Legal Medicine xxx (2014) xxx–xxx
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Brief Communication
Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case Kiyotaka Usui ⇑, Tomomi Aramaki, Masaki Hashiyada, Yoshie Hayashizaki, Masato Funayama Division of Forensic Medicine, Department of Public Health and Forensic Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan
a r t i c l e
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Article history: Received 16 January 2014 Received in revised form 27 March 2014 Accepted 27 March 2014 Available online xxxx Keywords: 3,4-Dimethylmethcathinone Synthetic cathinones Designer drugs Drugs of abuse QuEChERS LC–MS/MS
a b s t r a c t We report here the quantitative analysis of cathinone-type designer drug 3,4-dimethylmethcathinone (3,4-DMMC) in blood and urine using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in a fatal case. Abuse of 3,4-DMMC is widespread and a global issue. However, to date, there have been no reports of 3,4-DMMC-related deaths. We encountered a death in which 3,4-DMMC was thought to play a causative role, and successfully identified this designer drug from biological samples by using LC–MS/MS and QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method. For standard samples, detection of 3,4-DMMC in human blood and urine samples in the calibration range (5–400 ng/ mL) was successful with recoveries of 85.989.4% (blood) and 95.8101% (urine), limits of detection of 1.03 (blood) and 1.37 ng/mL (urine) and limits of quantification of 5.00 (blood) and 5.38 ng/mL (urine). The concentrations of 3,4-DMMC in blood (external iliac vein) and urine in the case were 27 mg/L and 7.6 mg/L, respectively. Some metabolites, including 3,4-dimethylcathione (DMC) and b-ketone reduced metabolites (b-OH-DMMC and b-OH-DMC), were detected in both blood and urine. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Abuse of designer drugs as recreational, rave, or club drugs, is widespread. These drugs, including tryptamines, phenethylamines, cannabinoids, and cathinones, are often found in Japan. Recently, abuse of cathinone-type designer drugs has rapidly spread throughout the world. In Japan, cathinone, methcathinone, ethcathinone, 4-methylmethcathinone (mephedrone), 3,4-methylenedioxypyrovalerone (MDPV), 3,4-methylenedioxy-N-methcathinone (methylone), 3,4-methylenedioxy-N-ethylcathinone (ethylone), apyrrolidinopentiophenone (a-PVP), pyrovalerone and amfepramone are restricted by the Narcotics and Psychotropics Control Act. Furthermore, 4-methylethcathinone, 3-fluoromethcathinone, 4-fluoromethcathinone, b-keto-N-methylbenzodioxolylbutanamine (butylone), 4-methoxymethcathinone (methedrone), and many other substances are restricted as scheduled drugs by the Pharmaceutical Affairs Act. Some fatal cases involving these drugs have been reported [1–9]. Other uncontrolled drugs are also abused in Japan, and are generally sold as herbs, bath salts, aromatic powders, or incense. It is
⇑ Corresponding author. Address: Division of Forensic Medicine, Department of Public Health and Forensic Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan. Tel.: +81 22 717 8110; fax: +81 22 717 8112. E-mail address: [email protected] (K. Usui).
easy for anyone to purchase legally these substances from an illegal dealer or using an online network. Because the use of these drugs is not regulated, many acute poisonings and fatal cases have been reported in mass media. 1-(3,4-Dimethylphenyl)-2-(methylamino) propane-1-one (3,4DMMC, Fig. 1) is a cathinone-type drug controlled by the Pharmaceutical Affairs Act in Japan. According to a report released by the European Monitoring Centre for Drugs and Drug Addiction – Europol, 3,4-DMMC first appeared in Hungary in October 2010 [10]. It is now abused throughout Europe and worldwide. In the present study, we report a method for quantifying 3,4-DMMC in blood and urine and results of its application to a forensic case. 2. Materials and methods 2.1. Chemicals and reagents 3,4-DMMC monohydrochloride (>98%) was purchased from Cayman Chemical Co. (Ann Arbor, MI). Diazepam-d5 (99.9%) was purchased from Cerilliant Co. (Round Rock, TX). LC–MS grade acetonitrile and methanol were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ammonium formate was purchased from Kanto Chemical Company Inc. (Tokyo, Japan). All other chemicals were of analytical grade. A QuEChERS pre-packed extraction packet containing 6 g of magnesium sulfate (MgSO4) and 1.5 g of
http://dx.doi.org/10.1016/j.legalmed.2014.03.008 1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
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K. Usui et al. / Legal Medicine xxx (2014) xxx–xxx
O H N
Fig. 1. The structure of 3,4-dimethylmethcathinone (3,4-DMMC).
