An analysis of post-mortem toxicology practices in drug-related death cases in Europe : technical report.
Date: 2019-04-29
Institutions: European Monitoring Centre for Drugs and Drug Addiction
Subjects: Europe, drug addiction, mortality, policy analysis, research report, sampling, toxicology

TECHNICAL REPORT
An analysis of post-mortem toxicology 
practices in drug-related death cases  
in Europe

April, 2019

 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Legal notice 

This publication of the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) is 
protected by copyright. The EMCDDA accepts no responsibility or liability for any consequences 
arising from the use of the data contained in this document. The contents of this publication do not 
necessarily reflect the official opinions of the EMCDDA’s partners, any EU Member State or any 
agency or institution of the European Union.  

Luxembourg: Publications Office of the European Union, 2019 

ISBN 978-92-9497-408-2  

doi 10.2810/81554 

TD-01-19-354-EN-N 

© European Monitoring Centre for Drugs and Drug Addiction, 2019  
Reproduction is authorised provided the source is acknowledged.  

Recommended citation:  
European Monitoring Centre for Drugs and Drug Addiction (2019), An analysis of post-mortem 
toxicology practices in drug-related death cases in Europe, Technical report, Publications Office of the 
European Union, Luxembourg. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Contents 

Acknowledgements ..................................................................................................................................... 4 

Executive summary ..................................................................................................................................... 6 

Background and objectives ..................................................................................................................... 6 

Key findings ............................................................................................................................................ 6 

Limitations ............................................................................................................................................... 7 

Conclusions ............................................................................................................................................ 7 

Introduction ................................................................................................................................................. 9 

Part  1:  Scoping  study  on  national  reference  documents  that  address  drug-related  death  toxicology 
investigations in Europe (28 EU Member States plus Norway and Turkey) ................................................. 11 

1.1. Objective and methods ...................................................................................................................... 11 

1.2. Findings ............................................................................................................................................. 12 

1.2.1. Coverage of post-mortem toxicology in national and international guidelines ............................ 12 

1.2.2.  Decision-making  on  the  application  of  post-mortem  forensic  toxicological  investigations  in 
suspected drug-related deaths ............................................................................................................. 16 

1.2.3.  Pre-analytical  management:  sample  collection,  preservatives,  storage  of  post-mortem 
specimens, preparation and extraction ................................................................................................. 16 

1.2.4 Calibration in the case of post-mortem specimens ...................................................................... 18 

1.2.5. Standards for post-mortem screening and confirmatory analytical methods .............................. 18 

1.2.6. Analytical methods for new psychoactive substances in post-mortem specimens ..................... 21 

1.2.7. Interpretation and reporting of findings in forensic toxicology ..................................................... 23 

1.2.8. Certification of drug-related deaths ............................................................................................. 24 

1.3. Discussion .......................................................................................................................................... 25 

Part  2:  Survey  of  practices  —  mapping  the  ‘typical’  or  ‘standard’  toxicology  practices  in  place  in  each 
country .......................................................................................................................................................... 26 

2.1. Objective ........................................................................................................................................... 26 

2.2. Methods ............................................................................................................................................ 26 

2.3. Results .............................................................................................................................................. 27 

2.3.1 Characteristics of participating laboratories ................................................................................. 27 

2.3.2. Collection of test material for toxicological analysis .................................................................... 28 

2.3.3. Laboratory equipment ................................................................................................................. 29 

2.3.4. Analytical strategy ....................................................................................................................... 31 

2.3.5. Analytical strategy: classification ................................................................................................ 33 

2.3.6. Special analytical considerations and reporting .......................................................................... 34 

2.3.7. Substance group coverage ......................................................................................................... 35 

2.3.8. Collaboration/information exchange ........................................................................................... 38 

2.3.9. Coverage of new psychoactive substances ................................................................................ 39 

2.3.10. Suggestions for improvements ................................................................................................. 41 

3. Discussion and conclusions ...................................................................................................................... 43 

3.1 Interpreting drug-related death prevalence data against the background of toxicology standards and 
capacity in European countries ................................................................................................................. 43 

3.2. Conclusions ....................................................................................................................................... 43 

3.2.1. Consequences and priorities for public health ............................................................................ 44 

References .................................................................................................................................................... 46 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Acknowledgements 

Authors: Axel Heinemann and Stefanie Iwersen-Bergmann (Institute for Legal Medicine, University 
Medical Center Hamburg-Eppendorf, Germany) 

The authors, as well as the project coordinator at the European Monitoring Centre for Drugs and Drug 
Addiction, Isabelle Giraudon, would like to extend their sincere thanks and appreciation to the experts 
who participated in the survey, to the drug-related deaths national experts and to the Reitox national 
focal points. We also wish to thank Ana Gallegos, Chara Spiliopoulou and Pirkko Kriikku for peer 
reviewing this report, as well as Nicola Singleton for editing the report and Pamela Nfondja for 
supervising the survey data management. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Tables 

Table 1 Relevant international and national guidance/reference documents 

Table 2 Recently published methods for the analysis of NPS in post-mortem specimens (examples) 

Table 3 Survey response: laboratories per country 

Table 4 Laboratories reporting using specimens from ‘needle autopsies’ 

