AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
2nd annual NAFT meeting and general assembly
Aarhus, June 16th-17th 2016
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AARHUS
UNIVERSITY
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DEPARTMENT OF FORENSIC MEDICINE
Welcome
The organising committee and the staff at the Department of Forensic Medicine, Section for Forensic
Chemistry welcome you all to the 2nd annual NAFT meeting and general assembly.
We are very pleased to give you 23 oral presentations representing almost all the laboratories
working with forensic toxicology in the Nordic countries. The abstracts describe many different
aspects of modern forensic toxicology and represent high international quality and cutting edge
research within our field.
The six workshops cover a wide range of areas within forensic toxicology addressing analytical,
metabolism methodologies, interpretation of results and practical improvements in the laboratory.
The intention with the three parallel workshop sessions is to create an informal atmosphere open
for sharing experiences and discussions.
We would like to thank all the vendors present at the meeting. Their presence contributes to qualify
the new technical solutions as well as making the meeting possible. The majority have a booth in the
hotel foyer and you are more than welcome to pay them a visit.
We are most grateful to all our colleagues who have contributed to this meeting with scientific and
workshop presentations as well as arranging and chairing the sessions. In total 39 of you are
contributing at the meeting which is very impressive. Thank you!
Finally we will like to encourage you all to participate actively in networking and discussions thus
creating a successful NAFT meeting. We hope you will enjoy your stay in Aarhus.
The organising committee
Mette Findal Andreasen Senior Researcher, PhD
Jakob Ross Jornil Forensic Chemist, PhD
Mogens Johannsen Professor, PhD
Jørgen Bo Hasselstrøm Senior Researcher, PhD
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Contents
Schedule…………………………………………………………………………………………………………………………………………. 4
Workshops..……………………………………………………………………………………………………………………………………. 6
Abstracts…………………………………………………………………………………………………………………………………………. 8
Participants…………………………………………………………………………………………………………………………………….. 31
NAFT 2017………………………………………………………………………………………………………………………………………. 33
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DEPARTMENT OF FORENSIC MEDICINE
Schedule - Thursday 16th June 2016
Time Title
09.00 - 10.00 Registration and coffee
10.00 - 10.10 Welcome
10.10 - 12.10 Scientific session I: Analysis and pharmacology Chairman: Robert Kronstrand, National Board of Forensic Medicine, Linköping, Sweden
O1 Ion-source -independent library-based identification and concurrent quantification of new psychoactive substances without authentic reference standards by gas chromatography –atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometry coupled to nitrogen chemiluminescence detection (GC-NCD-APCI-QTOFMS) Samuel Mesihää, University of Helsinki, Finland
O2 Parent drug calibration for the determination of metabolite to parent drug ratios in post-mortem blood by UPLC-DAD-CAD Jenni Viinamäki, University of Helsinki, Finland
O3 Method development for determination of new psychoactive substances in urine using solid phase extraction and LC-QTOF Sofia Lindahl, St. Olav University Hospital, Trondheim, Norway
O4 Screening for Therapeutic and Illicit Drugs in Airplane Wastewater – a New Matrix for Mapping Patterns of Drug Use and Abuse Marie Mardal, University of Copenhagen, Denmark
O5 Quantitative screening of synthetic cannabinoids in urine using UHPLC-QTOF-MS Per Ole M. Gundersen, St Olav University Hospital, Trondheim, Norway
O6 The INTOx project: quantification of ten sedatives and analgesics in blood from patients undergoing intensive care treatment Anna Johansson, National Board of Forensic Medicine, Linköping, Sweden
O7 Analysis of hair in post mortem cases can reveal use of drugs not detected in blood Marianne Arnestad, Norwegian Institute of Public Health, Norway
O8 Deposition of diazepam and its metabolites in hair following a single dose of diazepam Sys Stybe Johansen, University of Copenhagen, Denmark
O9 Targeted proteomics of CYP1A2 and CYP3A4 in post-mortem liver tissue Jakob Hansen, Aarhus University, Denmark
12.10 - 13.20 Lunch
13.20 - 15.20 Scientific session II: pharmacology and interpretation Chairman: Kristian Linnet, University of Copenhagen, Denmark
O10 In vitro metabolism studies on new designer benzodiazepines Carolina Noble, University of Copenhagen, Denmark
O11 Metabolic profiling using retrospective UPLC-HR-TOF-MS data from forensic toxicology screenings Kirstine Lykke Nielsen, Aarhus University, Denmark
O12 Ethanol elimination at low concentrations in drunk drivers Gudrun Høiseth, Norwegian Institute of Public Health, Norway
O13 Post-mortem levels and tissue distribution of codeine, codeine-6-glucuronide, nor-codeine, morphine and morphine glucuronides in a series of codeine-related deaths Joachim Frost, St Olav University Hospital, Trondheim, Norway
O14 Distribution of QT-prolonging drugs and metabolites between cardiac tissue and femoral blood in a psychiatric population Christian Reuss Mikkelsen, Aarhus University, Denmark
O15 Comparison of antidepressiva concentrations in different post mortem media Åse Marit Leere Øiestad, Norwegian Institute of Public Health, Norway
O16 Testosterone levels in forensic cases compared to testosterone levels in long-acting replacement therapy with intramuscular testosterone undecanoate Yvonne Lood, National Board of Forensic Medicine, Linköping, Sweden
O17 Reference values of Lithium in post-mortem blood Carl Söderberg, National Board of Forensic Medicine, Linköping, Sweden
O18 Beta-hydroxybutyrate and the post mortem diagnosis of ketoacidosis Elli Tyrkkö, National Board of Forensic Medicine, Linköping, Sweden
15.20 - 15.50 Coffee
15.50 - 16.50 General assembly
19.00 - Social dinner (Restaurant, Scandic Aarhus City Hotel)
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DEPARTMENT OF FORENSIC MEDICINE
Schedule – Friday 17th June 2016
Time Title
08.30 - 09.30 Scientific session III: interpretation and miscellaneous Chairman: Svava Thordardottir, University of Iceland, Iceland
O19 Deaths attributed to 1-phenyl-2-(1-pyrrolidinyl)-1-pentanone (α-PVP) Robert Kronstrand, National Board of Forensic Medicine, Linköping, Sweden
O20 An increase in fentanyl-related deaths during the winter 2015/2016 Gunilla Thelander, National Board of Forensic Medicine, Linköping, Sweden
O21 Trends in fatal poisonings by prescription drugs in Finland since 2000 lkka Ojanperä, University of Helsinki, Finland
O22 Reoffending in drugrelated crimes in Sweden, 1993 – 2013 Johan Ahlner, National Board of Forensic Medicine, Linköping, Sweden
O23 Experiences in an EU project “Strengthening and modernizing the forensic services in Kosovo” Ilpo Rasanen, National Institute for Health and Welfare (THL), Helsinki, Finland
09.30 - 09.45 Coffee
09.45 - 11.00 Parallel workshops 1-3
W1 Keeping the TOF library updated
W2 Forensic applications of in vitro metabolism
W3 Improvements in the laboratory I
11.00 - 11.30 Coffee
11.30 - 12.45 Parallel workshops 4-6
W4 Interpretation of post-mortem results
W5 Metabolomics in forensic toxicology
W6 Improvements in the laboratory II
12.45 - Lunch to go
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Workshops
Workshop no. 1: Keeping the TOF library updated Chairman: Petur Dalsgaard, University of Copenhagen, Denmark
This workshop will focus on how to keep our TOF libraries updated. New psychoactive substances
continuously appear on the illegal market in the Nordic countries and the rest of the world.
Therefore, it is increasingly important to collaborate and share information on new substances. The
following four lectures will tell how they keep their TOF libraries updated using different HR-TOF
instruments e.g. from Waters, Bruker, Agilent, Sciex and Thermo.
Petur Weihe Dalsgaard, University of Copenhagen, Denmark
Mette Findal Andreasen, Aarhus University, Denmark
Per Ole M. Gundersen, St. Olavs Hospital, Trondheim, Norway
Christian Brinch Mollerup, University of Copenhagen, Denmark.
Workshop no. 2: Forensic applications of in vitro metabolism Chairman: Jakob Ross Jornil, Aarhus University, Denmark
The workshop is an opportunity to get introduced to or to expand knowledge of in vitro metabolism in a forensic context.
Overview on different in vitro techniques for metabolite identification Ariane Wohlfarth, National Board of Forensic Medicine, Division of Drug Research, Department of Medical and Health Sciences, Linköping University
In vitro metabolism of synthetic cannabinoids Niels Bjerre Holm, Department of Forensic Medicine, Section of Forensic Chemistry, University of Copenhagen
In vitro - in vivo correlations Svante Vikingsson, National Board of Forensic Medicine, Division of Drug Research, Department of Medical and Health Sciences, Linköping University
In vitro - in vivo extrapolation, using in silico techniques to predict ADME Jakob Ross Jornil, Section for Forensic Chemistry, Department of Forensic Medicine, Aarhus University
Workshop no. 3: Improvements in the laboratory I Chairman: Jørgen Bo Hasselstrøm, Aarhus University, Denmark.
