Purpose The 4-chloromethcathinone (4-CMC) is a synthetic derivative of cathinone and belongs to new psychoactive substances. Neither data on the effects of 4-CMC on the human body, nor on nontoxic, toxic and lethal concentrations in biological materials have been published in the literature. This paper describes the results of an analysis of the blood con- centrations of 4-CMC determined in 15 forensic cases related to nonfatal intoxication including driving under the influence, and fatalities including overdoses, suicide and traffic accidents. Methods A new method for the quantification of 4-CMC using gas chromatography–mass spectrometry (GC–MS) was developed. The symptoms of 4-CMC use were also studied based on an analysis of the documents prepared during the col- lection of samples or at autopsies. Results The limits of detection and quantification of the method for blood samples were 0.3 and 1 ng/mL, respectively. The calibration curve was linear in the studied concentration range (1–500 ng/mL) with the correlation coefficient at 0.9979. The extraction recoveries varied in the range of 94.3–98.8%. The accuracy and precision were acceptable. The determined concentrations in nonfatal cases ranged from 1.3 to 75.3 ng/mL, and in fatalities from 56.2 to 1870 ng/mL. Conclusions Our study can assist in the recognition of the possible effects caused by 4-CMC and can be helpful during the preparation of forensic toxicological opinions for courts of law. The validation parameters indicate the sensitivity and accu- racy of the method. This is the first work presenting a validated method for the determination of 4-CMC in blood samples by GC–MS. Keywords 4-Chloromethcathinone · 4-CMC · Clephedrone · Blood concentration · Fatal and nonfatal intoxication · GC– MS Introduction on Drugs and Crime (UNODC), stated that 348 new psy- choactive substances (NPS) were identified between 2008 In the last decade, the black market for drugs has evolved and 2013, while only 97 were identified in 2013 [ 1]. Since enormously and offered both recreational and highly psy - then, a total of 101 new substances were reported in 2014 chostimulating products. The report on Global Synthetic for the first time to the EU Early Warning System (EWS) Drugs Assessment, published by the United Nations Office , while according to the “The World Drug Report” by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) approximately 1 in 20 people between the ages Ewa Tomczak and Mateusz Kacper Woźniak contributed equally of 15 and 64 used at least one drug in 2014 . as first authors. New psychoactive substances, such as synthetic cathi- * Mateusz Kacper Woźniak nones and synthetic cannabinoids, remain the most com- firstname.lastname@example.org monly used drugs nowadays. They were introduced to illicit trade in 2006/2007 and are commonly sold under slang Department of Forensic Medicine, Faculty of Medicine, Medical University of Gdańsk, 3A Marii Skłodowskiej-Curie terms such as “legal highs”, “designer drugs”, “research Str., 80-210 Gdańsk, Poland chemicals”, “bath salts”, “plant food” or “room odorizers” Department of Analytical Chemistry, Faculty of Chemistry, [4–6]. They are usually produced in China and India, from Gdańsk University of Technology, 11/12 Narutowicza Str., where they are packed and exported to target countries, to be 80-233 Gdańsk, Poland Vol:.(1234567890) 1 3 Forensic Toxicology (2018) 36:476–485 477 sold in the black market or via the Internet . Because they patients’ symptoms described by the authors were rather are absolutely not designed for human consumption without considered to be those by the 25B-NBOMe use, rather than any information on the dosage for safe use , they comprise by 4-CMC. Currently, the majority of information concern- a serious danger for human health. ing the model of consumption of 4-CMC and its impact on The 4-chloromethcathinone (1-(4-chlorophenyl)-2- the body originates from the anecdotal statements of users (methylamino)-1-propanone), also known as 4-CMC or published on Internet forums . Similarly to other syn- clephedrone, is a synthetic p-chlorine substituted deriva- thetic cathinones, 4-CMC is typically snorted or ingested tive of cathinone and belongs to the NPS (Fig. 1). It has orally. Depending on the route of administration and the been commercially available on the black market since 2014 user’s tolerance, the dose of 4-CMC resulting in the desired and is sold via the Internet in the form of white powder [4, effects ranges from 100 to 300 mg for oral ingestion and 