sodium acetate (CH3COONa), and a dispersive-solid phase extraction kit containing 25 mg of primary secondary amine, 25 mg of end-capped octadecylsilane (C18EC), and 150 mg of MgSO4 were purchased from Agilent Technologies, Inc. (Santa Clara, CA). Frozen human whole blood was purchased from Biopredic International (Rennes, France). Drug-free human urine was obtained from healthy volunteers with informed consent. 2.2. Preparation of standard solutions A standard stock solution of 3,4-DMMC (1 mg/mL) was prepared in ethanol and stored at 30 °C in the dark. Mixed working solutions (0.5, 1.0, 2.5, 5.0, 20, and 40 lg/mL) of 3,4-DMMC were prepared by serial dilution of the stock solution with distilled water. Calibration standards were prepared at 5, 10, 25, 50, 200, and 400 ng/mL by spiking whole blood and urine with the appropriate volume of mixed working solution. Spiked whole blood and urine solutions were stored frozen at 30 °C. Diazepam-d5 in acetonitrile at a concentration of 50 ng/mL was prepared as an internal standard (IS) and stored at 30 °C. 2.3. Analytical conditions for 3,4-DMMC 2.3.1. Extraction method Sample preparation was carried out according to the modified QuEChERS method reported by Usui et al. [11]. Briefly, 0.5 mL of whole blood or urine was diluted threefold with distilled water. The diluted sample was placed in a plastic tube with 0.5 g of the pre-packed extraction preparation, a stainless steel bead, and 1 mL of the IS (diazepam-d5) solution. Then, the mixture was vigorously shaken for 30 s by hand and centrifuged at 3000g for 5 min. The supernatant (600 lL) was transferred to a 2.0 mL centrifuge tube containing the solid-phase extraction sorbent for sample cleanup. The tube was mixed for 10 s and centrifuged at 3000g for 1 min. Next, the purified upper layer was transferred into a clean vial, and 5 lL was injected directly into the liquid chromatography–tandem mass spectrometer (LC–MS/MS). 2.3.2. LC–MS/MS conditions LC was performed with a Shimadzu Nexera LC system (Kyoto, Japan). Chromatographic separation was achieved on a Shim-pack XR-ODS III column (50 mm 2.0 mm i.d., 1.6 lm particle size; Shimadzu, Kyoto, Japan) with a UHPLC guard column (Optimize EXP™ 5 mm 2.1 mm i.d., sub 2 lm; Optimize Technologies, Inc., Oregon City, OR). The mobile phase was a mixture of 95% 10 mmol/L ammonium formate–5% methanol (solvent A) and 5% 10 mmol/L ammonium formate–95% methanol (solvent B). The sample was applied to the guard column in 20% solvent B (volume fraction) for 0.5 min. Then, the analytes were eluted with a linear gradient with the solvent B volume fraction increased from 20% to 80% over 3.5 min, followed by elution with 80% (volume fraction) solvent B for 1 min. The total run time was less than 6 min, including column equilibration. The solvent flow rate for analysis was 0.5 mL/min, and the column temperature was maintained at 50 °C. MS/MS detection was performed with a 3200 QTRAP (AB SCIEX, Framingham, MA) equipped with an electrospray ionization probe. The electrospray ionization conditions were as follows: curtain gas
(N2), 10 psi; ion spray voltage, 5500 V; ion source temperature, 500 °C; nebulizer gas (GS1), 50 psi; and turbo gas (GS2), 90 psi. The product ion spectra of the compounds were measured in multiple reaction monitoring (MRM)-enhanced product ion scan mode (EPI). In MRM-EPI mode, the product ion spectra were measured as EPI triggered by a threshold MRM signal intensity. The threshold for EPI was set to 1000 counts per second, the EPI scan range was m/z 50–750, and the collision energies were 20 V, 35 V, and 50 V. The dwell time was 150 ms for each MRM transition. Quantitative analysis was performed in MRM mode. The MS/MS transitions and the instrument parameters are listed in Table 1. MRM transition 192 > 174, which gave the strongest signal, was used for quantitation and the others were used as qualifier ions. All experiments were conducted in positive ion mode. 2.4. Analytical conditions for metabolites 2.4.1. Extraction and hydrolysis Metabolites in blood (external iliac vein) were directly extracted by the modified QuEChERS method described above. Urine (1 mL) was adjusted to pH 5.5 with acetic acid and incubated at 37 °C for 18 h with b-glucuronidase Type IX-A from Escherichia coli (Sigma–Aldrich Co., St. Louis, MO), 1675 U/mL urine. After incubation, 100 lL of hydrolyzed urine was mixed with 600 lL of methanol and centrifuged at 3000g for 5 min. The supernatant was directly injected onto the LC–MS/MS. 2.4.2. LC–MS/MS conditions for metabolites Chromatographic separation was achieved on a L-column 2 ODS (150 mm 1.5 mm i.d., 5 lm; Chemicals Inspection and Testing Institute, Tokyo, Japan) with mobile phases of 10 mmol/L ammonium formate pH 5.0 (solvent A) and methanol (solvent B). The solvent gradient increased linearly from 5% to 50% solvent B over 15 min and was maintained at 50% for 5 min. The total run time was 30 min including column equilibration. The solvent flow rate was 0.1 mL/min. The MS/MS conditions were the same as those in Section 2.3.2. 2.5. Method validation Six point (5, 10, 25, 50, 200, and 400 ng/mL) calibration curves were constructed by plotting the peak area ratio (3,4-DMMC/diazepam-d5) against the nominal concentrations of the calibration standards, and were fitted by weighted least squares linear regression with a weighting factor of 1/x2. The limit of detection (LOD) and limit of quantification (LOQ) were determined using the calibration curve. Briefly, the LOD and LOQ were determined according to the formulae: LOD = 3.29 SD/S and LOQ = 10 SD/S, where SD is the standard deviation of the y-intercept and S is the slope of the calibration curve. If the calculated LOQ values were lower than the lowest calibrator
Table 1 MS/MS transitions and instrument parameters. Analyte
Transitions
DP (v)a
EP (v)b
CEP (v)c
CE (v)d
CXP (v)e
3,4-DMMC
192 > 174 192 > 159 192 > 158 192 > 144 290 > 198
26 26 26 26 66
6.5 6.5 6.5 6.5 6
8 8 8 8 14
17 25 35 35 43
4 4 4 4 2
Diazepamd5 a b c d e
Declustering potential. Entrance potential. Collision cell entrance potential. Collision energy. Collision cell exit potential.
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
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K. Usui et al. / Legal Medicine xxx (2014) xxx–xxx
concentration based on the above formulae, then the lowest calibrator concentration (5 ng/mL) was defined as the practical LOQ. The recovery and matrix effects for each analyte were determined at three concentrations according to the method reported by Matuszewski et al. [12]. Briefly, three sets of samples were used, one consisting of neat standards (set 1), one prepared in a blank matrix extract spiked after extraction (set 2), and one spiked before extraction (set 3). The recovery and matrix effect were calculated as follows:
Table 2 Calibration range, correlation coefficients, limit of detection (LOD) and limit of quantitation (LOQ) values of the devised method (n = 9). Calibration range (ng/mL)
Whole blood r
LOD (ng/mL)
LOQ (ng/mL)
r
Urine LOD (ng/mL)
LOQ (ng/mL)
5–400
0.994
1.03
5.00
0.998
1.37
5.38
r: Correlation coefficient.