This workshop will focus on the daily routines in the forensic toxicology laboratory and how to improve the production. The workshop will consist of a series of short informal presentations all open for discussion and questions. Issues could be stability of compounds, pre-analytical considerations (sample tubes and preservation), LEAN initiatives etc.
Studies on test tubes with different sodium fluoride content Gunnel Nilsson and Nina Rinquist, National Board of Forensic Medicine, Linköping, Sweden
Pre-analytical stability and sample handling Jørgen Bo Hasselstrøm, Aarhus University, Denmark
LEAN/LIMS/Automation Brian Rasmussen, University of Copenhagen, Denmark
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Workshops (continued)
Workshop no. 4: Interpretation of post-mortem results Chairman: Marianne Arnestad, Norwegian Institute of Public Health, Norway and Ljubica Vukelic Andersen, Aarhus University, Denmark.
The workshop will go through standard interpretation of toxicological results from autopsies with focus on pitfalls and the importance of clinical medical information.
Pitfalls in interpretation of toxicological results from autopsies – introduction
Liliana Bachs, Norwegian Institute of Public Health, Norway
Importance of clinical medical information
Ljubica Vukelic Andersen/Eva Sædder, Aarhus University, Denmark
Reference concentrations for interpreting drug related deaths
Ilkka Ojanperä, University of Helsinki, Finland
Importance of pharmacogenetics in toxicological interpretation
Pernilla Haage, National Board of Forensic Medicine, Linköping, Sweden
Use of alternative matrixes in toxicological interpretation
Joachim Frost, St. Olavs Hospital, Trondheim, Norway
General discussion
Workshop no. 5: Metabolomics in forensic toxicology Chairman: Mogens Johannsen, Aarhus University, Denmark This workshop will give a short introduction to untargeted metabolomics. Thereafter an introduction to how we apply a metabolomic analysis on routine forensic data to assess drug metabolism and toxicology.
Kirstine Lykke Nielsen and Mogens Johannsen, Aarhus University, Denmark
Workshop no. 6: Improvements in the laboratory II Chairman: Jørgen Bo Hasselstrøm, Aarhus University, Denmark This workshop will focus on the daily routines in the forensic toxicology laboratory and how to improve the production. The workshop will consist of a series of short informal presentations all open for discussion and questions. We will continue from workshop no. 3 with focus on automation and LIMS.
Automated blood pipetting to glass tubes and 96-well format with Hamilton robots
Martin Josefsson and Gunnel Nilsson, National Board of Forensic Medicine, Linköping, Sweden
LIMS and automation
Åse Marit Leere Øiestad, Norwegian Institute of Public Health, Norway
How low can you go? Using a microbalance for preparation of stock solutions
Jakob Ross Jornil, Aarhus University, Denmark
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Abstracts
O1
Ion-source -independent library-based identification and concurrent quantification of new
psychoactive substances without authentic reference standards by gas chromatography –
atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometry coupled to
nitrogen chemiluminescence detection (GC-NCD-APCI-QTOFMS)
Samuel Mesihääa, Ilkka Ojanperäa, Ilpo Rasanena, Anna Pelandera & Raimo Ketolaa
aUniversity of Helsinki, Department of Forensic Medicine
Contact information (e-mail(s)): [email protected]
Introduction Two novel concepts are presented
Comparison between GC-APCI and LC-ESI product ion spectra for drugs
We investigated the feasibility of using a commercial drug spectrum library produced by LC-ESI-
QTOFMS as a reference for the identification of spectra produced by GC-APCI-QTOFMS. An in-house
GC-APCI-QTOFMS library was built containing spectral data of 50 compounds belonging to different
classes of drugs, such as amphetamines, benzodiazepines, synthetic cathinones, synthetic
cannabinoids and opioids. The GC-APCI-QTOFMS spectra were measured at three different collision
energy levels similarly to the commercial spectral library.
Quantification of new psychoactive substances (NPS) without reference standards
A vast majority of psychoactive drugs (> 90%) contain nitrogen. The equimolar quantification
capability of the nitrogen chemiluminescence detector was demonstrated by analysing the following
five NPS at five concentration levels between 0.17-1.7 µg/ml spiked post-extraction in sheep blood:
bupropion, desoxypipradrol (2-DPMP), mephedrone, methylone and naphyrone.
Materials and methods A 7890B Series GC System equipped with a 7693 Automatic Liquid Sampler
and a split/split less injector was coupled through a G3180B Two-Way Splitter with Makeup Gas to
an APCI 6540 UHD Accurate-Mass QTOF mass analyser and a 255 Nitrogen Chemiluminescence
Detector (all Agilent Technologies).
Results APCI generated a protonated precursor ion for most of the 50 compounds. The protonated
MS/MS spectra were highly similar between the LC-ESI and GC-APCI libraries in terms of ion
abundance ratios and mass accuracy. We conclude that the LC-ESI commercial mass spectral library
can be used in connection with other platforms provided that the [M+H]+ is produced. Derivatized
compounds and compounds associated with neutral loss at GC-APCI require separate libraries.
The equimolarity of NCD was on average 98.7%, and the range of individual equimolarity
determinations was 76.7 – 130.1%, providing acceptable quantitative results in the present study
setting. However, extraction recovery was not considered at this point.
Conclusion The current analysis platform affords a promising approach to instant simultaneous
qualitative and quantitative analysis of drugs in the absence of authentic reference standards.
Future research will concentrate on standardizing the GC-APCI ionization, as well as improving the
sensitivity of the NCD determination.
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DEPARTMENT OF FORENSIC MEDICINE
O2
Parent drug calibration for the determination of metabolite to parent drug ratios in post-mortem
blood by UPLC-DAD-CAD
Jenni Viinamäkia, Ilkka Ojanperäa
aDepartment of Forensic Medicine, University of Helsinki, Finland
Contact information (e-mail(s)): [email protected]
Introduction There is a constant demand for the quantification of drug metabolites within post-
mortem toxicology. However, the information of metabolite concentrations and metabolite to
parent ratios in post-mortem blood is scarce due to limited availability and high price of primary
reference standards. In this study, ultra-high performance liquid chromatography coupled with
photodiode array detection and corona charged aerosol detection (UPLC-DAD-CAD) was utilized for
the concurrent quantification of 23 drug metabolites using the corresponding parent drug for
calibration. After validation of this secondary calibration method, 633 previously analysed post-
mortem blood samples were retrospectively re-processed to discover the metabolite to parent
ratios of six toxicologically relevant drugs.
Materials and methods Blood samples were extracted in basic conditions and chromatographic
separation was performed with a C18 column using a mobile phase gradient consisting of an
aqueous solution of trifluoroacetic acid and methanol. One-point calibration was performed with the
parent drug. The linearity was tested at concentration range from 0.05 to 5.0 mg/L.
Results Based on the secondary calibration, the quantitative results for the N-demethylated
metabolites by both detectors were similar to those obtained by the ordinary calibration using
primary reference standards. With O-demethylated metabolites, differences in UV spectra and
retention times caused larger biases. The metabolite to parent ratios measured in this study fell
within the normal ratios utilized in therapeutic drug monitoring and were comparable to those
reported in post-mortem material.
Conclusion This study presents a cost-effective and straightforward method for the quantification of
the parent drug and its metabolites concurrently, with use of the parent drug reference standard
only. The possibility to utilize historic one-point calibration reduces the calibration workload
compared to mass spectrometric methods. We anticipate that the readily accumulating data by the
presented method will add to the scarce literature of drug metabolites in post-mortem samples.
Connected to the cause-of-death information, the metabolite to parent ratios will provide an
additional reference for interpretation of toxicological results.
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O3
Method development for determination of new psychoactive substances in urine using solid phase
extraction and LC-QTOF
Sofia Lindahla and Andreas Westina
aDepartment of Clinical Pharmacology, St. Olav University Hospital, Trondheim, Norway
Contact information (e-mail): [email protected]
Introduction During the last decade there has been a rapid and continuous growth in the availability
and use of novel psychoactive substances (NPS) across the world. Forensic laboratories have to
continuously adapt their urinalysis procedures in order to keep track with the ever-changing NPS
landscape. The aim of your study was to develop a generic method for analysis of NPS in urine to
simplify the addition of new compounds with time.