Table 5 Classification of analytical strategies for screening and confirmation in the event of a 
suspected DRD 

Table 6 ‘What kind of substances does your analytical strategy usually cover?’ (examples) 

Figures 

Figure 1 Laboratory equipment for analytical oriented chromatography and mass spectrometry 
(percentage of laboratories equipped) 

Figure 2 Time of implementation of new techniques/methods (% of laboratories, unknown excluded) 

Figure 3 Substance groups that are at present included in immunological screening at present 
(percentage of laboratories with positive answer) 

Figure 4 Substance groups included in immunological screening in the past 5 years (percentage of 
laboratories with positive answer) 

Figure 5 ‘When questions arise about substances you cannot analyse in your laboratory, do you have 
the opportunity to send samples to a specialised laboratory?’ (%) 

Figure 6 Analytical strategy and capacity for established substance group: coverage in routine versus 
‘on request’ testing (percentage of laboratories) 

Figure 7 Analytical strategy and capacity for some exemplary opioids and other substances: coverage 
in routine versus ‘on request’ testing (percentage of laboratories) 

Figure 8 ‘Routine analytical strategy covers…’: laboratories with (n = 12) versus laboratories without 
(n = 42) routine GUS 

Figure 9 Level of satisfaction regarding the exchange of case-related information between institutions 

Figure 10 Analytical coverage of NPS in EU Member State laboratories 

Figure 11 Analytical coverage of NPS according to laboratory classification in Table 5 (laboratories 
with advanced multi-target methods = Groups 1 and 2; laboratories with conventional methods = 
Groups 3 and 4) (percentage of laboratories with positive answer) 

Boxes 

Box 1 Forensic toxicology definitions 

Box 2 Basic procedures in forensic toxicological analyses 

Box 3 Technological changes in forensic laboratories 

Box 4 Pitfalls in the interpretation of substance concentrations in post-mortem specimens 

Box 5 Limitations of the survey 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Executive summary 

Background and objectives 

In the past decade, between 7 000 and 9 000 drug-related deaths (DRDs) have been reported in 
Europe every year (EMCDDA, 2018). Most are classified as such on the basis of toxicological 
investigations (more than 85 % of overdose deaths in the European Monitoring Centre for Drugs and 
Drug Addiction (EMCDDA) Statistical Bulletin are reported with ‘known toxicology’). However, only 
fragmentary information is given concerning the limitations of the data, such as regional or national 
differences in the analytical capacity of forensic toxicology laboratories. The objective of this report is 
to provide an updated analysis of the post-mortem toxicology practices of DRD cases in Europe and 
to discuss the effect of these practices on the monitoring of DRDs. There were two components to 
this project: a scoping study and a mapping survey. 

The scoping study analyses the international and national guidance relating to the post-mortem 
investigation of suspected DRD cases. Criteria for analysis included minimum requirements, 
recommendations regarding sampling, processing, confirmatory testing, ‘general unknown screening’, 
technical specifications and reporting with special reference to new psychoactive substances (NPS). 

The mapping survey was conducted from May to August 2017, in which 54 forensic toxicology 
laboratories from 27 European Union (EU) Member States, plus Norway, Turkey and Switzerland, 
were asked about their technical equipment, analytical strategies and standards for post-mortem 
investigations, their technical coverage of typical drugs of abuse with special reference to NPS, their 
reporting standards and potential hindrances to their daily work. 

Key findings 

The scoping study found that at the European level, but also at the broader international level, there 
are no specific up-to-date guidelines on forensic toxicology investigations for DRDs, except for single 
substance groups such as fentanyl and its analogues. General forensic toxicology guidelines follow 
international accreditation standards. While these are applicable to post-mortems, which follow the 
same general principles of quality assurance, there are some specific aspects in the case of drug 
poisoning that are not covered, such as the collection of samples other than blood and urine. The 
guidelines are also generally quite limited in their statements on minimum standards. It is also 
important to note that screening for NPS in post-mortem specimens requires up-to-date technical 
equipment, and therefore it is generally limited to specialised laboratories. Non-targeted 
comprehensive/‘general unknown’ screening (GUS) methods are included in current international 
guidelines but are not generally recommended as a minimum requirement in post-mortem 
investigations. The survey demonstrated that targeted screening with second-step confirmation is still 
a relevant approach. The guidelines indicated some differences across countries with regard to the 
practices and, therefore, to the sensitivity of toxicological investigations. This affects the comparability 
of the available data and should be reflected in the analysis and presentation of the data on DRDs. 

The mapping survey studied 54 laboratories in 30 countries (27 EU Member States, Norway, Turkey 
and Switzerland) and found that, in 11 EU Member States and in Turkey, all or the majority of the 
national DRD-associated toxicological investigations are processed centrally in a single laboratory. 
During the past 5-10 years, the majority of laboratories in Europe have changed the most common 
combination of techniques used for the detection of drugs or drug metabolites. Instead of 
immunoassay, followed by confirmation of presumptive-positive specimens by gas chromatography-
mass spectrometry, laboratories are switching to multi-target methods using high-tech equipment, for 
example liquid chromatography-tandem mass spectrometry, high performance liquid chromatography 
time-of-flight mass spectrometry or ultrahigh-performance liquid chromatography high-resolution high-
accuracy mass spectrometry. A change from one method or standard to another will influence trends. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Two thirds (68 %) of participating laboratories are equipped with advanced technology allowing them 
to undertake comprehensive screenings in the case of poisoning deaths. Among those laboratories 
with limited technical equipment for some analytes, 47 % reported being able to send biological 
samples to a specialised laboratory. 

