Forensic Science International 183 (2009) 91–96
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A fatal poisoning involving Bromo-Dragonfly Mette Findal Andreasen a,*, Rasmus Telving a, Rune Isak Dupont Birkler a, Bente Schumacher b, Mogens Johannsen a a b
Section for Toxicology and Drug Analysis, Institute of Forensic Medicine, University of Aarhus, Brendstrupgaardsvej 100, DK-8200 Aarhus N, Denmark Section for Forensic Pathology and Clinical Forensic Medicine, Institute of Forensic Medicine, University of Aarhus, Brendstrupgaardsvej 100, DK-8200 Aarhus N, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 May 2008 Received in revised form 31 October 2008 Accepted 3 November 2008 Available online 17 December 2008
This paper reports a fatal overdose case involving the potent hallucinogenic drug Bromo-Dragonfly (1-(8bromobenzo[1,2-b; 4,5-b0 ]difuran-4-yl)-2-aminopropane). In the present case, an 18-year-old woman was found dead after ingestion of a hallucinogenic liquid. A medico-legal autopsy was performed on the deceased, during which liver, blood, urine and vitreous humour were submitted for toxicological examination. Bromo-Dragonfly was identified in the liver blood using UPLC–TOFMS, and was subsequently quantified in femoral blood (0.0047 mg/kg), urine (0.033 mg/kg) and vitreous humour (0.0005 mg/kg) using LC–MS/MS. Calibration standards were prepared from Bromo-Dragonfly isolated from a bottle found next to the deceased. The structure and purity of the isolated compound were unambiguously determined from analysis of UPLC–TOFMS, GC–MS, HPLC–DAD, 1H and 13C NMR data and by comparison to literature data. The autopsy findings were non-specific for acute poisoning. However, based on the toxicological findings, the cause of death was determined to be a fatal overdose of Bromo-Dragonfly, as no ethanol and no therapeutics or other drugs of abuse besides Bromo-Dragonfly were detected in the liver, blood or urine samples from the deceased. To our knowledge, this is the first report of quantification of BromoDragonfly in a biological specimen from a deceased person. This case caused the drug to be classified as an illegal drug in Denmark on 5th December 2007. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Bromo-Dragonfly LC–MS/MS UPLC–TOFMS Intoxication
1. Introduction The consumption of synthetic drugs, usually of the phenethylamine class, has increased over the last decade and has become a serious public health problem in many European countries (http:// www.emcdda.europa.eu/). These drugs are often synthesized illegally in underground laboratories, where new variants may be produced by modifying the molecular structure of a known stimulant or hallucinogenic compound (e.g., amphetamine or different tryptamines). These novel designer drugs often have pharmacological properties very different from their chemical cousins [1]. Bromo-Dragonfly is one of these new substances on the drug market, and the first reports of its consumption appeared in 2005–2006 on informal internet pages. Bromo-Dragonfly is the common name for 1-(8-bromobenzo[1,2-b; 4,5-b0 ]difuran-4-yl)-2aminopropane (Fig. 1). Structurally, it is closely related to phenylethylamines like 2C-B (4-bromo-2,5-dimethoxyphenethylamine) and DOB (2,5-dimethoxy-4-bromoamphetamine). Its synth-
* Corresponding author. Tel.: +45 89429844; fax: +45 86175003. E-mail address:
[email protected] (M.F. Andreasen). 0379-0738/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2008.11.001
esis and potency as an agonist of the serotonin 5-HT2A receptor were described in 1998 [2]. It is considered a potent hallucinogen, only slightly less potent than LSD, and it has a very long duration of action. Bromo-Dragonfly has a single stereocenter, and R-()-BromoDragonfly is the more active stereoisomer [3]. The drug has recently entered the Scandinavian drug scene, and herein we describe the first death related to the intake of Bromo-Dragonfly in Denmark. In addition to the death detailed in this report, two other deaths presumably related to the intake of Bromo-Dragonfly have appeared in Scandinavia during 2007 [4]. To our knowledge, this is the first report of the identification and quantification of Bromo-Dragonfly in a biological specimen from a deceased person. 2. Case history In the present case, an 18-year-old woman was found dead after ingestion of a hallucinogenic LSD-like liquid. The woman and her boyfriend had presumably both ingested around 1 mL of the liquid on the preceding evening (between 10 and 11 pm), and their intention was to have sex during the high. At 12 pm they started feeling the ‘‘LSD-trip’’. However, they both fell asleep, and when the boyfriend woke up at 5 am the following morning, he found his
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About 5000 Fishman units of b-glucuronidase (containing sulfatase activity) were added to each sample and incubated in a shaking water bath for 16 h at 37 8C [6]. The hydrolysed and non-hydrolysed urine samples were extracted as described above for autopsy blood samples. Calibration standards containing BromoDragonfly were prepared from drug free urine at concentrations of 0, 0.5, 1.0, 5.0, 10, 25 and 50 mg/kg. Vitreous humour (0.5 g) was handled in the same manner as the autopsy blood samples. Calibration standards were prepared in MilliQ-water at the following concentrations: 0, 0.5, 1.0, 5.0, 10, 25 and 50 mg/kg.