7]. The 4-CMC is currently a controlled drug in Germany, from 50 to 150 mg for snorting. Additionally, the effects of China and Virginia (USA), but until today it has not been 4-CMC can be felt much faster after snorting than after oral classified as an illegal substance in the Act of Counteract- ingestion (2–3 and 30–60 min, respectively), but they last for ing Drug Addition in Poland [9, 10]. Due to the novelty of a shorter time. Also, the profile of effects of 4-CMC on the the drug, there is no data in scientific literature considering body depends on the route of administration; oral ingestion the pharmacokinetics, pharmacodynamics, pharmacological results in a euphoric effect, while snorting in a speed-like and toxicological effects, and the potential of acute over - effect is accompanied by agitation, increased concentration, dose effects and addiction in a long period of time. Wier - and increased self-confidence . gowski et al.  reported three cases of 4-CMC intake. There are only two studies available in scientific litera - However, these intoxication cases were considered also with ture describing procedures for the determination of 4-CMC a high dose administration of 25B-NBOMe. Therefore, the by gas chromatography–mass spectrometry (GC–MS). O R2 R1 R3 R2 R1 Pyrrolidinocathinones Methylenedioxy- cathinones NH Cathinone O R3 R1 O R4 R2 Cathinone Methylenedioxypyrrolidino- derivatives cathinones O H Cl 4-CMC (4-chlorometcathinone) Fig. 1 Structures of cathinone derivatives and 4-chloromethcathinone (4-CMC) 1 3 478 Forensic Toxicology (2018) 36:476–485 Recently, Taschwer et al.  characterized 4-CMC as an authorities to carry out routine toxicological analyses for the NPS by the analysis of crystals, which were bought via the presence of psychoactive substances and prescription drugs Internet, and presented its spectrum in electron ionization were also provided. In a number of cases, there were high (EI) mode . In another study, Klavz et al.  developed suspicions of drug administration, revealed by observations a method for the quantification of 4-CMC and four other of patients’ behaviours during the police control or medical NPS in seized materials (powders) found at a scene, and interview before blood collection. These documents were also in human urine and stomach contents. However, 4-CMC attached to most of the collected samples, which allowed was not detected in the biological materials investigated . us to discuss some symptoms of 4-CMC use. Preliminary Therefore, to the best of our knowledge, there is a lack of toxicology analyses for common drugs of abuse and pre- accurate studies concerning reference concentrations of scription drugs, being routinely performed in our laboratory, 4-CMC in biological materials in particular types of cases. involved the use of an enzyme-linked immunosorbent assay, Therefore, the aim of this study was to present and summa- headspace gas chromatography–flame ionization detection, rize the results of an analysis of 15 cases in which 4-CMC high-performance liquid chromatography–diode array detec- was detected and quantified in blood. This paper also dis- tion and GC–MS with electron/chemical ionization. Sam- cusses the concentrations of 4-CMC in different types of ples positive for 4-CMC were quantitatively analysed by the forensic cases related mainly to driving under the influence method developed in this study and described below. (DUI) and traffic accidents, as well as fatalities including The blood used as blank samples for the development and overdoses and suicides. The results of our study can assist validation of the method was obtained from a regional blood in the recognition of possible effects caused by this drug and donation centre (Gdańsk, Poland) and was stored in − 20 °C can be helpful during the preparation of reports of toxicol- prior to analysis. Blank blood was screened to be negative ogy analysis for courts of law for proper data interpretation. for drugs of abuse (including 4-CMC). A validation of the new methodology developed for 4-CMC determination in blood by GC–MS in electron ionization Preparation of stock solutions, calibrators mode is also presented. and quality control samples Stock solutions of 4-CMC were prepared in methanol by Materials and methods diluting the certified standard solution to reach concentra - tions at 50 and 5 µg/mL. The purchased standard solution Standards and chemicals of mAMP-d was used as the stock solution of the internal standard (IS). All solutions were used for calibration and A