Cathinones derivativesrelated deaths as exemplified

Drug Testing and Analysis

Correspondence case report Received: 25 February 2013

Revised: 26 November 2013

Accepted: 28 December 2013

Published online in Wiley Online Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1615

Cathinones derivatives-related deaths as exemplified by two fatal cases involving methcathinone with 4-methylmethcathinone and 4-methylethcathinone Sebastian Rojek,* Małgorzata Kłys, Martyna Maciów-Głąb, Karol Kula and Marcin Strona Introduction In association with sociocultural changes that have occurred in European and worldwide societies within the last decades, the consumption of amphetamine-related substances in order to achieve altered states of consciousness has become a popular practice. New stimulants with chemical structures similar to the amphetamine group – the cathinones – were synthesized in order to bypass legislation, which is constantly being updated.[1] It can be assumed that despite their illegal status, cathinone-related compounds will continue to be the prevalent drugs of abuse for the foreseeable future. They are considered ‘new designer drugs’ or ‘legal highs’, sold as ‘bath salts’ or ‘plant food’ and labelled ‘not for human consumption’. Such compounds are often sold as ‘research chemicals’ by several on-line distributors;[2] seen from the perspective of the most recent changes in the legislature, as possession of an increasing amount of new designer drugs is penalized, the term ‘legal high’ is going out of use. In view of the method and rate of producing new compounds, the term ‘research chemicals’ seems to be an apt description of newly emerging chemicals. Yet, following reports presented by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), the authors are inclined to accept the term ‘novel psychoactive substances’ (NPSs).[3] Cathinone is the principal active constituent of the khat plant (Catha edulis) responsible for the stimulant effects that have led khat to be known as a natural amphetamine.[4] Synthetic cathinones are derivatives of this compound demonstrating both amphetamine-like properties and the ability to inhibit reuptake of serotonin, causing distinct psychotropic effects. In addition to the desired effects, such as increased energy, empathy, openness, and increased libido, cathinone usage triggers adverse effects, such as cardiac, psychiatric, and neurological symptoms. Searching for new sensations leads to creating mixtures of various recreational substances; at a certain stage, the outcome becomes unpredictable and may finally lead to death.[5–8] The emergence of new substances on the worldwide market has led to the emergence of novel research fields, including analytical, clinical, and medico-legal studies that favour compilation of systemic databases. In the face of the thus formulated problem, in the present paper the authors discuss complex intoxication with substances belonging to the cathinone group as exemplified by two fatalities represented by 4-methylmethcathinone (mephedrone, 4-MMC) with

Drug Test. Analysis (2014)

methcathinone (ephedrone) poisoning in one case, and 4-methyethcathinone (4-MEC) with amphetamine intoxication in the other. In Poland, fatalities involving designer drugs that have been recorded by forensic medicine institutions are fortunately rare. Thus, the presented cases in their comprehensive form may provide a multi-parameter element that will enrich the database addressing analytical and toxicological investigations as well as medico-legal opinion given on designer drugs belonging to the cathinone group and make new contributions to knowledge in the field.

Case histories Case 1 A 29-year-old male was found in the street leaning against a local garage. As it followed from the depositions of witnesses, his lips, ears and face were bluish in color and he was foaming at the mouth. An ambulance was called for; resuscitation was initiated, which proved to be unsuccessful; and death was pronounced. His case history indicated that the man abused alcohol and additionally experimented with taking addictive substances, recently including research chemicals. Case 2 A 36-year-old male was found lying on the grass in a park, showing no signs of life. An ambulance was called for; resuscitation was initiated, which proved to be unsuccessful; and death was pronounced. His case history indicated he was a HIV and HCV carrier. For 10 years he had been on methadone drug replacement therapy. He found maintaining abstinence difficult, was in search of various alternative therapy forms, took medications, and experimented with designer drugs. A foil bag containing white powder was found by his body; subsequent analysis showed the powder to be 4-methylethcathinone (4-MEC).

* Correspondence to: Sebastian Rojek, Department of Forensic Medicine, Jagiellonian University Medical College, 31-531 Kraków, Grzegórzecka 16 Str., Poland. E-mail: [email protected] Department of Forensic Medicine, Jagiellonian University Medical College, 31-531 Kraków, Grzegórzecka 16 Str. Poland

Copyright © 2014 John Wiley & Sons, Ltd.