As would be expected, laboratories with advanced technical equipment are able to detect a greater 
range of substance groups. The drugs of abuse most related to deaths, such as opiates, cocaine, 
amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine 
(MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA) and methamphetamine, are able to be 
determined by nearly 100 % of forensic laboratories. However, only around 75 % of the laboratories 
include tests for buprenorphine, fentanyl or the antiepileptic pregabalin in their routine analysis. 
Among the NPS, synthetic cathinones (82 %) and phenethylamines (71 %) were most commonly 
tested for, followed by synthetic cannabinoids and piperazines. In addition to forensic toxicology 
testing, the exchange of case-related information between laboratories and institutions such as the 
police, hospitals and forensic pathologists is necessary for the proper interpretation of findings, and 
one third of the participating laboratories were unsatisfied (‘very unsatisfied’, 21 %, and ‘not satisfied’, 
13 %) with the extent to which this currently occurs. 

Limitations 

The representativeness of the survey results was questionable in some large EU Member States, 
where post-mortem analyses are distributed across many laboratories. The private sector was not 
involved in the study; however, in most countries it is unlikely that many private laboratories handle 
the quite specialised and economically less attractive post-mortem analyses. In addition, the survey 
asked about groups of psychoactive substances, with more specific information obtained for only a 
small number of single substances. 

In general, it was not possible to gain a detailed insight into laboratory processes and to validate 
ambiguous data, and expert panel recommendations and regulatory instructions by responsible 
authorities in national languages were not provided systematically. Finally, the study could not reveal 
distinct timelines for the past 20 years in terms of the development and implementation of new 
methods and new devices for individual laboratories. 

Conclusions 

At the interface between national regulations for inquests into cause-of-death investigations and 
guidelines for toxicological examinations, it would be beneficial to have specific medico-legal 
recommendations for decision-making on ordering toxicological examinations following autopsies. 
They should include guidance for cases in which findings at autopsy suggest any ambiguity over 
cause of death. 

There remains an urgent need to increase the screening capabilities of many toxicology laboratories. 
In countries with a decentralised organisation of forensic laboratories, establishing national reference 
laboratories to determine certain NPS groups in biological samples could be a strategic option. 
National or international guidelines on analytical laboratory standards need to be updated to take 
account of new developments in multi-targeted toxicological analysis. 

Although rarely investigated, the capacity of an analytical laboratory is an important factor to consider 
when interpreting the role of different substances in DRDs. Laboratory capacity has gained even more 
importance given the rise in NPS and the high prevalence of fatal polydrug poisonings.  

Importantly, over time, with the technical developments in terms of laboratory capacity, cases that 
would have been missed before can be identified. This improvement in detection should be kept in 
mind and documented when analysing data on direct or indirect DRDs associated with certain 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

substance groups, such as prescription opioids and NPS. Indeed, some of the observed increase 
might reflect the improved detection of cases that would have otherwise remained unnoticed. National 
longitudinal analyses could be used for a comparative analysis between countries with similar 
incidences of newly occurring substances. Such comparisons are particularly promising in multi-city 
studies or in comparisons between smaller countries with clearly assigned regional responsibilities for 
forensic laboratories. Analysis of DRDs will be enhanced by including toxicological laboratories — 
where this is not yet the case — more sustainably in information networks, for example through the 
Reitox national focal points. 

The technical capacity of toxicological laboratories has already been adjusted to meet the new 
requirements in many countries. However, new analytical methods inevitably lag behind the first 
appearance of an NPS. The exchange of mass spectrometry libraries of new analytes and of 
reference standards between institutions is promoted by the European information system and the 
European Database on New Drugs (EDND). There is an urgent need to promote the use of existing 
databases (such as the EDND) and accelerate the exchange of information between specialised 
national (reference) laboratories on NPS in order to increase the speed of analytical development. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Introduction 

High rates of drug-related deaths (DRDs) are a key area of concern for drug policy across Europe. 
However, monitoring these deaths, identifying actions to reduce them and evaluating their impact are 
hampered by concerns about the reliability and comparability of data on DRDs. For example, diversity 
in the national country-level structures and processes are likely to have an impact on the 
comparability of DRD data across Europe. On the one hand, the number of registered DRDs depends 
on the prevalence of actual overdose deaths, which is influenced by different regional, national and 
international triggers, such as the number of problem drug users and diverse risk factors for those 
who are at risk. On the other hand, changes in the detection rate of actual deaths can influence the 
number of reported DRDs. This report investigates the variability in guidance, standards and practice 
in post-mortem toxicological investigation of DRDs across the European Union (EU) in order to 
provide insights into the extent to which these may influence reported trends. This will help those 
tasked with interpreting the data to inform policymaking and those tasked with improving the 
structures and processes in place for the investigation, recording of and reporting of DRDs. 