Materials and methods Fifty NPS/NPS metabolites from different drug classes (e.g. benzodiazepines,
cathinones, phenethylamines and tryptamines) were selected based on the NPS prevalence in
seizures made by Norwegian police and customs. For sample preparation solid phase extraction was
selected, since then removal of interfering matrix components and up-concentration of low
abundance analytes can be combined. Two different SPE materials were evaluated; cation exchange
(Evolute Express CX, Biotage) and hydrophilic lipophilic balanced copolymer (Oasis Prime HLB,
Waters). Standards and spiked urine samples were analysed with UHPLC-QTOF (Agilent). Separation
was performed using an HSS T3 analytical column (2.1 x 100 mm, 1.8 µm, Waters), with gradient
elution (0.025 % formic acid and 5 mM ammonium format in water and 0.05% formic acid in
acetonitrile).
Preliminary results Since this project is still in the method development phase the results presented
will be focused on comparison of the extraction yield and matrix effects using the different SPE
materials and on the development of the LC-QTOF method. Discussion of advantages and
disadvantages of using a generic method compared to several targeted methods will also be
discussed.
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O4
Screening for Therapeutic and Illicit Drugs in Airplane Wastewater – a New Matrix for Mapping
Patterns of Drug Use and Abuse
Marie Mardala, Frank M. Aarestrupb, Brian Schou Rasmussena, Christian Brinch Mollerupa, Petur
Weihe Dalsgaarda, Kristian Linneta
aSection of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of
Copenhagen, Copenhagen, Denmark, bNational Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark.
Contact information (e-mail(s)): [email protected], [email protected]
Introduction There is limited knowledge on the global prescription and consumption patterns of
therapeutic drugs (TD) and illicit drugs (ID). Pooled urine analysis (PUA) is valuable for obtaining
snap-shots of where and when TD and ID are consumed. However, PUA fails to cover targets
excreted through faeces. Wastewater has previously been used for local-based wastewater-based
epidemiology (WBE) and drug screening. It is however difficult to study the global epidemiology due
to difficulties in obtaining samples.
The aim of the study was to test the detectability of TD and ID in airplane wastewater samples
categorized according to their geographical origin.
Materials and methods Wastewater samples (n= 17) were collected from long-distance flights
arriving at Copenhagen airport. The samples were prepared according to a previously described fully
automated setup (Andersen et al. JAT, 2012), with enzymatic conjugate cleaving followed by either
precipitation or solid phase extraction (SPE). Aliquots from precipitated samples were analysed by
UPLC-QTOF in data-independent acquisition mode (MSE) controlled by UNIFI software (Waters) with
a 3,145 compound library for target and suspect screening. SPE samples aliquots were injected onto
UPLC-MS/MS systems with previously validated methods. TD were grouped according to their
Anatomical Therapeutic Chemical (ATC) codes. Principal component analysis (PCA) was performed
with therapeutic subgroup ATC codes as variables.
Results Identification confidence was assigned to three levels based on: retention time error, mass
error, and number of identifying fragments, detection on both instruments and targets per
compound, with level 1 being the highest level of confidence. More than 74 % of the detected TD
belonged to the anatomical main groups for drugs acting on the neurological, respiratory, or
cardiovascular systems. Distributed on 89 different compounds, the accumulated number of TD and
ID detected in the wastewater samples were: 84 (level 1), 234 (level 2), and 433 (level 3). Cocaine
and methamphetamine were both identified in two samples. The screening also identified drugs
mainly excreted in faeces, e.g. dipyramidol. The first two principle components in the PCA separated
three clusters of wastewater samples based on geographical origin of the airplanes.
Conclusion Airplane wastewater analysis is a useful addition to PUA for identifying targets for WBE
and toxicological analysis.
mailto:[email protected]
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DEPARTMENT OF FORENSIC MEDICINE
O5
Quantitative screening of synthetic cannabinoids in urine using UHPLC-QTOF-MS
Per Ole M. Gundersena, Olav Spigseta,b, Martin Josefssonc,d
aDepartment of Clinical Pharmacology, St Olav University Hospital, Trondheim, Norway
bDepartment of Laboratory Medicine, Children’s and Women’s Health, Norwegian University of Science and Technology,
Trondheim, Norway cDepartment of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden
dDepartment of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden
Contact information (e-mail(s)): [email protected]
Introduction Synthetic cannabinoids share structural and pharmacodynamical properties with the
psychoactive substance Δ9-tetrahydrocannabinol (THC) but they often have higher affinity to the
cannabinoid receptors. This makes them potentially more toxic than THC. Designer drug
manufacturers bypass the legislations by continuously introducing new analogues to the market.
This calls for drug screening methods with a panel of analytes that can easily be updated.
Materials and methods High resolution mass spectrometry technology such as liquid
chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS) has been proven
sensitive and accurate for drug screening purposes and methods can relatively easily be updated.
Urine is the sample matrix of choice for screening due to the ease of sampling and the time window
of detection.
The aim of this study was to develop a sensitive quantitative screening method for a selection of
nine synthetic cannabinoids (AB-Fubinaca, AB-Pinaca, AB-Chminaca, BB-22, PB-22, 5F-PB-22, AKB48,
5F-AKB48 and UR-144) with different core structures and chemical properties. The target was to
reach a lower limit of quantification (LOQ) of 0.1 ng/ml for many of the analytes using an automated
preparation. Samples were prepared by 96 well-plate supported liquid extraction (SLE) followed by
analysis on an Agilent 6550 iFunnel QTOF combined with a 1290 LC system.
Results The advantage of SLE is an easy sample protocol but the technique has limitation in the
clean-up efficiency. Together with a two times pre-concentration this caused a pronounced matrix
effect with up to 45 % suppression (between 45 % and -15 % for the different metabolites) of the
analyte signal. This illustrates the analytical difficulties reaching the desired LOQs for these
substances.
Conclusion Analytical and practical issues particularly regarding choice of consumables and reagents
in the analytical method will be discussed. Thoughts around confirming findings and updating the
panel of the method will be covered.
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O6
The INTOx project: quantification of ten sedatives and analgesics in blood from patients
undergoing intensive care treatment
Anna Johanssona, Ulrica Lennbornb, Robert Kronstranda c, Markus Romana, Håkan Sandlerd e, Elisabet
Nielsenf, Johan Ahlnera,c, Fredrik C Kugelberga,c, Sten Rubertssonb
aDepartment of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine; Linköping, Sweden;
bDepartment of Surgical Sciences/Anaesthesiology and Intensive Care Medicine, Uppsala University, Uppsala, Sweden;
cDepartment of Medical and Health Sciences, Division of Drug Research, Linköping University, Linköping, Sweden;
dDepartment of Forensic Medicine, National Board of Forensic Medicine, Uppsala, Sweden;
eDepartment of Surgical
Sciences/Forensic Medicine, Uppsala University, Uppsala, Sweden; fDepartment of Pharmaceutical Biosciences, Uppsala
University, Uppsala, Sweden
Contact information (e-mail(s)): [email protected]
Introduction The INTOx project is a collaborative project between the general intensive care unit (ICU) at
the University Hospital of Uppsala, Sweden and the Swedish National Board of Forensic Medicine. The
aim of this project is to study blood concentrations of ten sedatives and analgesics regularly used in the
ICU and also relate the concentrations to dosage, degree of organ failure and clinical response. It will also
be studied whether intoxicated patients have taken drugs that initially were not suspected. In patients
that die during intensive care, antemortem and post-mortem blood drug concentrations will be
compared.
Patients and methods All patients over the age of seven, admitted to the general ICU at Uppsala
University Hospital, were included in the study. The first study period lasted for two months and the
second study period for three months. Blood samples were collected, according to standardized
procedures, when the patient arrived to the ICU and then twice daily until discharged. The samples were
screened for pharmaceuticals and drugs of abuse using liquid chromatography/time-of-flight mass
spectrometry (LC-TOF-MS). Propofol, thiopental, midazolam, dexmedetomidine, clonidine, ketamine,
fentanyl, morphine, ketobemidone and paracetamol were quantified using gas chromatography (GC), GC-
MS or LC-MS/MS.
Results During the first study period 125 patients, including 14 intoxications, were treated at the general
ICU. Ten patients died. During the second study period 174 patients, including 20 intoxications were
treated and 12 patients died. The most commonly administrated sedatives and analgesics were propofol
and fentanyl. The mean, median and range concentrations of propofol in 935 samples were 1.13 µg/g, 0.9
µg/g and 0.05-16.5 µg/g, respectively. The mean, median and range concentrations of fentanyl in 1031
samples were 0.99 ng/g, 0.71 ng/g and 0.05-9.4 ng/g, respectively.
Conclusion Quantification of sedatives and analgesics routinely used to treat patients in the ICU can
possibly result in more individualized dosing strategies. With an optimized dose the patient need less
time in the ICU and in the long run this lowers health care costs. Furthermore, both antemortem and
post-mortem reference concentrations for such drugs could be of great aid to the forensic pathologist.