S. Rojek et al.

Material and methods Biological materials Post-mortem autopsy specimens – samples of femoral blood were collected in the course of the autopsy. The samples were kept frozen ( 20°C) until the analyses were performed. Other materials were not collected, therefore they were not examined. Control blood samples for development and validation of the analytical method were taken from the Regional Center of Blood Donation and Blood Treatment in Kraków. Control autopsy blood samples for development and validation of the analytical method were taken from a non-poisoned subjects.

amphetamine, methcathinone, mephedrone, and 4-MEC. Calibrator and QC working solutions were made from different source lots. All working solutions were stored at 20°C when not in use. Daily calibration samples were prepared by fortifying 0.1 mL of blank blood with known amounts of amphetamine, methcathinone, mephedrone and 4-MEC at concentrations ranging from 50 to 2000 ng/mL. Low, medium, and high QC specimens were also prepared daily at concentrations of 50, 500, 2000 ng/mL for amphetamine, methcathinone, mephedrone, and 4-MEC. For the deuterated internal standard (IS), a working solution of 1.0 ng amphetamine-d3 and mephedrone-d3 in methanol was prepared and stored at 20°C when not in use. Fifty microlitres of this working solution was added to each sample prior to extraction, giving a final deuterated IS concentration of 500 ng/mL.

Standards and chemicals The standard solutions of 6-acetylcodeine, 6-acetylmorphine, 7-aceta midoclonazepam, acetaminophen, 7-aminoclonazepam, 7-aminofl unitrazepam, 7-aminonitrazepam, acetylsalicylic acid, alpha-hydroxy alprazolam, alprazolam, aminophenazone, amisulpiride, amitriptyline, amphetamine benzoylecgonine, bk-MBDB, bk-MDEA, bro mazepam, 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-bromo-2, 5-dimethoxyphenethylamine (2CB) buprenorphine, caffeine, carbamazepine, cathinone, chlorpromazine, citalopram, clobazam, clomipramine, clomipramine, clonazepam, clozapine, cocaethylene, cocaine, codeine, codeine-6-glucuronide, cothinine, desmethylcl omipramine, desmethylclozapine, diazepam, diclofenac, 10,11-dih ydrocarbamazepine, 2,5-dimethoxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-methylamphetamine (DOM), diphenhydramine, osulepin, doxepine, drotaverine, ephedrine, ephedrone, 10,11-ep oxycarbamazepine, estazolam, 2-ethyl-1,5-dimethyl-3,3-diphenylpi rrolidine (EDDP), flephedrone, flunitrazepam, fluoxetine, flurazepam, fluvoxamine, hydrocodone, hydromorphone, hydroxyamphetamine, ibuprofen, imipramine, ketamine, ketoprofen, levomepromazine, lorazepam, lormetazepam, methadone, methamphetamine, methcath inone, methedrone, 4-methoxyamphetamine (PMA), 4-methoxyme thamphetamine (PMMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDEA), 3,4-methylenediox ymethamphetamine (MDMA), 4-methylethcathinone (4-MEC) 4-me thylmethcathinone (4-MMC, mephedrone), methylone, mianserine, midazolam, moclobemide, morphine, morphine-3-glucuronide, morphine-6-glucuronide, nicotine, nitrazepam, nitrazepam, norbupren orphine, nordiazepam, nordoxepine, norephedrine, norfluoxetine, norketamine, normorphine, O-desmethylvenlafaxine, oxazepam, ox carbazepine, oxycodone, oxymorphone, paroxetine, pentazocine, pethidine, promazine, promethazine, propranolol, pseudoephedrine, reboxetine, sertraline, sulpiride, temazepam, temazepam, thiory dazine, tramadol, venlafaxine, zaleplone, zolpidem, zopiclone and mepehdrone-d3 and amphetamine-d3 used as internal standards (IS), were purchased from LGC Standards (Warsaw, Poland), ammonium formate, ammonium carbonate, formic acid, acetic acid – 99.9% for liquid chromatography-mass spectrometry (LC–MS) were purchased from SIGMA (Poznań, Poland), acetonitrile, methanol from Fluka (Poznań, Poland). Solid-phase extraction (SPE) columns LiChrolut RP-18 (Merck, Darmstadt, Germany) containing 500 mg C18-RP bonded silica were used. For the calibrator samples, two working solutions were prepared in methanol at the following concentrations: 1.0 and 10 ng amphetamine, methcathinone, mephedrone, and 4-MEC/ μl. Also methanolic solutions were prepared for quality control (QC) samples at concentrations of 1.0 and 10 ng/μL for