The recognition of a death as drug related depends on the organisation of the post-mortem 
examination at the scene of death, which varies across countries and can — depending on 
competence — be considerably influenced by legal regulations and decisions by the police as well as 
by the judiciary or the health sector. A detailed investigation of a fatality may produce different results 
depending on the technical resources used. These resources relate to the methods and procedures 
for obtaining findings from the corpse by autopsy or, more recently, imaging techniques for obtaining 
information on items or samples collected at a scene of death but also for including information from 
the social environment, relatives or treating physicians. 

The quality of the investigation of a suspected death, however, is greatly affected by the quality of 
toxicological investigations of biological materials. It is known that, in the case of suspected DRDs, 
the choice of toxicological investigations is not obvious. Even if an autopsy is performed and the 
macroscopic and histological results do not definitively clarify the cause of death, it is often the case 
that a separate decision needs to be made by the contracting authority about whether or not a 
toxicological examination should take place. Even if toxicological analyses are ordered, differences in 
the analytical strategy between laboratories can lead to different results owing to variation in the 
substances tested for, as well as to different limits of detection (LODs). The technical equipment of a 
laboratory, the competence of the personnel, the financial resources for the maintenance and quality 
assurance of the results, and the development of methods for the analysis of new psychoactive 
substances are all important influencing factors on toxicology results. 

Such differences may influence statistics on the number of registered DRDs and may lead to 
differences between countries in the ability to detect new causes of poisoning deaths. Last but not 
least, a toxicological analysis can contribute to the epidemiologically reported prevalence only if it is 
integrated into the results of the post-mortem examination. The ultimate cause of death then needs to 
be reported in a timely manner to the General Mortality Registries or Special Registers. Finally, the 
rules for the interpretation of toxicological results and their coding should be consistent, and they 
should conform to the relevant national and European (EMCDDA) definition of a DRD. 

The objective of harmonising the registration of DRDs in EU Member States has been accompanied 
in the past 20 years by numerous analyses of the various factors influencing the process of 
registering cases at national level. However, relatively little is known about the extent of regional or 
national differences in the performance of toxicological analyses. Those differences did not appear to 
be a relevant factor for established drugs of misuse, such as heroin or ‘established’ opiates. However, 
the issue remains because of the increased prevalence of use of some prescription opioids and the 
analytical challenges relating to the constantly evolving NPS on the market, some of which may 
contribute directly or indirectly to DRDs. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

The first section of this report describes a scoping study (Part 1), which analyses the international and 
national guidance with regard to toxicological post-mortem investigations of suspected DRD cases. 
This includes minimum requirements, recommendations by national expert associations regarding 
sampling, processing, confirmatory testing, general unknown screening (GUS), technical 
specifications and reporting with special reference to NPS. 

In the second section, the findings of a mapping survey of the typical or standard toxicology practices 
in place in each EU Member State and in Norway and Turkey are presented (Part 2). Experts on 
toxicological analysis based in laboratories undertaking post-mortem analyses were identified and 
asked to participate in a survey on laboratory performance in suspected DRD cases, guidelines and 
standards for laboratory practice and reporting, analytical strategies, technical equipment, and 
potential hindrances/challenges to their daily work on DRD cases. 

Finally, based on these results, some general conclusions are drawn and the potential implications 
are discussed with regard to how to interpret drug-induced deaths prevalence data, taking into 
consideration the background of toxicology standards and capacities in different countries. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Part 1: Scoping study on national reference documents that 
address drug-related death toxicology investigations in Europe 
(28 EU Member States plus Norway and Turkey) 

1.1. Objective and methods 

The scoping study analyses the international and national guidance on the post-mortem investigation 
of suspected DRD cases. It includes guidelines published by international and national professional 
associations or national toxicologist working groups, as well as documents published by national 
authorities or international organisations regarding scientific or expert recommendations. The 
evaluation of these documents took account of decision-making about post-mortem forensic 
toxicological investigations, minimum requirements regarding the sampling and storage of post-
mortem specimens, the processing of samples, technical equipment, standards for screening and 
confirmatory testing with special reference to NPS, interpretation and reporting (prioritising polydrug 
poisonings). Implications for the monitoring of DRDs at a European level are briefly discussed and 
areas for improvement are suggested. 

Search strategies for relevant documents were carried out between April and August 2017 and 
included: 

  Sources: PubMed, TOXNET, Web of Science 
  Searching mode with the following filters: 

o 

o 

o 

forensic 

toxicology’[MeSH  Terms]  OR 

(‘forensic 
toxicology[Text  Word])  AND 
((‘autopsy’[MeSH  Terms]  OR  ‘autopsy’[All  Fields]  OR  ‘post-mortem’[All  Fields])  OR 
post-mortem[All Fields]) AND (‘standards’[Subheading] OR ‘standards’[All Fields] OR 
‘reference  standards’[MeSH  Terms]  OR  (‘reference’[All  Fields]  AND  ‘standards’[All 
Fields]) OR ‘reference standards’[All Fields]) AND drug[All Fields] 
(‘forensic 
standards AND drug OR ‘drugs of abuse’ AND death OR poisoning 
(‘forensic 
standards AND drugs OR drugs of abuse AND death OR poisoning 

toxicology’[MeSH  Terms]  OR 

toxicology’[MeSH  Terms]  OR 

toxicology[Text  Word])  AND 

toxicology[Text  Word])  AND 

forensic 

forensic 

  A Google browser-based search: ‘Forensic toxicology’ AND (guidelines OR standards) AND 
drugs  OR  ‘drugs  of  abuse’  OR  ‘illegal  drugs’  OR  ‘controlled  substances’  OR  ‘new 
psychoactive substances’ OR ‘legal highs’ OR drug- related AND death OR poisoning 

