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Medicolegal aspects of PMA-related deaths
Legal Medicine 21 (2016) 64–72
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Case Report
Medicolegal aspects of PMA-related deaths Sebastian Rojek ⇑, Filip Bolechała, Karol Kula, Martyna Maciów-Gła˛b, Małgorzata Kłys Department of Forensic Medicine, Jagiellonian University Medical College, Grzegórzecka 16 Str., 31-531 Kraków, Poland
a r t i c l e
i n f o
Article history: Received 26 November 2015 Received in revised form 16 May 2016 Accepted 6 June 2016 Available online 6 June 2016 Keywords: PMA Fatal poisonings PMA in blood LC–MS Medicolegal opinion
a b s t r a c t Unlike amphetamine, amphetamine-like substances accessible on the drug market are less expensive and more easily available; they also produce hallucinogenic effects expected by the users. Such properties render them more attractive as compared to amphetamine. On the other hand, the knowledge of the toxicity of these compounds is very limited, what in consequence generates problems that create everexpanding research areas, including analytical, clinical and medicolegal issues, thus leading to development of systemic databases. An example here is paramethoxyamphetamine (PMA), which appeared on the drug market in recent years as a result of creative inventiveness of producers of psychoactive substances, who aimed at PMA replacing the popular ecstasy (MDMA) as a less expensive and more available product. It is more potent than MDMA, but has a slower onset of action, which encourages users to take more. The problem is illustrated in the present paper by three fatal cases involving PMA, which were comprehensively investigated taking into consideration case histories, pathological and toxicological findings obtained with the use of LC–MS-MS method. In blood samples taken from all the three victims, very high concentrations of PMA were found (in the range of 10–27 mg/L) and thus the cause of deaths was determined as overdoses of PMA with the underlying mechanism of acute cardiorespiratory failure. Ó 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction A search for new sensations leading to experiencing a seemingly better reality results in an increasing worldwide demand for novel psychoactive substances. Their manufacturers encourage the users to try various substances, increasingly less expensive, more exciting, with longer action and in forms that are more attractive. Amphetamine-like substances are abused in several score countries worldwide and the number of their users exceeds 6 million [1]. Official Polish sources estimate the number of opiate addicts to be above 30,000, while the number of users of amphetamine and its derivatives is reported as several thousands. As it follows from polls carried out among various social groups, the number of individuals who regularly or occasionally take ecstasy-like substances may be as high as several thousand users [1]. Taking amphetamine derivatives resulted in numerous disasters. At the end of the 20th century in Canada [2], the United States [3], Australia [4,5] and in Spain [6], a score or so of fatalities were noted, which led to justified concerns. Their cause appeared to be a narcotic substance advertised as ecstasy, which in truth was another amphetamine derivative (paramethoxyamphetamine – PMA), ⇑ Corresponding author. E-mail address: [email protected] (S. Rojek). http://dx.doi.org/10.1016/j.legalmed.2016.06.002 1344-6223/Ó 2016 Elsevier Ireland Ltd. All rights reserved.
the street name of which – ‘‘death” – suggested danger. Later, poisonings with this substance were noted in various countries – again in Canada [7] and Australia [8], as well as in Denmark [9]. PMA appeared on the drug market as a result of creative inventiveness of producers of psychoactive substances, who aimed at paramethoxyamphetamine replacing the popular ecstasy (MDMA). Substrates for PMA production are cheaper (e.g. the substrate anethole) and more easily (legally) available as compared to substrates necessary to produce MDMA (e.g. the substrate safrole) [10]. In addition to their lower price and greater availability, amphetamine-like substances may evoke hallucinogenic effects, what makes them more attractive as compared to amphetamine [13]. In Poland, fatal poisonings with PMA appeared at the threshold of the third millennium [11–14] and were associated with introduction of the UFO pill, composed of approximately 15% PMA (40 mg PMA) and minimal amounts of amphetamine, MDMA, methamphetamine and other diluents and having a tablet mass of approximately 270 mg [11]. Victims of the UFO pill focused public concern on the issue of the fight against drug abuse. However, it is emphasized that preventive measures employed to date, which consist in a fight against drug supply and attempts at decreasing demand, are of a limited effectiveness. Although in Poland to date, severe complications following ecstasy ingestion have been infrequent and fatalities resulting from MDMA overdosing are extremely rare
S. Rojek et al. / Legal Medicine 21 (2016) 64–72
[15], attempts at replacing the substance by another drug have led to numerous accidental deaths being a consequence of manufacturers’ frauds and casual approach of the users. Evidence in support of this statement can be found in the recent (2012) Polish fatalities in consequence of PMA abuse. Deaths caused by new narcotic substances generate problems that create ever-expanding research areas, including analytical, clinical and medicolegal issues, thus leading to development of systemic databases. The problem is illustrated in the present paper by three fatal cases representing accidental poisonings involving PMA, which were the subject of complex and detailed investigations in the analytical, pathological, toxicological and medico- legal aspect. 2. Cases histories 2.1. Case 1 A male friend found KŁ, a 19-year-old female, with no vital signs and lying on the floor of a room in a flat they rented in Krakow. She was pronounced dead by an emergency medical service team. PB was interrogated as a witness; he reported that he had spent some time with KŁ during the night and they both had inhaled approximately 0.5 g amphetamine. KŁ had not drunk alcohol; the witness did not report her possibly having taken any other substances. In the past, they occasionally had used amphetamine, but not on a regular basis. PB described the effects of amphetamine as normal, stating he had not experienced any unusual consequences. At approximately 5:00 a.m., they both had gone to bed. PB had waked at approximately 4:30 p.m. and found KŁ lying on the floor with her face down; she did not react to any external stimuli. The victim was autopsied within 24 h after death. The postmortem examination showed minor skin abrasions and bruising of the hip, knee and thigh, pulmonary edema and congestion of the internal organs. The above-described macroscopic lesions combined with subsequent toxicology allowed for determining the cause of death as acute cardiorespiratory failure and acute multiorgan failure resulting from a complex poisoning with PMA and amphetamine. 2.2. Case 2 Late at night, an emergency medical service team was summoned to a flat in Krakow, where DS, a 23-year-old male, had fainted. The emergency physician pronounced him dead and did not attempt resuscitation. DS and his two colleagues were determined to have been drinking alcohol (beer and vodka) starting on the preceding day. As it followed from the case history, in the evening hours, DS had been in full contact but excited, he had been walking around the flat and suddenly had fallen to the floor, convulsed and lost consciousness. During the police inspection and searching of the premises, particles of dry plant material and a cigarette butt had been secured and referred to identification tests. The autopsy performed within 24 h after death showed minor skin abrasions involving the head, trunk and right upper extremity, disseminated bruising of the upper and lower extremities, pulmonary edema, subepicardial blood extravasation and congestion of the internal organs. Histopathology of the collected sections demonstrated cardiac muscle congestion, disseminated perivascular fibrosis, parenchymal degeneration of single muscle fibers, interstitial edema; pulmonary congestion, edema and unequal aeration; hepatic congestion, small droplet steatosis of disseminated hepatocytes; nephrotic congestion, hyalinization and fibrosis of single glomeruli; cerebral congestion. Comprehensive toxicology did not demonstrate blood and urine ethyl alcohol. The above-described macroscopic lesions combined with subsequent toxicology allowed for determining the cause of death as acute cardiorespiratory failure
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and acute multiorgan failure resulting from a complex poisoning with PMA and amphetamine. 2.3. Case 3 A male friend found MZ, a 21-year-old female, with no vital signs and lying on the floor in a flat situated in the center of Krakow. She was pronounced dead by an emergency medical service physician. JK testified that they had spent the previous evening together, had eaten dinner and drunk wine. Several hours apart, they had also taken two pills each of a drug JK purchased several days earlier (approximately 80 tablets) as ‘‘something akin to ecstasy”. JK had been feeling well, had been in high spirits, but had felt somewhat weak and tired. MZ had been reputedly behaving ‘‘strange”, been exuberant in expressing her feelings, restless and animated. JK had fallen asleep at approximately 5:00–6:00 a.m. Around noon, he had heard MZ taking on the phone and saying she was not feeling well and was feverish. The police inspection of the premises disclosed a bag containing portions of dry plant material wrapped in aluminum foil and another bag with a large number of small, round pills. The material was referred to toxicology. The autopsy of MZ demonstrated minor skin abrasions and bruising involving the back and extremities, pulmonary edema and congestion of the internal organs. Histopathology of the collected sections demonstrated cardiac muscle congestion, interstitial edema, disseminated foci of fiber fragmentation; profound pulmonary congestion with blood extravasation to the alveoli, edema; hepatic congestion; nephrotic congestion; cerebral congestion. Comprehensive toxicology did not demonstrate blood and urine ethyl alcohol. The above-described macroscopic lesions combined with subsequent toxicology allowed for determining the cause of death as acute cardiorespiratory failure and acute multiorgan failure resulting from a complex poisoning with PMA. 3. Material and methods 3.1. Non-biological materials ▪ Two pills with a logo representing a serpent, confiscated by the prosecution and referred to identification analysis. 3.2. Biological materials ▪ Postmortem autopsy specimens – samples of femoral blood were collected in the course of autopsies carried out in the Department of Forensic Medicine, Jagiellonian University Medical College. The samples were kept frozen ( 20 °C) until the analyses were performed. ▪ 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 (n = 2) and nonpoisoned dead subjects (n = 4). 3.3. Standards and chemicals The standard solutions of 6-acetylcodeine, 6-acetylmorphine, 7-acetamidoclonazepam, acetaminophen, 7-aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, acetylsalicylic acid, alpha-hydroxyalprazolam, alprazolam, aminophenazone, amisulpride, amitriptyline, amphetamine (AMF), benzoylecgonine, bk-MBDB, bk-MDEA, bromazepam, 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-bromo-2,5-dimethoxyphenethylamine (2CB, Nexus), buprenorphine, caffeine, carbamazepine, cathinone, chlorpromazine, citalopram, clobazam, clomipramine, clonazepam, clozapine, cocaethylene, cocaine, codeine, codeine-6-glucuronide,