Recovery ð%Þ ¼ set 3area =set 2area 100
value of the correlation coefficients showed acceptable linearity in both blood (r > 0.994) and urine (r > 0.998). The LOD and LOQ values are also shown in Table 2. The analyte was spiked at three different concentrations into blood and urine samples, and the recoveries for these tests are summarized in Table 3. The recoveries at different analyte concentrations ranged from 85.9% to 89.4% in blood and 95.8% to 101% in urine. The matrices in blood and urine had little influence on the 3,4-DMMC measurement at all three concentrations of the analyte (5, 50, and 400 ng/mL) (Table 3). The results for intra- and interday assay precision, and the accuracy are shown in Table 3. The precision and accuracy were acceptable in both blood and urine.
Matrix effect ð%Þ ¼ set 2area =set 1area 100 The precision, expressed as the relative standard deviation, and the accuracy, expressed as relative error, were determined by analyzing the three samples at different time intervals on the same day (intra-day assay) and on three different days (inter-day assay). 3. Results and discussion 3.1. Product ion spectra of 3,4-DMMC As shown in Fig. 2, we obtained product ion spectra at three different collision energies (20, 35, and 50 V) using the protonated molecule [M+H]+ as the precursor ion.
3.4. A forensic case
3.2. MRM chromatograms
3.4.1. Case history A male in his thirties was found dead by his parents in his apartment. There was a disposable syringe beside his left arm. The police found plastic packets with the printed letters ‘‘LOOP 3’’ scattered all around the room. These packets contained a fine white powder. Research showed that LOOP 3 could be purchased by an online network. However, it was initially unclear what kind of designer drug was contained in the LOOP 3 package. Forensic investigations by the police revealed that the white powder was 3,4-DMMC. In addition to the routine drug screening, we then conducted a targeted analysis for 3,4-DMMC at our institute.
We confirmed that there were no interference peaks at the retention times of the analyte and IS by analyzing non-spiked whole blood and urine samples. The analyte and IS eluted in less than 6 min. 3.3. Method validation The validation parameters of the developed procedure for blood and urine analysis are summarized in Tables 2 and 3. The mean
174.1
Intensity, cps
2.0e6
20 V
159.1
[M+H]+ 133.2 100
105
110
115
120
125
130
135
140
145
192.2
158.0
144.1 150
155
160
165
170
175
180
185
190
195
200
205
m/z, Da 159.1
158.0
9.3e5 Intensity, cps
35 V 144.1 174.1
133.4 100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
165
170
175
180
185
190
195
200
205
m/z, Da
2.5e5 Intensity, cps
158.2
144.0
50 V 105.1 100
105
110
115
120
125
130
135
140
145
150
155
160
m/z, Da
Fig. 2. Product ion spectra of 3,4-DMMC at three different collision energies (20, 35, and 50 V).
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
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K. Usui et al. / Legal Medicine xxx (2014) xxx–xxx
Table 3 Recovery, matrix effects, precision and accuracy at three different analyte concentrations (n = 9). Spiked conc. (ng/mL)
Whole blood Recovery (%)
5 50 400
86.9 89.4 85.9
Urine ME (%)
103 100 102
Precision (RSD%)
Accuracy (RE%)
Intraday
Interday
Intraday
Interday
5.3 5.5 4.8
3.8 8.1 6.6
0.5 5.3 4.8
0.78 7.2 2.2
Recovery (%)
ME (%)
95.8 100 101
99.7 100 99
Precision (RSD%)
Accuracy (RE%)
Intraday
Interday
Intraday
Interday
3.0 6.4 7.1
2.4 8.5 4.1
0.1 1.1 0.9
0.2 1.7 1.3
RSD, relative standard deviation; RE, relative error; ME, matrix effect.
(d)
7.9
1.0e4
162
100%
OH
8
10
(e) 12
min
16
(b)
20
24
28
18.5 176
100% Intensity, cps
5
OH
min
4
50%
224 m/z
5
10
8
12
min
O
H N
3.6e4
194>176
20.9 min
16
20
24
28
224>206
15
14.7
20
160 172
100%
208
50%
HO
145
190
13.5
m/z
208>172
(c) 5
1.4e5
160
100%
10
min
O
15
20 OH
NH2
145
20.0
50%
1.1e5
[M+H]+ 178
133
4
(g)
8
178>160 12
min
16
20
24
Intensity, cps
Intensity, cps
H N
HOOC
188
(f) 194
105 130
OH
206
7.2
H N
[M+H]+
20
6.0e4
50% 161 145
222>204
15
100%
Intensity, cps
4
4.0e5
m/z
[M+H]+ 180
180>162
HOOC
204
50%
105 130 117
H N
222
119
NH2
145
145
50%
Intensity, cps
Intensity, cps
17.8
O
186
Intensity, cps
(a) 1.3e6
160
100%
OH
192
100%
192
100%
H N
12.4
174
H N
HO
50%
HO 50%
162
105
210
133 162
210 m/z
m/z
10.0
28 5
10
10.6
210>192 min
15
20
Fig. 3. MRM chromatograms and product ion spectra of metabolites detected in the decedent’s urine. (a) b-OH-DMC, (b) b-OH-DMMC, (c) DMC, (d) 3-carboxyl-4methylmethcathinone and/or 4-carboxyl-3-methylmethcathinone, (e) 3-carboxyl-4-methylephedrine (diastereomers) and/or 4-carboxyl-3-methylephedrine (diastereomers), (f) 4-hydroxymethyl-3-methylmethcathinone and 3-hydroxymethyl-4-methylmethcathinone, (g) 4-hydroxymethyl-3-methylephedrine (diastereomers) and 3hydroxymethyl-4-methylephedrine (diastereomers).
3.4.2. Autopsy findings At autopsy, there were no specific findings except for multiple needle marks observed in the decedent’s cubital fossa of both arms and the inside of both ankles. No other pathological abnormalities were noted except for congestion of abdominal organs and pulmonary edema. On the basis of the autopsy findings and the police information, the postmortem interval was estimated to be approximately 1.5 days. For toxicological analysis, we collected urine from the bladder and blood from external iliac vein to minimize the influence of the postmortem redistribution and diffusion of drugs. Samples were stored at 30 °C until analysis. No preservative agents were added to the collected samples.