Fig. 1. Structure of Bromo-Dragonfly.
girlfriend dead. A medico-legal autopsy was done on the deceased, and liver, blood, urine and vitreous humour were submitted for toxicological examination. Furthermore, a bottle containing the hallucinogen LSD-like liquid was subjected to analysis. The autopsy findings included oedema of the lungs, slight oedema of the brain, enlargement of the spleen, irritation of the mucous membrane in the stomach and ischemic changes in the kidneys. 3. Materials and methods 3.1. Chemicals and reagents 1-(8-Bromobenzo[1,2-b; 4,5-b0 ]difuran-4-yl)-2-aminopropane (Bromo-Dragonfly) was extracted and purified from the bottle containing 38 mL of an ‘‘LSD-like liquid’’ found next to the deceased (Section 3.3.1). b-Glucuronidase from Helix pornatia (EC 3.2.1.31) was supplied from Sigma (St. Louis, MO) (100,000 per millilitre for b-glucuronidase and some sulfatase activities). All other chemicals and reagents used were of the highest commercially available quality. 3.2. Biological materials Post-mortem specimens were collected at autopsy 3 days after the death (within the 12 h after death, the deceased was stored at 4 8C). Samples (liver blood, femoral vein blood, urine, vitreous humour), mixed with NaF (5 mg/g), were frozen at 18 8C until use. Blood samples from healthy volunteers and autopsy urine free from any xenobiotics were used as blank samples, and for the preparation of standards and quality control (QC) samples. 3.3. Analytical procedures 3.3.1. Identification, extraction, purification and confirmation of Bromo-Dragonfly from liquid Liquid (1 mL) from the bottle containing the colourless hallucinogenic liquid was evaporated to dryness and analysed using UPLC–TOFMS (ultra performance liquid chromatography–time-of-flight mass spectrometry), GC–MS (gas chromatography–mass spectrometry), HPLC–DAD (high performance liquid chromatography– diode array detector) and 1H NMR (proton nuclear magnetic resonance spectroscopy). The spectra showed no trace of LSD, but instead an almost pure solution of Bromo-Dragonfly. The drug was purified according to the previously published general procedure [5]: 2.5 mL 1 M KOH was added to 25 mL of an aqueous solution containing the hallucinogenic substance. The aqueous solution was extracted three times with 25 mL of diethyl ether, and the combined organic phases were washed with 10 mL of a saturated aqueous solution of sodium chloride and dried over MgSO4. After drying and filtering, 2 mL of a 2 M HCl solution in diethyl ether was added, whereby a white precipitate was formed. After 2 h at 18 8C, the ether phase was carefully removed, and the precipitate was washed twice with a mixture of hexane and ether. After drying, 11.5 mg of analytical pure Bromo-Dragonfly was obtained. Another 10 mg was isolated by adding hexane to the ether solution and repeating the final steps of the above procedure. Concomitant GC–MS, HPLC–DAD, LC–TOFMS, 1H and 13C NMR analyses were used to confirm the structure and purity of the isolated compound as 1-(8-bromobenzo[1,2-b; 4,5-b0 ]difuran-4-yl)-2aminopropane (Bromo-Dragonfly). 3.3.2. Extraction and analysis from biological specimens Autopsy blood samples (1.0 g) and the internal standard (MDMA-d5, 10 ng) were mixed with 250 mL carbonate buffer (1 M Na2CO3 adjusted to pH 11 with HCl) and extracted twice using 2 mL hexane/butylchloride (50:50). The organic extracts were combined with 50 mL 0.1 M HCl in methanol and evaporated to dryness under a stream of nitrogen at 30 8C. The residue was dissolved in 200 mL mobile phase (9:1 solvent A:solvent B, Section 3.3.5), and a 10 mL sample was automatically injected onto the LC– MS/MS system. Double determination in two analytical series was performed, and calibration standards containing Bromo-Dragonfly were prepared from drug free blood at the following concentrations: 0, 0.5, 1.0, 5.0, 10, 25 and 50 mg/kg. Autopsy urine samples (1.0 g) were added to screw-capped tubes, and the pHs of the samples were adjusted to 5 by adding 1.0 mL of 1.1 M acetate buffer (pH 5.2).