certified standard solution of 4-CMC monohydrochlo- validation, and were stored at − 20 °C prior to analysis. ride in methanol at a concentration of 1 mg/mL (as a free The calibrators (n = 3) were prepared by spiking 1 mL base) was manufactured by Cayman Chemicals (Ann Arbor, of blank blood samples with an appropriate amount of the MI, USA); a methanolic solution of racemic-methamphet- stock solution to obtain the concentrations of 1, 5, 10, 25, amine-d (rac-mAMP-d ) at a concentration of 0.1 mg/mL 50, 100, 250, and 500 ng/mL. The concentration of the IS 5 5 was manufactured by LGC Standards (London, UK). Both in each calibrator was maintained at 500 ng/mL by adding solutions were delivered by a subdivision of LGC Standards 5 µL of the IS stock solution. Finally, the extraction and (Dziekanów Leśny, Poland). derivatization procedure was performed. Acetonitrile (HPLC grade purity) was purchased from Quality control (QC) samples were prepared in triplicate Merck Millipore (Warsaw, Poland); 1-chlorobutane (HPLC at three concentration levels within the range of concentra- grade) and pentafluoropropionic anhydride (PFPA) (99% tions of calibration solutions: low—5 (LQC), medium—100 purity) from Sigma-Aldrich (Warsaw, Poland); hydrochloric (MQC) and high 500 (HQC) ng/mL. QC samples were used acid (HCl) at a concentration of 35–38%, potassium carbon- for the evaluation of the repeatability and recoveries of the ate (K CO ) and sodium chloride (NaCl) powder (all analyti- method. 2 3 cal grade) from POCH (Gliwice, Poland). Ultrapure water Samples with concentrations higher than 500 ng/mL were was produced by a Millipore Synergy 185 ultra-pure water diluted with drug-free blood to fit into the linear range to system (Millipore, Warsaw, Poland). allow quantification. Blood samples Extraction Blood samples were sent to the Department of Forensic To a blood sample (1 mL) placed in a 15 mL screw-capped Medicine, Gdańsk, Poland or collected during an ongo- glass tube, 5 μL of the IS solution was added followed by ing autopsy. The provision documents from investigative 2 mL of 5 M K CO solution and 2 mL of saturated NaCl 2 3 1 3 Forensic Toxicology (2018) 36:476–485 479 solution. Protein precipitation was performed with 2 mL in the field of our study [13, 14] in terms of selectivity, of acetonitrile and by mixing the sample for 1 min. Sub- linearity, sensitivity, limit of detection (LOD) and limit of sequently, after the addition of 2 mL of 1-chlorobutane, quantification (LOQ), matrix effect, carry-over, recovery mechanical stirring was carried out for 2 min. Finally, the and repeatability. sample was centrifuged for 3 min at 3500 rpm, and the The selectivity experiments were performed to verify organic layer was transferred to a clean glass tube. Then, the presence of endogenous or exogenous compounds at an additional 2 mL of 1-chlorobutane was added to the the retention time of the analyte or the IS. For this pur- blood residuals after the first extraction, and the sample pose, ten blood samples obtained from different subjects was vortex-mixed for 2 min and re-centrifuged for 3 min who are not involved in drugs of abuse were carefully at 3500 rpm. The organic layer was combined with the first checked by our GC–MS-based screening method after the extract. After the addition of 100 µL of HCl solution in extraction/derivatization procedure. methanol (1:9, v/v), the extraction solvent was evaporated To compensate the variability of the detector signal dur- to dryness under a gentle stream of nitrogen at 40 °C, and the ing different analyses and losses of analyte in the extrac- dry residue was dissolved in 50 μL of ethyl acetate. Then, tion and derivatization step (correction of recovery), the 50 µL of PFPA was added to perform derivatization (20 min, IS calibration was performed. An eight-point calibration 55 °C). Finally, the solution was evaporated to dryness and curve was constructed using the peak area ratio (4-CMC the dry residue was dissolved in 50 µL of ethyl acetate. A vs. IS) plotted against the concentration (the number of 2-µL aliquot was injected into the GC–MS apparatus. replicates for each level, n = 3). In order to increase the accuracy at the low concentration levels, the weighing fac- tor of 1/x was applied to the calibration curve. The linear- GC–MS conditions ity of the weighted calibration curve was evaluated in the range of 1–500 ng/mL of blood and was expressed as the All analyses were performed using a 7890A GC System (gas correlation coefficient (r ). The LOD of the method