  A targeted document search on the following websites: International Association of Forensic 
Toxicologist  (TIAFT),  United  Nations  Office  on  Drug  and  Crime  (UNODC),  American 
Association  of  Forensic  Toxicologist  (AAFS),  Scientific  Working  Group  for  Forensic 
Toxicology  (SWGFTOX),  Nordic  Association  of  Forensic  Toxicologists,  Society  of  Forensic 
Toxicologists (SOFT), US Food and Drug Administration (FDA), European Medicines Agency 
and International Union of Pure and Applied Chemistry 

  Personal communication with relevant experts taking part in the survey (Part 2). 

The following components were explored: 

sample preparation; 
calibration; 
standards for post-mortem screening and confirmatory analytical methods; 

  decision-making on post-mortem forensic toxicology investigations; 
  pre-analytical management; 
 
 
 
  analytical methods for NPS; 
 
 

interpretation and reporting of findings; 
toxicological reporting and certification. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

1.2. Findings 

1.2.1. Coverage of post-mortem toxicology in national and international guidelines 

Box 1 Forensic toxicology definitions 

Toxicology involves the analysis of how chemical substances affect living organisms. The most common 
applications are workplace drug testing, doping control in sport and human performance toxicology (detecting the 
presence or absence of drugs or alcohol in the human body, which may be necessary in impaired driving, road 
traffic accident and sexual assault cases). 

Forensic toxicology is concerned with cases in which adverse effects of chemical substances could have 
administrative or medico-legal consequences. Standards for toxicological DRD investigation fall into the field of 
post-mortem forensic toxicology (death investigation toxicology), defined as a methodology for the determination 
of drugs and their metabolites, chemicals such as ethanol and other volatile substances, carbon monoxide and 
other gases, metals and other toxic chemicals in human fluids and tissues, and for evaluating their role as a 
determinant or contributory factor in the cause and manner of death. 

The toxicology report provides key information to a pathologist, who considers it in the context of the findings of 
medical conditions at autopsy and the investigative history of a case. 

Toxicology results from biological samples are examined in many countries by medico-legal institutions. Seized 
drugs are typically analysed in police laboratories. 

In line with international accreditation standard ISO/IEC 17025 (General requirements for the 
competence of testing and calibration laboratories), there are a number of guidance documents that 
represent all fields of forensic toxicology. They include requirements for laboratory staff 
responsibilities, running of a quality management system, method calibration and validation, 
specimen collection, labelling and handling including security, and chain-of-custody of specimens. 
External proficiency programmes monitor both the assay and the staff performing the work (United 
Nations International Drug Control Programme, 1995, 1997, 1999; SOFT/AAFS, 2006; GTFCh, 2009; 
Cooper et al., 2010; Drummer, 2010). 

These guidelines are fully applicable to post-mortem investigations, which follow the same general 
principles of quality assurance and methods and involve validation according to standard parameters: 
selectivity, calibration model, stability, accuracy, precision, lower limits of quantification (LOQs), 
LODs, recovery, reproducibility and robustness (Peters et al., 2007; Scientific Working Group for 
Forensic Toxicology, 2013). 

Specimen collection at autopsy, the potential impact of post-mortem matrix-related effects on 
standard analytical methods and the interpretation of recommendations are examples of issues 
relating to post-mortem investigations that need to be addressed specifically in some documents. 
With regard to analytical methods, guidelines addressing systematic toxicological analysis (STA) 
involving GUS are particularly relevant to post-mortem toxicology (where the confirmation or exclusion 
of expected substances is of minor importance) and have increased in importance as markets for 
increasingly diverse NPS are steadily growing (EMCDDA and Europol, 2016; Guillou, 2017). 

In total, 17 references were identified, from 8 countries and 6 international institutions, dating from 
1995 to 2017 (Table 1). There were specific recommendations for post-mortem toxicology (and, 
therefore, indirectly applicable to DRDs) in 12 out of 17 references identified. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

TABLE 1 
Relevant international and national guidance/reference documents 

Author/editor 

Title (reference) 

No  Country/orga
nisation 
(alphabetical 
order) 

1  Czechia 

Czech Society for 
Legal Medicine and 
Forensic Toxicology 

(in Czech) Metodický 
pokyn pro postup p i 
toxikologickém vyšet 
ení speci kovaných 
návykových látek v krvi 
a nebo v mo i. 
[Methodical guidance 
for the process of 
toxicological testing of 
specified substances of 
the substance or in the 
blood] 
Czech Society for Legal 
Medicine and Forensic 
Toxicology, expert 
personal 
communication 2012) 
Recommandations 
pour la réalisation des 
analyses toxicologiques 
dans les cas de décès 
impliquant des NPS 
(SFTA, 2017) 
(in German) Anhang D 
zur Richtlinie der 
GTFCh zur 
Qualitätssicherung bei 
forensisch-
toxikologischen 
Untersuchungen 
Empfehlungen zur 
Asservierung von 
Obduktionsmaterial für 
forensisch-
toxikologische 
Untersuchungen und 
spezielle Aspekte der 
Post-mortem-Analytik 
(attachment to 2) 
(GTFCh, 2004)  
(in German) Richtlinie 
der GTFCh zur 
Qualitätssicherung bei 
forensisch-
toxikologischen 
Untersuchungen 
(GTFCh, 2009)  