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O7
Analysis of hair in post mortem cases can reveal use of drugs not detected in blood
Gudrun Høisetha, Marianne Arnestada, Ritva Karinena, Luca Morinib, Sidsel Rogdea,c, Cristina Sempioa,
Vigdis Vindenesa,c, Åse Marit Leere Øiestada
aNorwegian Institute of Public Health, Domain for Forensic Sciences, Oslo, Norway
bDepartment of Public Health, Experimental and Forensic Medicine, University of Pavia, Italy
cInstitute of Clinical Medicine, Faculty of Medicine, University of Oslo, Norway
Contact information (e-mail(s)): [email protected]
Introduction: In post mortem cases, detection of drugs in blood is most relevant with regard to
determining cause of death. However, it is sometimes also of interest to gain information regarding
the deceased´s use of drugs in the period before death. The aim of this study was to compare results
from analyses of a broad repertoire of psychoactive medicinal drugs in blood and hair samples from
post mortem cases.
Materials and Methods: Forensic autopsy cases in which intake of drugs was suspected were
included. Post mortem blood collected from the femoral vein and hair samples from the posterior
vertex were collected simultaneously, and analysed for a number of psychoactive medicinal drugs,
mainly benzodiazepines, antidepressants and antipsychotics. Blood was analysed using validated GC-
MS, LC-MS or UPLC-MS/MS methods, and 3 cm long proximal hair segments were analysed by a
validated LC-MS/MS method. Substances not included in both the blood and hair methods were
excluded. Cases where external contamination of hair was suspected were also not included in the
study.
Results: Blood and hair samples from 55 post mortem cases were included in the study. Drug(s)
were detected in hair or blood in 53 of the 55 cases (96%). The most frequently detected drugs in
hair or blood was diazepam, followed by clonazepam. In total, hair analyses gave information of use
of drugs not detected in blood in 47 of the 55 cases (85%). In these cases, additional use of 1-10
drugs (median 2) was revealed. In only two cases (4%), benzodiazepines were detected in blood,
with no benzodiazepines detected in hair.
Conclusions: In a large proportion of cases, past use of drugs in addition to those detected in blood,
was confirmed. Post mortem hair analysis thus gave additional information about the use of
medicinal drugs in the period before death. In only a very few cases, drugs were detected in blood
and not in hair.
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O8
Deposition of diazepam and its metabolites in hair following a single dose of diazepam
Sys Stybe Johansena, Xin Wanga and Kristian Linneta
aSection of Forensic Chemistry, Department of Forensic Medicine, University of Copenhagen, Denmark
Contact information (e-mail(s)): [email protected]
Introduction Diazepam is one of the most prevalent drug-facilitated crime drugs. Only sporadic data
are available on hair concentrations of diazepam and some metabolites following a single controlled
dose. In this study, the deposition of diazepam and its phase I and II metabolites in hair following a
single dose of diazepam were investigated.
Materials and methods Eight participants (four women and four men, ages 24–26, Chinese)
consumed 10 mg diazepam each. Hair was collected one month after exposure, and also two months
post-exposure from the men and 10 months post-exposure from the women. Diazepam and its
metabolites were measured after solvent extraction from hair by ultra-high pressure liquid
chromatography–tandem mass spectrometry (UHPLC–MS/MS).
Results There were no differences by gender in the amounts of diazepam or metabolites found. The
proximal segment from 0–2 cm collected one month after administration gave detectable diazepam
and nordazepam concentrations ranging from 4.0 to 11.8 pg/mg and 9.4 to 24.3 pg/mg, respectively.
Oxazepam and temazepam traces were found in some segments. In the hair samples collected from
the four men two months after administration, the highest concentrations of diazepam and
nordazepam were detected in the 1–3 cm segment corresponding to the time from ingestion with an
average growth rate of 1 cm/month. The concentrations of both drugs in the 1–3 cm segment
collected two months post-exposure were similar to that in the 0–2 cm segment collected one
month after. Diazepam and nordazepam concentrations in the hair collected from the 4 women 10
months after exposure were near the LOQ, indicating drug loss by personal hygiene and physical
handling. Oxazepam glucuronide and temazepam glucuronide were not detected in any hair
segments from the subjects.
Conclusion Diazepam and nordazepam are detectable at pg/mg level in hair collected one and two
month after intake of a single dose of diazepam. However, after ten months only traces are found.
mailto:[email protected]
16
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O9
Targeted proteomics of CYP1A2 and CYP3A4 in post-mortem liver tissue
Jakob Hansena, Anette Daa Fundera, Jørgen Hasselstrøma, Jakob Ross Jornila
aAarhus University, Department of Forensic Medicine, Bioanalytical Unit
Contact information (e-mail(s)): [email protected]
Introduction Members of the cytochrome P450 (CYP) enzyme family metabolize a great number of
drugs that are responsible for intoxications and drug-related fatalities in forensic settings. Inter-
individual differences in drug metabolizing capacity and thus susceptibility to adverse drug effects is
partly based on the expression level and activity of different CYP-isoforms. The activity of specific
CYP-isoforms is influenced by multiple factors including genetic (polymorphisms and gene-dosage
effects) and exogenous circumstances such as intake of certain drugs and food ingredients that act
as both inducers and inhibitors of activity. For the specific CYP-isoforms CYP1A2 and CYP3A4,
genotyping analyses lack predictive value for enzyme activities and methods for estimating the CYP
activities in post-mortem materials have not been successful. We have pursued the idea that
quantification of the protein levels of CYP1A2 and CYP3A4 in post-mortem liver tissue is a valid
approximation of the corresponding ante-mortem metabolic enzyme capacities.
The aim of the project was to develop a quantitative method for CYP1A2 and CYP3A4 in hepatic
tissue and investigate the post-mortem stability of the two CYP-isoforms.
Materials and methods CYP 1A2 and CYP3A4 proteins were quantified using a targeted proteomics
approach based on analysis of proteotypic peptides by UPLC-MS/MS. A method for enrichment of
CYP proteins in microsomal fractions of homogenized post-mortem liver tissue using differential
centrifugation was established. Peptides generated by trypsin cleavage were separated by reverse
phase chromatography (C18 column) and detected by multiple-reaction-monitoring. Synthetic stable
isotopic variants of the target peptides were added and analysed simultaneously with the
proteotypic CYP peptides to ensure correct identification and accurate quantification. The post-
mortem stability of CYP enzymes was studied by incubating sample specimens from newly deceased
persons at different temperatures for up to 7 days. CYP1A2 and CYP3A4 quantification data from
these stability samples will be presented and discussed.
Conclusion Post-mortem CYP quantitation may potentially develop into a valuable tool for forensic
toxicological interpretation by disclosing possible pharmacokinetic causes of low or high drug
concentrations.
17
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O10
In vitro metabolism studies on new designer benzodiazepines
Carolina Noblea, Niels Bjerre Holma, Sys Stybe Johansena and Kristian Linneta
aUniversity of Copenhagen, Faculty of Health and Medical Sciences, Dept. of Forensic Medicine, Section of Forensic
Chemistry
Contact information (e-mail(s)): [email protected]
Introduction Designer benzodiazepines are new psychoactive substances recently introduced in the
illegal drug market. Little is known about metabolism of this group of substances so far.
Materials and methods In this study, in vitro metabolism of two designer benzodiazepines,
flubromazolam and clonazolam, was investigated in NADPH-fortified pooled human liver
microsomes (HLM) and baculovirus-expressed cytochrome CYP-enzymes: CYP1A2, 2A6, 2B6, 2D6,
2C8, 2C9, 2C18, 2C19, 2E1, 3A4 and 3A5, followed by inhibitory reactions in HLM with 1 and 5 µM
ketoconazole (CYP3A4/5 inhibitor), 5 µM sulfaphenazole (CYP2C9 inhibitor), 5 µM nootkatone
(CYP2C19 inhibitor), and 5 µM quinidine (CYP2D6 inhibitor). The parent compound and its
metabolites were detected by LC-MS on a high-resolution Q-Exactive mass spectrometer, with
positive electrospray ionization (ESI+) in data-dependent acquisition mode (DDA). Metabolic
pathways for both compounds in HLM included hydroxylation, and reduction of the nitro group for
clonazolam.