was chromatograph) equipped with a G4567A autosampler and assessed using the MSD ChemStation software and calcu- a split/splitless injection port, and connected with a 5975C lated (by extrapolation) based on the analysis of samples single quadrupole mass spectrometer (MS) with an electron (n = 3) at the lowest concentration level from the calibra- ionization ion source (all Agilent Technologies, Santa Clara, tion curve (1 ng/mL). The signal-to-noise ratio equal to 3 CA, USA). The separation of analytes was carried out on a reported for the least abundant among qualifier ions was Phenomenex ZB-5 MS capillary column (30 m × 0.25 mm considered as LOD. The lowest point of the calibration i.d., 0.25 µm film thickness; Phenomenex, Izabelin, Poland) curve was assumed as the LOQ. with helium at a purity of 99.999% as the carrier gas at a The matrix effect (ME) of the developed method was constant flow of 1 mL/min. The split injection mode (10:1) evaluated by comparing the slopes of the eight-point cali- was used. The oven temperature was programmed at 50 °C bration curves (n = 3) prepared in extracts obtained from for 1 min, then increased to 160 °C at 30 °C/min, finally drug-free blood with those prepared in a solution (metha- ramped up to 250 °C at 5 °C/min and held for 1 min. The nol) and calculated using the following equation : temperatures of the injection port, MS transfer line, ion source and detector were set at 280, 285, 280 and 200 °C, slope of calibration curve in matrix ME (%) = − 1 × 100%. respectively. The MS was operated in positive mode (elec- slope of calibration curve in solvent tron energy 70 eV). Full-scan acquisition was performed The potential for carry-over of the analyte and the IS to with the mass detection range set at m/z 40–380. For the the subsequent sample in the autosampler sequence was identification and quantification of the derivatized analyte/ evaluated by injecting 2 µL of ethyl acetate solution after IS, selected ion monitoring (SIM) mode was used with the analysis of blank blood samples fortified with the IS and ions at m/z 204, 139, 160 for 4-CMC and 208, 163, 119 for the analyte at the highest concentration level from the cali- the IS. The bolded ions were used for quantification. All the bration curve (500 ng/mL). The test was performed in six ions were chosen due to their specificity and abundance. replicates. Data acquisition and analysis were accomplished with MSD The extraction recoveries (in %) were determined by ChemStation software by Agilent Technologies (version comparing the analyte-to-IS peak area ratios for the spiked E.02.02.1431). (analyte only without IS) and extracted/derivatized blank blood samples according to the above procedure with Method validation the corresponding analyte-to-IS peak area ratios of the matrix extracts fortified with the reference standard after The developed GC–MS-based method for 4-CMC quantifi- the extraction (just before derivatization) at concentrations cation was validated according to international guidelines 1 3 480 Forensic Toxicology (2018) 36:476–485 of QC samples (n = 3 each). In this test, the IS was exclu- precision and can be used for the analysis of real samples. sively added after extraction (just before derivatization). Moreover, the low LOD and LOQ demonstrate that our The repeatability of the method was estimated as intra- and method is sensitive and is well suited for the quantification interassay accuracy and precision. Intraday assay experi- of the blood concentration of 4-CMC in forensic cases at ments were carried out by analysing QC samples (n = 3). even small concentrations of this drug. To evaluate the interday assay repeatability (as between- day averages), the tests were repeated over three consecu- tive days. The accuracy of the method was calculated as Toxicological cases the mean ratio of the measured and the nominal concentra- tions, while precision was assessed as percent coefficients In Poland, the popularity of 4-CMC as an NPS has increased of variation (% CVs) of these measurements. year by year. In the first half of 2015, it was the eighth psy - choactive substance most frequently found in “designer drugs”, and in the second half of 2015, it ranked second. Results and discussion Taking into account the data for 2016, 4-CMC was the psy- choactive substance most frequently found in these types of GC–MS and validation products . At the time when this article was written, it was not yet under legal control in Poland. The developed method includes three ions (one quantifier