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cothinine, desmethylclomipramine, desmethylclozapine, diazepam, diclofenac, 10,11-dihydrocarbamazepine, 2,5-dimethoxy4-ethylamphetamine (DOET), 2,5-dimethoxy4-methylamphetamine (DOM), diphenhydramine, dosulepin, doxepine, drotaverine, ephedrine, ephedrone, 10,11epoxycarbamazepine, estazolam, 2-ethyl-1,5-dimethyl-3,3-diphe nylpirrolidine (EDDP), flephedrone, flunitrazepam, fluoxetine, flurazepam, fluvoxamine, hydrocodone, hydromorphone, hydroxyamphetamine, ibuprofen, imipramine, ketamine, ketoprofen, levomepromazine, lorazepam, lormetazepam, methadone, methamphetamine (methAMF), methcathinone, methedrone, 4-methoxyamphetamine (PMA), 4-methoxymethamphetamine (PMMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylene dioxyethylamphetamine (MDEA), 3,4-methylenedioxymethamphe tamine (MDMA), 3,4-methylenedioxy-N-methyl-butanphenamine (MBDB), 4-methylethcathinone (4-MEC) 4-methylmethcathinone (4-MMC, mephedrone), methylone, mianserine, midazolam, moclobemide, morphine, morphine-3-glucuronide, morphine-6glucuronide, nicotine, nitrazepam, norbuprenorphine, nordiazepam, nordoxepine, norephedrine, norfluoxetine, norketamine, normorphine, O-desmethylvenlafaxine, oxazepam, oxcarbazepine, oxycodone, oxymorphone, paroxetine, pentazocine, pethidine, promazine, promethazine, propranolol, reboxetine, sertraline, sulpiride, temazepam, thiorydazine, tramadol, venlafaxine, zaleplone, zolpidem, zopiclone and amphetamine-d3 (AMF-d3), ephedrine-d3, MDA-d5, methamphetamine-d5 (methAMF-d5), MDMA-d5, MDEAd5 used as internal standards (IS), were purchased from LGC Standards (Warszawa, Poland), ammonium formate, ammonium carbonate, formic acid, acetic acid – 99.9% for LC–MS were purchased from SIGMA (Poznan´, Poland), acetonitrile, methanol from Fluka (Poznan´, Poland). Solid-phase extraction columns LiChrolut RP-18 (Merck, Darmstadt, Germany) containing 500 mg C18-RP bonded silica were used. 3.4. Calibrators and controls For the calibrator samples, two working solutions were prepared in methanol at the following concentrations: 1.0 and 10 ng AMF, ephedrine, MDA, PMA, methAMF, MDMA, PMMA, MBDB and MDEA/ll. Also methanolic solutions were prepared for quality control (QC) samples at concentrations of 1.0 and 10 ng/ll for amphetamines. 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 1 ml of blank blood with known amounts of AMF, ephedrine, MDA, PMA, methAMF, MDMA, PMMA, MBDB and MDEA at concentrations ranging from 10 to 500 ng/ml. Low, medium and high QC specimens were also prepared daily at concentrations of 10, 50, 500 ng/ml for amphetamines. For the deuterated internal standard (IS), a working solution of 1.0 ng AMF-d3, ephedrine-d3, MDA-d5, methAMF-d5, MDMA-d5 and MDEA-d5 in methanol was prepared and stored at 20 °C when not in use. Hundred microliters of this working solution was added to each sample prior to extraction, giving a final deuterated IS concentration of 100 ng/ml. 3.5. Analytical methods 3.5.1. Preliminary screening The screening test included an enzyme-linked immunosorbent assay (ELISA, Neogen, Ayr, Scotland, UK) of blood for amphetamine, methamphetamine/MDMA, opiates, cocaine, cannabinoids, barbiturates, benzodiazepines, tricyclic antidepressants, 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 (case 1) and amphetamine, methamphetamine (case 2 and 3). The HPLC-DAD analysis paved a path for further analysis. Comparing the UV spectra found in postmortem specimens suggested that it could be some amphetamine derivative, which has idiosyncratic spectra. 3.5.2. Extraction The autopsy blood sample (1 ml) was subjected to solid-phase extraction. The samples were put into a clean 15 ml Falcon tubes and diluted five times with 0.1 M carbonate buffer to pH 9.3. AMF-d3, ephedrine-d3, MDA-d5, methAMF-d5, MDMA-d5 and MDEA-d5 at the concentration of 100 ng/ml were added to the samples. Afterwards, the samples were vortexed and centrifuged for 7.5 min at 1500g. 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 0.1 ml of mixture (95% phase A + 5% phase B) and injected into LC–MS system. Forensic blood samples were diluted. 3.5.3. Separation by liquid chromatography An Agilent Technologies 1200 liquid chromatograph (Santa Clara, CA, USA) equipped with a binary pump (G1312 A) and an autosampler (G1329 A) 15 ll injection loop were used in gradient mode. The chromatographic separation was performed with a Allure PFP-C18 column (50 mm 3 mm, 2.7 lm, Restec, USA). The phase A was water, which contained 0.02% formic acid and 0.002 M of ammonium formate and phase B was acetonitrile with 0.02% formic acid and 0.002 M of ammonium formate. The gradient for samples concerning PMA and IS’s 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. 3.5.4. Detection by tandem mass spectrometry A 6410 triple quad mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), an atmospheric pressure electrospray ion source (ESI), operated under positive mode was used. The operational parameters of the ESI source were as follows: vaporizing temperature 350 °C; pressure of the nebulising gas 40 psi; flow of the drying gas 9 l/min; capillary potential 3500 V. Fragmentation parameters of analyzed compounds are listed in Table 1. 3.6. Validation of LC–ESI-MS–MS method 3.6.1. Selectivity To evaluate peak-purity and selectivity, two blank blood samples from living person and four from non-poisoned dead subjects (no analyte or IS added) were analyzed with each batch to check for peaks that might interfere with detection of AMF-d3, AMF, ephedrine-d3, ephedrine, MDA-d5, MDA, PMA, methAMF-d5, methAMF, MDMA-d5, MDMA, PMMA, MBDB, MDEA-d5 and MDEA. To assess possible interferences of other drugs on abuse and pharmaceuticals quality control samples were spiked individually to contain 10,000 ng/ml of 6-acetylcodeine, 6-acetylmorphine, 7-acetamidoclonazepam, acetaminophen, 7-aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, acetylsalicylic acid, alpha-hydroxyalprazolam, alprazolam, aminophenazone, amisulpride, amitriptyline, benzoylecgonine, bk-MBDB, bk-MDEA, bromazepam, 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-bro
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S. Rojek et al. / Legal Medicine 21 (2016) 64–72 Table 1 Fragmentation parameters of the analyzed compounds. Compound name
Precursor ion
Product ion
Fragmentor voltage [V]
Collision energy [V]
Retention time [min]
AMF-d3
139.2 136.1
Ephedrine-d3
169.2
Ephedrine
166.2
MDA-d5
185.1
MDA
180.1
PMA
166.2
methAMF-d5
155.2
methAMF
150.1
MDMA-d5
199.2
MDMA
194.2
PMMA
180.1
MBDB
208.2
MDEA-d5
213.2
MDEA
208.2
80 80 80 80 90 90 90 90 70 70 70 70 70 70 50 50 90 90 95 95 95 95 70 70 90 90 70 70 90 90
5 15 5 15 10 20 10 20 5 15 5 15 5 20 9 21 5 15 5 20 10 20 5 20 20 10 9 21 15 20
4.7
AMF
122.1 92.1 119.1 91.1 151.1 136.1 148.1 133.1 168.1 138.1 163.2 135.1 149.1 121.1 121.1 92.1 119.1 91.1 165.2 135.0 163.1 135.0 149.1 121.1 135.0 177.1 163.1 135.0 163.1 135.1
4.7 5.0 5.0 5.2 5.2 5.3 5.2 5.2 5.5 5.5 5.5 5.8 5.7 5.7
Bold indicated quantitative ions.
mo-2,5-dimethoxyphenethylamine (2CB), buprenorphine, caffeine, carbamazepine, cathinone, chlorpromazine, citalopram, clobazam, clomipramine, clonazepam, clozapine, cocaethylene, cocaine, codeine, codeine-6-glucuronide, cothinine, desmethylclomipramine, desmethylclozapine, diazepam, diclofenac, 10,11dihydrocarbamazepine, 2,5-dimethoxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-methylamphetamine (DOM), diphenhydramine, dosulepin, doxepine, drotaverine, ephedrone, 10,11epoxycarbamazepine, estazolam, 2-ethyl-1,5-dimethyl-3,3-diphe nylpirrolidine (EDDP), flephedrone, flunitrazepam, fluoxetine, flurazepam, fluvoxamine, hydrocodone, hydromorphone, hydroxyamphetamine, ibuprofen, imipramine, ketamine, ketoprofen, levomepromazine, lorazepam, lormetazepam, methadone, methcathinone, methedrone, 4-methylethcathinone (4-MEC) 4methylmethcathinone (4-MMC, mephedrone), methylone, mianserine, midazolam, moclobemide, morphine, morphine-3glucuronide, morphine-6-glucuronide, nicotine, nitrazepam, norbuprenorphine, nordiazepam, nordoxepine, norephedrine, norfluoxetine, norketamine, normorphine, O-desmethylvenlafaxine, oxazepam, oxcarbazepine, oxycodone, oxymorphone, paroxetine, pentazocine, pethidine, promazine, promethazine, propranolol, reboxetine, sertraline, sulpiride, temazepam, thiorydazine, tramadol, venlafaxine, zaleplone, zolpidem and zopiclone. There were no interferences with the said compounds. 3.6.2. Linearity, limit of quantitation, carry – over Calibration curves were prepared daily by spiking blank blood with corresponding analytical working solutions to obtain calibration concentrations of 10, 20, 50, 100 and 500 ng/ml AMF, ephedrine, MDA, PMA, methAMF, MDMA, PMMA, MBDB and MDEA. Validation samples were prepared in triplicate at the following concentrations: 1, 2, 3, 5, 750, 1000, 1250 ng/ml AMF, ephedrine, MDA, PMA, methAMF, MDMA, PMMA, MBDB and MDEA to assess the method’s accuracy above and below the calibration curve.