wileyonlinelibrary.com/journal/dta

Analytical procedure Preliminary screening tests The screening test included an enzyme-linked immunosorbent assay (ELISA, Neogen, Ayr, Scotland, UK) of blood for amphetamine, methamphetamine, opiates, cocaine, cannabinoids, barbiturates, benzodiazepines, tricyclic antidepressants, and HPLC-DAD Tox Screening method (MTSS) by Merck (Darmstadt, Germany) for the presence of acidic, neutral, and basic drugs in the blood. The result of the ELISA test was positive for methamphetamine (Cases 1 and 2) and amphetamine (Case 2). The HPLC-DAD analysis paved a path for further analysis. Comparing the UV spectra found in postmortem specimens and records delivered by Takahashi et al.[9] suggested that it could be some cathinone derivative, which has idiosyncratic spectra. Extraction The autopsy whole blood sample (0.1 mL) was subjected to SPE. The samples were put into a clean 1.5-mL Eppendorf tubes and diluted five times with 0.1 M carbonate buffer to pH 9.3. Mephedrone-d3 and amphetamine-d3 at the concentration of 500 ng/mL were added to the samples. Afterwards, the samples were vortexed and centrifuged for 7.5 min at 1500 × g. The cartridge were firstly equilibrated with 1 mL of methanol, distilled water, and carbonate buffer pH 9.3, then the supernatants of blood samples were applied on the columns and slowly passed through. In the next step, matrix interferences were cleaned with 2 mL of carbonate buffer pH 9.3 and then vacuum was applied to the cartridges for 30 min to remove residual moisture. The analytes were eluted with 2 mL of 1 M acetic acid in methanol (1:9, v:v). The elution solvents were evaporated to dryness under nitrogen gentle stream at 40°C and the residues were resolved in 0.09 mL of mixture (95% phase A + 5% phase B) and 15 μL of biological extracts was injected into the LC-MS system. Validation of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method Selectivity To evaluate peak-purity and selectivity, blank blood samples (no analyte or IS added) were analyzed with each batch to check for peaks that might interfere with detection of amphetamine, methcathinone, mephedrone, 4-MEC or mephedrone-d3, amphetamine-d3. To assess possible interferences of other cathinones, quality control samples were spiked individually to contain 10 μg/mL of bk-MBDB, bk-MDEA,




Cathinones derivatives-related deaths ephedrone, flephedrone, cathinone, methedrone, and methylone. There were no interferences with the said compounds. Linearity, limits of quantitation and detection Calibration curves were constructed after the analysis of drug-free blood containing known amounts of amphetamine, methcathinone, mephedrone, and 4-MEC. To prepare these standards, blood samples were spiked with the studied compounds to the following concentrations: 0, 50, 100, 500, 1000, and 2000 ng/mL for blood. Each level was prepared three times. The samples were extracted according to the procedure described. Calibration curves were constructed by plotting the peak-area ratios of the analytes (methcathinone, mephedrone, 4-MEC)/internal standard (mephedrone-d3) and amphetamine/amphetamine-d3. Validation samples were prepared in triplicate at the following concentrations: 5, 10, 25, 2500, 5000, 10 000 ng/mL of amphetamine, methcathinone, mephedrone, 4-MEC to assess the accuracy of the method above and below the calibration curve. Negative quality control samples were analyzed after each linearity sample to evaluate potential carry-over. The limit of detection (LOD) of the method was determined by analyzing validation samples (n = 5) to determine if acceptance criteria were met for each analyte. The LOD was defined as the lowest concentration at which the analyte ion signal-to-noise ratio (determined by peak height) was ≥10/1, and chromatography (peak shape and resolution) and relative retention time (±2% of target RT) were acceptable. The LOQ was defined as the lowest concentration that met LOD criteria and had analyte quantification within ±20% of the target value. Accuracy and precision Inter- and intra-assay accuracy and precision data for amphetamine, methcathinone, mephedrone and 4-MEC were determined with the low, medium, and high QC samples. Intra-assay data were assessed by comparing data from within one run (n = 5) and interassay data were determined between five separate runs (n = 15). Data were evaluated using one-way analysis of variance with day as the grouping variable. Accuracy, expressed as a percentage, was calculated by taking the difference between mean calculated concentrations and target concentrations, dividing by the calculated mean and multiplying by 100. Precision, expressed as percent relative standard deviation (%R.S.D.), was determined by calculating the percent ratio of the standard deviation divided by the calculated mean concentration times 100. Extraction efficiency, matrix effect, process efficiency Extraction efficiency, matrix effect and process efficiency were evaluated via three sets of samples as described by Matuszewski et al. (n = 4 for each set).[10] Extraction efficiency for each analyte was measured at each QC concentration. Blank blood (5 different lots) was fortified with QC and IStd solution before and after SPE. Percent extraction efficiency from blood was expressed as mean analyte area of samples (n = 5) fortified with control solution before extraction divided by mean area of samples (n = 5) with control solution added after SPE. Matrix effect was assessed by comparing analyte peak areas in 10 different blank extracted blood specimens fortified with QC and IStd solutions after SPE to peak areas of samples at the same nominal concentrations prepared in an 95:5 mixture of mobile phase A and mobile phase B (neat). Matrix suppression or enhancement was calculated as follows: (100 x mean peak area of fortified blood after SPE/mean peak area of