The cases (n = 15) in which 4-CMC was detected, were and two qualifiers) for the determination of 4-CMC in blood analysed between 2015 and 2017 (Table 3). In 93.3% of samples. The total ion current chromatogram of 4-CMC and cases of 4-CMC users, there were mostly males (14 cases), the IS prepared in methanol at a concentration of 50 µg/ and only a single case was a woman (6.7%). Other authors mL after derivatization with PFPA is presented in Fig. 2a. described a similar gender scheme for synthetic cathi- A fragmentation pattern of derivatized 4-CMC was also nones . The age range for the cases under study was suggested (Fig. 2c). The increased sensitivity and the better 18–38 years (n = 12; mean 24.5 years; median 23.5 years). signal-to-noise ratio was enabled by careful optimization Therefore, young men are the main users of drugs, which is of chromatographic conditions, such as the temperature of comparable to data from relevant literature [5, 17]. the injector, the initial and final column temperatures, the In a just single case (No. 6), 4-CMC was determined as temperature ramping up rate and carrier gas flow rate, as the only psychoactive substance, and in the other cases, other well as the temperature elements of the MS. pharmacologically active substances and/or their metabolites No interfering peaks due to endogenous/exogenous sub- were also detected. The most frequent combination found stances that could have obstructed the identification and in our study was the combination with THC/THC-COOH quantification of the compounds of interest were observed (n = 6). The combination of another synthetic cathinone in the drug-free samples investigated for selectivity at the (α-PVP) with cannabinoids was also reported as the most retention times of the analyte and the IS (Fig. 2b). Therefore, popular combination in other studies . The other deter- the developed method can be considered as highly specific mined compounds included amphetamine (n = 5), ethyl alco- and selective for the determination of 4-CMC. The data of hol (n = 4), benzodiazepines (n = 3, diazepam, nordazepam, the parameters of calibration curves to establish the ME and estazolam), MDMA (n = 1), MDA (n = 1), 3-MMC are summarized in Table 1. A negative value of the matrix (n = 1), and cocaine metabolites (n = 1, ecgonine methyl effect was obtained, which indicates the suppression of the ester and benzoylecgonine). This indicates that young peo- detector signal by co-extracted compounds as compared ple have a tendency to use several psychoactive substances, to the signal of the analyte injected in the solvent. How- often marked by a profile of similar effects, at the same time. ever, MEs in the range of − 20 to + 20% are perceived as This may lead to drug-drug interactions (DDI), including soft and can be neglected . A carry-over effect was not life- or health-threatening additive or superadditive syner- observed. The method was shown to be linear within the gism, and, in consequence, some effects which could not tested range (1–500 ng/mL). The correlation coefficient (r ) be foreseen by the users [18, 19]. Such a situation may take of the weighted calibration curve was 0.9979. The LOD and place, for example, in the case of the simultaneous use of LOQ of the method were 0.3 and 1 ng/mL, respectively. The synthetic cathinones (including 4-CMC) and amphetamine- accuracy, precision and recoveries data for intra- and inter- type stimulants, since both classes of substances are essen- day measurements are summarized in Table 2. tially phenethylamine derivatives. The substances have a All the validation parameters fulfilled the established similar mechanism of action; they inhibit monoamine trans- international criteria for bioanalytical methods. Therefore, porters, which are responsible for the reuptake of mono- the developed method for the determination of 4-CMC amines in the synaptic cleft of the neurons . This may in blood samples is characterized by high accuracy and explain the symptoms observed in case 9, clearly indicating 1 3 Forensic Toxicology (2018) 36:476–485 481 Fig. 2 a Total ion current chromatogram of a mixture of derivatized m/z 204 for the selectivity test, and c mass spectrum plus proposed 4-CMC and internal standard (IS) at a concentration of 50 µg/mL, b fragmentation pattern of derivatized 4-CMC. Rt retention time selected ion monitoring chromatogram for drug-free blood sample at 1 3 482 Forensic Toxicology (2018) 36:476–485 Table 1 Regression equations for neat spiked solvent and spiked Beside 