Negative quality control samples were analyzed after each linearity sample to evaluate potential carry-over. The samples were extracted according to the procedure described. Calibration curves were constructed by plotting the peak-area ratios analytes/internal standard. 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 P10/1, 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. 3.6.3. Extraction efficiency, matrix effects and process efficiency Extraction efficiency for each analyte was measured at each QC concentration. Blank blood was fortified with QC and internal standard 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 ten unique blank extracted bloods fortified with QC and internal standards 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 mean peak area of fortified bloods 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.
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3.6.4. Accuracy and precision Inter- and intra-assay accuracy and precision data for amphetamines were determined with the low, medium and high QC samples. Intra-assay data were assessed by comparing data from within one run (n = 10) and inter-assay data were determined between five separate runs (n = 34). 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.
4. Results In the course of the developed analysis performed by the LC– ESI-MS–MS method (Table 1) the validation parameters for amphetamines were determined. All six blood samples were free of co-eluting peaks at the retention times of amphetamines and their respective deuterated IS. Analysis of negative blood samples in each assay also demonstrated that the IS did not contain relevant amounts of native amphetamines. Of the 120 interference compounds added to a low validation sample (10 ng/ml) at a concentration of 10,000 ng/ ml for 120 drugs, none yielded analyte concentrations outside the ±20% limits of expected concentration. Due to, the presence one of the same 208.2/135.1 MRM pair and co-eluting problem in the case of MDEA and MBDB it was impossible simultaneous their determined. An overview of characteristic calibration data over a dynamic range from the LOQs to 500 ng/ml for amphetamines is presented in Table 2. A linear relationship between concentration and peak area was demonstrated. Additional quality control samples (n = 3) were analyzed to evaluate the upper limit of linearity and potential carryover. The 750 ng/ml samples quantified within the acceptable criteria of ±20% of target concentration. Negative samples were analyzed between samples of increasing analyte concentration. No detectable carryover occurred following the 750 ng/ml amphetamines sample. Precision and accuracy of the method were evaluated at three concentrations over the linear dynamic range (low, medium and high). Data for both intra-assay (n = 10) and inter-assay (n = 34) are presented in Table 3. Intra-assay precision (%R.S.D.) and accuracy ranged from 2.1 to 5.2 and 8.7 to 7.9%, respectively. Intra-assay precision (% R.S.D.) and accuracy ranged from 2.2 to 6.1 and 9.0 to 7.9%, respectively. Extraction efficiencies ranged from 73.6% to 136.1%. No significant matrix effect was detected for any compound. Matrix effect, suppression or enhancement, ranged from 46.0 to 57.0% with variation between 6 different bloods <16.7%. Process efficiencies ranged from 61.1 to 139.7%. All these data are shown in Tables 4 and 5. The qualitative and quantitative analysis of the two pills demonstrated:
▪ PMA – 23% of the tablet mass, what corresponded to 42.8 mg; ▪ PMA – 18.7% of the tablet mass, what corresponded to 44.6 mg. Additionally, the two pills demonstrated trace concentrations of dimethylcathinone (DMC), 4-metylo-N-ethylcathinone (4-MEC), 1-(4-fluorophenyl)piperazine (pFPP), 1-phenyl-2-(pyrrolidyne-1-yl)1-propanone (PPP) and caffeine. Autopsy samples of peripheral blood taken from all the three cases were submitted for a toxicological analysis. PMA at a very high concentration value was revealed in all the three cases. Additionally, in Case 1 and 2, amphetamine was determined. Toxicological findings obtained are presented in Table 6. The analytical documentation of the results obtained in quality control sample and autopsy blood sample for one case is shown in Figs. 1 and 2, respectively.
5. Discussion PMA is one of the most toxic amphetamine derivatives showing hallucinogenic properties and thus a substance characterized by a high lethal potential. A dose that triggers the expected effect (approximately 50–75 mg) is only slightly lower from the toxic dose (approximately 150 mg), and the effect of PMA is completely unforeseeable. It has been reported that the effects of PMA are similar to MDMA but with a slower onset, which encourages the user to take more. PMA acts as potent selective serotonin releasing agent (SSRA) with only very weak effects on dopamine and norepinephrine. However, relative to MDMA, it is considerably less effective as a releaser of serotonin and behaves more like a reuptake inhibitor in comparison [16]. This leads to the user developing malignant serotonin syndrome manifested by an increase in blood concentration, body temperature and blood pressure that occur much more rapidly and with higher severity than in case of MDMA [9]. Patients with a core body temperature over 40 °C tend to have a poor prognosis. Moreover, agitation, confusion and convulsions have been also reported in overdoses. Cardiac function disturbances and arrhythmia combined with respiratory dysfunction lead in consequence to acute cardiorespiratory failure, multiorgan failure and death. In certain cases of PMA overdose, hypoglycemia and hypercalcemia have been observed [17]. While PMA alone may cause significant toxicity, the combination of PMA with MDMA has a synergistic effect that seems to be particularly hazardous [18]. Since PMA has a slow onset of effects, several deaths have occurred where individuals have taken a PMAcontaining pill, followed by a pill containing MDMA some time afterwards due to their conviction that the first pill was not active [19]. Generally, investigations of psychoactive substances concentrate on identification of components of complex preparations based on analytic procedures developed as an ongoing process and on determination of concentration values of such compounds
Table 2 Parameters of calibration curves for the analyzed compounds. Compound name
Calibration curve (n = 3)
Coefficient of determination (R2)
Limit of detection (LOD) [ng/ml]
Limit of quantitation (LOQ) [ng/ml]
AMF Ephedrine MDA PMA methAMF MDMA PMMA MBDB MDEA
y = 0.014x 0.018 y = 0.013x 0.012 y = 0.012x 0.025 y = 0.048x+0.032 y = 0.030x 0.113 y = 0.015x 0.043 y = 0.022x+0.013 y = 0.096x 0.222 y = 0.046x 0.179
0.9999 0.9993 0.9999 0.9981 0.9996 0.9999 0.9992 0.9984 0.9996
3 3 3 1 1 3 2 1 1
10 10 10 10 10 10 10 10 10
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S. Rojek et al. / Legal Medicine 21 (2016) 64–72 Table 3 The precision and accuracy of developed method for the determination of amphetamine and its derivatives in the blood. Compound name
Concentration in blood [ng/ml]
Intra-assay precision [%RSD] (n = 7)
Intra-assay accuracy [%] (n = 7)
Inter-assay precision [%RSD] (n = 21)
Inter-assay accuracy [%] (n = 21)
AMF
10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500
2.4 2.3 2.1 3.7 4.2 5.4 3.4 4.8 4.2 4.6 4.3 3.8 3 2.9 3.2 2.8 2.5 2.4 5.2 5.1 4.3 5.2 4.7 4.2 4.1 3.4 3.4
2.8 0.8 2.1 6.4 3.6 2.1 4 2.7 3.5 7.9 6.8 8.7 4.7 4.8 3.1 2.4 2.2 1.5 4.3 8.4 5.8 7.2 5.6 4.3 4.7 3.3 0.4
2.6 2.3 2.2 5.1 4.8 3.8 4.8 4.9 5.5 4.9 4.4 3.8 4.1 3.3 4 3.7 3.1 2.6 5.1 5.9 4.5 6.1 4.7 5.2 4.7 3.3 4.8
3.1 1 2.1 6.4 4.1 2.1 3.9 3 3.5 7.9 7 6.7 4.6 4.9 2.9 2.7 2.2 1.7 4.4 9 6 7.2 5.5 4.9 5 2.9 1.4
Ephedrine
MDA
PMA
methAMF
MDMA
PMMA
MBDB
MDEA
Table 4 The extraction efficiency, process efficiency and matrix effect of developed method for the determination of amphetamine and its derivatives in the blood. Matrix effect (n = 18)
Compound name
Concentration in blood [ng/ml]
Extraction efficiency [%RSD] (n = 7)
Process efficiency [%] (n = 7)
Effect [%]
%RSD
AMF
10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500
77.9 87.7 91 97.4 90.6 99.8 94 87.5 101.5 81 78.4 84.9 83.2 99.6 93.2 114 84.4 136.1 73.6 78.2 82.4 84.7 83.4 91.3 122.4 101.9 105.7
97.4 91.9 103.8 98.1 73.8 88.1 96 79 95.2 66.1 73.5 61.6 74.1 110.8 95.6 91.5 67.8 73.2 94 122.9 74.8 72.2 76.5 79.1 85 63.9 62.2
25 5 14 1 19 12 2 10 6 18 6 27 11 11 3 20 20 46 28 57 9 15 8 13 31 37 41
3.1 5.9 12.4 2.4 4.1 15.1 3.9 6.6 13.5 3.9 7 16.7 1.6 3.9 9.9 2.7 4.2 11.7 4.4 9 16 2.2 3.5 9.9 1.8 2.9 6.4
Ephedrine
MDA
PMA
methAMF
MDMA
PMMA
MBDB
MDEA
in body fluids and poisoned tissues; the objective of such investigations is development of toxicology databases. In the present investigations, PMA and amphetamine were revealed in two cases. The developed analytical procedure based on the LC–MS-MS method has allowed for obtaining toxicology
Table 5 The extraction efficiency, process efficiency and matrix effect of developed method for the determination of deuterated amphetamine and its derivatives in the blood. Compound name
Concentration in blood [ng/ml]
Extraction efficiency [%RSD] (n = 7)
Process efficiency [%] (n = 7)
AMF-d3 ephedrine-d3 MDA-d5 MDMA-d5 methAMF-d5 MDEA-d5
100 100 100 100 100 100
104.8 116.6 118.0 104.4 126.9 98.9
139.7 115.1 123.8 111.1 117.5 124.9
Matrix effect (n = 6) Effect [%]
%RSD
33.0 1.0 5.0 6.0 7.0 26.0
3.0 4.2 4.0 4.4 3.9 7.2
Table 6 Toxicological findings in PMA fatalities. Case
1
2
3
PMA in blood concentration [mg/L]
Other xenobiotics in blood concentration
KŁ – 19 years old took narcotic substances, drank alcohol, accidental death DS – 23 years old took narcotic substances, occasionally drank alcohol, accidental death
27.1
amphetamine – 0.14 mg/L
10.0
MZ – 21 years old took narcotic substances, accidental death
10.6
amphetamine – 0.02 mg/L THC – 1.3 ng/ml THC-COOH – 35 ng/ml –
Case History
results that justified determination of poisoning with PMA as the cause of death. In all the three presented cases, the postmortem findings were very similar. Both macroscopically and microscopically, the authors noted profound congestion of the internal organs with blood extravasation and pulmonary edema. Such observations are seen in acute cardiorespiratory failure and although they are
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AMF-d3 AMF-d3 AMF AMF MDA-d5 MDA-d5 MDA MDA PMA PMA metAMF-d5 metAMF-d5 metAMF metAMF MDMA-d5 MDMA-d5 MDMA MDMA PMMA PMMA MDEA-d5 MDEA-d5 MBDB MBDB MDEA MDEA
Fig. 1. MRM chromatograms of AMF, MDA, PMA, metAMF, MDMA, PMMA, MBDB and MDEA and their deuterated internal standards from extracted quality control blood sample by LC–ESI-MS–MS. Quality control sample fortified with 10 and 100 ng/ml analytes and internal standards, respectively.
