Journal of Analytical Toxicology, 2016;40:12–16

doi: 10.1093/jat/bkv108
Advance Access Publication Date: 25 September 2015
Article

Article

Determination of Synthetic Cathinones in Urine

Using Gas Chromatography–Mass Spectrometry

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Techniques
Wei-Yin Hong, Ya-Chun Ko, Mei-Chih Lin, Po-Yu Wang, Yu-Pen Chen,
Lih-Ching Chiueh, Daniel Yang-Chih Shih, Hsiu-Kuan Chou, and
Hwei-Fang Cheng*
Food and Drug Administration, Ministry of Health and Welfare, Taipei, Taiwan

*Author to whom correspondence should be addressed. Email: eteredu@gmail.com

Abstract
In recent years, the abuse of synthetic cathinones has increased considerably. This study proposes a
method, based on gas chromatography/mass spectrometry (GC–MS), to analyze and quantify six
synthetic cathinones in urine samples: mephedrone (4-MMC), methylone (bk-MDMA), butylone,
ethylone, pentylone and methylenedioxypyrovalerone (MDPV). In our procedure, the urine samples
undergo solid-phase extraction (SPE) and derivatization prior to injection into the GC–MS device.
Separation is performed using a HP-5MS capillary column. The use of selective ion monitoring
(SIM mode) makes it is good sensitivity in this method, and the entire analysis process is within
18 min. In addition, the proposed method maintains linearity in the calibration curve from 50 to
2,000 ng/mL (r 2 > 0.995). The limit of detection of this method is 5 ng/mL, with the exception of
MDPV (20 ng/mL); the limit of quantification is 20 ng/mL, with the exception of MDPV (50 ng/mL).
In testing, the extraction performance of SPE was between 82.34 and 104.46%. Precision and accur-
acy results were satisfactory <15%. The proposed method was applied to six real urine samples, one
of which was found to contain 4-MMC and bk-MDMA. Our results demonstrate the efficacy of the
proposed method in the identification of synthetic cathinones in urine, with regard to the limits of
detection and quantification. This method is highly repeatable and accurate.

Introduction popular as ‘legal highs’ (2–4). A number of these derivatives have

Cathinone ((S)-2-amino-1-phenyl-1-propanone) is a naturally occur-
ring product of beta-ketone amphetamine congener, which is found
in the leaves of the Catha edulis (Khat) plant. Cathinone induces
amphetamine-like effects, including psychoactive euphoria and tachy-
cardia, and therefore belongs to the class of stimulants that affect the
central nervous system (1). New designer drugs, such as synthetic cath-
inones, are continually being developed in order to circumvent existing
legislation and avoid detection. Synthetic cathinone analogs are be-
coming increasingly common, and their derivatives, such as butylone
(bk-MBDB), dimethylcathinone, ethcathinone, ethylone (bk-MDEA),
mephedrone (4-MMC), methedrone, methylenedioxypyrovalerone
(MDPV), methylone (bk-MDMA) and pyrovalerone, have become
also been investigated for potential medical use. Bupropion is the
only synthetic cathinone currently used in the USA or Europe (2, 5).
Synthetic cathinones can be detected in blood, urine and stomach
content in both pre- and postmortem specimens. They can be charac-
terized qualitatively and quantitatively using gas chromatography–
mass spectrometry (GC–MS) or liquid chromatography–mass
spectrometry (LC–MS) techniques. Zaitsu et al. (6) established a
method to detect bk-MBDB in urine using GC–MS and LC–MS. In
2011, Frison et al. (7) developed a GC–MS method to identify
4-MMC following derivatization with 2,2,2-trichloroethyl chlorofor-
mate. In that same year, Wissenbach et al. (8) established a database of
abused drugs to facilitate the screening of urine samples using LC–MS.