3.3.3. Calibration standards and quality control samples Stock standard solutions (20 mg/mL) of Bromo-Dragonfly were prepared in methanol. Working solutions at concentrations from 0.02 mg/mL to 2 mg/mL were prepared by dilution of the stock standards with methanol. The IS working solution was used at a concentration of 0.5 mg/mL. Calibration standards containing BromoDragonfly in concentrations from 0.5 to 50 mg/kg were prepared daily for each analytical batch by adding suitable amounts of the methanol working solutions to 1 g pre-checked drug-free blood pool sample. The samples were extracted according to the procedure described previously (Section 3.3.2). Calibration curves were constructed by plotting the peak-area ratios Bromo-Dragonfly/IS (MDMA-d5). A weighted (1/concentration) least-squares regression analysis was used for slopes and intercepts. Quality control (QC) samples of 1 mg/kg (LOQ), 5 mg/kg (low), 10 mg/kg (medium) and 50 mg/kg (high) were prepared in drug-free blood, aliquoted and stored at 18 8C. The QC samples were included in each analytical batch to check calibration, accuracy and precision. 3.3.4. Validation procedures The blood extraction method was validated by assaying the selectivity, recovery, matrix effect, linearity, precision and accuracy, limit of detection (LOD) and lower limit of quantification (LLOQ) according to accreditation demands [7,8]. Selectivity was tested using 10 different drug-free autopsy blood samples. These were extracted and analysed for interfering substances. Extraction recovery was calculated by comparing the peak areas of blood standards to the peak areas of post-extracted spiked samples at 0.01 mg/kg (n = 5). Ion suppression/enhancement was studied using the protocol described by King et al. [9]. The ion suppression/ enhancement was evaluated by post-column infusion of Bromo-Dragonfly at 500 mg/L at a flow rate of 10 mL/min and continuously measurement of the MRM transition 294 ! 277. Simultaneously, blank post-mortem blood, urine and vitreous humour extracted according to the assay were injected (10 mL) with the same chromatographic conditions as used in the method. The mobile phase was tested in a similar way. Finally, the matrix effect (ME) was also investigated in five post-mortem blood samples negative on the drug screening. The five blood samples were extracted according to the assay and spiked after extraction with BromoDragonfly at 0.01 mg/kg. ME% was calculated by comparing the response of these samples with the response of standard solutions at 0.01 mg/kg dissolved in mobile phase. ME% = ((area samples spiked after extraction/area standard solution) 1) 100. Calibration curves were tested over the concentration range from the LOD to 50 mg/kg. Peak area ratios between compounds and IS were used for calculations. A weighted (1/concentration) least-squares regression analysis was used for slopes and intercepts. The standard deviation (S.D.) of 10 spiked blood samples at 0.5 mg/ kg (Signal/Noise 10) was used to determine the detection limit (LOD = 3S.D.) and lower quantification limit (LLOQ = 10S.D.). Double determination of each of the four QC samples analysed in three independent experimental assays were used for determination of precision and accuracy. Precision (repeatability and factordifferent intermediate precision) was calculated using one-way analysis of variance (ANOVA) with day as the grouping factor and expressed as the relative S.D. (R.S.D.) of the concentrations calculated for QC samples. Accuracy (bias) was expressed as a percent deviation from the spiked concentrations. 