3.4.3. Toxicological analysis We applied our method to the forensic case and successfully detected 3,4-DMMC in all of the specimens. We also detected desalkylflurazepam, quazepam, and its metabolite 2-oxoquazepam, but no other illegal drugs or designer drugs were found in the external iliac vein blood. The concentrations of 3,4-DMMC in the blood and urine were 27 mg/L and 7.6 mg/L, respectively. Although there have been no reports of fatal poisoning with 3,4-DMMC, several fatal poisoning cases with structurally similar cathinone-derivative drugs have been reported [1–9]. For example, Pearson et al. reported peripheral blood methylone concentrations in three fatal cases of 0.84, 3.3, and 0.56 mg/
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
K. Usui et al. / Legal Medicine xxx (2014) xxx–xxx
L, and methylone concentrations in cardiac blood of 1.0 and 0.58 mg/L [7]. Dickson et al. reported accidental death through the combined use of mephedrone, 4-methylmethcathinone, and heroin [4]. In this case, the concentrations of mephedrone in the decedent’s blood and urine were 0.50 and 198 mg/L, respectively. Adamowicz et al. also reported a fatal case of mephedrone intoxication and detected 5.5 mg/L of mephedrone in the decedent’s blood [1]. Maskell et al. also reported four deaths related to mephedrone, where the concentrations of mephedrone in femoral venous blood ranged from 0.13 to 2.24 mg/L; it should be noted that the official cause of deaths in one of these cases was vehicular collision [6]. Furthermore, Torrance et al. reported that mephedrone was detected in four fatalities [8]. The concentrations of mephedrone detected in the femoral blood of the four cases were 22, 3.3, 5.7, and 1.2 mg/L, and in two of these cases mephedrone use was suspected to be a cause of death. Wikström et al. reported two fatalities involving methedrone and 4-methoxymethcathinone [9]; the concentrations of methedrone detected in femoral blood were 8.4 and 9.6 mg/L. The blood concentration of 3,4-DMMC detected in our case was much higher than those observed in the cases involving cathinone-type drugs described above. In this forensic case, the concentration of 3,4-DMMC was higher in blood than in urine. As a syringe was found beside the left arm of the corpse, it was strongly suspected that the decedent injected 3,4-DMMC intravenously into his left arm and died relatively soon after injection. Shima et al. provided a detailed report on the metabolism of 3,4-DMMC in humans [13]. They identified three metabolites, 3,4-dimethylcathione (DMC), 1-(3,4-dimethylphenyl)-2-methylaminopropan-1-ol (b-OH-DMMC diastereomers), and 2-amino-1(3,4-dimethylphenyl)-2-methylaminopropan-1-ol (b-OH-DMC diastereomers) in urine from 3,4-DMMC users. In addition, Shima et al. detected other putative metabolites, such as glucuronide conjugates of 3,4-DMMC and glucuronide conjugates of all of the identified metabolites, as well as oxidative metabolites of the xylyl group (carboxylic and alcohol form) and reductive metabolites of the ketone group (diastereomers). In our case, we detected three peaks considered to be DMC, b-OH-DMMC, and b-OH-DMC in the decedent’s blood and urine. Furthermore, in agreement with Shima’s report, we detected the putative metabolites in the urine
5
sample (Fig. 3). However, because we could not obtain standards for these metabolites, it was difficult to accurately identify them. A more detailed investigation of the metabolites of 3,4-DMMC is required. Acknowledgments We would like to thank Dr. Noriaki Shima, of the Forensic Science Laboratory of the Osaka Prefectural Police Department, for his helpful discussions and advice regarding data analysis. This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number Grant-in-Aid for Young Scientists (B) 24790637. References [1] Adamowicz P, Tokarczyk B, Stanaszek R, Slopianka M. Fatal mephedrone intoxication – a case report. J Anal Toxicol 2013;37(1):37–42. [2] Aromatario M, Bottoni E, Santoni M, Ciallella C. New, ‘‘lethal highs’’: a case of a deadly cocktail of GHB and mephedrone. Forensic Sci Int 2012;223(1– 3):e38–41. [3] Cawrse BM, Levine B, Jufer RA, Fowler DR, Vorce SP, Dickson AJ, et al. Distribution of methylone in four postmortem cases. J Anal Toxicol 2012;36(6):434–9. [4] Dickson AJ, Vorce SP, Levine B, Past MR. Multiple-drug toxicity caused by the coadministration of 4-methylmethcathinone (mephedrone) and heroin. J Anal Toxicol 2010;34(3):162–8. [5] Kovacs K, Toth AR, Kereszty EM. A new designer drug: methylone related death. Orv Hetil 2012;153(7):271–6 (in Hungarian with English abstract). [6] Maskell PD, De Paoli G, Seneviratne C, Pounder DJ. Mephedrone (4methylmethcathinone)-related deaths. J Anal Toxicol 2011;35(3):188–91. [7] Pearson JM, Hargraves TL, Hair LS, Massucci CJ, Frazee 3rd CC, Garg U, et al. Three fatal intoxications due to methylone. J Anal Toxicol 2012;36(6):444–51. [8] Torrance H, Cooper G. The detection of mephedrone (4-methylmethcathinone) in 4 fatalities in Scotland. Forensic Sci Int 2010;202(1–3):e62–3. [9] Wikström M, Thelander G, Nystrom I, Kronstrand R. Two fatal intoxications with the new designer drug methedrone (4-methoxymethcathinone). J Anal Toxicol 2010;34(9):594–8. [10] European Monitoring Centre for Drugs and Drug Addiction, EMCDDA Europol 2010 annual report on the implementation of council decision 2005/387/JH; 2010. [11] Usui K, Hayashizaki Y, Hashiyada M, Funayama M. Rapid drug extraction from human whole blood using a modified QuEChERS extraction method. Leg. Med. (Tokyo) 2012;14(6):286–96. [12] Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS/MS. Anal Chem 2003;75(13):3019–30. [13] Shima N, Katagi M, Kamata H, Matsuta S, Nakanishi K, Zaitsu K, et al. Urinary excretion and metabolism of the newly encountered designer drug 3,4dimethylmethcathinone in humans. Forensic Toxicol 2013;31:101–12.