neat) 100. Process efficiency examined the overall effect of SPE extraction efficiency and matrix effect on the quantification of analytes of interest. It was determined by comparing mean analyte peak areas of five samples fortified before SPE with mean peak areas of five neat samples prepared in mobile phase at the same concentration. Instrumentation

LC-ESI-MS/MS system

Liquid chromatography An Agilent Technologies 1200 liquid chromatograph (Santa Clara, CA, USA) equipped with a binary pump (G1312 A) and an autosampler (G1329 A) were used in gradient mode. The chromatographic separation was performed with a Poroshell 120 C18 column (100 mm x 3 mm, 2.7 μm, Agilent, Santa Clara, CA, USA). The phase A was water, which contained 0.2% formic acid and 0.002 M of ammonium formate and phase B was acetonitrile with 0.2% formic acid and 0.002 M of ammonium formate. The gradient for samples containing amphetamine, methcathinone, mephedrone, 4-MEC, and ISs was programmed as follows: 95% [A] and 5% [B] at a flow rate 0.5 mL/min, followed by a linear change to 10% [A] and 90% [B] at a flow rate of 1 mL/min in 10 min. Detection by mass spectrometry A 6410 triple quad mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), an atmospheric pressure electrospray ion (ESI) source operated under positive mode was used. The operational parameters of the ESI source were as follows: vaporizing temperature 350° C; pressure of the nebulizing gas 40 psi; flow of the drying gas 9 l/min; capillary potential 3500 V. Fragmentation parameters of the analyzed compounds are listed in Table 1.

Results The autopsies were carried out at the Department of Forensic Medicine, Jagiellonian University Medical College in Krakow, within 24 h of death. Macroscopic investigation in Case 1 revealed small skin abrasions on the posterior surface of the body, as well as such pathological lesions as splenomegaly, considerable venous congestion in the internal organs, fatty degeneration of the hepatic parenchyma, and pulmonary oedema. Microscopic investigation of formalin-fixed sections of the internal organs subjected to standard hematoxylin and eosin staining disclosed the following findings: in the cardiac muscle – vascular wall thickening, proliferation of perivascular connective tissue walls, profound congestion and disseminated small petechiae, fibre dissociation; in the lungs – congestion, foci of oedema, focally, in the lumen of the alveoli, masses of red blood cells; in the liver – stasis-associated congestion and pronounced disseminated, smalldrop fatty degeneration; in the kidney – considerable congestion; in the brain – congestion; the spleen presentation was typical of Gaucher’s disease. The above-described macro and microscopic lesions combined with the subsequent toxicology allowed for accepting death in consequence of acute circulatory and respiratory failure resulting from complex intoxication with cathinone derivatives, including mephedrone and methcathinone, in an alcohol-addicted man who was under the influence at the time of death. Macroscopic investigation in Case 2 disclosed the following findings: needle marks in the skin, pulmonary oedema,