3.3.5. Instrumentation—LC–MS/MS Quantification was performed using reverse phase LC–MS/MS analysis. A Quattro Micro (Waters Micromass Ltd.) tandem mass spectrometer fitted with a Zspray ion source was used for the analysis. Ionization was achieved using electrospray in the positive ionization mode (ES+). LC separation was achieved using a Waters 2695 Alliance HPLC system equipped with an analytical column from Phenomenex (C18 Mercury MS Synergi 2m Hydro-RP, 20 mm 4.0 mm). Chromatography was performed at 35 8C at a flow rate of 0.3 mL/min using a gradient solvent system consisting of solvent A (5 mM aqueous ammonium acetate with 0.1% formic acid) and solvent B (methanol:acetonitrile (50:50, v/v) with 0.1% formic acid), with the following elution profile: 0–1 min linear from 10 to 50% B; 1– 5.5 min linear from 50 to 80% B; 5.5–6 min linear from 80 to 10% B; 6–10 min isocratic at 10% B (before the first analysis, the column was conditioned 3 min with 10% B). Quantification of Bromo-Dragonfly and IS was performed using multiple reaction monitoring (MRM). The following conditions were found to be optimal for the analysis of Bromo-Dragonfly and the IS: capillary voltage, 3.0 kV; source block temperature, 140 8C; desolvation gas (nitrogen) heated to 300 8C and delivered at a flow rate of 800 L/h. The MRM-transitions and collision energies used for quantifying Bromo-Dragonfly and MDMA-d5 are listed in Table 1.
M.F. Andreasen et al. / Forensic Science International 183 (2009) 91–96 Table 1 MRM transition and collision energies for the measurement of Bromo-Dragonfly and MDMA-d5. MS/MS-transition (m/z)
Collision energies (V)
Cone voltage (V)
Bromo-Dragonfly
294 ! 277a 294 ! 198b
21 12
18 18
MDMA-d5
199 ! 165a
12
21
a b
Used as quantifiers. Used as a qualifier.
3.3.6. Instrumentation—UPLC–TOFMS The UPLC–TOFMS analysis was performed on an Acquity Ultra Performance Liquid Chromatograph coupled with an LCT Premiere XE Time-Of-Flight instrument (Waters Micromass Ltd.). Chromatographic separation was performed at 35 8C on an analytical column from Waters (Acquity UPLCTM BEH C18, 1.7 mm, 2.1 mm 100 mm) using gradient elution. A solvent system consisting of solvent A (ammonium bicarbonate 5 mM, pH 10) and solvent B (100% acetonitrile) was used with the following elution profile: 0–3 min linear from 95 to 55% A; 3–3.8 min linear from 55 to 5% A; 3.8–6.5 min isocratic at 5% A; 6.5–7.5 min immediately up to 95% A. The flow rate was 0.6 mL/min and 5 mL samples were injected. The eluent was directly introduced into the mass spectrometer by electrospray. Mass spectrometry was performed on the TOF instrument operating in positive electrospray ionization mode (W+). The desolvation gas flow was set to 1000 L/h at a temperature of 350 8C with the cone gas set to 20 L/h and the source temperature set to 120 8C. The capillary and cone voltages were set to 3.0 kV and 50 V, respectively. Leucine-enkephalin was used as the mass standard (m/z 556.2771) for accurate mass calibration, and was introduced using the LockSprayTM interface at 20 mL/min. In MS scanning, data were acquired in centroid mode from 50 to 1000 m/z. Following data acquisition, UPLC–MS chromatograms and spectra were further analyzed by MassLynx application software (Waters). 3.3.7. Instrumentation—GC–MS The GC–MS analysis was performed on an HP6890N-5973 GC–MS from Agilent Technologies using a HP-5MS capillary column from Agilent (30 m 0.25 mm 0.25 mm). Samples were injected in splitless mode at 225 8C. The GC was temperature programmed for the screening of unknowns; the samples were held at 60 8C for 1 min before a temperature ramp at 20 8C/min to 210 8C, and a further temperature ramp at 12 8C/min to 310 8C, and were then held at 310 8C for 8.67 min. The flow rate of the carrier gas (helium) was 1.0 mL/min. The analysis was performed in full scan mode over the range 40–500 (m/z) with an ionisation energy of 70 eV. 3.3.8. Instrumentation—HPLC–DAD The HPLC–DAD analysis was performed on an Agilent 1100 series equipped with a Diode Array Detector. LC separation was achieved using an analytical column from Varian (OmniSpher 5 C18, 250 mm 4.6 mm) using a gradient solvent system consisting of solvent A (phosphate buffer, 0.012 mol/L, pH 3.2 with 10% acetonitrile) and solvent B (acetonitrile) with the following elution profile: 0–10 min isocratic 100% A; 10–14 min linear to 30% A; 14–16 min isocratic at 30% B; 16–20 min immediately up to 100% A. Separation was conducted at 25 8C at a flow rate of 1.0 mL/min, with detection at 210 nm. 3.3.9. Instrumentation—NMR NMR spectra were recorded on a Varian 400 MHz instrument. The solvent used was deuterated water (D2O). 3.3.10. Screening for ethanol, therapeutic and abused drugs Screening for ethanol, therapeutic and abused drugs were performed on liver, liver blood, urine and femoral blood from the deceased using analytical methods