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
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Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Brief Communication
Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case Kiyotaka Usui ⇑, Tomomi Aramaki, Masaki Hashiyada, Yoshie Hayashizaki, Masato Funayama Division of Forensic Medicine, Department of Public Health and Forensic Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan
a r t i c l e
i n f o
Article history: Received 16 January 2014 Received in revised form 27 March 2014 Accepted 27 March 2014 Available online xxxx Keywords: 3,4-Dimethylmethcathinone Synthetic cathinones Designer drugs Drugs of abuse QuEChERS LC–MS/MS
a b s t r a c t We report here the quantitative analysis of cathinone-type designer drug 3,4-dimethylmethcathinone (3,4-DMMC) in blood and urine using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in a fatal case. Abuse of 3,4-DMMC is widespread and a global issue. However, to date, there have been no reports of 3,4-DMMC-related deaths. We encountered a death in which 3,4-DMMC was thought to play a causative role, and successfully identified this designer drug from biological samples by using LC–MS/MS and QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method. For standard samples, detection of 3,4-DMMC in human blood and urine samples in the calibration range (5–400 ng/ mL) was successful with recoveries of 85.989.4% (blood) and 95.8101% (urine), limits of detection of 1.03 (blood) and 1.37 ng/mL (urine) and limits of quantification of 5.00 (blood) and 5.38 ng/mL (urine). The concentrations of 3,4-DMMC in blood (external iliac vein) and urine in the case were 27 mg/L and 7.6 mg/L, respectively. Some metabolites, including 3,4-dimethylcathione (DMC) and b-ketone reduced metabolites (b-OH-DMMC and b-OH-DMC), were detected in both blood and urine. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Abuse of designer drugs as recreational, rave, or club drugs, is widespread. These drugs, including tryptamines, phenethylamines, cannabinoids, and cathinones, are often found in Japan. Recently, abuse of cathinone-type designer drugs has rapidly spread throughout the world. In Japan, cathinone, methcathinone, ethcathinone, 4-methylmethcathinone (mephedrone), 3,4-methylenedioxypyrovalerone (MDPV), 3,4-methylenedioxy-N-methcathinone (methylone), 3,4-methylenedioxy-N-ethylcathinone (ethylone), apyrrolidinopentiophenone (a-PVP), pyrovalerone and amfepramone are restricted by the Narcotics and Psychotropics Control Act. Furthermore, 4-methylethcathinone, 3-fluoromethcathinone, 4-fluoromethcathinone, b-keto-N-methylbenzodioxolylbutanamine (butylone), 4-methoxymethcathinone (methedrone), and many other substances are restricted as scheduled drugs by the Pharmaceutical Affairs Act. Some fatal cases involving these drugs have been reported [1–9]. Other uncontrolled drugs are also abused in Japan, and are generally sold as herbs, bath salts, aromatic powders, or incense. It is
⇑ Corresponding author. Address: Division of Forensic Medicine, Department of Public Health and Forensic Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan. Tel.: +81 22 717 8110; fax: +81 22 717 8112. E-mail address: [email protected] (K. Usui).
easy for anyone to purchase legally these substances from an illegal dealer or using an online network. Because the use of these drugs is not regulated, many acute poisonings and fatal cases have been reported in mass media. 1-(3,4-Dimethylphenyl)-2-(methylamino) propane-1-one (3,4DMMC, Fig. 1) is a cathinone-type drug controlled by the Pharmaceutical Affairs Act in Japan. According to a report released by the European Monitoring Centre for Drugs and Drug Addiction – Europol, 3,4-DMMC first appeared in Hungary in October 2010 [10]. It is now abused throughout Europe and worldwide. In the present study, we report a method for quantifying 3,4-DMMC in blood and urine and results of its application to a forensic case. 2. Materials and methods 2.1. Chemicals and reagents 3,4-DMMC monohydrochloride (>98%) was purchased from Cayman Chemical Co. (Ann Arbor, MI). Diazepam-d5 (99.9%) was purchased from Cerilliant Co. (Round Rock, TX). LC–MS grade acetonitrile and methanol were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ammonium formate was purchased from Kanto Chemical Company Inc. (Tokyo, Japan). All other chemicals were of analytical grade. A QuEChERS pre-packed extraction packet containing 6 g of magnesium sulfate (MgSO4) and 1.5 g of
http://dx.doi.org/10.1016/j.legalmed.2014.03.008 1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
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K. Usui et al. / Legal Medicine xxx (2014) xxx–xxx
O H N
Fig. 1. The structure of 3,4-dimethylmethcathinone (3,4-DMMC).