S. Rojek et al.

Table 1. Fragmentation parameters of analyzed compounds by LCESI-MS/MS Analyte 4-MECa mephedrone methcathinone amphetamine amphetamine-d3 mephedrone-d3

+b

PIc

FVd (V)

CIDe (V)

174.1 91.0 160.1 91.1 146.1 77.1 119.1 91.1 122.1 92.1 163.1 91.1

70 70 80 80 75 75 35 35 40 40 50 50

9 41 9 41 9 53 5 17 9 17 9 41

[M+H]

192.1 192.1 178.1 178.1 164.1 164.1 136.1 136.1 139.1 139.1 181.1 181.1

a

4-methylethcathinone Parent ion c Product ion d Fragmentor voltage e Collision-induced dissociation Bold font denotes quantifier transition b

congestion of internal organs and mild atherosclerosis. Microscopic investigation of formalin-fixed sections of the internal organs subjected to standard hematoxylin and eosin staining disclosed the following findings: in the cardiac muscle – congestion, stromal loosening and hypertrophy of isolated cardiomyocytes; in the lungs – congestion, edema, a single granuloma encapsulating a foreign body, dispersed minute ‘hyaline’ reactive material; in the liver – profuse infiltrates of mononuclear cells in the enlarged portal spaces resembling the picture seen in chronic hepatitis; in the kidney – congestion; in the brain – congestion. The above-described macro and microscopic lesions and subsequent toxicology allowed for accepting death in consequence of acute circulatory and respiratory failure resulting from complex poisoning with the cathinone derivative 4-methylethcathinone (4-MEC) in an addicted individual who was under the influence of amphetamine at the time of death. The toxicological analyses were preceded by development and validation of the LC-ESI-MS/MS method of amphetamine, methcathinone, 4-methylmethcathinone, and 4-methylethcathinone determination. Validation parameters for amphetamine, methcathinone, 4-meth ylmethcathinone and 4-methylethcathinone determined by the LC-

ESI-MS/MS method were obtained. They are presented in Tables 2–4. Autopsy samples of femoral blood taken from both cases were subjected to a toxicological analysis. Toxicological findings are illustrated in Table 5. The analytical documentation of the results obtained for autopsy blood in both cases is presented in Figures 1 and 2.

Discussion The habit of chewing khat by the inhabitants of Eastern Africa and Arabian Peninsula has a long history. It is estimated that in our times, there are five to ten million regular khat users.[4] Identification of active substances in khat led Wolfes[11] to establish the presence of norpseudoephedrine (cathine), and only in 1975, was cathinone identified.[12] Synthetic cathinones have recently emerged and grown to be popular drugs of abuse. As the chemical structure of cathinone is similar to that of amphetamine and it produces an amphetaminelike effect, so the mechanism underlying the activity of both compounds is based on the release of neurotransmitters at the catecholaminergic synapses, especially at the dopaminergic and serotonergic synapses.[4] In relatively low doses, some of the cathinones have an entactogenic effect – they boost the mood, evoke a need to talk with other people, and promote emotional openness. Nevertheless, as it follows from Kalix’s investigations,[13] cathinone is three times less potent than amphetamine in causing this release, while studies carried out by Glennon and Liebowitz[14] indicate that affinity for serotonin receptors by cathinone was found to be about four times higher than that of amphetamine. The main difference between cathinones and their amphetamine equivalents lies in the presence of the ketone group in the beta position (beta-ketones). The ketone group is responsible for increased solubility of cathinone as compared to amphetamines and by the same token, it hinders crossing the blood-brain barrier, what in turn should be manifested by a decreased activity potential.[1] According to Simmler, cathinone, methcathinone, and flephedrone, similarly to amphetamine and methamphetamine, acted as preferential dopamine and noradrenaline uptake inhibitors and induced the release of dopamine.[15] On the other hand, pyrovalerone and 3,4-methylenedioxypyrovalerone (MDPV) are highly potent and selective dopamine and noradrenaline transporter inhibitors, but unlike amphetamines do not evoke the release of monoamines. The high potency at the noradrenaline transporter and dopamine transporter is more likely to be responsible for the