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Determination of Synthetic Cathinones
Strano-Rossi conducted in vitro studies on the processing of MPDV,
using GC–MS and liquid chromatography/quadrupole time-of-flight
mass spectrometry (LC–MS-MS) (3).
A total of 32 types of ‘designer drugs’ were discovered in Taiwan
between 2004 and 2011, including ‘bath salts’ that were found to con-
tain synthetic cathinones (9). This study developed a method to quan-
tify six designer drugs in urine using GC–MS, including 4-MMC,
bk-MDMA, bk-MBDB, bk-MDEA, pentylone (bk-MBDP) and
MDPV. The chemical structures of these compounds are presented
in Figure 1. In the proposed method, the urine samples undergo deri-
vatization following solid-phase extraction (SPE). Samples are then in-
jected into the GC–MS device for analysis. We expect that this
research will benefit official drug examination techniques by providing
repeatable and accurate analytical methods that improve current limits
of detection and quantification.
Experimental
Chemicals and reagents
4-MMC, bk-MDMA, bk-MBDB, bk-MDEA, bk-MBDP, MDPV and
stable isotopically labeled internal standards (IS) mephedrone-d3,
methylone-d3, butylone-d3 and ethylone-d5 were purchased from Cer-
illiant Corporation (Austin, TX, USA). Methanol, dichloromethane,
ethyl acetate (EA), sodium hydroxide, isopropanol and ammonium
hydroxide were obtained from E. Merck. Phosphate and heptafluoro-
butyric anhydride (HFBA) were obtained from Sigma–Aldrich. Artifi-
cial human urine (certified drug negative containing 0.01% sodium
azide) was obtained from UTAK Laboratories.
GC–MS analysis
GC–MS was performed using an Agilent 6890N GC system with
7683B series autosampler, 2 μL injection, splitless mode −4 mm
liner (deactivated tapered borosilicate, glass wool), with HP-5MS col-
umn (30 m × 0.25 mm i.d., 0.25 μm film thickness) at a constant flow
(1 mL/min of helium). This was coupled to an Agilent 5975B mass se-
lective detector. The GC-MS operating parameters were as follows:
inlet temperature set at 260°C, interface temperature set at 280°C,
MS source set at 230°C and MS Quad set at 150°C EI and ionization
Figure 1. Chemical structures of mephedrone (A); methylone (B); butylone (C);
ethylone (D); pentylone (E); MDPV (F).
13
energy under 70-eV. The analytes were monitored in selective ion
monitoring (SIM) mode. The program governing GC temperature
began with an initial temperature of 140°C, where it was held for
2 min before being ramped up as follows: 140–180°C at 50°C/min,
180–195°C at 2°C/min, 195–220°C at 5°C/min and 220–320°C at
50°C/min. The final temperature was held for 2 min. The total run
time was 18 min.
Sample preparation and extraction
Drug-free urine samples were bought for use as negative controls.
Urine real samples totally stored at −20°C until use. Drug-free urine
samples were purchased from UTAK Laboratories, Inc. (Valencia,
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CA, USA).
A SPE technique was used to prepare the urine samples. This pro-
cedure was conducted as outlined in the following four steps using
Agilent SPEC® DAU:
Step 1: Conditioning involved loading 1 mL of methanol and
1 mL of water.
Step 2: Samples comprising 2 mL of urine and 1 mL of 0.1 M
phosphate buffer solution ( pH 6.0) were loaded into car-
tridges (SPEC® DAU 3 mL, Agilent Technologies).
Step 3: Sorbent was washed using 0.1 M of acetic acid and 1 mL
of methanol and subsequently dried using an air stream
for 1 min.
Step 4: Analytes were eluted in 1 mL of methylene chloride:iso-
propanol:ammonium hydroxide solution at a ratio of
80:20:2.
Derivatization of the samples for GC was performed using acylation.
The SPE elution was evaporated until dry under a gentle stream of ni-
trogen, whereupon acylation was conducted in a mixture of HFBA
(50 μL) and EA (50 μL) at 90°C for 15 min. Following derivatization,
the solution was evaporated to dryness and added 100 μL of EA. The
resulting mixture was analyzed by GC–MS with an injection volume
of 2 μL.
Extraction recovery
The drug-free were divided into two groups: A and B. In Group A, six
standards and four isotope-labeled internal standards were respective-
ly added, whereupon the samples were analyzed following SPE and de-
rivatization. In Group B, six standards were respectively added prior
to SPE extraction. Derivatization and analysis were then performed
after the addition of four isotope-labeled internal standards. B/A
(%) is calculated as the recovery rate of extraction.
Evaluation of method precision and accuracy
The efficacy of the proposed method was evaluated according to the
following criteria: limit of detection (LOD), limit of quantification
(LOQ), selectivity, carryover, accuracy and precision.
To estimate intraday and interday precision and accuracy, blank
urine samples were fortified using analytes at concentrations of 100,
500 and 1,000 ng/mL. Each concentration was repeated three times
in a single day to determine intraday precision and accuracy. The con-
centrations were also repeated on 3 consecutive days to determine in-
terday precision and accuracy.
Prior to each analytical batch, a blank was inserted to eliminate the
possible carryover. To determine the selectivity of the proposed meth-
od, we analyzed non-fortified (control) samples as well as samples for-
tified with the aforementioned six cathinones. Interfering peaks were
not observed at the retention time of some of the analytes in the
14 Hong et al.