not typical for a single, tangible cause of death, yet they fully correlate with the toxic mechanism of PMA effect exerted on a human body. It should be emphasized that in each of the presently
described cases, minor skin abrasions were observed; their location may point to severe motor hyperactivity of the victims immediately before death.
S. Rojek et al. / Legal Medicine 21 (2016) 64–72
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AMF-d3 AMF-d3 AMF AMF PMA PMA
Fig. 2. MRM chromatograms of PMA, AMF and AMF-d3 from extracted autopsy blood sample in the forensic case of 2 by LC–ESI-MS–MS.
According to Felgate et al. [4], concentration of PMA in blood or plasma after hallucinogenic doses does not exceed 0.4 mg/L. Levels above 0.5 mg/L appear likely to be associated with toxic effects, which result in hyperthermia, but symptoms of malignant serotonin syndrome and respiratory depression have been also observed. Such symptoms are intensified in the presence of amphetamine. In fatal poisonings, the xenobiotic has been observed in a relatively broad concentration range: in blood – 0.2–4.9 mg/L following ingestion of 50–450 mg doses [2,4,20,21]. Apart from hyperthermia occurring in all instances, in some cases, rhabdomyolysis and hemorrhages have been described. Comparing blood PMA concentration values in the victims described in the presented cases with data reported in previously quoted publications authored by other investigators, a striking feature are relative high PMA concentrations that justify the violent course of poisoning. In the first case, the present authors demonstrated an extremely high PMA concentration – 27.1 mg/L, as well as the amphetamine concentration amounting to 145 ng/ ml, i.e. the level encountered in amphetamine addicts. In the second case, the level of PMA was more than two times lower, equaling 10 mg/L. In the course of the analysis, in addition to PMA, low levels of amphetamine were detected, as well as cannabinoids (1.3 ng/ml THC and 35 ng/ml THC-COOH). In Case 3, the level of PMA was similar to that noted in Case 2 (10.6 mg/L). It might be surmised that in all the three analyzed cases, the victims took high single PMA doses or ingested several doses over a short period. Such a mode of the drug use is confirmed by other investigators of the subject [7–9,17]. However, the doses of the ingested narcotic substance are unknown. The present analysis of PMA-containing pills that appeared on the Polish market has indicated that the product contained an average active dose – approximately 40 mg. It might be also assumed that the doses taken by the victims were very high, most likely above 450 mg [4], what would correspond to more than 10 PMA pills ingested over a relatively short period. Poisonings with unknown mixtures of narcotic substances pose problems in various areas. In clinical diagnostic management, only in a scarce few institutions outside specialist toxicology centers is the staff sufficiently experienced in diagnosing and treatment of severe conditions associated with poisoning with an unknown substance. Yet even a prompt diagnosis of the causes of ailments does not always increase the chances of survival. There is no causative treatment available; the only intervention is an attempt at alleviating the symptoms.
Problems in clinical diagnostic management are associated with laboratory diagnostics. A long time needed for performing analytical procedures while searching for a xenobiotic substance is, unfortunately, not helpful for clinicians. From this viewpoint, development of databases of new substances being introduced on the market is fully justified. Justification can be also found for methodological papers on adjusting analytical procedures to meet clinical requirements. A database enriched by introduction of subsequent cases of fatal poisonings with various substances that would comprehensively present toxicological findings supported by the range of postmortem macroscopic and microscopic lesions and meet medicolegal purposes is of a profound cognitive value in understanding the toxicity of such compounds. References _ [1] B. Habrat, H. Baran – Furga, K. Chmielewska, Z ufo w zas´wiaty, Wiedza i Zycie, http://archiwum.wiz.pl/2001/01033700.asp. [2] G. Cimbura, PMA deaths in Ontario, Can. Med. Assoc. J. 110 (1974) 1263–1267. [3] Erowid, http://www.erowid.org/chemicals/pma/pma.shtml. [4] H.E. Felgate, P.D. Felgate, R.A. James, D.N. Sims, D.C. Vozzo, Recent paramethoxyamphetamine death, J. Anal. Toxicol. 22 (1998) 169–172. [5] R.A. James, A. Dinan, Hyperpyresia associated with fatal paramethoxyamphetamine (PMA) abuse, Med. Sci. Law 38 (1) (1998) 83–85. [6] C. Lora-Tamayo, T. Tena, A. Rodriguez, Amphetamine derivative related death, Forensic Sci. Int. 85 (1997) 149–157. [7] T.L. Martin, Three cases of fatal paramethoxyamphetamine overdose, J. Anal. Toxicol. 25 (2001) 649–651. [8] L.H. Ling, C. Marchant, N.A. Buckley, M. Prior, R.J. Irvine, Poisoning with the recreational drug methoxyamphetamine (‘‘death”), Med. J. Aust. 174 (9) (2001) 453–455. [9] S.S. Johansen, A.C. Hansen, I.B. Muller, J.B. Lundemose, M.B. Franzmann, Three fatal cases of PMA and PMMA poisoning in Denmark, J. Anal. Toxicol. 27 (2003) 253–256. [10] D. Waumas, N. Bruneel, J. Tytgat, Anise oil para-methoxyamphetamine (PMA) precursor, Forensic Sci. Int. 133 (2003) 159–170. [11] Z. Chodorowski, M. Wiergowski, J. Sein Anand, Fatal intoxication with paramethoxyamphetamine, Przeg. Lek. 59 (4-5) (2002) 379–380. [12] W. Lechowicz, E. Chudzikiewicz, E. Janowska, R. Stanaszek, Zatrucia p-metoksyamfetamin1 (PMA) oraz innymi fenyloalkiloaminami, Alkoholizm i Narkomania 15 (1) (2002) 29–36. _ [13] M. Kłys, Z. Jankowski, B. Bystrowska, B. Bujak - Gizycka, G. Nowak, A significance of toxic interaction in medicolegal evidence. Complex fatal poisoning with amphetamine derivatives, Arch. Med. Sa˛d. i Krym. 51 (2001) 133–143. [14] I. Ptaszyn´ska-Sarosiek, Z. Wardaszka, A. Sackiewicz, M. Okłota, M. Niemcunowicz Janica, Cases of fatal paramethoxyamphetamine (PMA) poisoning in the material of the Forensic Medicine Department, Medical University of Bialystok, Poland, Arch. Med. Sa˛d. i Krym. 59 (2009) 190–193. [15] M. Kłys, S. Rojek, K. Woz´niak, E. Rzepecka Woz´niak, Fatality due to the use of a designer drug MDMA (Ecstasy), Leg. Med. 9 (4) (2009) 185–191.
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[16] L.C. Daws, R.J. Irvine, P.D. Callaghan, N.P. Toop, J.M. White, F. Bochner, Differential behavioral and neurochemical effects of paramethoxyamphetamine and 3,4-methylenedioxymethamphetamine in the rat, Prog. Neuropsychopharmacol. Biol. Psychiatry 24 (6) (2000) 955–977. [17] D.G. Caldicott, N.A. Edwards, A. Kruys, K.P. Kirkbride, D.N. Sims, R.W. Byard, M. Prior, R.J. Irvine, Dancing with ‘‘death: p-methoxyamphetamine overdose and its acute management”, J. Toxicol. Clin. Toxicol. 41 (2) (2003) 143–154. [18] C. Lora Tamayo, T. Tena, A. Rodriguez, D. Moreno, J.R. Sancho, P. Ensenat, F. Muela, The designer drug situation in Ibiza, Forensic Sci. Internat. 140 (2004) 195–206.