Table 2. Calibration curves parameters Analyte amphetamine 4-MECa mephedrone methcathinone

Regression equation of calibratorsb y=5.561x-0,079 y=1.183x-0.004 y=1.166x-0.013 y=0.596x+0.025

Rc

LODd (ng/ml)

LOQe (ng/ml)

LOLf (ng/ml)

0.9996 0.9986 0.9993 0.9953

25

50

50-2000

a

4-methylethcathinone n=3 c Correlation coefficient d Limit of detection e Limit of quantitation f Limit of linearity b





Cathinones derivatives-related deaths Table 3. The mean accuracy and precision of the developed for the determination of analytes in blood by LC-ESI-MS/MS Concentration of analytes in blood (ng/ml) amphetamine 50 500 2000 4-MECa 50 500 2000 mephedrone 50 500 2000 methcathinone 50 500 2000

Intra - day (n=5) b

Accuracy (%)

Inter - day (n=15) c

Precision (RSD%)

Accuracyb (%)

Precisionc (RSD%)

2.3 0.2 1.8

2.5 1.4 2.8

6.4 1.7 1.8

2.8 2.9 2.6

8.4 4.0 5.1

10.2 6.3 4.0

17.2 0.2 9.1

12.2 16.8 4.9

2.1 2.3 0.7

0.5 1.9 1.5

3.7 1.9 1.4

5.0 1.5 2.1

15.6 2.4 7.9

12.4 2.7 4.9

17.6 9.9 3.2

12.2 10.8 11.3

a

4-methylethcathinone Percent difference between mean and target concentration c Percent relative standard deviation b

psychotropic effects at low doses in humans. Simmler found that the potencies to inhibit the norepinephrine transporter and dopamine transporter were significantly correlated with the doses reported to produce psychotropic effects in recreational users.[15] Based on descriptions provided by users of cathinone derivatives, it may be concluded that there is an entire spectrum of adverse somatic effects, such as tachycardia, elevated arterial blood pressure, trismus, nystagmus, sweating, headache and dizziness, hand tremor, nausea, and in extreme cases also seizures, convulsions, and respiratory problems. Mental symptoms have been additionally described, such as short-term memory disturbances, flight of ideas and lapse in concentration, irritability, insomnia, anxiety that may progress to paranoid states, mood swings, dysphoria and depressive states, and hallucinations.[1,7,16–18] Generally, investigations of designer drugs concentrate on identification of components of complex preparations based on analytical procedures developed concurrently and on determining the content of the said compounds in body fluids and tissues collected from poisoned and deceased individuals; their purpose is compilation of toxicological databases. In this paper, three cathinones derivatives are involved: methcathinone, mephedrone, and 4-MEC. Synthesis of cathinone derivatives has been reported since the late 1920s. Methcathinone was synthesized in 1928 in Germany. Attempts were made to use the compound – as many other cathinone derivatives – for medicinal purposes, for example in

Table 4. Matrix effect, extraction efficiency and process efficiency Compound

amphetamine

mephedrone

4-MECa

methcathinone

amphetamine-d3

mephedrone-d3

QC (ng/ml)

50 500 2000 50 500 2000 50 500 2000 50 500 2000 50 500 2000 50 500 2000

Absoluteb

Matrix effect Relativec

Extraction efficiencyd (%)

Process efficiencye (%)

19.3 24.0 115 83.0 54.0 15.0 80.7 31.0 56.6 88.9 59.4 131 10.0 19.0 81.0 83.0 52.0 43.0

0.81 1.24 2.15 0.17 0.46 0.85 0.19 0.69 1.57 11.1 0.41 2.31 0.90 1.19 1.81 0.17 0.48 0.57

85.2 97.6 94.3 131 65.7 63.1 112 46.5 48.1 97.9 56.6 36.6 90.0 95.2 91.1 128 65.2 95.6