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routinely used in the laboratory. The laboratory is accreditated by an external, independent organization, DANAK (Danish Accreditation), which handles the administration of accreditation and metrology in Denmark. Most methods used are validated and accredited in accordance with the requirements of the DS/EN ISO/IEC 17025 standard. Blood and urine were analysed for ethanol using a HS-GC method (Head Space Gas Chromatography) based on Machata [10]. Liver blood was screened for benzodiazepines using SPE (solid phase extraction) and LC–MS/MS [11]. Urine and blood were screened for amphetamines, cocaine (benzoylecgonine), cannabinoids, opioids, methadone and buprenorphine using EMIT (enzyme multiplied immunoassay technique) and CEDIA (cloned enzyme donor immunoassays). Homogenised liver tissue was screened for basic compounds using LLE (liquid liquid extraction) with diethyl ether and dichloromethane and subsequent GC–MS and HPLC–DAD analysis on the extract. Screening for acidic and neutral compounds were conducted using a precipitation method with acetonitrile and subsequent HPLC–DAD analysis. The laboratory participates in different screening and quantification proficiency tests.
4. Results and discussion Preliminary analysis (UPLC–TOFMS, GC–MS, HPLC–DAD and 1H NMR) of the residue obtained by careful evaporation of an aliquot of the liquid contained in the bottle found at the scene showed no trace of LSD. Instead, another major compound was present, which gave spectral data in accordance with the structure of 1-(8-bromobenzo[1,2-b; 4,5-b0 ]difuran-4-yl)-2-aminopropane, also known as Bromo-Dragonfly. The compound was finally extracted and purified from the liquid and concomitant GC–MS, HPLC–DAD, 1H and 13C NMR as well as UPLC–TOFMS analysis confirmed the structure and purity of the isolated compound as Bromo-Dragonfly. GC and HPLC chromatograms as well MS and UV–vis spectra of Bromo-Dragonfly are shown in Figs. 2 and 3. Figs. 4 and 5 show 1H and 13C NMR data. The proton NMR spectrum corresponds to the one given in the literature [2], and the 13C NMR spectrum corresponds to the 13 carbons in the structure. The chemical shifts also correspond to the calculated theoretical chemical shifts for the 13 carbons. Liver, blood and urine from the deceased were screened for ethanol, therapeutic and abused drugs using methods used routinely in the laboratory. No ethanol, therapeutic or abused drugs were detected in biological specimens from the deceased. However, when screening specific for Bromo-Dragonfly using UPLC–TOFMS, the drug was identified in liver blood from the deceased. The extracted ion chromatogram (EIC) obtained from extracted liver blood is shown in Fig. 6, and identification was based on the compound’s accurate mass. Bromo-Dragonfly was subsequently quantified by LC–MS/MS in femoral blood, urine, hydrolysed urine and vitreous humour using the isolated BromoDragonfly as a reference and for calibration standards. Identification was based on retention time, two daughter ions and ion ratios monitored between the daughter ions. Fig. 7 shows an extracted ion chromatogram obtained after extraction of femoral blood from the deceased. The concentration of Bromo-Dragonfly in femoral blood was 4.7 0.7 mg/kg (double determination in two analytical series). In urine the concentration was 22 2 mg/kg and in bglucuronidase-treated urine the concentration was 33 3 mg/kg, so
Fig. 2. GC–MS chromatogram and mass spectrum (EI 70 eV) of Bromo-Dragonfly. The retention time of Bromo-Dragonfly is 11.87 min. The peak at 11.65 min is a formyl derivative of Bromo-Dragonfly created in the inlet [12]. When dissolved in toluene only one peak appear in the chromatogram.
