4-substituted MA analogs
with or without TFA derivatization
Compound
Free base
TFA derivative
MA
1188
1445
1199
1391
1487
1605
1669
1411
1652
1419
1607
1703
1821
1882
4-MMA
4-FMA
4-CMA
4-BMA
4-IMA
4-NMA
Figure 2. Totalionandextractedmasschromatogramsofmixedsolutionof
MA and six 4-substituted analogs of MA with or without TFA derivatization.
Peak identification: 1, MA; 2, 4-FMA; 3, 4-CMA; 4, 4-MMA; 5, 4-BMA; 6, 4-IMA
and 7, 4-NMA.
automatically injected into the GC–MS system. Seven calibration curves
were established in the range 1–100 µg/ml by mass chromatography; the
peak-area ratios of the base peak ions, m/z 154 or 121 to 91, were used for
quantitation.
MA and six 4-substituted MAs were analyzed by GC–MS with and
without TFA derivatization. The retention indices of each compound
under the abovementioned conditions are listed in Table 1. Without TFA
derivatization, all seven compounds could be separated from each other
within 10 min in the following order: MA, 4-FMA, 4-CMA, 4-MMA, 4-BMA,
4-IMA and 4-NMA (Fig. 2). However, all were eluted with somewhat tailing
peaks.
To improve the shape of the peaks, we subsequently performed the
TFA derivatization of the compounds. As shown in Fig. 2, after TFA
derivatization, all compounds could be separated within 11 min. Most
importantly, the tailing peaks of the analytes observed for the free bases
were overcome.
58, produced by an α-cleavage reaction.[13] With the exception of 4-NMA,
fragment ions corresponding to the aromatic part of the molecule were
exhibited by MA at m/z 91; 4-FMA at m/z 109; 4-CMA at m/z 125 and 127; 4-
MMA at m/z 121; 4-BMA at m/z 90 and 4-IMA at m/z 90. In the mass spectra
of 4-BMA and 4-IMA, the fragment ions at m/z 90 may be generated by an
α-cleavage benzyl bond and subsequent halogen elimination. The mass
spectraofthesevennonderivatizedcompoundswerealmostindistinguish-
able because the fragment ions derived from the aromatic part exhibited
an abundance of ∼10% or less. Therefore, the mass spectral informa-
tion was insufficient to unambiguously identify the seven nonderivatized
compounds.
Figure 3 shows the fragmentation patterns of MA and the six
4-substituted MAs. Each EI mass spectrum of the seven nonderiva-
tized compounds was characterized by the predominant ions at m/z
In previous studies, TFA derivatization has been successfully applied
to the discrimination of the phenethylamine-type drugs. Therefore, we
Table 2. Validation data based on total ion chromatograms for quantitative analysis of MA and six 4-substituted MA analogs by GC–MS after TFA
derivatization
Intra-day CV (%)b
Inter-day CV (%)b
Compound
Correlation coefficienta
1 µg/ml
10 µg/ml
100 µg/ml
1 µg/ml
10 µg/ml
100 µg/ml
MA
0.9993
0.9984
0.9984
0.9988
0.9987
0.9988
0.9898
6.2
6.7
3.1
12.5
5.1
0.7
11.8
3.0
4.3
17.1
3.0
7.0
16.3
10.8
11.4
15.5
21.4
27.7
5.8
9.7
4-MMA
4-FMA
4-CMA
4-BMA
4-IMA
4-NMA
1.2
6.2
3.4
10.3
13.5
16.7
20.7
8.7
9.5
5.1
21.1
13.8
20.4
11.9
14.6
17.5
20.7
26.9
7.6
10.6
15.2
13.2
CV, coefficient of variation.
a Linearity range: 1–100 µg/ml.
b Each value was obtained from three determinations.
c
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JMS Letter
Figure 3. EI mass spectra of nonderivatized MA and six 4-substituted analogs of MA and background at 6.5 min.
Figure 4. EI mass spectra of TFA derivatized MA and six 4-substituted analogs of MA and background at 8.0 min.
explored the TFA derivatization of MA and its analog compounds. As
shown in Fig. 4, the ions at m/z 110 and 154 were common in the mass
spectra of the seven trifluoroacetylated compounds. The diagnostic ions
derived from the substituted aromatic part, exhibited by MA-TFA at m/z 91
and 118; 4-FMA-TFA at m/z 109 and 136; 4-CMA-TFA at m/z 125, 127 and
152; 4-MMA-TFA at m/z 121 and 148; 4-BMA-TFA at m/z 169, 171, 196 and
198; 4-IMA-TFA at m/z 217 and 244 and 4-NMA-TFA at m/z 137, made it
easy to distinguish the seven trifluoroacetylated compounds. These results
show that TFA derivatization is highly effective for differentiating the mass
spectra of MA and the six 4-substituted MAs.