chromatograms of the non-fortified samples. The concentration of Results and discussion

LOQ was the lowest concentration of analytes in the calibration stand-
ard. LOQ was defined as a signal-to-noise (S/N) ratio exceeding 10
and LOD was defined as an S/N ratio exceeding 3. These limits are
viewed as an acceptable degree of precision and accuracy (i.e. within
±15%) for GC–MS in urine samples.
Chromatographic analysis of cathinones
To achieve sensitivity sufficient for the simultaneous detection of
4-MMC, bk-MDMA, bk-MBDB, bk-MDEA, bk-MBDP and MDPV
in human urine, we selected the specific fragments for each compound.

Table I. Derivatization and No Derivatization SIM Mode Parameter

Compound No derivatization Derivatization

Retention time (min) Monitored ions (ratio %) Retention time (min) Monitored ions (ratio %)

Mephedrone 4.36 58, 91 (19), 119 (8) 5.38 254, 210 (36), 255 (8)

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Methylone 7.06 58, 149 (12), 121 (7) 8.81 254, 210 (45), 255 (8)
Butylone 8.10 72, 57 (15), 121 (12) 9.80 268, 210 (29), 269 (9)
Ethylone 7.88 72, 149 (16), 121 (10) 10.05 268, 240 (43), 269 (9)
Pentylone 9.57 86, 149 (20), 121 (15) 11.23 149, 282 (26), 240 (25)
MDPV 15.85 126, 127 (11), 149 (4) 15.85 126, 127 (11), 149 (4)

The italic values are quantitative ions.

Figure 2. The chromatograms of standard solutions containing (10 μg/mL) six synthetic (10 µg/mL) cathinones with HFBA derivatization (A) and without
derivatization (B).
Determination of Synthetic Cathinones 15

Table I lists the ions monitored in this study as well as the retention
time with and without derivatization. Analysis based on derivatization
(Figure 2A) provided higher sensitivity and separation than did direct
analysis (Figure 2B); therefore, this method was adopted for all subse-
quent testing. It should be noted that MDPV cannot be derivatized
using HFBA; therefore, we selected the ions found in a prototype
drug ion for SIM mode.
It proved impossible to completely separate the analytes and their
IS in the retention; therefore, we evaluated the cross-contributions of
detected ion fragments. The cross-contributions were <2%, indicating
that the selected ion fragments were suitable for both quantitative and
qualitative analyses.
Urine sample treatment
Selecting an appropriate preparation method is important for urine
analysis. This study compared two kinds of SPE for urine pretreat-
ment: Thermo HyperSep C18 3 mL and Agilent SPEC® 3 mL DAU
cartridges. Figure 3A presents chromatograms for analytes in urine
samples obtained using a Thermo HyperSep C18 SPE cartridge. As
shown in Figure 3B, the Agilent SPEC cartridge provided better recov-
ery than that of the Thermo HyperSep C18 cartridge. The Agilent
SPEC cartridge proved to be the best; therefore, this cartridge was
used for all subsequent testing of the estimated recovery rate. The re-
covery values (averaged from three measurements) ranged from 82.34
to 104.46% (Table II).

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Figure 3. The chromatograms for the separation of the six cathinones in urine samples using solid-phase extraction with Thermo HyperSep C18 (A); Agilent SPEC®
DAU (B) (100 ng/mL).