[19] R. Dams, F.A. De Letter, K.A. Mortier, J.A. Cordonnier, W.E. Lambert, M.H. Piette, S. Van Calenbergh, A.P. De Leenheer, Fatality due to combined use of designer drugs MDMA and PMA; a distribution study, J. Anal. Toxicol. 27 (2003) 318– 322. [20] A.C. Moffat, M.D. Osselton, B. Widdop (Eds.), Clarke’s Analysis of Drugs and Poisons, The Pharmaceutical Press, London, 2013. [21] R.C. Baselt, Disposition of Toxic Drugs and Chemicals in Man, fifth ed., Chemical Toxicology Institute, Foster City, CA, USA, 2000.
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Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Case Report
Medicolegal aspects of PMA-related deaths Sebastian Rojek ⇑, Filip Bolechała, Karol Kula, Martyna Maciów-Gła˛b, Małgorzata Kłys Department of Forensic Medicine, Jagiellonian University Medical College, Grzegórzecka 16 Str., 31-531 Kraków, Poland
a r t i c l e
i n f o
Article history: Received 26 November 2015 Received in revised form 16 May 2016 Accepted 6 June 2016 Available online 6 June 2016 Keywords: PMA Fatal poisonings PMA in blood LC–MS Medicolegal opinion
a b s t r a c t Unlike amphetamine, amphetamine-like substances accessible on the drug market are less expensive and more easily available; they also produce hallucinogenic effects expected by the users. Such properties render them more attractive as compared to amphetamine. On the other hand, the knowledge of the toxicity of these compounds is very limited, what in consequence generates problems that create everexpanding research areas, including analytical, clinical and medicolegal issues, thus leading to development of systemic databases. An example here is paramethoxyamphetamine (PMA), which appeared on the drug market in recent years as a result of creative inventiveness of producers of psychoactive substances, who aimed at PMA replacing the popular ecstasy (MDMA) as a less expensive and more available product. It is more potent than MDMA, but has a slower onset of action, which encourages users to take more. The problem is illustrated in the present paper by three fatal cases involving PMA, which were comprehensively investigated taking into consideration case histories, pathological and toxicological findings obtained with the use of LC–MS-MS method. In blood samples taken from all the three victims, very high concentrations of PMA were found (in the range of 10–27 mg/L) and thus the cause of deaths was determined as overdoses of PMA with the underlying mechanism of acute cardiorespiratory failure. Ó 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction A search for new sensations leading to experiencing a seemingly better reality results in an increasing worldwide demand for novel psychoactive substances. Their manufacturers encourage the users to try various substances, increasingly less expensive, more exciting, with longer action and in forms that are more attractive. Amphetamine-like substances are abused in several score countries worldwide and the number of their users exceeds 6 million [1]. Official Polish sources estimate the number of opiate addicts to be above 30,000, while the number of users of amphetamine and its derivatives is reported as several thousands. As it follows from polls carried out among various social groups, the number of individuals who regularly or occasionally take ecstasy-like substances may be as high as several thousand users [1]. Taking amphetamine derivatives resulted in numerous disasters. At the end of the 20th century in Canada [2], the United States [3], Australia [4,5] and in Spain [6], a score or so of fatalities were noted, which led to justified concerns. Their cause appeared to be a narcotic substance advertised as ecstasy, which in truth was another amphetamine derivative (paramethoxyamphetamine – PMA), ⇑ Corresponding author. E-mail address: [email protected] (S. Rojek). http://dx.doi.org/10.1016/j.legalmed.2016.06.002 1344-6223/Ó 2016 Elsevier Ireland Ltd. All rights reserved.
the street name of which – ‘‘death” – suggested danger. Later, poisonings with this substance were noted in various countries – again in Canada [7] and Australia [8], as well as in Denmark [9]. PMA appeared on the drug market as a result of creative inventiveness of producers of psychoactive substances, who aimed at paramethoxyamphetamine replacing the popular ecstasy (MDMA). Substrates for PMA production are cheaper (e.g. the substrate anethole) and more easily (legally) available as compared to substrates necessary to produce MDMA (e.g. the substrate safrole) [10]. In addition to their lower price and greater availability, amphetamine-like substances may evoke hallucinogenic effects, what makes them more attractive as compared to amphetamine [13]. In Poland, fatal poisonings with PMA appeared at the threshold of the third millennium [11–14] and were associated with introduction of the UFO pill, composed of approximately 15% PMA (40 mg PMA) and minimal amounts of amphetamine, MDMA, methamphetamine and other diluents and having a tablet mass of approximately 270 mg [11]. Victims of the UFO pill focused public concern on the issue of the fight against drug abuse. However, it is emphasized that preventive measures employed to date, which consist in a fight against drug supply and attempts at decreasing demand, are of a limited effectiveness. Although in Poland to date, severe complications following ecstasy ingestion have been infrequent and fatalities resulting from MDMA overdosing are extremely rare
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[15], attempts at replacing the substance by another drug have led to numerous accidental deaths being a consequence of manufacturers’ frauds and casual approach of the users. Evidence in support of this statement can be found in the recent (2012) Polish fatalities in consequence of PMA abuse. Deaths caused by new narcotic substances generate problems that create ever-expanding research areas, including analytical, clinical and medicolegal issues, thus leading to development of systemic databases. The problem is illustrated in the present paper by three fatal cases representing accidental poisonings involving PMA, which were the subject of complex and detailed investigations in the analytical, pathological, toxicological and medico- legal aspect. 2. Cases histories 2.1. Case 1 A male friend found KŁ, a 19-year-old female, with no vital signs and lying on the floor of a room in a flat they rented in Krakow. She was pronounced dead by an emergency medical service team. PB was interrogated as a witness; he reported that he had spent some time with KŁ during the night and they both had inhaled approximately 0.5 g amphetamine. KŁ had not drunk alcohol; the witness did not report her possibly having taken any other substances. In the past, they occasionally had used amphetamine, but not on a regular basis. PB described the effects of amphetamine as normal, stating he had not experienced any unusual consequences. At approximately 5:00 a.m., they both had gone to bed. PB had waked at approximately 4:30 p.m. and found KŁ lying on the floor with her face down; she did not react to any external stimuli. The victim was autopsied within 24 h after death. The postmortem examination showed minor skin abrasions and bruising of the hip, knee and thigh, pulmonary edema and congestion of the internal organs. The above-described macroscopic lesions combined with subsequent toxicology allowed for determining the cause of death as acute cardiorespiratory failure and acute multiorgan failure resulting from a complex poisoning with PMA and amphetamine. 2.2. Case 2 Late at night, an emergency medical service team was summoned to a flat in Krakow, where DS, a 23-year-old male, had fainted. The emergency physician pronounced him dead and did not attempt resuscitation. DS and his two colleagues were determined to have been drinking alcohol (beer and vodka) starting on the preceding day. As it followed from the case history, in the evening hours, DS had been in full contact but excited, he had been walking around the flat and suddenly had fallen to the floor, convulsed and lost consciousness. During the police inspection and searching of the premises, particles of dry plant material and a cigarette butt had been secured and referred to identification tests. The autopsy performed within 24 h after death showed minor skin abrasions involving the head, trunk and right upper extremity, disseminated bruising of the upper and lower extremities, pulmonary edema, subepicardial blood extravasation and congestion of the internal organs. Histopathology of the collected sections demonstrated cardiac muscle congestion, disseminated perivascular fibrosis, parenchymal degeneration of single muscle fibers, interstitial edema; pulmonary congestion, edema and unequal aeration; hepatic congestion, small droplet steatosis of disseminated hepatocytes; nephrotic congestion, hyalinization and fibrosis of single glomeruli; cerebral congestion. Comprehensive toxicology did not demonstrate blood and urine ethyl alcohol. The above-described macroscopic lesions combined with subsequent toxicology allowed for determining the cause of death as acute cardiorespiratory failure
65