(in German) Anhang A 
zur Richtlinie der 
GTFCh zur 
Qualitätssicherung bei 
forensisch-
toxikologischen 
Untersuchungen 
Qualitätsanforderungen 
an die Bestimmung 
spezieller Analyten aus 
biologischen Matrices 

Last 
edition 

Scope: post-mortem 
applications (PA)/general 
recommendations (GR) 

2012 

GR: analysis, interpretation, 
quality assurance, 
documentation 

2017 

PA: sample collection, 
analysis, minimum list of 
NPS to be included 

Version 
1 2004 

PA: sample collection 

GR: container type, labelling, 
logging, shipping, storage 
(no specific DRD 
recommendations) 

Version 
1 2009 

PA: analysis (standard 
addition method) 

Version 
1 2009 

GR: analysis, quality 
assurance and control, 
reporting, interpretation (no 
specific DRD 
recommendations) 
GR: analysis of 
amphetamines, 
methamphetamines, 
methylendioxyamphetamines
, cocaine, benzoylecgonine, 
opiates/opioids. 

LOD, LOQ for drugs of 
abuse (no specific DRD 
recommendations) 

13 

2 

France 

Société Française 
de Toxicologie 
Analytique (SFTA) 

3  Germany 

Gesellschaft für 
Toxikologische und 
Forensische 
Chemie Arbeitskreis 
Qualitätssicherung 
(GTFCh) [Society 
for Toxicological 
and Forensic 
Chemistry Working 
Group Quality 
Assurance]  

4  Germany 

GTFCh 

5  Germany 

GTFCh 

 
 
 
 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

No  Country/orga
nisation 
(alphabetical 
order) 

Author/editor 

Title (reference) 

Last 
edition 

Scope: post-mortem 
applications (PA)/general 
recommendations (GR) 

mit Tabellenanhang 
(aktuelle Vorgaben zu 
Bestimmungsgrenzen) 
(attachment to 2) 
(GTFCh, 2009) 
(in Polish) Zalecenia w 
sprawie pobierania 
materialu sekcyjnego 
do badań 
toksykologcznych 
[On the collection of 
autopsy material for 
toxicological 
investigations] 
(Polish Society of 
Forensic Medicine and 
Criminology, 2017) 
Specific 
recommendations for 
the unification of 
judicial autopsies 
(Institut de Medicina 
Legal de Catalunya, 
2013)  
Forensic toxicology 
laboratory guidelines 
(Cooper et al., 2010) 

6 

Poland 

Polish Society of 
Forensic Medicine 
and Criminology 

7 

Spain 

8  United 

Kingdom 

Generalitat de 
Catalunya/Departa
ment de 
Justícia/Institut de 
Medicina Legal de 
Catalunya 

UK and Ireland 
Association of 
Forensic 
Toxicologists 

2012 

PA: sample collection 

2013 

PA: sample 
collection/recommended 
amounts 

2010 

Guidelines for drugs of 
abuse testing 
(SCDAT/AGSA, 2012) 

2012 

9 

Switzerland 

Work Group on 
Drugs of Abuse 
Testing 
(SCDAT/AGSA) 
Swiss Association 
of Pharmacists 
(pharmaSuisse) 
• Swiss Society of 
Clinical Chemistry 
• Swiss Society of 
Legal Medicine 
• Swiss Association 
of the Diagnostic 
Equipment and 
Product Industry 
• University of Bern 

PA: sample 
collection/recommended 
amounts, post-mortem and 
sample changes; external 
proficiency testing 
programme; analysis of 
alcohol, review of data 
before reporting, 

GR: sample collection, 
container type, labelling, 
chain-of-custody, logging, 
transport, storage, analysis, 
quality assurance and 
control, reporting, 
interpretation 
PA: sampling/specimen to be 
taken; technical 
recommendations for 
quantitative analysis of 
substance groups 

GR: analysis, interpretation, 
quality assurance, 
documentation ((no specific 
DRD recommendations) 

14 

 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Author/editor 

Title (reference) 

No  Country/orga
nisation 
(alphabetical 
order) 

10  USA 

11  Council of 

Europe 

Laboratory 
Guidelines 
Committee of the 
Society of Forensic 
Toxicologists 
(SOFT) and the 
Toxicology Section 
of the American 
Academy of 
Forensic Sciences 
(AAFS) 
Committee of 
Ministers to 
Member States  

12 

13 

International 
Association of 
Forensic 
Toxicologists 

TIAFT Committee of 
Systematic 
Toxicological 
Analysis 

International 
Association of 
Forensic 
Toxicologists 

Scientific Working 
Group for Forensic 
Toxicology 
(SWGTOX) 

14  United Nations 

International 
Drug Control 
Programme 
(UNDCP) 