Results Reaction phenotyping with CYP-enzymes showed that monohydroxylated metabolites for
both compounds were primarily produced by CYP3A4 and 3A5. This fact was further supported in
incubations with ketoconazole in HLM. Flubromazolam was also further metabolized to dihydroxy-
flubromazolam only by CYP3A4. The reduction of the nitro group of clonazolam to yield the primary
amine was detected after incubation with CYP2D6, while for CYP3A4 and 2C19 a combination of
hydroxylation and reduction of the nitro group was observed. Interestingly, formation of this
metabolite was not observed in HLM. CYP3A4 metabolized clonazolam to monohydroxy-clonazolam
at the first stage of the reaction (0-60 minutes) and after 120 minutes to monohydroxyamine-
clonazolam. For CYP2C19, however, only monohydroxyamine-clonazolam was detected after 120
minutes of incubation. No glucuronides were identified in HLM with the cofactor UDPGA for neither
of the two drugs, even with the addition of NADPH to produce phase-I metabolites previously
described. In addition, we identified in vivo metabolites for both compounds in different forensic
cases and corresponded to those formed in vitro.
mailto:[email protected]
18
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O11
Metabolic profiling using retrospective UPLC-HR-TOF-MS data from forensic toxicology screenings
Kirstine Lykke Nielsena, Rasmus Telvinga, Mette Findal Andreasena, Jørgen Hasselstrøma, Mogens
Johannsena
aDept. of Forensic Medicine, Section for Forensic Chemistry, Aarhus University, Palle Juul-Jensens Boulevard 99, DK-8200
Aarhus N, Denmark
Contact information (e-mail(s)): [email protected]
Introduction Whole blood samples collected by the Danish police from drivers suspected driving
under the influence of drugs are routinely screened by UPLC-HR-TOF-MS to determine the nature of
potential drugs.
Materials and methods Data collected within a two-year period was used for metabolic profiling
(metabolomics) to evaluate the drug metabolism of 3,4-methylenedioxymethamphetamine (MDMA,
“Ecstasy”). Metabolomics is an unbiased, untargeted, and very comprehensive method to detect
changes of low molecular weight metabolites in biological samples in relation to various exposures
and diseases; thus, it may be useful to profile drug metabolism, mechanism and toxicology.
Metabolomics usually requires a very controlled setup to minimize biological and external
confounders to extract useful information. However, the use of such retrospective data involves a
very heterogeneous population with different concentrations of MDMA/kg blood weight, as well as
unknown information about amount and time of administration in relation to blood sampling.
Results Collection of data covering two years may additionally introduce variation from sample
handling and analysis. Despite of this, it was found possible to extract meaningful information.
Various statistical methods were tested and their predictability was validated by the positive
identification of MDMA blood metabolites. In addition, endogenous metabolites that may be related
to energy metabolism, the serotonergic syndrome, and drug induced neurotoxicity could be
identified.
Conclusion The use of retrospective UPLC-HR-TOFMS forensic data is a potential new approach for
identifying changes in human drug metabolism and endogenous biomarkers related to licit and illicit
drug administration and toxicology, especially in relation to new designer drugs.
19
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O12
Ethanol elimination at low concentrations in drunk drivers
G. Høisetha, E. Wiika, L. Kristoffersena, J. Mørlanda
aNorwegian Institute of Public Health, Division of Forensic Medicine
Contact information (e-mail(s)): [email protected]
Introduction For ethanol, the elimination curve change from apparent zero to first order kinetics at
falling blood alcohol concentrations (BACs). This is less studied than elimination at higher BACs, and
knowledge about this low BAC elimination is especially missing in drunk drivers. The aim of this study
was to investigate the point at which elimination rates turns from zero to first order kinetics and the
exact elimination rates at the very low BAC intervals in this population with a high frequency of
heavy drinkers.
Materials and methods Two consecutively collected samples from suspected drunk drivers were
used. All samples were analysed by two headspace gas chromatography flame ionisation detector
methods (limit of quantification=0.04 g/kg). The elimination rates at BACs below 0.25 g/kg (study
group, n=175) was studied in detail, and compared to the elimination rates in a moderate BAC
reference group (n=789) as well as a high BAC reference group (n=4435).
Results There were no differences in age, gender and driving’s occurring during the night-time
between the study group and the reference groups. The elimination rates were stable at 0.18-0.19
g/kg/h from a BAC of 4.0 g/kg and until BAC in the first blood sample fell below 0.19 g/kg. At BACs
below 0.19 g/kg, the mean elimination rate gradually declined from 0.163 g/kg/h to the lowest
elimination rate of 0.083 g/kg/h. There was no relation between the concentration of ethanol and
elimination rate at BACs above 0.19 g/kg (Pearson´s r=0.035, p=0.3), but there was a strong relation
between concentration of ethanol and elimination rate at BACs below 0.19 g/kg (Pearson´s r=0.56,
p
20
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O13
Post-mortem levels and tissue distribution of codeine, codeine-6-glucuronide, norcodeine,
morphine and morphine glucuronides in a series of codeine-related deaths
Joachim Frosta,b, Trine Nordgård Løkkena, Arne Hellanda,b, Ivar Skjåk Nordruma,c, Lars Slørdala,b
aDepartment of Laboratory Medicine, Children’s and Women’s Health, Norwegian University of Science and Technology
(NTNU), Trondheim, Norway bDepartment of Clinical Pharmacology, St. Olav University Hospital, Trondheim, Norway
cDepartment of Pathology and Medical Genetics, St. Olav University Hospital, Trondheim, Norway
Contact information (e-mail(s)): [email protected]
Introduction The toxicodynamics and, to a lesser degree, toxicokinetics of the widely used opiate
codeine remain a matter of controversy. We present levels and tissue distribution of codeine,
codeine-6-glucuronide (C6G), norcodeine, morphine and the morphine metabolites morphine-3-
glucuronide (M3G) and morphine-6-glucuronide (M6G) in post-mortem blood, vitreous fluid, muscle,
and fat and brain tissue in a series of 23 codeine-related fatalities. CYP2D6 genotype is also
determined and taken into account.
Materials and methods Quantification was performed with a validated SPE-LC-MS method. The
series comprise 19 deaths (83%) attributed to mixed drug intoxication, 4 deaths (17%) attributed to
other causes of death, and no cases of unambiguous mono-intoxication with codeine. The typical
peripheral blood concentration pattern in individual cases was
C6G>>codeine>>norcodeine>morphine, and M3G>M6G>morphine. In matrices other than blood,
the concentration pattern was similar, although in a less systematic fashion. Measured
concentrations were generally lower in matrices other than blood, especially in brain and fat, and in
particular for the glucuronides (C6G, M3G and M6G) and, to some extent, morphine. In brain tissue,
the presumed active moieties morphine and M6G were both below the LLOQ (0.0080 mg/L and
0.058 mg/L, respectively) in a majority of cases.
Conclusion In general, there was a large variability in both measured concentrations and calculated
blood/tissue concentration ratios. There was also a large variability in calculated ratios of morphine
to codeine, C6G to codeine and norcodeine to codeine in all matrices, and CYP2D6 genotype was not
a reliable predictor of these ratios. The different blood/tissue concentration ratios showed no
systematic relationship with the post-mortem interval. No coherent degradation or formation
patterns for codeine, morphine, M3G and M6G were observed upon reanalysis in peripheral blood
after storage.
Keywords: Forensic toxicology; codeine; toxicity; post-mortem; tissue distribution
mailto:[email protected]
21
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O14
Distribution of QT-prolonging drugs and metabolites between cardiac tissue and femoral blood in
a psychiatric population
Christian Reuss Mikkelsena, Jakob Ross Jornila, Ljubica Vukelic Andersena, Jytte Bannerb, Jørgen Bo
Hasselstrøma
aDepartment of Forensic Medicine, Aarhus University, DK-8200, Denmark
bDepartment of Forensic Medicine, University of Copenhagen, DK-2100, Denmark
Contact information (e-mail(s)): [email protected]
Introduction The Danish forensic autopsy-based prospective study, named SURVIVE, aims at
elucidating the causes of the increased mortality and morbidity among mentally ill patients.
Mentally ill patients are a heavily medicated group, and many of these drugs have been found to
prolong the QT interval of the ECG. This can lead to the ventricular arrhythmia condition Torsades de
Pointes which can further lead to sudden cardiac death. In forensic post-mortem toxicology, blood
concentrations are usually used for estimating a possible death by cardiac arrhythmia. However,
animal studies and post-mortem case studies have shown that some QT-prolonging drugs (QTD) can
be found in higher concentration in cardiac tissue when compared to peripheral blood.
The aim of this project is to characterize the distribution of seven frequently used QTD and their
metabolites in cardiac tissue and femoral blood to investigate, if the cardiac tissue concentration
could be a tool for estimating cardiac arrhythmia as a potential cause of death.
Materials and methods Among the 500 cases included in SURVIVE, cases with at least one of QTD
reported was selected for this project. A protein precipitation method was used to extract the QTD
from cardiac homogenate and femoral whole blood. Extracts were analysed on a UPLC-MS/MS.
Demographic, physiological and forensic results were obtained from police, toxicology and autopsy
reports for the descriptive analysis.
211 cases were included in this project, 121 male and 90 female. The compound concentrations in
whole blood and cardiac tissue were found to be statistically correlated (p0.8), one strong correlated (R^2=0.76) and one moderate correlated
(R^2= 0.62). Across all 14 compounds the heart-to-blood ratio (HB-ratio) ranged from 0.093 to 43
and the median HB-ratio ranged from 1.6 to 14. Each compound showed a high variance in HB-ratio
between cases which was addressed statistically. Only age showed to be statistically significant in
explaining the high HB-ratio variance of each compound. No difference in HB-ratios was detected
due to extended post-mortem interval.