sodium acetate (CH3COONa), and a dispersive-solid phase extraction kit containing 25 mg of primary secondary amine, 25 mg of end-capped octadecylsilane (C18EC), and 150 mg of MgSO4 were purchased from Agilent Technologies, Inc. (Santa Clara, CA). Frozen human whole blood was purchased from Biopredic International (Rennes, France). Drug-free human urine was obtained from healthy volunteers with informed consent. 2.2. Preparation of standard solutions A standard stock solution of 3,4-DMMC (1 mg/mL) was prepared in ethanol and stored at 30 °C in the dark. Mixed working solutions (0.5, 1.0, 2.5, 5.0, 20, and 40 lg/mL) of 3,4-DMMC were prepared by serial dilution of the stock solution with distilled water. Calibration standards were prepared at 5, 10, 25, 50, 200, and 400 ng/mL by spiking whole blood and urine with the appropriate volume of mixed working solution. Spiked whole blood and urine solutions were stored frozen at 30 °C. Diazepam-d5 in acetonitrile at a concentration of 50 ng/mL was prepared as an internal standard (IS) and stored at 30 °C. 2.3. Analytical conditions for 3,4-DMMC 2.3.1. Extraction method Sample preparation was carried out according to the modified QuEChERS method reported by Usui et al. [11]. Briefly, 0.5 mL of whole blood or urine was diluted threefold with distilled water. The diluted sample was placed in a plastic tube with 0.5 g of the pre-packed extraction preparation, a stainless steel bead, and 1 mL of the IS (diazepam-d5) solution. Then, the mixture was vigorously shaken for 30 s by hand and centrifuged at 3000g for 5 min. The supernatant (600 lL) was transferred to a 2.0 mL centrifuge tube containing the solid-phase extraction sorbent for sample cleanup. The tube was mixed for 10 s and centrifuged at 3000g for 1 min. Next, the purified upper layer was transferred into a clean vial, and 5 lL was injected directly into the liquid chromatography–tandem mass spectrometer (LC–MS/MS). 2.3.2. LC–MS/MS conditions LC was performed with a Shimadzu Nexera LC system (Kyoto, Japan). Chromatographic separation was achieved on a Shim-pack XR-ODS III column (50 mm 2.0 mm i.d., 1.6 lm particle size; Shimadzu, Kyoto, Japan) with a UHPLC guard column (Optimize EXP™ 5 mm 2.1 mm i.d., sub 2 lm; Optimize Technologies, Inc., Oregon City, OR). The mobile phase was a mixture of 95% 10 mmol/L ammonium formate–5% methanol (solvent A) and 5% 10 mmol/L ammonium formate–95% methanol (solvent B). The sample was applied to the guard column in 20% solvent B (volume fraction) for 0.5 min. Then, the analytes were eluted with a linear gradient with the solvent B volume fraction increased from 20% to 80% over 3.5 min, followed by elution with 80% (volume fraction) solvent B for 1 min. The total run time was less than 6 min, including column equilibration. The solvent flow rate for analysis was 0.5 mL/min, and the column temperature was maintained at 50 °C. MS/MS detection was performed with a 3200 QTRAP (AB SCIEX, Framingham, MA) equipped with an electrospray ionization probe. The electrospray ionization conditions were as follows: curtain gas
(N2), 10 psi; ion spray voltage, 5500 V; ion source temperature, 500 °C; nebulizer gas (GS1), 50 psi; and turbo gas (GS2), 90 psi. The product ion spectra of the compounds were measured in multiple reaction monitoring (MRM)-enhanced product ion scan mode (EPI). In MRM-EPI mode, the product ion spectra were measured as EPI triggered by a threshold MRM signal intensity. The threshold for EPI was set to 1000 counts per second, the EPI scan range was m/z 50–750, and the collision energies were 20 V, 35 V, and 50 V. The dwell time was 150 ms for each MRM transition. Quantitative analysis was performed in MRM mode. The MS/MS transitions and the instrument parameters are listed in Table 1. MRM transition 192 > 174, which gave the strongest signal, was used for quantitation and the others were used as qualifier ions. All experiments were conducted in positive ion mode. 2.4. Analytical conditions for metabolites 2.4.1. Extraction and hydrolysis Metabolites in blood (external iliac vein) were directly extracted by the modified QuEChERS method described above. Urine (1 mL) was adjusted to pH 5.5 with acetic acid and incubated at 37 °C for 18 h with b-glucuronidase Type IX-A from Escherichia coli (Sigma–Aldrich Co., St. Louis, MO), 1675 U/mL urine. After incubation, 100 lL of hydrolyzed urine was mixed with 600 lL of methanol and centrifuged at 3000g for 5 min. The supernatant was directly injected onto the LC–MS/MS. 2.4.2. LC–MS/MS conditions for metabolites Chromatographic separation was achieved on a L-column 2 ODS (150 mm 1.5 mm i.d., 5 lm; Chemicals Inspection and Testing Institute, Tokyo, Japan) with mobile phases of 10 mmol/L ammonium formate pH 5.0 (solvent A) and methanol (solvent B). The solvent gradient increased linearly from 5% to 50% solvent B over 15 min and was maintained at 50% for 5 min. The total run time was 30 min including column equilibration. The solvent flow rate was 0.1 mL/min. The MS/MS conditions were the same as those in Section 2.3.2. 2.5. Method validation Six point (5, 10, 25, 50, 200, and 400 ng/mL) calibration curves were constructed by plotting the peak area ratio (3,4-DMMC/diazepam-d5) against the nominal concentrations of the calibration standards, and were fitted by weighted least squares linear regression with a weighting factor of 1/x2. The limit of detection (LOD) and limit of quantification (LOQ) were determined using the calibration curve. Briefly, the LOD and LOQ were determined according to the formulae: LOD = 3.29 SD/S and LOQ = 10 SD/S, where SD is the standard deviation of the y-intercept and S is the slope of the calibration curve. If the calculated LOQ values were lower than the lowest calibrator
Table 1 MS/MS transitions and instrument parameters. Analyte
Transitions
DP (v)a
EP (v)b
CEP (v)c
CE (v)d
CXP (v)e
3,4-DMMC
192 > 174 192 > 159 192 > 158 192 > 144 290 > 198
26 26 26 26 66
6.5 6.5 6.5 6.5 6
8 8 8 8 14
17 25 35 35 43
4 4 4 4 2
Diazepamd5 a b c d e
Declustering potential. Entrance potential. Collision cell entrance potential. Collision energy. Collision cell exit potential.
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
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K. Usui et al. / Legal Medicine xxx (2014) xxx–xxx
concentration based on the above formulae, then the lowest calibrator concentration (5 ng/mL) was defined as the practical LOQ. The recovery and matrix effects for each analyte were determined at three concentrations according to the method reported by Matuszewski et al. [12]. Briefly, three sets of samples were used, one consisting of neat standards (set 1), one prepared in a blank matrix extract spiked after extraction (set 2), and one spiked before extraction (set 3). The recovery and matrix effect were calculated as follows:
Table 2 Calibration range, correlation coefficients, limit of detection (LOD) and limit of quantitation (LOQ) values of the devised method (n = 9). Calibration range (ng/mL)
Whole blood r
LOD (ng/mL)
LOQ (ng/mL)
r
Urine LOD (ng/mL)
LOQ (ng/mL)
5–400
0.994
1.03
5.00
0.998
1.37
5.38
r: Correlation coefficient.
Recovery ð%Þ ¼ set 3area =set 2area 100
value of the correlation coefficients showed acceptable linearity in both blood (r > 0.994) and urine (r > 0.998). The LOD and LOQ values are also shown in Table 2. The analyte was spiked at three different concentrations into blood and urine samples, and the recoveries for these tests are summarized in Table 3. The recoveries at different analyte concentrations ranged from 85.9% to 89.4% in blood and 95.8% to 101% in urine. The matrices in blood and urine had little influence on the 3,4-DMMC measurement at all three concentrations of the analyte (5, 50, and 400 ng/mL) (Table 3). The results for intra- and interday assay precision, and the accuracy are shown in Table 3. The precision and accuracy were acceptable in both blood and urine.