UNDCP Scientific 
Section 

15  UNDCP 

UNDCP Scientific 
Section 

16  UNDCP 

UNDCP Scientific 
Section 

17  UNODC 

UNODC 

Forensic toxicology 
laboratory guidelines 
(SOFT/AAFS, 2006) 

Recommendation no 
R (99) 3 on the 
harmonization of 
medico-legal autopsy 
rules 
(Committee of Ministers 
to Member States, 
2000) 
Recommendations on 
sample preparation of 
biological specimens 
for systematic 
toxicological analysis 
(Stimpfl et al., 2011) 
Standard practices for 
method validation in 
forensic toxicology 
(Scientific Working 
Group for Forensic 
Toxicology, 2013) 
Recommended 
methods for detection 
and assay of heroin, 
cannabinoids, cocaine, 
amphetamines, 
methamphetamine and 
ring-substituted 
amphetamines in 
biological specimen 
(UNDCP, 1995) 
Recommended 
methods for detection 
and assay of 
barbiturates and 
benzodiazepines in 
biological specimen 
(UNDCP, 1997) 
Recommended 
methods for detection 
and assay of lysergide, 
phencyclidine, 
psilocybin, 
methaqualon in 
biological specimen 
(UNDCP, 1999) 
Recommended 
methods for the 
identification and 
analysis of fentanyl and 
its analogues in 
biological specimens 

Last 
edition 

Scope: post-mortem 
applications (PA)/general 
recommendations (GR) 

2006 

PA: sample collection 

GR: sample collection, 
container type, labelling, 
chain-of-custody, logging, 
transport, storage, analysis, 
quality assurance and 
control, reporting, 
interpretation 

1999 

PA: sample collection 

2011 

PA: sample collection 

GR: sample pre-analytic 
treatment, sample extraction 
methods 

2013 

GR: method validation 

1995 

1997 

1999 

2017 

GR: sample collection, 
container type, labelling, 
chain-of-custody, logging, 
transport, storage, analysis, 
quality assurance and 
control, reporting, 
interpretation 

GR: sample collection, 
container type, labelling, 
chain-of-custody, logging, 
transport, storage, analysis, 
quality assurance and 
control, reporting, 
interpretation 
GR: sample collection, 
container type, labelling, 
chain-of-custody, logging, 
transport, storage, analysis, 
quality assurance and 
control, reporting, 
interpretation 

Specific to fentanyls: PA: 
sample collection, post-
mortem and sample 
changes, interpretation 

GR: sample collection, 

15 

 
 
 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

No  Country/orga
nisation 
(alphabetical 
order) 

Author/editor 

Title (reference) 

Last 
edition 

Scope: post-mortem 
applications (PA)/general 
recommendations (GR) 

(UNODC, 2017) 

stabiliser, storage, screening 
and confirmation methods 

1.2.2. Decision-making on the application of post-mortem forensic toxicological investigations 
in suspected drug-related deaths 

There are no generally accepted principles for decision-making regarding the ordering of toxicological 
examinations after autopsies in EU countries. Such guidelines could be used at the interface of the 
jurisdiction of the police, legal medicine and prosecutor. Recent US recommendations specify 
inclusion criteria for the toxicological analysis for controlled substances if one or more of the following 
apply to a case (Davis, 2014): 

 
known history of prescription opioid or illicit drug use, misuse or abuse; 
  evidence of opioid or illicit drug abuse revealed by scene investigation; 
  autopsy findings suggesting a history of illicit drug abuse; 
  massive lung oedema and froth in airways present with no grossly visible explanation 
(e.g. heart disease) or other non-toxicological explanation (e.g. epileptic seizure) 
(Dinis-Oliveira et al., 2012); 

  potential or suspected smugglers of illicit drugs (mules); 
  no unequivocal cause for death identified at autopsy; 
  decedents with a potential natural cause of death visible at autopsy whenever a drug 
may have precipitated or contributed to death by an additive mechanism, such as 
opioid-induced respiratory depression; 
traumatic deaths. 

 

There are currently no comparable specific recommendations from EU Member States available. 
Guidelines for toxicological investigations have their first point of application in principle during the first 
pre-analytical measures, but they do not refer to whether case-related toxicological investigations 
should be initiated at all. The decision here depends on the recommendation of forensic pathologists 
after autopsy but apparently follows a general algorithm for the clarification of the cause of death 
rather than DRD-specific considerations. 

1.2.3. Pre-analytical management: sample collection, preservatives, storage of post-mortem 
specimens, preparation and extraction 

Box 2 describes the basic procedures in forensic toxicological analyses. These have an impact on the 
ability of these analyses to contribute to the identification of DRDs and include pre-analytical 
management as well as the analytical procedures themselves. In general, urine and blood are the 
most frequently used liquid specimens in post-mortem analysis. Urine is less sensitive in the event of 
rapid death after drug use (Stimpfl et al., 2011). 