4-CMC, psychostimulants and/or their metabo- blood extract for assessment of matrix effect (ME) of the proposed lites, including amphetamine, MDMA, MDA and 3-MMC method (eight point calibration curves, n = 3) for quantification of as well as ecgonine methyl ester and benzoylecgonine were 4-chloromethcathinone (4-CMC) also determined in four cases under study (cases 2, 4, 7 and Matrix Curve’s equation r ME (%) 9). The determination of the relatively high concentrations of the aforementioned stimulants in blood, along with 4-CMC Solvent y = 0.00233x − 0.00048 0.9993 − 11.2 (cases 2, 4 and 7), as well as the clearly observable symp- Blood extract y = 0.00207x − 0.0010 0.9996 toms of intoxication, did not make it possible for the authors r correlation coefficient to recognize 4-CMC as the only substance responsible for the symptoms. In case 9, despite the effects on the central the use of psychoactive substances, despite the detection of nervous system observed during medical examination, rela- relatively low concentrations of 4-CMC and amphetamine. tively low concentrations of amphetamine and 4-CMC as well as the presence of psychologically inactive cocaine Nonfatal cases metabolites were also detected. This is puzzling, but we may suspect that prior to analysis, cocaine in blood underwent For nonfatal cases (1–9), the concentrations of 4-CMC in degradation in vitro as a result of the improper storage of blood ranged between 1.3 and 75.3 ng/mL (n = 9; mean con- biological material before its delivery to the laboratory. centration 21.1 ng/mL; median 11.8 ng/mL). These cases In just a single case (case 6), only 4-CMC was detected. mainly concerned drivers stopped upon traffic control due However, the concentration of 4-CMC in the blood (5.5 ng/ to the suspected consumption of alcohol or psychoactive mL) was several times lower than the mean concentration, substances (driving under the influence, DUI; 77.8%, n = 7), but we should take into account that as many as 3 h had traffic accident (11.1%, n = 1) or burglary (11.1%, n = 1). passed between the car journey and the collection of the Among DUI, the concentration of 4-CMC ranged from blood sample. The concentration of 4-CMC may have been 1.3 to 75.3 ng/mL (mean 22.3 ng/mL; median 16.7 ng/mL). considerably higher when the patient was in the car. The sub- In the case of a burglary, 11.8 ng/mL of 4-CMC was deter- ject was agitated, displayed increased drive, and, according mined. In the case of the driver who caused a fatal acci- to a witness, behaved “like a monkey in a cage” or as if he dent involving two passengers (driver: case 3; passengers: had experienced an epileptic seizure. Additionally, the pupils cases 10 and 11), the concentration of 4-CMC amounted to of this driver were dilated and tachycardia was detected. The 33.4 ng/mL. It was the only case with no symptoms indicat- patient was restrained with belts during transportation from ing effects of psychoactive substances. In the other eight the place in which he was stopped by traffic control to the nonfatal cases, symptoms indicating the consumption of hospital. However, the abovementioned symptoms could not alcohol or ingestion of psychoactive substances were iden- be clearly identified as specific to 4-CMC, because in the tified during traffic control and/or medical examination. other eight cases, all patients took at least one psychoactive The most frequently observed disturbances of psychomotor substance except 4-CMC. In case 5, beside 4-CMC, ben- functions in the cases under analysis included dilated pupils zodiazepines (diazepam, nordazepam and estazolam) were (n = 5), slow or absent pupillary light reflex (n = 4), slurred also quantified. Diazepam and nordazepam are frequently speech (n = 2), agitation, increased drive (n = 2), tachycardia used for patient sedation and these medicines might have (n = 2), difficulty in walking (n = 2), difficulty in picking up been administered by the medical staff. Estazolam is not objects from the ground (n = 2), positive Romberg’s sign administered for medical purposes in Poland, and, there- (n = 1), disorientation as to time, place, and surroundings fore, it must have been taken for abusing purposes. However, (n = 1), drowsiness (n = 1), logorrhea (n = 1), and slowed this has not been confirmed by the investigating authorities behaviour (n = 1). Enforced body position was applied in (no relevant packaging was found next to the patient, and two cases (nos. 1 and 6). he did not confirm having taken estazolam). Furthermore, Table 2 Validation