Matrix effect ð%Þ ¼ set 2area =set 1area 100 The precision, expressed as the relative standard deviation, and the accuracy, expressed as relative error, were determined by analyzing the three samples at different time intervals on the same day (intra-day assay) and on three different days (inter-day assay). 3. Results and discussion 3.1. Product ion spectra of 3,4-DMMC As shown in Fig. 2, we obtained product ion spectra at three different collision energies (20, 35, and 50 V) using the protonated molecule [M+H]+ as the precursor ion.
3.4. A forensic case
3.2. MRM chromatograms
3.4.1. Case history A male in his thirties was found dead by his parents in his apartment. There was a disposable syringe beside his left arm. The police found plastic packets with the printed letters ‘‘LOOP 3’’ scattered all around the room. These packets contained a fine white powder. Research showed that LOOP 3 could be purchased by an online network. However, it was initially unclear what kind of designer drug was contained in the LOOP 3 package. Forensic investigations by the police revealed that the white powder was 3,4-DMMC. In addition to the routine drug screening, we then conducted a targeted analysis for 3,4-DMMC at our institute.
We confirmed that there were no interference peaks at the retention times of the analyte and IS by analyzing non-spiked whole blood and urine samples. The analyte and IS eluted in less than 6 min. 3.3. Method validation The validation parameters of the developed procedure for blood and urine analysis are summarized in Tables 2 and 3. The mean
174.1
Intensity, cps
2.0e6
20 V
159.1
[M+H]+ 133.2 100
105
110
115
120
125
130
135
140
145
192.2
158.0
144.1 150
155
160
165
170
175
180
185
190
195
200
205
m/z, Da 159.1
158.0
9.3e5 Intensity, cps
35 V 144.1 174.1
133.4 100
105
110
115
120
125
130
135
140
145
150
155
160
165
170
175
180
185
190
195
200
205
165
170
175
180
185
190
195
200
205
m/z, Da
2.5e5 Intensity, cps
158.2
144.0
50 V 105.1 100
105
110
115
120
125
130
135
140
145
150
155
160
m/z, Da
Fig. 2. Product ion spectra of 3,4-DMMC at three different collision energies (20, 35, and 50 V).
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
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K. Usui et al. / Legal Medicine xxx (2014) xxx–xxx
Table 3 Recovery, matrix effects, precision and accuracy at three different analyte concentrations (n = 9). Spiked conc. (ng/mL)
Whole blood Recovery (%)
5 50 400
86.9 89.4 85.9
Urine ME (%)
103 100 102
Precision (RSD%)
Accuracy (RE%)
Intraday
Interday
Intraday
Interday
5.3 5.5 4.8
3.8 8.1 6.6
0.5 5.3 4.8
0.78 7.2 2.2
Recovery (%)
ME (%)
95.8 100 101
99.7 100 99
Precision (RSD%)
Accuracy (RE%)
Intraday
Interday
Intraday
Interday
3.0 6.4 7.1
2.4 8.5 4.1
0.1 1.1 0.9
0.2 1.7 1.3
RSD, relative standard deviation; RE, relative error; ME, matrix effect.
(d)
7.9
1.0e4
162
100%
OH
8
10
(e) 12
min
16
(b)
20
24
28
18.5 176
100% Intensity, cps
5
OH
min
4
50%
224 m/z
5
10
8
12
min
O
H N
3.6e4
194>176
20.9 min
16
20
24
28
224>206
15
14.7
20
160 172
100%
208
50%
HO
145
190
13.5
m/z
208>172
(c) 5
1.4e5
160
100%
10
min
O
15
20 OH
NH2
145
20.0
50%
1.1e5
[M+H]+ 178
133
4
(g)
8
178>160 12
min
16
20
24
Intensity, cps
Intensity, cps
H N
HOOC
188
(f) 194
105 130
OH
206
7.2
H N
[M+H]+
20
6.0e4
50% 161 145
222>204
15
100%
Intensity, cps
4
4.0e5
m/z
[M+H]+ 180
180>162
HOOC
204
50%
105 130 117
H N
222
119
NH2
145
145
50%
Intensity, cps
Intensity, cps
17.8
O
186
Intensity, cps
(a) 1.3e6
160
100%
OH
192
100%
192
100%
H N
12.4
174
H N
HO
50%
HO 50%
162
105
210
133 162
210 m/z
m/z
10.0
28 5
10
10.6
210>192 min
15
20
Fig. 3. MRM chromatograms and product ion spectra of metabolites detected in the decedent’s urine. (a) b-OH-DMC, (b) b-OH-DMMC, (c) DMC, (d) 3-carboxyl-4methylmethcathinone and/or 4-carboxyl-3-methylmethcathinone, (e) 3-carboxyl-4-methylephedrine (diastereomers) and/or 4-carboxyl-3-methylephedrine (diastereomers), (f) 4-hydroxymethyl-3-methylmethcathinone and 3-hydroxymethyl-4-methylmethcathinone, (g) 4-hydroxymethyl-3-methylephedrine (diastereomers) and 3hydroxymethyl-4-methylephedrine (diastereomers).
3.4.2. Autopsy findings At autopsy, there were no specific findings except for multiple needle marks observed in the decedent’s cubital fossa of both arms and the inside of both ankles. No other pathological abnormalities were noted except for congestion of abdominal organs and pulmonary edema. On the basis of the autopsy findings and the police information, the postmortem interval was estimated to be approximately 1.5 days. For toxicological analysis, we collected urine from the bladder and blood from external iliac vein to minimize the influence of the postmortem redistribution and diffusion of drugs. Samples were stored at 30 °C until analysis. No preservative agents were added to the collected samples.