Conclusion In this project, we present the distribution of frequent used QTD’s in the SURVIVE
population between cardiac tissue and femoral blood.
mailto:[email protected]
22
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O15
Comparison of antidepressiva concentrations in different post mortem media
Åse Marit Leere Øiestada, Ritva Karinena, Sidsel Rogdea, Kari Beate Boye Eldora, Gerd Wenche
Brochmanna, Marianne Arnestada, Elisabeth Leere Øiestada, Mariana Dadalto Peresa, Lena
Kristoffersena, and Vigdis Vindenesa
aNorwegian Institute of Public Health, Domain for Forensic Sciences, Oslo, Norway
Contact information (e-mail(s)): [email protected]
Introduction Analyses of concentrations of different compounds in alternative media to blood or
urine can improve the understanding of the ante mortem concentrations. In Norway, alternative
media are mostly used when blood and urine are not available. Several studies comparing different
media can be found in the literature, but for many compounds the knowledge available for
interpretation of the results is sparse at best, thus making interpretation difficult. More studies are
therefore needed, and we have initiated a study aiming to compare concentrations in six different
media; i.e. peripheral blood, cardiac blood, pericardial fluid, vitreous humour, and two different
muscles – m vastus lateralis and m psoas. We wish to compare concentrations for a large number of
drugs and medicinal products in different sampling media, collected in connection with medicolegal
autopsies.
Aim The aim of the project is to study how other media than blood/urine can be of use in potential
intoxication deaths, accidents, and when the deceased’s treatment compliance is questioned.
Materials and methods So far we have included almost 160 forensic autopsy cases in which intake
of drugs were suspected. All six media were collected on the same day and were analysed using
validated GC-MS, LC-MS or UHPLC-MS/MS methods. Care was taken to analyse all media from each
case on the same series to minimise differences in concentrations due to experimental factors.
Result We here present results from cases where antidepressants were found.
23
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O16
Testosterone levels in forensic cases compared to testosterone levels in long-acting replacement
therapy with intramuscular testosterone undecanoate
Yvonne Looda, Bertil Ekmanb, Jeanette Wahlbergb, Johan Ahlnera
aNational Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Artillerigatan 12, S-587 58
Linköping, Sweden and Department of Medical and Health Sciences, Linköping University, S-581 85 Linköping, Sweden. bDepartment of Endocrinology and Department of Medical and Health Sciences, Linköping University, S-581 85 Linköping,
Sweden.
Contact information (e-mail(s)): [email protected]
Introduction Testosterone is the most important endogenous anabolic androgenic steroid (AAS) in
men and next to nandrolone the most frequently abused AAS. The use of AAS is prohibited in
Sweden since 1999. Testosterone is therapeutically used in the treatment of hypogonadism in men
and in cross-sex hormone treatment of biological female transsexual individuals to develop and
maintain male secondary sex characteristics. Determination of AAS in forensic cases and doping
controls is mainly performed in urine and detection of exogenous testosterone administration is
complex to discriminate from the state of normality or testosterone levels achieved by conventional
replacement therapy.
Aim To study urinary testosterone levels detected in forensic cases comparing to testosterone levels
in standard treatment with testosterone undecanoate.
Subjects Forensic cases (n=1155), males with hypogonadism (n=23) and transsexuals with male
identity (n=15) at the Department of Endocrinology in Linköping and a healthy control group of men
(n=32) were investigated.
Methods A national forensic database was used to retrieve toxicological analytical results. Peak and
trough values of urinary testosterone were measured during testosterone treatment by GC-MS.
Results Urinary testosterone were by mean significantly higher in the forensic cases (380 ± 397
ng/mg creatinine) compared to the patient group (62 ± 60 ng/mg creatinine) and controls (33 ± 26
ng/mg creatinine). The mean T/E ratio was 53 in the forensic cases, 24 in the patients and 1.1 in the
controls. Peak urinary testosterone, 7-14 days after injection, (1.4-548 ng/mg creatinine) were
significantly lower compared to the concentrations measured in the forensic cases (10.4-3620 ng/mg
creatinine). No significant differences were found in trough testosterone levels in treated patients
comparing to controls.
Conclusion It seems reliable to discriminate between abuse of testosterone and conventional
replacement therapy by the significantly higher urinary testosterone concentrations achieved after
illegal testosterone administration.
24
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O17
Reference values of Lithium in post-mortem blood
Carl Söderberg, Emma Wernvik, Anna Jönsson, Henrik Druid
National Board of Forensic Medicine, Linköping, Sweden
Contact information (e-mail(s)): [email protected]
Introduction The main recipients of lithium, people diagnosed with bipolar disorder, show an
increased mortality in both natural and unnatural causes of death. Based on international data
persons diagnosed with bipolar disorder comprise 2.3-9.6 % of all suicidal deaths. In cases of suicide
among those suffering from bipolar disorder, 17-53 % are due to fatal intoxications. Diagnosing fatal
intoxications is often challenging in the forensic pathological setting, particularly when the reference
information needed to evaluate the concentration of a drug is lacking or scarce.
The aim of this study was to establish post-mortem femoral blood reference concentrations of
lithium, providing both fatal and “normal” concentrations.
Materials and methods In Sweden, forensic autopsies are performed in unnatural and obscure
deaths. This study included all autopsies, in which lithium was found during 1992-2010. Lithium was
not included in the regular drug screen, but analysed upon request using flame photometry, ion-
selective electrode detection or atomic absorption spectrophotometry. Each case was evaluated
according to an established strategy, with strict inclusion and exclusion criteria followed by a multi-
observer manual review. Included cases were classified as single intoxications (Group A), multi drug
intoxications (Group B) or control (Group C). The control group only included cases where death by
intoxication and ante-mortem incapacitation by drugs were ruled out.
Results During the study period, toxicological analysis of femoral blood samples was performed in
93,623 autopsies. Lithium was found in 124 (< 1 %) of these. After application of inclusion and
exclusion criteria and the subsequent manual review, 21 cases were included in the study (Group A,
n=4; Group B, n=7; Group C, n=10). The femoral blood lithium concentrations in Group A (median
2.69 mmol/l) and Group B (median 2.10 mmol/l) were significantly different (p=0.01) compared to
Group C (median 0.2 mmol/l). There were, however no statistically significant difference between
the concentrations in Group A and Group B.
Conclusion Although a relatively small number of cases met the inclusion criteria in this study, we
believe that the presented post-mortem concentrations may be used as a reference when
interpreting toxicological results involving lithium.
mailto:[email protected]
25
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O18
Beta-hydroxybutyrate and the post mortem diagnosis of ketoacidosis
Elli Tyrkköa, Anna Jönssona, Robert Kronstranda
aThe National Board of Forensic Medicine, Linköping, Sweden
Contact information (e-mail(s)): [email protected]
Introduction Ketoacidosis, resulting from increased blood beta-oxidative ketone bodies caused by
alcoholism (alcoholic ketoacidosis, AKA) or diabetes (diabetic ketoacidosis, DKA), is a pathological
condition that features in many fatalities. The analyses of beta-hydroxybutyrate (BHB), acetone, and
glucose are essential for identification and differentiation between AKA, DKA and hyperosmolar
hyperglycaemic state (HHS). The analysis of BHB in blood by GC-MS was introduced in 2013 at the
laboratory of forensic toxicology in Linköping, Sweden. This study aims at evaluating how the
analysis of BHB assists in identification of the conditions causing ketoacidosis.
Materials and methods In the Swedish forensic toxicology and pathology database we identified all
cases including a BHB analysis, the term “acidosis”, and the ICD9 codes 250C (diabetes with coma)
and/or 276C (disorders in acid-base balance), for two time periods, 2012-2013 and 2014-2015. The
AKA, DKA and HHS cases were identified and scrutinized.
Results The total number of identified cases was 241. The number of cases with AKA as the
immediate cause of death increased from 5 to 19 between 2012-2013 and 2014-2015. The number
of BHB analyses increased from 11 to 49 during these time periods. The results from the BHB
analyses supported the diagnosis of AKA in 18 of the total number of 19 cases reported in 2014-
2015. The number of DKA cases decreased by 10% (from 93 to 84) between 2012-2013 and 2014-
2015. The toxicological findings support the diagnosis of HHS in 20 cases in each time period.
However, HHS was mentioned in the death certificate in only 4 of these 40 cases. The most common
cause of death assigned in these cases was diabetes with coma.
Conclusion The study shows that the diagnosis of AKA remarkably benefits from the analysis of BHB.