Table II. Precision and Accuracy of GC–MS Analysis

Compound Fortified concentration (ng/mL) Recovery (%, mean SD) Intraday (n = 3) Interday (n = 3)
Precision Accuracy Precision Accuracy

Mephedrone 100 95.5 ± 5.2 8.1 5.0 2.4 −2.5

500 93.0 ± 3.6 2.2 2.3 2.2 3.6
1,000 85.6 ± 10.8 2.5 −0.6 0.2 0.6
Methylone 100 95.0 ± 4.9 2.8 3.5 5.6 −3.4
500 91.2 ± 4.9 2.1 1.7 1.8 3.9
1,000 83.6 ± 10.5 2.7 −1.5 0.9 0.6
Butylone 100 95.1 ± 1.9 4.8 4.2 8.0 −3.2
500 98.5 ± 4.9 2.8 1.4 4.8 −0.8
1,000 88.5 ± 11.8 3.0 −1.0 1.8 −1.4
Ethylone 100 95.5 ± 6.2 1.8 1.8 9.3 −6.0
500 91.0 ± 5.9 2.6 1.4 6.3 0.1
1,000 84.3 ± 10.7 2.0 −1.7 1.9 0.0
Pentylone 100 99.2 ± 4.4 2.2 −2.2 11.3 −6.5
500 92.8 ± 2.6 2.7 3.5 7.2 2.2
1,000 82.3 ± 10.6 3.8 −1.1 2.4 1.4
MDPV 100 104.5 ± 9.0 5.9 −1.4 8.2 −9.5
500 95.2 ± 8.1 1.1 3.6 1.4 3.2
1,000 86.4 ± 14.8 3.6 1.2 1.0 −1.1
16
Table III. Results from GC–MS Analysis of Real Samples (ng/mL)
Sample Mephedrone Methylone
Sample 1 16,001 4,153
Sample 2 ND ND
Sample 3 ND ND
Sample 4 ND ND
Sample 5 ND ND
Sample 6 ND ND
ND, not detected; <LOQ, below the limit of quantification (LOQ).
Method validation
Selectivity
We examined the extracted ion chromatograms and retention times of
each compound in order to identify interfering peaks. For this ana-
lysis, we compared results from fortified urine samples with results
from non-fortified samples. Interfering peaks were not observed at
the retention time of the analytes in the chromatograms of non-
fortified samples.
Carryover
Initial carryover was rectified by implementing a wash program in the
autosampler. No carryover was noted during method validation (i.e.,
when solvent blanks were analyzed before and after the analysis of
samples).
LOD and LOQ
The LOD of this method is 5 ng/mL, with the exception of MDPV
(20 ng/mL) and the limit of quantification (LOQ) is 20 ng/mL, with
the exception of MDPV (50 ng/mL). These limits indicate that our
method can effectively quantify drugs in human urine. The correlation
coefficients of calibration curve for all compounds are above 0.999.
Accuracy and precision
The accuracy and precision of the proposed method were determined
using human urine samples fortified at 100, 500 and 1,000 ng/mL.
Intra- and interday results were obtained from three tests performed
on a single day and over 3 consecutive days, respectively. Both RE
and RSD results were satisfactory <15% (Table II).
Application using real urine samples
We also sought to evaluate the ability of the proposed method to iden-
tify six synthetic cathinones in routine urine tests. The real samples
were from a druggy in Taiwan. The quantitative results of this analysis
are presented in Table III. The cathinones’ cutoff value was not defined
by law; therefore, it was defined by LOQ concentrations. Sample 1 was
found to contain high concentrations of 4-MMC (16,001 ng/mL) and
bk-MDMA (4,153 ng/mL). When the samples’ concentration went be-
yond the calibration curve, we would dilute samples. In Samples 2–6,
all synthetic cathinones were below the LOQ or were undetected. This
analysis method can be used to analyze six synthetic cathinones. This
could be useful for forensic science laboratory in analyzing biological
samples containing six synthetic cathinones.
Hong et al.
Butylone Ethylone Pentylone MDPV
<LOQ ND ND <LOQ
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
Conclusions
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This study developed a method to detect six synthetic cathinones using
GC–MS with SPE sample preparation and was derivatized sensitive
enough to provide LOQs reaching lower cutoff value. The reproduci-
bility and high rate of recovery associated with the proposed method
make it suitable for the processing of large numbers of urine samples
in forensic science laboratories. In future work, we hope that the meth-
od would facilitate administration and be an important basis for the
development of policy by the government.
Funding
The authors wish to thank the Food and Drug Administration (Taipei,
Taiwan) for funding part of this work (DOH102-FDA-72023).
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