parameters Concentration Intraday assay (%) Interday assay (%) Recovery (%) of the method: accuracies, (ng/mL) (mean ± SD) precisions and recovery rates Day 1 Day 2 Day 3 (%) (n = 3) for quantification of 5 96.8 (0.3) 102 (0.6) 92.7 (0.7) 97.3 (4.4) 98.2 ± 2.5 4-CMC 100 95.3 (3.6) 99.9 (5.6) 102 (1.1) 98.9 (5.2) 98.8 ± 3.1 500 96.1 (1.8) 94.8 (1.4) 98.7 (1.2) 96.5 (2.2) 94.3 ± 4.1 The precision expressed as percent coefficient of variation is shown in parenthesis SD standard deviation 1 3 Forensic Toxicology (2018) 36:476–485 483 Table 3 Fatal and nonfatal intoxication cases involving 4-CMC Case number Sex/age (years) Symptoms Type of case/case description 4-CMC blood Other drug blood concen- concentration (ng/ tration (ng/mL; alcohol mL) in g/L) Nonfatal case 1 M/35 Slurred speech, slowed DUI 2.0 Diazepam: 14.3 behaviour, drowsiness, dilated pupils, disorientation as to time, “obligatory body posture” 2 M/23 Facial redness, tachycardia DUI 7.9 MDMA: 755 MDA: 50 Ethyl alcohol: 1.29 3 M/20 No abnormalities discovered Road accident (driver) 33.4 THC: < LOQ THC-COOH: 37 4 M/U Slurred speech, eye redness, DUI 25.4 Amphetamine: 15 facial redness 3-MMC: 450 THC-COOH: 8 5 M/24 Markedly agitated and DUI and drug possession 75.3 Estazolam: 8.1 distracted, logorrhea, pupils Nordazepam: 28.8 unreacted to light Diazepam: 14.4 6 M/26 The patient was hospital- DUI 5.5 – ized: he was restrained while being transported to the hospital. The patient was joyful, and exhibited increased drive, tachycardia, dilated pupils, and difficulty in picking up objects from the ground 7 M/25 Slurred speech, dilated pupils DUI 27.4 Amphetamine: 249 with poor light reflex, dif- ficulty in walking 8 M/20 Dilated pupils with poor light Burglary 11.8 THC: < LOQ reflex. Difficulty in picking THC-COOH: 15 up objects from the ground. Positive Romberg’s sign 9 M/18 Dilated pupils with poor light DUI 1.3 Amphetamine: 9.8 reflex. Disturbed orientation BZE: 297 as to time, place and sur- EME: 23 roundings Fatal case 10 M/20 n/a Death in road accident (pas- 183 THC: 13.5 senger) THC-COOH: 160 Ethyl alcohol: 0.67 11 M/20 n/a Death in road accident (pas- 78.4 THC: 3.8 senger) THC-COOH: 57 Ethyl alcohol: 0.72 12 M/U n/a Death in train accident - 56.2 Ethyl alcohol: 2.68 suicide 13 M/25 n/a Body found in an apartment 394 Amphetamine: 2200 14 M/38 n/a Body found in an apartment 698 Nordazepam: 308 15 F/U n/a Fall from a height - suicide 1870 Amphetamine: 861 THC: 12 THC-COOH: 85 DUI driving under the influence, n/a not available, U unknown, 3-MMC 3-methylmethcathinone (3-mephedrone), BZE benzoylecgonine, EME ecgonine methyl ester, THC tetrahydrocannabinol, THC-COOH 11-nor-9-carboxy-tetrahydrocannabinol, LOQ limit of quantification 1 3 484 Forensic Toxicology (2018) 36:476–485 the symptoms observed in this case were typical of psy- only, because only nordazepam at therapeutic concentration chostimulants (marked agitation, logorrhea, no pupillary was additionally determined. light reflex), and the concentration of 4-CMC was highest Case 15 concerns a suicidal death of a young woman of all nonfatal cases under analysis. Diazepam was also caused by a fall from a height. What is noteworthy, a much determined in the blood in case 1, but it was most probably higher concentration of 4-CMC was determined in the administered during medical intervention. blood in this case than in the case of deaths caused by acute intoxication with psychoactive substances (cases 13 and 14). Fatal cases Therefore, the determined concentration of 4-CMC at a level many times higher than in the other cases of fatal intoxica- In the majority of fatal cases (n = 5), the concentration tions presented may be an argument in favor of the prob- of 4-CMC was markedly higher than in nonfatal ones able addictive effect of 4-CMC, and her body might have (56.2–1870 ng/mL; mean concentration 547 ng/mL, median developed tolerance to this substance, resulting in the users 288 ng/mL). However, four of the fatal cases were con- increasing the taken doses of the substance. This effect is nected with events possibly resulting from risky behaviour observed for the majority of amphetamine-type stimulants. being a typical effect of a class of stimulants. In the cases of deaths resulting from car accidents, 4-CMC concentra- tions amounted to 78.4 and 183 ng/mL (cases 10 and 11), Conclusions respectively. THC, THCCOOH and ethyl alcohol were also detected in these cases. Furthermore, ethyl alcohol and can- The 4-CMC is a psychostimulating substance very danger- nabinoids plus