Therefore, the analysis of BHB is strongly recommended in cases, where glucose is low and acetone
is positive in either blood or urine, and there is no other competing cause of death. Analysis of BHB
also aids in differentiation of DKA from HHS, as acetone might be negative in blood in some cases.
We also found that HHS is clearly an under-identified condition.
Keywords beta-hydroxybutyrate, alcoholic ketoacidosis, diabetic ketoacidosis, hyperosmolar
hyperglycaemic state
26
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O19
Deaths attributed to 1-phenyl-2-(1-pyrrolidinyl)-1-pentanone (α-PVP)
Robert Kronstranda
aNational Board of Forensic Medicine, Linköping, Sweden
Contact information (e-mail(s)): [email protected]
Introduction The psychoactive substance 1-phenyl-2-(1-pyrrolidinyl)-1-pentanone or α-PVP emerged
on the European market around 2011. It is a cathinone without substitutions on the phenyl ring
structure. The toxidromes commonly encountered after ingestion of cathinones are mainly of
sympathomimetic and hallucinogenic character with a risk of excited delirium and life-threatening
cardiovascular effects. Reported severe side-effects from α-PVP are tachycardia, hypertension,
hyperthermia, and rhabdomyolysis. Since 2011, α-PVP has been a fairly common finding in both
cases from the living and the dead even after it was scheduled February 1st 2013.
Materials and methods To investigate the occurrence of acute fatal toxicity of α-PVP a database
search was performed. All cases found positive for α-PVP between 2011 and 2015 were included.
The ante-mortem and post mortem findings in cases of fatal intoxication were evaluated.
Results The search resulted in 641 positive findings in the living and 22 positive findings in autopsy
cases. There was a steep increase in positive cases with a peak of 221 and 9 cases during 2014. Ten
of the autopsy cases were signed out as fatal intoxications by the medical examiner. Of these, one
was unrelated to α-PVP, 7 had α-PVP as contributing, and two had α-PVP as the primary cause of
death. The first case involved a 43 year old male that after having injected α-PVP was found
unconscious by a friend. Arriving at hospital he was still unconscious, had tachypnea and was
shaking. He presented with brain edema, brain haemorrhages and an examination of the brain
revealed severe anoxic brain injury and life support was stopped one day after arrival. The femoral
blood concentration of α-PVP was 0.008 µg/g which seem low but can partly be explained by
metabolism during supportive treatment. The second case was not witnesses and the autopsy
revealed moderate brain edema, pulmonary congestion, lung emphysema and needle marks. The
femoral blood concentration of α-PVP was 0.04 µg/g. Among the other deaths one showed signs of
excited delirium with excitation, confusion, and hyperthermia but died from multiple injuries after a
fall from a six story building.
Conclusion In conclusion, among the 22 findings of α-PVP in autopsy cases 10 were intoxications and
7 had α-PVP as contributing or primary cause of death.
mailto:[email protected]
27
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O20
An increase in fentanyl-related deaths during the winter 2015/2016
Gunilla Thelandera and Robert Kronstranda
aThe National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden
Contact information (e-mail(s)): [email protected]
Introduction A sudden increase in fentanyl positive cases prompted us to investigate this further.
The aim of our study was to show the Swedish situation of fentanyl-related deaths and describe the
demographics, circumstances, and post mortem findings.
Material and methods Autopsy cases from 2015 to March 2016 with positive findings of fentanyl
were reviewed regarding concentrations, cause of death, age, gender, location in Sweden and
availability of fentanyl in different forms.
Results We found a significant increase in deaths related to fentanyl from December 2015 and
onwards. Many of the decedents had a history of drug abuse and several cases indicated frequent
poly-drug use. Prior to December no more than 5 cases were seen per month 2015 (mean= 3 cases).
During December we encountered 22 fatal intoxications, in January 2016 there were 16 and in
February 8 fentanyl deaths. The preliminary figure for March is 9; however cause of death has not
been assigned to all cases yet. Of these decedents 85 % were male and the median age was 34 years.
Geographically, the cases were from all regions of the country. During December to February, the
most common forms of fentanyl were powder, nasal sprays, and tablets even though a few cases of
transdermal patches were seen. The blood concentrations of fentanyl (December to February)
revealed; range 0.06-142 ng/g, median 18 ng/g and mean 26 ng/g.
Conclusion The increased availability of fentanyl has generated a large peak of fentanyl-related
deaths in Sweden. The data suggest that the fentanyl derived, not from pharmaceutical fentanyl, but
from illegal sources.
28
AARHUS
UNIVERSITY
HEALTH
DEPARTMENT OF FORENSIC MEDICINE
O21
Trends in fatal poisonings by prescription drugs in Finland since 2000
Ilkka Ojanperäa
aDepartment of Forensic Medicine, University of Helsinki and Forensic Toxicology Unit, National Institute for Health and
Welfare, Helsinki, Finland
Contact information (e-mail(s)): [email protected]
Introduction The present paper reviews the trends in fatal poisonings by prescription drugs in
Finland during 2000 – 2013.
Materials and methods The post-mortem toxicology database included for each case the forensic
pathologist's referral, laboratory analysis results, and information extracted from the death
certificate issued by forensic pathologist. The prevalences of fatal prescription drug poisonings were
based on the most important single drug finding in each fatality, as stated in the death certificate.
Results The number of fatal poisonings due to drugs during the period 2000 – 2013 has varied from
450 to 600 cases. The annual number of poisonings by antipsychotics has stabilized at 60 cases after
the withdrawal of promazine that still caused tens of poisonings 2000 – 2003. Quetiapine and
levomepromazine formed the major part of antipsychotics poisonings in 2013. The annual number
of antidepressant poisonings averaged 120 cases during 2000 – 2009, but since then the number has
steadily declined to 80 cases. In 2013, amitriptyline and venlafaxine were the most frequent major
antidepressant drugs but bupropion was on the increase in 2013 and should be carefully monitored.
The annual number of poisonings by prescription opioids has steadily increased from 50 to 200 cases
during 2000 – 2011 and, since then, decreased to 160 cases in 2013. Buprenorphine and tramadol
formed the major part of opioid poisonings in 2013. The proportion of prescription opioids from all
drug poisonings was 33% in 2013, as opposed to only 9.5% in 2000. The annual number of
poisonings by hypnotics and sedatives since 2000 has varied from 40 to 80 cases; being 65 cases in
2013. Pregabalin was associated with the highest number of poisonings among hypnotics and
sedatives, with 26 cases in 2013.
Conclusion When the number of fatal poisonings was divided by the associated sales using the
number of defined daily doses (summed from years 2005, 2009 and 2013), the following prescription
drugs possessed the highest risk of fatal poisonings in descending order of risk: methadone,
dextropropoxyphene, levomepromazine, doxepin, chlorprothixene, oxycodone, amitriptyline,
trimipramine, tramadol, morphine, bupropion, clomipramine, propranolol, quetiapine, fentanyl,
codeine, and pregabalin. Buprenorphine was not evaluated because of the frequency of illicit
trafficking associated with the drug.
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DEPARTMENT OF FORENSIC MEDICINE
O22
Re-offending in drug-related crimes in Sweden, 1993 – 2013
Johan Ahlnera, Anita Holmgrena, A. Wayne Jonesa
aNational Board of Forensic Medicine and Institution of Medicine and Care, Linköping University, Linköping, Sweden
Contact information (e-mail(s)): [email protected]
Introduction The National Laboratory of Forensic Toxicology in Linköping receives annually ~30 000
suspected cases of illegal drug use along with ~12 500 cases of driving under the influence of drugs.
Our statistics show that during the period 1993-2013 over 600 000 drug-related cases were
registered and toxicological analysis of blood and/or urine samples were performed.
Materials and methods Information about all cases of illegal drug use or driving under the influence
of drugs were extracted from a national forensic toxicology database (TOXBASE) for scrutiny. The
database contained a personal identification number, age, and sex of the suspect and results of a
comprehensive toxicological analysis of blood and urine for licit and illicit drugs.
Results During the period 1993-2013 around 630 000 cases were registered at our laboratory. We
found that 80% of those arrested were repeat offenders, that is, the same suspect appeared at least
once before in the database. We found that 503 260 positive cases belonged to only 88 067 different
individuals, which makes an average of 5.7 arrests per person. The central stimulant amphetamine
was the most commonly encountered illicit drug, followed by abuse of cannabis/marijuana. Among
reoffenders more than 87 % were men. One individual had been arrested 154 times over the 21 year
study period and in one year he was arrested on 21 occasions. Around 300 different individuals were
arrested at least 50 times each.
Conclusion Reoffending frequency was high in people arrested in Sweden for use of illegal drugs or
driving under the influence of drugs. This probably reflects an increased activity by the police
authorities and ineffectiveness of the legal sanctions for those convicted of such drug-related
crimes. Much might be gained by sentencing to a mandatory treatment programme for substance
abuse disorder rather than monetary fines or short terms of imprisonment.