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Fig. 3. HPLC chromatogram and UV–vis spectra (inset) of Bromo-Dragonfly. The HPLC trace was detected at 210 nm.
Bromo-Dragonfly is presumably glucuronated to some extend. In vitreous humour the concentration of Bromo-Dragonfly was 0.5 0.1 mg/kg, however it should be kept in mind that the calibration standards were prepared in MilliQ-water and that the concentration measured is close to the method LLOQ. The toxicological significance of these concentrations could not be determined due to a lack of similar data in the literature. However, from the deceased’s boyfriend, we know that they both ingested 1 mL of the liquid. We have quantified the liquid found with the deceased and found a concentration of 0.69 mg/mL, which means that they ingested approximately 700 mg Bromo-Dragonfly. According the Internet drug culture database EROWID (www.erowid.org), the usual Bromo-Dragonfly dose in humans has been reported to be in the range of 500–1000 mg. The dose ingested in this
Fig. 6. Extracted ion chromatogram of Bromo-Dragonfly from a liquid-liquid extracted liver blood sample (2 g) acquired by LC–TOFMS. Measured mass: 294.0132; calculated mass: 294.0130; formula: C13H13NO2Br; i-FIT: 11.9.
case seems to be in the usual dose region, and the boyfriend also survived the dose. However, a lethal concentration of BromoDragonfly may have been attained in the deceased girl because of a different individual response. She might be a poor metabolizer or has actually ingested more than 1 mL of liquid. The analysed drug may also have been degraded to some extent, because 3 days had elapsed after death before the autopsy was performed on the deceased. Blood, liver blood and urine from the deceased women were also analysed for metabolites using the software Metabolynx from Waters. No metabolites were detected, however the concentration of the metabolites may have been below limit of detection, as the concentration of Bromo-Dragonfly were low. The autopsy findings revealed oedema of the lungs, slight oedema of the brain, enlargement of the spleen, irritation of the mucous membranes in
Fig. 4. 1H NMR spectrum of Bromo-Dragonfly as hydrochloride. The chemical shifts d are given in ppm relative to the HDO signal assigned to 4.79 ppm. Coupling constants J are given in Hz. 1H NMR (D2O) d 1.19 (t, EtOH solvent residual peak), 1.28 (d, 3H, CH3, J = 6.4 Hz), 3.27 (m, 2H, ArCH2), 3.57 (q, EtOH solvent residual peak), 3.80 (sextet, 1H, ArCH2CHCH3, J = 6.4 Hz), 6.90 (d, 1H, Ar–CH CH–O–), J = 2.0 Hz), 7.02 (d, 1H, Ar–CH CH–O–, J = 2.0 Hz) 7.79 (d, 1H, Ar–CH CH–O–, J = 2.4 Hz), 7.81 (d, 1H, Ar–CH CH–O–, J = 2.4 Hz).
Fig. 5. 13C NMR spectrum of Bromo-Dragonfly. The chemical shifts d are given in ppm. 13C NMR (D2O) 17.7 (–CH(NH2)CH3), 31.7 (Ar–CH2–) 48.1 (–CH2CH(NH2)CH3), 92.7 (Br–C(Ar)) 106.1, 106.6 (Ar–CH CH–O–), 109.9 (C–CH2CH(NH2)CH3), 126.0 (C–CH H–O–), 26.3 (C–CH CH–O–), 146.7, 147.0, 148.6, 149.9 (C–O–CH CH–).