68.8 121 203 23.0 30.0 53.0 21.6 32.1 75.3 10.9 23.0 84.6 80.9 113 165 21.5 31.5 54.6

a4

-methylethcathinone Matrix effect (absolute) = matrix suppression or enhancement was calculated as follows: (100 x mean peak area of fortified plasma after SPE/ mean peak area of neat) 100 c Matrix effect (relative) was assessed by by comparing analyte peak areas in blank extracted blood specimens fortified with QC and IStd solutions after SPE to peak areas of samples at the same nominal concentrations prepared in an 95:5 mixture of mobile phase A and mobile phase B (neat) d Extraction efficiency was expressed as mean analyte area of samples fortified with control solution before extraction divided by mean area of samples with control solution added after SPE e Process efficiency was determined by comparing mean analyte peak areas of samples fortified before SPE with mean peak areas of neat samples prepared in mobile phase at the same concentration b





S. Rojek et al.

Table 5. Toxicological findings in autopsy blood in fatal cases of poisoning Case No. 1 2

Cathinones

methcathinone mephedrone 4-MEC

Concentration (ng/ml) 210 1300 1200

Other xenobiotics ethanol - 2.80 ‰ amphetamine – 230 ng/ml

Russia as an antidepressant in the 1930s and 1940s.[2] Later data originating from Russia and dating back to the 1960s indicate that many methcathinone addicts suffer permanent brain damage and exhibit Parkinson-like symptoms. Findings from investigations carried out in the US suggest that methcathinone is highly addictive.[4] Prominent findings of acute toxicity include hallucinations, fever, and tachycardia followed by periods of bradycardia and moderate hypotension as acute symptoms resolve. Chronic binge use may result in development of paranoid psychosis and brief periods with withdrawal tremors.[19] The synthesis of mephedrone was first described in 1929 by Saem de Burnaga Sanchez. The first online mention of mephedrone synthesis seems to have appeared around 2003, but forum chatter about the substance as a recreational drug seems to have really begun in 2007. Mephedrone appeared as capsules made by Neorganics in Israel. Today it seems to be the fourth most commonly used drug after cannabis, ecstasy, and cocaine[20] and at the same time the most widely experienced legal high. Mephedrone is usually available as a powder or tablet so it can be used orally, by means of nasal insufflation, in intramuscular and intravenous injections and in rectal insertion. Because of its physical characteristics (instability), mephedrone is not suitable for smoking. There are report studies that mephedrone is

sometimes used in conjunction with alcohol or controlled substances.[21] The information on the acute toxicity (harm) associated with the use of mephedrone collected by Wood et al., suggest that the pattern of acute toxicity is similar to that previously described for amphetamine/MDMA.[22] A synthetic stimulant 4-MEC has an extremely short history of human use. It became available online in 2010, sold as a replacement for mephedrone, and has since appeared in some samples of NRG-2.[23] The effects of 4-methylethcathinone are often compared to those of 4-methylmethcathinone with less euphoria. In contrast to new medications which are usually extensively studied with respect to their metabolism prior to their introduction to the market, designer drugs are consumed without any safety testing. Information on their metabolism in humans, as well as the knowledge of the activity and toxicity of their metabolites, which usually greatly help in investigating the cause of acute intoxication in forensic and clinical toxicology, is incomplete or even not available at all.[7] An increasingly serious challenge faced by clinical toxicologists is their awareness of the fact that the majority of commonly used designer drugs cannot be possibly detected in routine in-patient toxicological diagnostic management, especially when medical history is uncertain or incomplete, thus rendering physical examination the basic tool in the diagnostic process. ŁukasikGłębocka et al.[24] draw attention to the poorly characteristic symptomatology of intoxications in patients examined with respect to designer drugs usage. The above investigators studied a group of six individuals and observed mainly symptoms involving the central nervous system (mental confusion, hallucinations, agitation, anxiety, phobias, logorrhea, weakness, dizziness and somnolence) and the circulatory system (hypertension, tachycardia, chest pain) and dilated pupils. Although contemporary analytics allows for determination of designer drugs in various biological

Figure 1. Selected reaction monitoring chromatograms of mephedrone, methcathinone and mephedrone-D3 (IS) from extracted autopsy blood by LCESI-MS-MS in the case 1.