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DEPARTMENT OF FORENSIC MEDICINE
O23
Experiences in an EU project “Strengthening and modernizing the forensic services in Kosovo”
Ilpo Rasanena
aNational Institute for Health and Welfare (THL), Helsinki, Finland
Contact information (e-mail(s)): [email protected]
Introduction A consortium formed by the National Institute for Health and Welfare (THL) and the
University of Helsinki implemented an EU funded project “Strengthening and modernizing the
forensic services in Kosovo” starting in February 2014. Later, the project received a one-year
extension and will continue until February 2017. The project team consists of a team leader (410
working days) and a key expert II (220 working days), as well as several short term experts
implementing the training programme.
The purpose of the project is to strengthen and modernize the forensic services at the Department
of Forensic Medicine (DFM) in Pristina by enhancing its technical and administrative capacity.
Materials and methods The terms of reference of the project were written in the EU Office in
Kosovo. After the inception period, it was found that the goals of the project were set too high
compared to the current status of DFM. In the inception report, the project team evaluated the
current status of DFM and presented the training needs and a proposal for the new equipment
purchased by the project. These issues were sharpened during the project.
My main role in the project was to develop the laboratory for toxicology in DFM. The laboratory was
founded in 2006 by the United Nations when also the new building of DFM was build. The personnel
of the laboratory consist of a laboratory manager, a chemist and a toxicologist. In 2014, the
analytical equipment of the laboratory consists of immunoassay (ELISA) equipment, a CO-oximeter,
an HS-GC and two GC-MS instruments.
The autopsy rate in Kosovo is very low, being approximately 300-350 cases annually, compared with
the population of about 1.7 million. The laboratory has received samples from about 250 autopsy
cases annually. Only qualitative screenings were performed before the project started. My goal was
to develop new methods and to exploit the quantitative methods used in Helsinki for alcohols,
carbon monoxide and most common drugs of abuse (opiates, amphetamines, cannabis,
benzodiazepines and cocaine), as well as for individual therapeutic drugs. However, for example the
lack of reference standards and the difficulty to purchase them complicated the goal considerably.
Conclusion Developing the laboratory operation has been challenging, such as many other parts of
the project as well. I have encountered a multitude of surprises and setbacks during the project that
I could not imagine take place in the 2000s. All things seem to be more difficult in developing
countries.
The training programme has been implemented fairly successfully. The challenges, difficulties and
major achievements of the project will be discussed.
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DEPARTMENT OF FORENSIC MEDICINE
Participants
Johan Ahlner The National Board of Forensic Medicine, Sweden Aðalheiður Dóra Albertsdóttir University of Iceland Ingeborg Amundsen Folkehelseinstituttet, Norway Ljubica Vukelic Andersen Department of Forensic Medicine, Aarhus University, Denmark Nina Zacho Andersen University of Copenhagen, Denmark Mette Findal Andreasen Department of Forensic Medicine, Aarhus University, Denmark Marianne Arnestad Norwegian Institute of Public Health, Norway Liliana Bachs Norwegian Institute of Public Health, Norway Eirin Bakke Norwegian Institute of Public Health, Norway Jesper Berggreen Department of Forensic Medicine, Aarhus University, Denmark Willy Bjørklund Thermo Fisher Scientific, Sweden Jenny Button Chiron / CAS Referensmaterial AB, Norway Søren Dalby Bruker Daltonics, Denmark Petur Dalsgaard University of Copenhagen, Denmark Henrik Druid Rättsmedicinalverket, Norway Anne Dyrdal Waters A/S, Denmark Elin Eliassen Institute of Public Health, Norway Eva Ericsson The National Board of Forensic Medicine, Sweden Tina Eriksen Syddansk University, Denmark Liselotte Frantzen Department of Forensic Medicine, Aarhus University, Denmark Jens Glastrup MSCi ApS, Denmark Per Ole M. Gundersen St. Olavs Hospital, Trondheim University Hospital, Norway Kristin Irene Gaare Institute of Public Health, Norway Solveig Sif Halldorsdottir University of Iceland An-Magritt Haneborg Institute of Public Health, Norway Jakob Hansen Department of Forensic Medicine, Aarhus University, Denmark Tore Hardlei Department of Forensic Medicine, Aarhus University, Denmark Jørgen Bo Hasselstrøm Department of Forensic Medicine, Aarhus University, Denmark Solfrid Hegstad St. Olavs Hospital, Trondheim University Hospital, Norway Niels Bjerre Holm University of Copenhagen, Denmark Gudrun Høiseth Institute of Public Health, Norway Pernilla Haage The National Board of Forensic Medicine, Sweden Gerd Jakobsson The National Board of Forensic Medicine, Sweden Brian Sonne Jensen Department of Forensic Medicine, Aarhus University, Denmark Mogens Johannsen Department of Forensic Medicine, Aarhus University, Denmark Sys Johansen University of Copenhagen, Denmark Unni Johansen Institute of Public Health, Norway Anna Johansson The National Board of Forensic Medicine, Sweden Jakob Ross Jornil Department of Forensic Medicine, Aarhus University, Denmark Martin Josefsson The National Board of Forensic Medicine, Sweden Aino Kankaanpää National Institute for Health and Welfare, Helsinki, Finland Inge Korup Department of Forensic Medicine, Aarhus University, Denmark Robert Kronstrand The National Board of Forensic Medicine, Sweden Fredrik Kugelberg The National Board of Forensic Medicine, Sweden Susan Marie Jøker Lambertsen Department of Forensic Medicine, Aarhus University, Denmark Veronica Horpestad Liane Institute of Public Health, Norway Sofia Lindahl St. Olavs Hospital, Trondheim University Hospital, Norway Lone Lindal University of Southern Denmark
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HEALTH
DEPARTMENT OF FORENSIC MEDICINE
Christian Lindholst Department of Forensic Medicine, Aarhus University, Denmark Kristian Linnet University of Copenhagen, Denmark Yvonne Lood The National Board of Forensic Medicine, Sweden Samuel Mesihää University of Helsinki, Finland Gerrit J Middelkoop FHI Christian Reuss Mikkelsen Department of Forensic Medicine, Aarhus University, Denmark Sisse Elle Mikkelsen Department of Forensic Medicine, Aarhus University, Denmark Peter Milland Bruker Daltonics, Denmark Christian Brinch Mollerup University of Copenhagen, Denmark Kirstine Lykke Nielsen Department of Forensic Medicine, Aarhus University, Denmark Louise Stride Nielsen Department of Forensic Medicine, Aarhus University, Denmark Gunnel Nilsson The National Board of Forensic Medicine, Sweden Andrea Carolina Noble University of Copenhagen, Denmark Ilkka Ojanperä University of Helsinki, National Institute Health & Welfare, Finland Mads Lundgren Petersen Agilent Technologies, Sweden Emma Rapp The National Board of Forensic Medicine, Sweden Ilpo Rasanen University of Helsinki, National Institute Health & Welfare, Finland Brian Schou Rasmussen University of Copenhagen, Denmark Nina Ringquist The National Board of Forensic Medicine, Sweden Ingrid Rosendal Department of Forensic Medicine, Aarhus University, Denmark Tim Salbert Advanced Chemistry Development Germany GmbH Mette-Lise Simonsen Department of Forensic Medicine, Aarhus University, Denmark Tina Slots Department of Forensic Medicine, Aarhus University, Denmark Dag Helge Strand Institute of Public Health, Norway Lena Ström The National Board of Forensic Medicine, Sweden Eva Sædder Department of Forensic Medicine, Aarhus University, Denmark Carl Söderberg National Board of Forensic Medicine, Sweden Lauri Saastamoinen Sigma-Aldrich subsidiary of Merck, England Helena Tell The National Board of Forensic Medicine, Sweden Rasmus Telving Department of Forensic Medicine, Aarhus University, Denmark Lone Støvring Rattenborg Department of Forensic Medicine, Aarhus University, Denmark Cecilie Thaulow Institute of Public Health, Norway Gunilla Thelander The National Board of Forensic Medicine, Sweden Svava Thordardottir University of Iceland Inga Dahl Sigma-Aldrich subsidiary of Merck, Denmark Elli Tyrkkö The National Board of Forensic Medicine, Sweden Jenni Viinamäki University of Helsinki, Finland Svante Vikingsson Linköping University, Sweden Denise Wallworth Sigma-Aldrich subsidiary of Merck, England Linda Widar Chiron / CAS Referensmaterial AB, Sweden Ariane Wohlfarth The National Board of Forensic Medicine, Sweden Åse Marit Leere Øiestad Institute of Public Health, Norway
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DEPARTMENT OF FORENSIC MEDICINE
Welcome to Trondheim in 2017!
3rd annual NAFT meeting and general assembly
De Department of Clinical Pharmacology St. Olavs Hospital - Trondheim University Hospital, Norway
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