amphetamine were also quantified in two sui- ous for health but not banned in many countries, including cidal death cases (12 and 15). Moreover, two cases (13 and Poland. Intoxication cases with NPS and their unpredictable 14) may be identified with acute intoxication with psychoac - effects are very difficult to recognize, which makes these tive substances (including 4-CMC), and this hypothesis was drugs an important health issue, especially when other drugs also suggested by forensic pathologists. are used in combination. This is an effect of a rapid evolu- Case 13 concerns a 25-year-old man who was found tion in the availability of NPS and a large number of their dead in his flat. The autopsy revealed that the immediate possible structures. Therefore, there is still an urgent need cause of death was acute cardiac failure in the mechanism of for the development of new methods for the determination arrhythmias due to chronic focal lesions in the myocardium. of NPS in various biological materials. The results can be Moreover, according to the forensic pathologist, considering useful for the proper interpretation of toxicological results. the pathomechanism of his death, the acute cardiac failure To the best of the authors’ knowledge, this is the first work does not contradict the possibility of an unknown substance presenting blood reference concentrations of 4-CMC in dif- being co-responsible for the intoxication. This is suggested ferent toxicological cases and a validated method for the by the presence of gastric contents in his respiratory tract determination of this drug in blood samples by GC–MS. as a result of vomiting. Therefore, this case may be consid- Finally, our developed method can be an alternative for most ered death resulting from a mixed causes of myocardium toxicological laboratories, which do not include a liquid lesions with 4-CMC and amphetamine intoxication, as the chromatography–tandem mass spectrometry system, which concentrations determined in his blood (394 and 2200 ng/ is usually used for NPS analysis nowadays. mL, respectively) are relatively high, and the detected con- centration of amphetamine can be fatal to a nonaddict . Compliance with ethical standards Case 14 concerns a 38-year-old man who was found dead in his flat. During autopsy, blood and a fragment of abdomi- Conflict of interest The authors declare that they have no conflict of interest. nal wall containing a pill were collected for toxicological tests. Analyses showed that the pill contained disulfiram, Ethical approval All blood collections from living suspects were made although it was not detected in the blood. Ethyl alcohol was by judicial authorities, and the samples were sent to our Forensic Sci- not detected either. The autopsy did not reveal any bodily ence Department for routine toxicology analysis. Blood collections from the deceased were made by judicial authorities during autopsy injuries. However, signs of sudden death were found, includ- either in our Department or in other external dissecting-rooms to be ing recent passive congestion of internal organs, presence sent to us. All analyses were made according to the request of judicial of liquid blood in the heart and great vessels, and intensive authorities. When blank blood samples were collected from healthy livor mortis. Moreover, the forensic pathologist indicated the subjects for validation experiments, informed consent was obtained from each subject. action of a psychoactive substance on the body probably as the primary cause of his death. Therefore, it is highly prob- able that the death resulted from an overdose of 4-CMC Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creat iveco 1 3 Forensic Toxicology (2018) 36:476–485 485 mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- 11. Wiergowski M, Aszyk J, Kaliszan M, Wilczewska K, Anand JS, tion, and reproduction in any medium, provided you give appropriate Kot-Wasik A, Jankowski Z (2017) Identification of novel psycho- credit to the original author(s) and the source, provide a link to the active substances 25B-NBOMe and 4-CMC in biological material Creative Commons license, and indicate if changes were made. using HPLC-Q-TOF-MS and their quantification in blood using UPLC-MS/MS in case of severe intoxications. J Chromatogr B 1041–1042:1–10. https://doi.or g/10.1016/j.jchromb.2016.12.018 12. 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Forensic Toxicology – Springer Journals
Published: May 28, 2018
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