Cathinones derivatives-related deaths

Figure 2. Selected reaction monitoring chromatograms of 4-MEC, amphetamine, mephedrone-D3 (IS) and amphetamine-D3 (IS) from extracted autopsy blood by LC-ESI-MS-MS in the case 2.

and non-biological matrices using highly sensitive and highly specific instrumental methods, chiefly GC-MS and LC-MS/MS, yet, in a clinical (emergency) setting, the procedure is of a low usefulness due to the lengthy process and high costs.[6] In view of the above information, the history and comprehensive post-mortem examinations performed in the described cases of fatal intoxication in the two male victims indicate accidental deaths with the underlying mechanism of acute circulatory and respiratory failure, most likely resulting from disturbances in serotonin and dopamine transport in consequence of experimenting with narcotic substances that was directed towards designer drugs. In Case 1, as it follows from the medical history and the testimony of family, the man was supposed to be addicted to legal highs, among them to methcathinone and alcohol. Most likely, in order to potentiate the effect, he took a high dose of mephedrone that resulted in death. High blood mephedrone concentration levels were demonstrated, amounting to 1300 ng/mL. According to the publications on the subject, mephedrone shows a wide range of blood concentration values in various cases. In some of the cases under consideration, it was detected alone, but in the majority of other instances - in various combinations, including with ethanol.[20,21] The case discussed in the present report supports this observation; mephedrone, methcathinone and ethanol, as well as their concentration values found in blood are within the range noted by other investigators. The interaction between ethanol and mephedrone is not yet known. Therefore, we can only assess the risk based on classic non-b-keto analogue stimulants. Some studies show that MDMA and methamphetamine in combinations with alcohol can diminish the subjective feelings of alcohol intoxication. At the same time, a combination of methamphetamine and alcohol may increase cardiac toxicity. From an analytical point of view, it is suggested that alcohol may slightly increase the plasma level of ecstasy, while alcohol levels may be slightly reduced by ecstasy.[25]


Several cases of fatal poisonings with mephedrone were reported recently in Scotland and published by Torrance et al.,[26] with the range of concentrations in femoral blood of 1200–2200 ng/mL; Maskell et al.[27] determined the range of 130–2200 ng/mL. Some cases involved a combined use of mephedrone with other drugs, such as cannabis[8] and heroin.[28] In one described case of non-fatal poisoning, 150 ng/mL was determined and the dose taken orally was 0.2 g and later, per an intramuscular injection – another 3.8 g.[29] Other reports describe mephedrone conjunction with ketamine.[20] In Case 2, the man took a high dose of 4-MEC. As it follows from the medical history, he was addicted to amphetamine and previously to opiates and for this reason, he participated in methadone drug replacement therapy for 10 years. The analysis of his post-mortem blood samples did not demonstrated methadone, but did demonstrate amphetamine levels similar to those encountered in individuals addicted to the xenobiotic. The profile of opioid addiction is frequently associated with amphetamine. Addicts participating in methadone drug replacement therapy are to a considerable degree able to maintain abstinence from opioids, but not from amphetamine, what was demonstrated in our earlier studies.[30] An experiment consisting in taking a high dose of a ‘research chemical’ resulted in death; post-mortem blood samples showed a high concentration of the research chemical amounting to 1200 ng/mL, similarly as in the fatality involving mephedrone. However, the literature on the subject lacks reports on fatal cases involving this xenobiotic. Investigations carried out in the field of designer drugs concentrate on identification of their components based on analytical procedures developed concurrently and on determination of the content of these preparations in body fluids and tissues of intoxicated individuals. Modern analytics allows for determination of designer drugs in various biological and non-biological




S. Rojek et al.

matrixes by very sensitive and highly specific instrumental methods, predominantly GC-MS, GC-MS/MS, LC-MS, LC-MS/MS, and LC-MS-TOF. Based on the medico-legal practice of the authors, it can be inferred that abusing narcotic substances poses social dangers associated with crimes committed while under the influence (robberies, rapes, homicides) as well as with traffic road accidents. An easy accessibility of designer drugs for young people leads to damages that are difficult to assess in their social aspect, such as destruction of personality, mental diseases, degradation of social ties and social marginality. Analyzing the problem of designer drugs in its broader aspect, one may perceive two opposite directions of activities in this field. On the one hand, we have an unlimited inventiveness of designers of an ever increasing spectrum of new psychoactive xenobiotics that are very much in demand, especially on the Internet, thus fuelling the financial machine. On the other hand, there emerge social-legal and educational initiatives addressing the issues of public health and emergency management of intoxications; such initiatives constitute significant elements of the strategy of societal development in the contemporary world.

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