and acute multiorgan failure resulting from a complex poisoning with PMA and amphetamine. 2.3. Case 3 A male friend found MZ, a 21-year-old female, with no vital signs and lying on the floor in a flat situated in the center of Krakow. She was pronounced dead by an emergency medical service physician. JK testified that they had spent the previous evening together, had eaten dinner and drunk wine. Several hours apart, they had also taken two pills each of a drug JK purchased several days earlier (approximately 80 tablets) as ‘‘something akin to ecstasy”. JK had been feeling well, had been in high spirits, but had felt somewhat weak and tired. MZ had been reputedly behaving ‘‘strange”, been exuberant in expressing her feelings, restless and animated. JK had fallen asleep at approximately 5:00–6:00 a.m. Around noon, he had heard MZ taking on the phone and saying she was not feeling well and was feverish. The police inspection of the premises disclosed a bag containing portions of dry plant material wrapped in aluminum foil and another bag with a large number of small, round pills. The material was referred to toxicology. The autopsy of MZ demonstrated minor skin abrasions and bruising involving the back and extremities, pulmonary edema and congestion of the internal organs. Histopathology of the collected sections demonstrated cardiac muscle congestion, interstitial edema, disseminated foci of fiber fragmentation; profound pulmonary congestion with blood extravasation to the alveoli, edema; hepatic congestion; nephrotic congestion; cerebral congestion. Comprehensive toxicology did not demonstrate blood and urine ethyl alcohol. The above-described macroscopic lesions combined with subsequent toxicology allowed for determining the cause of death as acute cardiorespiratory failure and acute multiorgan failure resulting from a complex poisoning with PMA. 3. Material and methods 3.1. Non-biological materials ▪ Two pills with a logo representing a serpent, confiscated by the prosecution and referred to identification analysis. 3.2. Biological materials ▪ Postmortem autopsy specimens – samples of femoral blood were collected in the course of autopsies carried out in the Department of Forensic Medicine, Jagiellonian University Medical College. The samples were kept frozen ( 20 °C) until the analyses were performed. ▪ 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 (n = 2) and nonpoisoned dead subjects (n = 4). 3.3. Standards and chemicals The standard solutions of 6-acetylcodeine, 6-acetylmorphine, 7-acetamidoclonazepam, acetaminophen, 7-aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, acetylsalicylic acid, alpha-hydroxyalprazolam, alprazolam, aminophenazone, amisulpride, amitriptyline, amphetamine (AMF), benzoylecgonine, bk-MBDB, bk-MDEA, bromazepam, 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-bromo-2,5-dimethoxyphenethylamine (2CB, Nexus), buprenorphine, caffeine, carbamazepine, cathinone, chlorpromazine, citalopram, clobazam, clomipramine, clonazepam, clozapine, cocaethylene, cocaine, codeine, codeine-6-glucuronide,
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cothinine, desmethylclomipramine, desmethylclozapine, diazepam, diclofenac, 10,11-dihydrocarbamazepine, 2,5-dimethoxy4-ethylamphetamine (DOET), 2,5-dimethoxy4-methylamphetamine (DOM), diphenhydramine, dosulepin, doxepine, drotaverine, ephedrine, ephedrone, 10,11epoxycarbamazepine, estazolam, 2-ethyl-1,5-dimethyl-3,3-diphe nylpirrolidine (EDDP), flephedrone, flunitrazepam, fluoxetine, flurazepam, fluvoxamine, hydrocodone, hydromorphone, hydroxyamphetamine, ibuprofen, imipramine, ketamine, ketoprofen, levomepromazine, lorazepam, lormetazepam, methadone, methamphetamine (methAMF), methcathinone, methedrone, 4-methoxyamphetamine (PMA), 4-methoxymethamphetamine (PMMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylene dioxyethylamphetamine (MDEA), 3,4-methylenedioxymethamphe tamine (MDMA), 3,4-methylenedioxy-N-methyl-butanphenamine (MBDB), 4-methylethcathinone (4-MEC) 4-methylmethcathinone (4-MMC, mephedrone), methylone, mianserine, midazolam, moclobemide, morphine, morphine-3-glucuronide, morphine-6glucuronide, nicotine, nitrazepam, norbuprenorphine, nordiazepam, nordoxepine, norephedrine, norfluoxetine, norketamine, normorphine, O-desmethylvenlafaxine, oxazepam, oxcarbazepine, oxycodone, oxymorphone, paroxetine, pentazocine, pethidine, promazine, promethazine, propranolol, reboxetine, sertraline, sulpiride, temazepam, thiorydazine, tramadol, venlafaxine, zaleplone, zolpidem, zopiclone and amphetamine-d3 (AMF-d3), ephedrine-d3, MDA-d5, methamphetamine-d5 (methAMF-d5), MDMA-d5, MDEAd5 used as internal standards (IS), were purchased from LGC Standards (Warszawa, Poland), ammonium formate, ammonium carbonate, formic acid, acetic acid – 99.9% for LC–MS were purchased from SIGMA (Poznan´, Poland), acetonitrile, methanol from Fluka (Poznan´, Poland). Solid-phase extraction columns LiChrolut RP-18 (Merck, Darmstadt, Germany) containing 500 mg C18-RP bonded silica were used. 3.4. Calibrators and controls For the calibrator samples, two working solutions were prepared in methanol at the following concentrations: 1.0 and 10 ng AMF, ephedrine, MDA, PMA, methAMF, MDMA, PMMA, MBDB and MDEA/ll. Also methanolic solutions were prepared for quality control (QC) samples at concentrations of 1.0 and 10 ng/ll for amphetamines. 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 1 ml of blank blood with known amounts of AMF, ephedrine, MDA, PMA, methAMF, MDMA, PMMA, MBDB and MDEA at concentrations ranging from 10 to 500 ng/ml. Low, medium and high QC specimens were also prepared daily at concentrations of 10, 50, 500 ng/ml for amphetamines. For the deuterated internal standard (IS), a working solution of 1.0 ng AMF-d3, ephedrine-d3, MDA-d5, methAMF-d5, MDMA-d5 and MDEA-d5 in methanol was prepared and stored at 20 °C when not in use. Hundred microliters of this working solution was added to each sample prior to extraction, giving a final deuterated IS concentration of 100 ng/ml. 3.5. Analytical methods 3.5.1. Preliminary screening The screening test included an enzyme-linked immunosorbent assay (ELISA, Neogen, Ayr, Scotland, UK) of blood for amphetamine, methamphetamine/MDMA, opiates, cocaine, cannabinoids, barbiturates, benzodiazepines, tricyclic antidepressants, 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 (case 1) and amphetamine, methamphetamine (case 2 and 3). The HPLC-DAD analysis paved a path for further analysis. Comparing the UV spectra found in postmortem specimens suggested that it could be some amphetamine derivative, which has idiosyncratic spectra. 3.5.2. Extraction The autopsy blood sample (1 ml) was subjected to solid-phase extraction. The samples were put into a clean 15 ml Falcon tubes and diluted five times with 0.1 M carbonate buffer to pH 9.3. AMF-d3, ephedrine-d3, MDA-d5, methAMF-d5, MDMA-d5 and MDEA-d5 at the concentration of 100 ng/ml were added to the samples. Afterwards, the samples were vortexed and centrifuged for 7.5 min at 1500g. 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 0.1 ml of mixture (95% phase A + 5% phase B) and injected into LC–MS system. Forensic blood samples were diluted. 3.5.3. Separation by liquid chromatography An Agilent Technologies 1200 liquid chromatograph (Santa Clara, CA, USA) equipped with a binary pump (G1312 A) and an autosampler (G1329 A) 15 ll injection loop were used in gradient mode. The chromatographic separation was performed with a Allure PFP-C18 column (50 mm 3 mm, 2.7 lm, Restec, USA). The phase A was water, which contained 0.02% formic acid and 0.002 M of ammonium formate and phase B was acetonitrile with 0.02% formic acid and 0.002 M of ammonium formate. The gradient for samples concerning PMA and IS’s 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. 3.5.4. Detection by tandem mass spectrometry A 6410 triple quad mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), an atmospheric pressure electrospray ion source (ESI), operated under positive mode was used. The operational parameters of the ESI source were as follows: vaporizing temperature 350 °C; pressure of the nebulising gas 40 psi; flow of the drying gas 9 l/min; capillary potential 3500 V. Fragmentation parameters of analyzed compounds are listed in Table 1. 3.6. Validation of LC–ESI-MS–MS method 3.6.1. Selectivity To evaluate peak-purity and selectivity, two blank blood samples from living person and four from non-poisoned dead subjects (no analyte or IS added) were analyzed with each batch to check for peaks that might interfere with detection of AMF-d3, AMF, ephedrine-d3, ephedrine, MDA-d5, MDA, PMA, methAMF-d5, methAMF, MDMA-d5, MDMA, PMMA, MBDB, MDEA-d5 and MDEA. To assess possible interferences of other drugs on abuse and pharmaceuticals quality control samples were spiked individually to contain 10,000 ng/ml of 6-acetylcodeine, 6-acetylmorphine, 7-acetamidoclonazepam, acetaminophen, 7-aminoclonazepam, 7-aminoflunitrazepam, 7-aminonitrazepam, acetylsalicylic acid, alpha-hydroxyalprazolam, alprazolam, aminophenazone, amisulpride, amitriptyline, benzoylecgonine, bk-MBDB, bk-MDEA, bromazepam, 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-bro
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S. Rojek et al. / Legal Medicine 21 (2016) 64–72 Table 1 Fragmentation parameters of the analyzed compounds. Compound name
Precursor ion
Product ion
Fragmentor voltage [V]
Collision energy [V]
Retention time [min]
AMF-d3
139.2 136.1
Ephedrine-d3
169.2
Ephedrine
166.2
MDA-d5
185.1
MDA
180.1
PMA
166.2
methAMF-d5
155.2
methAMF
150.1
MDMA-d5
199.2
MDMA
194.2
PMMA
180.1
MBDB
208.2
MDEA-d5
213.2
MDEA
208.2
80 80 80 80 90 90 90 90 70 70 70 70 70 70 50 50 90 90 95 95 95 95 70 70 90 90 70 70 90 90
5 15 5 15 10 20 10 20 5 15 5 15 5 20 9 21 5 15 5 20 10 20 5 20 20 10 9 21 15 20
4.7
AMF
122.1 92.1 119.1 91.1 151.1 136.1 148.1 133.1 168.1 138.1 163.2 135.1 149.1 121.1 121.1 92.1 119.1 91.1 165.2 135.0 163.1 135.0 149.1 121.1 135.0 177.1 163.1 135.0 163.1 135.1
4.7 5.0 5.0 5.2 5.2 5.3 5.2 5.2 5.5 5.5 5.5 5.8 5.7 5.7
Bold indicated quantitative ions.