3.4.3. Toxicological analysis We applied our method to the forensic case and successfully detected 3,4-DMMC in all of the specimens. We also detected desalkylflurazepam, quazepam, and its metabolite 2-oxoquazepam, but no other illegal drugs or designer drugs were found in the external iliac vein blood. The concentrations of 3,4-DMMC in the blood and urine were 27 mg/L and 7.6 mg/L, respectively. Although there have been no reports of fatal poisoning with 3,4-DMMC, several fatal poisoning cases with structurally similar cathinone-derivative drugs have been reported [1–9]. For example, Pearson et al. reported peripheral blood methylone concentrations in three fatal cases of 0.84, 3.3, and 0.56 mg/
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
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L, and methylone concentrations in cardiac blood of 1.0 and 0.58 mg/L [7]. Dickson et al. reported accidental death through the combined use of mephedrone, 4-methylmethcathinone, and heroin [4]. In this case, the concentrations of mephedrone in the decedent’s blood and urine were 0.50 and 198 mg/L, respectively. Adamowicz et al. also reported a fatal case of mephedrone intoxication and detected 5.5 mg/L of mephedrone in the decedent’s blood [1]. Maskell et al. also reported four deaths related to mephedrone, where the concentrations of mephedrone in femoral venous blood ranged from 0.13 to 2.24 mg/L; it should be noted that the official cause of deaths in one of these cases was vehicular collision [6]. Furthermore, Torrance et al. reported that mephedrone was detected in four fatalities [8]. The concentrations of mephedrone detected in the femoral blood of the four cases were 22, 3.3, 5.7, and 1.2 mg/L, and in two of these cases mephedrone use was suspected to be a cause of death. Wikström et al. reported two fatalities involving methedrone and 4-methoxymethcathinone [9]; the concentrations of methedrone detected in femoral blood were 8.4 and 9.6 mg/L. The blood concentration of 3,4-DMMC detected in our case was much higher than those observed in the cases involving cathinone-type drugs described above. In this forensic case, the concentration of 3,4-DMMC was higher in blood than in urine. As a syringe was found beside the left arm of the corpse, it was strongly suspected that the decedent injected 3,4-DMMC intravenously into his left arm and died relatively soon after injection. Shima et al. provided a detailed report on the metabolism of 3,4-DMMC in humans [13]. They identified three metabolites, 3,4-dimethylcathione (DMC), 1-(3,4-dimethylphenyl)-2-methylaminopropan-1-ol (b-OH-DMMC diastereomers), and 2-amino-1(3,4-dimethylphenyl)-2-methylaminopropan-1-ol (b-OH-DMC diastereomers) in urine from 3,4-DMMC users. In addition, Shima et al. detected other putative metabolites, such as glucuronide conjugates of 3,4-DMMC and glucuronide conjugates of all of the identified metabolites, as well as oxidative metabolites of the xylyl group (carboxylic and alcohol form) and reductive metabolites of the ketone group (diastereomers). In our case, we detected three peaks considered to be DMC, b-OH-DMMC, and b-OH-DMC in the decedent’s blood and urine. Furthermore, in agreement with Shima’s report, we detected the putative metabolites in the urine
5
sample (Fig. 3). However, because we could not obtain standards for these metabolites, it was difficult to accurately identify them. A more detailed investigation of the metabolites of 3,4-DMMC is required. Acknowledgments We would like to thank Dr. Noriaki Shima, of the Forensic Science Laboratory of the Osaka Prefectural Police Department, for his helpful discussions and advice regarding data analysis. This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number Grant-in-Aid for Young Scientists (B) 24790637. References [1] Adamowicz P, Tokarczyk B, Stanaszek R, Slopianka M. Fatal mephedrone intoxication – a case report. J Anal Toxicol 2013;37(1):37–42. [2] Aromatario M, Bottoni E, Santoni M, Ciallella C. New, ‘‘lethal highs’’: a case of a deadly cocktail of GHB and mephedrone. Forensic Sci Int 2012;223(1– 3):e38–41. [3] Cawrse BM, Levine B, Jufer RA, Fowler DR, Vorce SP, Dickson AJ, et al. Distribution of methylone in four postmortem cases. J Anal Toxicol 2012;36(6):434–9. [4] Dickson AJ, Vorce SP, Levine B, Past MR. Multiple-drug toxicity caused by the coadministration of 4-methylmethcathinone (mephedrone) and heroin. J Anal Toxicol 2010;34(3):162–8. [5] Kovacs K, Toth AR, Kereszty EM. A new designer drug: methylone related death. Orv Hetil 2012;153(7):271–6 (in Hungarian with English abstract). [6] Maskell PD, De Paoli G, Seneviratne C, Pounder DJ. Mephedrone (4methylmethcathinone)-related deaths. J Anal Toxicol 2011;35(3):188–91. [7] Pearson JM, Hargraves TL, Hair LS, Massucci CJ, Frazee 3rd CC, Garg U, et al. Three fatal intoxications due to methylone. J Anal Toxicol 2012;36(6):444–51. [8] Torrance H, Cooper G. The detection of mephedrone (4-methylmethcathinone) in 4 fatalities in Scotland. Forensic Sci Int 2010;202(1–3):e62–3. [9] Wikström M, Thelander G, Nystrom I, Kronstrand R. Two fatal intoxications with the new designer drug methedrone (4-methoxymethcathinone). J Anal Toxicol 2010;34(9):594–8. [10] European Monitoring Centre for Drugs and Drug Addiction, EMCDDA Europol 2010 annual report on the implementation of council decision 2005/387/JH; 2010. [11] Usui K, Hayashizaki Y, Hashiyada M, Funayama M. Rapid drug extraction from human whole blood using a modified QuEChERS extraction method. Leg. Med. (Tokyo) 2012;14(6):286–96. [12] Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS/MS. Anal Chem 2003;75(13):3019–30. [13] Shima N, Katagi M, Kamata H, Matsuta S, Nakanishi K, Zaitsu K, et al. Urinary excretion and metabolism of the newly encountered designer drug 3,4dimethylmethcathinone in humans. Forensic Toxicol 2013;31:101–12.
Please cite this article in press as: Usui K et al. Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography–tandem mass spectrometry in a fatal case. Leg Med (2014), http://dx.doi.org/10.1016/j.legalmed.2014.03.008