All quantitative analyses were performed after TFA derivatization.
The validation data for the analysis of the seven compounds after
derivatization are listed in Table 2. Good linearity of the response was
confirmed between 1 and 100 µg/ml for all compounds. As shown in
the table, the precision of the quantitative analysis by GC–MS after TFA
derivatization was satisfactory. Further, Table 3 listing the analysis results
c
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JMS Letter
Table 3. Validation data based on mass chromatograms for quantitative analysis of blank urine samples spiked with MA and six 4-substituted MA
analogs by GC-MS after TFA derivatization
Intra-day CV (%)b
Inter-day CV (%)b
Compound
Correlation coefficienta
1 µg/ml
10 µg/ml
100 µg/ml
1 µg/ml
10 µg/ml
100 µg/ml
MAc
0.9994
0.9958
0.9976
0.9952
0.9945
0.9962
0.9977
4.4
13.4
8.3
5.7
4.7
1.5
9.2
28.6
14.2
11.3
13.5
11.4
16.4
11.7
6.9
17.2
7.7
4.7
13.5
10.1
13.3
12.6
13.4
15.8
4-MMAd
4-FMAc
4-CMAc
4-BMAc
4-IMAc
4-NMAc
3.9
5.9
13.8
12.6
19.5
16.8
7.8
8.8
15.0
15.8
21.4
25.2
7.3
9.9
5.4
10.2
11.9
13.7
CV, coefficient of variation.
a Linearity range: 1–100 µg/ml.
b Each value was obtained from three determinations.
c The peak area of the base peak ion at m/z 154 was used.
d The peak area of the base peak ion at m/z 121 was used.
of blankurine samples spiked with the seven compounds proves that there
is no interference from the urine matrix.
[2] P. Ro¨snera, B. Quednowb, U. Girreserc, T. Junge. Isomeric fluoro-
methoxy-phenylalkylamines: a new series of controlled-substance
analogues (designer drugs). Forensic Sci. Int. 2005, 148, 143.
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We synthesized five 4-substituted MAs and developed a reliable
GC–MS method for the discrimination of MA and six 4-substituted MAs.
Additionally, quantitative reliability was tested for the TFA-derivatized
forms of these compounds. TFA derivatization improved the peak shape
of the chromatograms and the fragmentation patterns in the mass spectra
of all the compounds, allowing both chromatographic as well as mass
spectrometric discrimination of MA and its six 4-substituted analogs.
The proposed GC–MS technique should be especially helpful in forensic
toxicological investigations.
[4] J. B. Ledgard. ALaboratory History of Narcotics, Vol. 1: Amphetamines
and Derivatives, Jared Ledgard: United States, 2007.
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its metabolism and toxicological detection in rat urine
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Acknowledgements
We wish to thank Mr. Noboru Urushiyama of Central Research Laboratory,
Shiga University of Medical Science, for his assistance with MALDI-TOF/MS
analyses. We are indebted to Dr Noriaki Minakawa, Dr Takashi Ooi and Miss
Mariko Yuge, Institute of Health Biosciences, The University of Tokushima
Graduate School, for their technical support in NMR analyses.
[7] A. Shulgin, A. Shulgin. Pihkal, A Chemical Love Story, D. Joy (Ed.).
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Supporting information
Supporting information may be found in the online version of this article.
Yours,
MASASHI TANIGUCHI,a,b∗ YOSHIO YAMAMOTOc AND KATSUJI NISHIb
a
Forensic Science Laboratory, Shiga Police Headquarters, 1-34-3, Karasaki, Otsu,
Shiga 520-0106, Japan
b
DepartmentofLegalMedicine, ShigaUniversityofMedicalScience, SetaTsukinowa-
cho, Otsu, Shiga 520-2192, Japan
c
Laboratory of Environmental Chemistry, Iga Research Institute of Mie University,
1-3-3, Yumegaoka, Iga, Mie 518-0131, Japan
[11] R. F. Borch, M. D. Bernstein, H. D. Durst. Cyanohydridoborate anion
as a selective reducing agent. J. Am. Chem. Soc. 1971, 93, 2897.
[12] R. S. Ftank. The Clandestine Drug Laboratory Situation in the United
States. J. Forensic Sci. 1983, 28, 18.
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c
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J. Mass. Spectrom. 2010, 45, 1473–1476