mo-2,5-dimethoxyphenethylamine (2CB), buprenorphine, caffeine, carbamazepine, cathinone, chlorpromazine, citalopram, clobazam, clomipramine, clonazepam, clozapine, cocaethylene, cocaine, codeine, codeine-6-glucuronide, cothinine, desmethylclomipramine, desmethylclozapine, diazepam, diclofenac, 10,11dihydrocarbamazepine, 2,5-dimethoxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-methylamphetamine (DOM), diphenhydramine, dosulepin, doxepine, drotaverine, ephedrone, 10,11epoxycarbamazepine, estazolam, 2-ethyl-1,5-dimethyl-3,3-diphe nylpirrolidine (EDDP), flephedrone, flunitrazepam, fluoxetine, flurazepam, fluvoxamine, hydrocodone, hydromorphone, hydroxyamphetamine, ibuprofen, imipramine, ketamine, ketoprofen, levomepromazine, lorazepam, lormetazepam, methadone, methcathinone, methedrone, 4-methylethcathinone (4-MEC) 4methylmethcathinone (4-MMC, mephedrone), methylone, mianserine, midazolam, moclobemide, morphine, morphine-3glucuronide, morphine-6-glucuronide, nicotine, nitrazepam, norbuprenorphine, nordiazepam, nordoxepine, norephedrine, norfluoxetine, norketamine, normorphine, O-desmethylvenlafaxine, oxazepam, oxcarbazepine, oxycodone, oxymorphone, paroxetine, pentazocine, pethidine, promazine, promethazine, propranolol, reboxetine, sertraline, sulpiride, temazepam, thiorydazine, tramadol, venlafaxine, zaleplone, zolpidem and zopiclone. There were no interferences with the said compounds. 3.6.2. Linearity, limit of quantitation, carry – over Calibration curves were prepared daily by spiking blank blood with corresponding analytical working solutions to obtain calibration concentrations of 10, 20, 50, 100 and 500 ng/ml AMF, ephedrine, MDA, PMA, methAMF, MDMA, PMMA, MBDB and MDEA. Validation samples were prepared in triplicate at the following concentrations: 1, 2, 3, 5, 750, 1000, 1250 ng/ml AMF, ephedrine, MDA, PMA, methAMF, MDMA, PMMA, MBDB and MDEA to assess the method’s accuracy above and below the calibration curve.
Negative quality control samples were analyzed after each linearity sample to evaluate potential carry-over. The samples were extracted according to the procedure described. Calibration curves were constructed by plotting the peak-area ratios analytes/internal standard. 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 P10/1, 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. 3.6.3. Extraction efficiency, matrix effects and process efficiency Extraction efficiency for each analyte was measured at each QC concentration. Blank blood was fortified with QC and internal standard 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 ten unique blank extracted bloods fortified with QC and internal standards 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 mean peak area of fortified bloods 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.
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3.6.4. Accuracy and precision Inter- and intra-assay accuracy and precision data for amphetamines were determined with the low, medium and high QC samples. Intra-assay data were assessed by comparing data from within one run (n = 10) and inter-assay data were determined between five separate runs (n = 34). 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.
4. Results In the course of the developed analysis performed by the LC– ESI-MS–MS method (Table 1) the validation parameters for amphetamines were determined. All six blood samples were free of co-eluting peaks at the retention times of amphetamines and their respective deuterated IS. Analysis of negative blood samples in each assay also demonstrated that the IS did not contain relevant amounts of native amphetamines. Of the 120 interference compounds added to a low validation sample (10 ng/ml) at a concentration of 10,000 ng/ ml for 120 drugs, none yielded analyte concentrations outside the ±20% limits of expected concentration. Due to, the presence one of the same 208.2/135.1 MRM pair and co-eluting problem in the case of MDEA and MBDB it was impossible simultaneous their determined. An overview of characteristic calibration data over a dynamic range from the LOQs to 500 ng/ml for amphetamines is presented in Table 2. A linear relationship between concentration and peak area was demonstrated. Additional quality control samples (n = 3) were analyzed to evaluate the upper limit of linearity and potential carryover. The 750 ng/ml samples quantified within the acceptable criteria of ±20% of target concentration. Negative samples were analyzed between samples of increasing analyte concentration. No detectable carryover occurred following the 750 ng/ml amphetamines sample. Precision and accuracy of the method were evaluated at three concentrations over the linear dynamic range (low, medium and high). Data for both intra-assay (n = 10) and inter-assay (n = 34) are presented in Table 3. Intra-assay precision (%R.S.D.) and accuracy ranged from 2.1 to 5.2 and 8.7 to 7.9%, respectively. Intra-assay precision (% R.S.D.) and accuracy ranged from 2.2 to 6.1 and 9.0 to 7.9%, respectively. Extraction efficiencies ranged from 73.6% to 136.1%. No significant matrix effect was detected for any compound. Matrix effect, suppression or enhancement, ranged from 46.0 to 57.0% with variation between 6 different bloods <16.7%. Process efficiencies ranged from 61.1 to 139.7%. All these data are shown in Tables 4 and 5. The qualitative and quantitative analysis of the two pills demonstrated:
▪ PMA – 23% of the tablet mass, what corresponded to 42.8 mg; ▪ PMA – 18.7% of the tablet mass, what corresponded to 44.6 mg. Additionally, the two pills demonstrated trace concentrations of dimethylcathinone (DMC), 4-metylo-N-ethylcathinone (4-MEC), 1-(4-fluorophenyl)piperazine (pFPP), 1-phenyl-2-(pyrrolidyne-1-yl)1-propanone (PPP) and caffeine. Autopsy samples of peripheral blood taken from all the three cases were submitted for a toxicological analysis. PMA at a very high concentration value was revealed in all the three cases. Additionally, in Case 1 and 2, amphetamine was determined. Toxicological findings obtained are presented in Table 6. The analytical documentation of the results obtained in quality control sample and autopsy blood sample for one case is shown in Figs. 1 and 2, respectively.
5. Discussion PMA is one of the most toxic amphetamine derivatives showing hallucinogenic properties and thus a substance characterized by a high lethal potential. A dose that triggers the expected effect (approximately 50–75 mg) is only slightly lower from the toxic dose (approximately 150 mg), and the effect of PMA is completely unforeseeable. It has been reported that the effects of PMA are similar to MDMA but with a slower onset, which encourages the user to take more. PMA acts as potent selective serotonin releasing agent (SSRA) with only very weak effects on dopamine and norepinephrine. However, relative to MDMA, it is considerably less effective as a releaser of serotonin and behaves more like a reuptake inhibitor in comparison [16]. This leads to the user developing malignant serotonin syndrome manifested by an increase in blood concentration, body temperature and blood pressure that occur much more rapidly and with higher severity than in case of MDMA [9]. Patients with a core body temperature over 40 °C tend to have a poor prognosis. Moreover, agitation, confusion and convulsions have been also reported in overdoses. Cardiac function disturbances and arrhythmia combined with respiratory dysfunction lead in consequence to acute cardiorespiratory failure, multiorgan failure and death. In certain cases of PMA overdose, hypoglycemia and hypercalcemia have been observed [17]. While PMA alone may cause significant toxicity, the combination of PMA with MDMA has a synergistic effect that seems to be particularly hazardous [18]. Since PMA has a slow onset of effects, several deaths have occurred where individuals have taken a PMAcontaining pill, followed by a pill containing MDMA some time afterwards due to their conviction that the first pill was not active [19]. Generally, investigations of psychoactive substances concentrate on identification of components of complex preparations based on analytic procedures developed as an ongoing process and on determination of concentration values of such compounds
Table 2 Parameters of calibration curves for the analyzed compounds. Compound name
Calibration curve (n = 3)
Coefficient of determination (R2)
Limit of detection (LOD) [ng/ml]
Limit of quantitation (LOQ) [ng/ml]
AMF Ephedrine MDA PMA methAMF MDMA PMMA MBDB MDEA
y = 0.014x 0.018 y = 0.013x 0.012 y = 0.012x 0.025 y = 0.048x+0.032 y = 0.030x 0.113 y = 0.015x 0.043 y = 0.022x+0.013 y = 0.096x 0.222 y = 0.046x 0.179
0.9999 0.9993 0.9999 0.9981 0.9996 0.9999 0.9992 0.9984 0.9996
3 3 3 1 1 3 2 1 1
10 10 10 10 10 10 10 10 10
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S. Rojek et al. / Legal Medicine 21 (2016) 64–72 Table 3 The precision and accuracy of developed method for the determination of amphetamine and its derivatives in the blood. Compound name
Concentration in blood [ng/ml]
Intra-assay precision [%RSD] (n = 7)
Intra-assay accuracy [%] (n = 7)
Inter-assay precision [%RSD] (n = 21)
Inter-assay accuracy [%] (n = 21)
AMF
10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500
2.4 2.3 2.1 3.7 4.2 5.4 3.4 4.8 4.2 4.6 4.3 3.8 3 2.9 3.2 2.8 2.5 2.4 5.2 5.1 4.3 5.2 4.7 4.2 4.1 3.4 3.4
2.8 0.8 2.1 6.4 3.6 2.1 4 2.7 3.5 7.9 6.8 8.7 4.7 4.8 3.1 2.4 2.2 1.5 4.3 8.4 5.8 7.2 5.6 4.3 4.7 3.3 0.4
2.6 2.3 2.2 5.1 4.8 3.8 4.8 4.9 5.5 4.9 4.4 3.8 4.1 3.3 4 3.7 3.1 2.6 5.1 5.9 4.5 6.1 4.7 5.2 4.7 3.3 4.8