M.F. Andreasen et al. / Forensic Science International 183 (2009) 91–96
Fig. 7. Extracted ion chromatogram of Bromo-Dragonfly (4.7 mg/kg 0.005 mg/L) obtained from an extracted blood sample by LC–MS/MS. Top: two transitions of Bromo-Dragonfly (277 loss of NH3, 198 loss of NH3 and Br). Bottom: one transition of the internal standard MDMA-d5 (10 ng). Retention times of Bromo-Dragonfly and the internal standard were 5.0 and 3.7 min, respectively.
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the stomach and ischemic changes in the kidneys. These findings are non-specific for acute poisoning. However, based on the autopsy and toxicological findings, the cause of death was most likely an overdose of Bromo-Dragonfly, although the mechanism of death could not be determined. Our laboratory has received blood samples from two young men who were hospitalised after ingestion of Bromo-Dragonfly at a techno party. The concentrations of BromoDragonfly in their blood were 0.7 and 0.6 mg/kg respectively, using the method described previously. Compared with these results the concentration of Bromo-Dragonfly in the deceased women was approx. 8 times higher. The blood samples from the two young men were taken before midnight the evening they ingested the drug. No other analysis beside quantification of Bromo-Dragonfly was performed on the blood. The analysis of Bromo-Dragonfly in blood was validated by assaying selectivity, recovery, linearity, precision and accuracy, limit of detection (LOD), lower limit of quantification (LLOQ) and matrix effect. The method was found to be selective for BromoDragonfly in post-mortem blood. No interfering peaks were observed in the extracts of ten different drug-free post-mortem blood samples. Interference with other compounds was minimized and checked due to LC–MS/MS identification; retention time, two daughter ions and ion ratios were monitored between the daughter ions. Extraction recovery varied from 71 to 96%. LOD and LLOQ were 0.2 mg/kg and 0.5 mg/kg, respectively. Precision was 10% and bias was 11% at LLOQ. The calibration curves were linear over the measurement interval from 0.5 to 50 mg/kg (r2 > 0.993y = 10.4x 0.001). Inter- and intra-assay precision for blood quality control samples (1, 5, 10 and 50 mg/kg) were in the range from 5 to 15% R.S.D., and accuracy was between 98 and 128% of the target amount. Post-Column infusion was used as a qualitative assessment of matrix effect. Fig. 8 shows injection of mobile phase (A), extract of blood (B), extract of urine (C) and extract of vitreous humour (D). All four matrixes show the same pattern; an increase in signal for approximately eight and a half minutes followed by reduction to the initial value. This pattern is
Fig. 8. MRM ion chromatograms for Bromo-Dragonfly (294 ! 277) during continuous post-column infusion of an 500 mL/mL aqueous solution of the compound at 10 mL/min and injection (10 mL) of mobile phase B (A), blank human blood extracted according to the assay (B), blank urine extracted according to the assay (C), vitreous humour extracted according to the assay (D). Overlay of MRM ion chromatograms for Bromo-Dragonfly (Rt 5.0 min) and internal standard MDMA-d5 (Rt 3.6 min).
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due to the LC-gradient used. Negative dips identify regions where ion suppression is likely to occur. There was no ion suppression present for Bromo-Dragonfly for any of the matrixes used at the time of elution (Rt 5 min). However, suppression was observed for extracts of blood at the following retention timer 1, 2 and 7– 8 min. For urine suppression was observed in the beginning of the chromatogram (Rt 2 min). Ion enhancement was observed in blood in the area where Bromo-Dragonfly is eluting. The quantitative assessment of the matrix effect in blood showed an ion enhancement lower than 20%. 5. Conclusion Bromo-Dragonfly was isolated, and its structure was determined from a submitted aqueous sample. Subsequently, the drug was identified in liver blood and quantified in femoral blood, urine and vitreous humour from an autopsy case. The autopsy findings were non-specific for acute poisoning. However, based on the toxicological findings, the cause of death was determined to be a fatal overdose of Bromo-Dragonfly, as no ethanol, therapeutic or other drugs of abuse were detected in any biological specimens from the deceased. To our knowledge, this is the first report of the quantification of Bromo-Dragonfly in biological specimens from a cadaver. This case caused the drug to be classified as an illegal drug in Denmark on 5th December 2007. Acknowledgements Rune I.D. Birkler thanks the Sexual Assault Centre, Aarhus University Hospital and Tryg Foundation for funding.
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