3.1 1 2.1 6.4 4.1 2.1 3.9 3 3.5 7.9 7 6.7 4.6 4.9 2.9 2.7 2.2 1.7 4.4 9 6 7.2 5.5 4.9 5 2.9 1.4
Ephedrine
MDA
PMA
methAMF
MDMA
PMMA
MBDB
MDEA
Table 4 The extraction efficiency, process efficiency and matrix effect of developed method for the determination of amphetamine and its derivatives in the blood. Matrix effect (n = 18)
Compound name
Concentration in blood [ng/ml]
Extraction efficiency [%RSD] (n = 7)
Process efficiency [%] (n = 7)
Effect [%]
%RSD
AMF
10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500 10 50 500
77.9 87.7 91 97.4 90.6 99.8 94 87.5 101.5 81 78.4 84.9 83.2 99.6 93.2 114 84.4 136.1 73.6 78.2 82.4 84.7 83.4 91.3 122.4 101.9 105.7
97.4 91.9 103.8 98.1 73.8 88.1 96 79 95.2 66.1 73.5 61.6 74.1 110.8 95.6 91.5 67.8 73.2 94 122.9 74.8 72.2 76.5 79.1 85 63.9 62.2
25 5 14 1 19 12 2 10 6 18 6 27 11 11 3 20 20 46 28 57 9 15 8 13 31 37 41
3.1 5.9 12.4 2.4 4.1 15.1 3.9 6.6 13.5 3.9 7 16.7 1.6 3.9 9.9 2.7 4.2 11.7 4.4 9 16 2.2 3.5 9.9 1.8 2.9 6.4
Ephedrine
MDA
PMA
methAMF
MDMA
PMMA
MBDB
MDEA
in body fluids and poisoned tissues; the objective of such investigations is development of toxicology databases. In the present investigations, PMA and amphetamine were revealed in two cases. The developed analytical procedure based on the LC–MS-MS method has allowed for obtaining toxicology
Table 5 The extraction efficiency, process efficiency and matrix effect of developed method for the determination of deuterated amphetamine and its derivatives in the blood. Compound name
Concentration in blood [ng/ml]
Extraction efficiency [%RSD] (n = 7)
Process efficiency [%] (n = 7)
AMF-d3 ephedrine-d3 MDA-d5 MDMA-d5 methAMF-d5 MDEA-d5
100 100 100 100 100 100
104.8 116.6 118.0 104.4 126.9 98.9
139.7 115.1 123.8 111.1 117.5 124.9
Matrix effect (n = 6) Effect [%]
%RSD
33.0 1.0 5.0 6.0 7.0 26.0
3.0 4.2 4.0 4.4 3.9 7.2
Table 6 Toxicological findings in PMA fatalities. Case
1
2
3
PMA in blood concentration [mg/L]
Other xenobiotics in blood concentration
KŁ – 19 years old took narcotic substances, drank alcohol, accidental death DS – 23 years old took narcotic substances, occasionally drank alcohol, accidental death
27.1
amphetamine – 0.14 mg/L
10.0
MZ – 21 years old took narcotic substances, accidental death
10.6
amphetamine – 0.02 mg/L THC – 1.3 ng/ml THC-COOH – 35 ng/ml –
Case History
results that justified determination of poisoning with PMA as the cause of death. In all the three presented cases, the postmortem findings were very similar. Both macroscopically and microscopically, the authors noted profound congestion of the internal organs with blood extravasation and pulmonary edema. Such observations are seen in acute cardiorespiratory failure and although they are
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AMF-d3 AMF-d3 AMF AMF MDA-d5 MDA-d5 MDA MDA PMA PMA metAMF-d5 metAMF-d5 metAMF metAMF MDMA-d5 MDMA-d5 MDMA MDMA PMMA PMMA MDEA-d5 MDEA-d5 MBDB MBDB MDEA MDEA
Fig. 1. MRM chromatograms of AMF, MDA, PMA, metAMF, MDMA, PMMA, MBDB and MDEA and their deuterated internal standards from extracted quality control blood sample by LC–ESI-MS–MS. Quality control sample fortified with 10 and 100 ng/ml analytes and internal standards, respectively.
not typical for a single, tangible cause of death, yet they fully correlate with the toxic mechanism of PMA effect exerted on a human body. It should be emphasized that in each of the presently
described cases, minor skin abrasions were observed; their location may point to severe motor hyperactivity of the victims immediately before death.
S. Rojek et al. / Legal Medicine 21 (2016) 64–72
71
AMF-d3 AMF-d3 AMF AMF PMA PMA
Fig. 2. MRM chromatograms of PMA, AMF and AMF-d3 from extracted autopsy blood sample in the forensic case of 2 by LC–ESI-MS–MS.
According to Felgate et al. [4], concentration of PMA in blood or plasma after hallucinogenic doses does not exceed 0.4 mg/L. Levels above 0.5 mg/L appear likely to be associated with toxic effects, which result in hyperthermia, but symptoms of malignant serotonin syndrome and respiratory depression have been also observed. Such symptoms are intensified in the presence of amphetamine. In fatal poisonings, the xenobiotic has been observed in a relatively broad concentration range: in blood – 0.2–4.9 mg/L following ingestion of 50–450 mg doses [2,4,20,21]. Apart from hyperthermia occurring in all instances, in some cases, rhabdomyolysis and hemorrhages have been described. Comparing blood PMA concentration values in the victims described in the presented cases with data reported in previously quoted publications authored by other investigators, a striking feature are relative high PMA concentrations that justify the violent course of poisoning. In the first case, the present authors demonstrated an extremely high PMA concentration – 27.1 mg/L, as well as the amphetamine concentration amounting to 145 ng/ ml, i.e. the level encountered in amphetamine addicts. In the second case, the level of PMA was more than two times lower, equaling 10 mg/L. In the course of the analysis, in addition to PMA, low levels of amphetamine were detected, as well as cannabinoids (1.3 ng/ml THC and 35 ng/ml THC-COOH). In Case 3, the level of PMA was similar to that noted in Case 2 (10.6 mg/L). It might be surmised that in all the three analyzed cases, the victims took high single PMA doses or ingested several doses over a short period. Such a mode of the drug use is confirmed by other investigators of the subject [7–9,17]. However, the doses of the ingested narcotic substance are unknown. The present analysis of PMA-containing pills that appeared on the Polish market has indicated that the product contained an average active dose – approximately 40 mg. It might be also assumed that the doses taken by the victims were very high, most likely above 450 mg [4], what would correspond to more than 10 PMA pills ingested over a relatively short period. Poisonings with unknown mixtures of narcotic substances pose problems in various areas. In clinical diagnostic management, only in a scarce few institutions outside specialist toxicology centers is the staff sufficiently experienced in diagnosing and treatment of severe conditions associated with poisoning with an unknown substance. Yet even a prompt diagnosis of the causes of ailments does not always increase the chances of survival. There is no causative treatment available; the only intervention is an attempt at alleviating the symptoms.
Problems in clinical diagnostic management are associated with laboratory diagnostics. A long time needed for performing analytical procedures while searching for a xenobiotic substance is, unfortunately, not helpful for clinicians. From this viewpoint, development of databases of new substances being introduced on the market is fully justified. Justification can be also found for methodological papers on adjusting analytical procedures to meet clinical requirements. A database enriched by introduction of subsequent cases of fatal poisonings with various substances that would comprehensively present toxicological findings supported by the range of postmortem macroscopic and microscopic lesions and meet medicolegal purposes is of a profound cognitive value in understanding the toxicity of such compounds. References _ [1] B. Habrat, H. Baran – Furga, K. Chmielewska, Z ufo w zas´wiaty, Wiedza i Zycie, http://archiwum.wiz.pl/2001/01033700.asp. [2] G. Cimbura, PMA deaths in Ontario, Can. Med. Assoc. J. 110 (1974) 1263–1267. [3] Erowid, http://www.erowid.org/chemicals/pma/pma.shtml. [4] H.E. Felgate, P.D. Felgate, R.A. James, D.N. Sims, D.C. Vozzo, Recent paramethoxyamphetamine death, J. Anal. Toxicol. 22 (1998) 169–172. [5] R.A. James, A. Dinan, Hyperpyresia associated with fatal paramethoxyamphetamine (PMA) abuse, Med. Sci. Law 38 (1) (1998) 83–85. [6] C. Lora-Tamayo, T. Tena, A. Rodriguez, Amphetamine derivative related death, Forensic Sci. Int. 85 (1997) 149–157. [7] T.L. Martin, Three cases of fatal paramethoxyamphetamine overdose, J. Anal. Toxicol. 25 (2001) 649–651. [8] L.H. Ling, C. Marchant, N.A. Buckley, M. Prior, R.J. Irvine, Poisoning with the recreational drug methoxyamphetamine (‘‘death”), Med. J. Aust. 174 (9) (2001) 453–455. [9] S.S. Johansen, A.C. Hansen, I.B. Muller, J.B. Lundemose, M.B. Franzmann, Three fatal cases of PMA and PMMA poisoning in Denmark, J. Anal. Toxicol. 27 (2003) 253–256. [10] D. Waumas, N. Bruneel, J. Tytgat, Anise oil para-methoxyamphetamine (PMA) precursor, Forensic Sci. Int. 133 (2003) 159–170. [11] Z. Chodorowski, M. Wiergowski, J. Sein Anand, Fatal intoxication with paramethoxyamphetamine, Przeg. Lek. 59 (4-5) (2002) 379–380. [12] W. Lechowicz, E. Chudzikiewicz, E. Janowska, R. Stanaszek, Zatrucia p-metoksyamfetamin1 (PMA) oraz innymi fenyloalkiloaminami, Alkoholizm i Narkomania 15 (1) (2002) 29–36. _ [13] M. Kłys, Z. Jankowski, B. Bystrowska, B. Bujak - Gizycka, G. Nowak, A significance of toxic interaction in medicolegal evidence. Complex fatal poisoning with amphetamine derivatives, Arch. Med. Sa˛d. i Krym. 51 (2001) 133–143. [14] I. Ptaszyn´ska-Sarosiek, Z. Wardaszka, A. Sackiewicz, M. Okłota, M. Niemcunowicz Janica, Cases of fatal paramethoxyamphetamine (PMA) poisoning in the material of the Forensic Medicine Department, Medical University of Bialystok, Poland, Arch. Med. Sa˛d. i Krym. 59 (2009) 190–193. [15] M. Kłys, S. Rojek, K. Woz´niak, E. Rzepecka Woz´niak, Fatality due to the use of a designer drug MDMA (Ecstasy), Leg. Med. 9 (4) (2009) 185–191.
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