Most guidelines do not make specific recommendations as regards the types of specimen to be 
collected in DRD cases. The general SOFT/AAFS (2006) guidelines suggested heart blood, 
peripheral blood, bile, urine, gastric contents, liver, kidney and brain for routine sample collection 
without prioritisation. German guidelines recommend peripheral and cardiac blood, stomach contents 
and urine as basic specimens, supplemented by hair, bile, liver, lung, brain and kidney in all cases of 
unclear cause of death at autopsy. UK/Irish guidelines suggest, in accordance with TIAFT guidelines 
for post-mortem collection in general, the routine sampling of peripheral blood and urine, and the 
sampling of ‘heart blood, peripheral blood, bile, urine, gastric contents, liver, kidney, brain and hair’ 
only after consultation with the laboratory. Lung and intestine specimens may be needed for unique 

16 

 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

poisons (not specified) (Cooper et al., 2010, Stimpfl et al., 2011). Recent US recommendations 
specify blood, urine and vitreous humour as minimum standard autopsy specimens for toxicological 
analysis in suspected opiate-related deaths (Davis, 2014).There are no DRD specifications regarding 
collection in the event of severe decomposition or skeletisation. 

Specifically in relation to DRDs, the European Council guidelines from 1999 (published in 2000) 
recommended that vitreous humour, brain tissue, injection marks and hair should be collected in 
addition to a basic standard sampling (Committee of Ministers to Member States, 2000). Deaths 
relating to opioids should include the sampling of blood, vitreous humour, urine, bile and gastric 
contents (Davis, 2014). Other tissue samples considered but not included in general 
recommendations are brain stem/cerebellum segments in relation to opiate-/opioid- and cocaine-
related deaths, which may reflect drug concentrations at their site of action (Stimpfl and Reichel, 
2007). Pericardial fluid was also proposed for cocaine-related deaths (Contreras et al., 2006; 
Contreras et al., 2007) but has not come into widespread use. 

With regard to the quantities of each specimen to be collected, guidelines specify quite consistently 
10 ml as the minimum amount for peripheral blood and whatever amounts are available for urine and 
vitreous humour (Cooper et al., 2010; Institut de Medicina Legal de Catalunya, 2013). Blood from the 
femoral vein is preferred over blood from other sites such as the subclavian vein, right atrium or any 
intact blood vessel (Dinis-Oliveira et al., 2010, Polish Society of Forensic Medicine and Criminology, 
2012; Davis, 2014). Peripheral blood should be collected in two different tubes with no air pockets and 
at least one of them should include sodium fluoride as a preservative (GTFCh, 2009) and potassium 
oxalate as an anticoagulant. Bile is recommended for its significance as an alternative route of 
elimination (Institut de Medicina Legal de Catalunya, 2013). Hair should be collected from the head or 
the armpit or pubic areas if this is not possible, primarily for storage and analysis in the event of 
positive blood and urine/bile results (Institut de Medicina Legal de Catalunya, 2013). 

Box 2 Basic procedures in forensic toxicological analyses 

Sample collection: urine and blood are the most frequently used liquid specimens from the human body but the 
condition of a corpse may require the collection of different liquids or tissues. 

Storage conditions: post-mortem metabolism needs to be controlled if possible, otherwise the active metabolites 
of drugs may be lost. 

Sample preparation: conventional approaches require the separation of a substance from its accompanying 
matrix (1). As there is no single extraction procedure for STA covering all relevant substances, complementary 
techniques have to be combined (Stimpfl et al., 2011). A full ‘clean-up’ of a sample is impossible; components 
from a sample matrix will be co-extracted. However, in laboratories with the latest technology (see Box 3), 
substances are identified through their elemental composition, so matrix components can be identified. 

Method calibration: the comparison of measurement values delivered by a device under test with those of a 
calibration standard of known accuracy. An adequate matrix for calibration is a challenge in post-mortem 
toxicology. If analyses are performed on unusual specimens (decomposed tissue, vitreous humour, etc.), 
appropriate matrix-matched calibrators should, when possible, be prepared and tested concurrently with the 
specimen (SOFT/AAFS, 2006). 

Accuracy, validity and reliability: this needs to be proved for each method before samples are measured in order 
to minimise any uncertainties in the measurement. 

(1) In chemical analysis, ‘matrix’ refers to the components of a sample other than the analyte of interest. The matrix can have a 
considerable effect on the way the analysis is conducted and the quality of the results obtained; such effects are called matrix 
effects. 

Blood sampling before opening the body minimises the risk of sample contamination. The site, time 
and date of blood sampling should always be recorded (SOFT/AAFS, 2006; Flanagan, 2012-2013). In 

17 

 
 
 
 
 
 
 
 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

the case of prolonged survival, blood samples from the date of hospital admission should be secured 
as soon as possible (GTFCh, 2009). Short-term storage conditions for specimens and the use of 
anticoagulants/preservatives for the stabilisation of liquids against in vitro metabolism must be 
defined. 

Recommendations for sample pre-treatment specify the following: sample hydrolysis, precipitation in 
the case of high protein contents and homogenisation (necessary for nearly all post-mortem 
specimens), followed by extraction (liquid-liquid extraction or solid-phase extraction), purification, 
concentration and derivatisation (if gas chromatography mass spectrometry (GC-MS) is applied) 
(UNDCP, 1995; Stimpfl et al., 2011). 

1.2.4 Calibration in the case of post-mortem specimens 

There are major challenges regarding the analysis of post-mortem specimens compared with the 
routine analysis of body fluids in living humans (e.g. linked to matrix effects) (Staeheli et al., 2015). 

In post-mortem analyses, a directly comparable reference matrix is often not available, so validation 
of these parameters needs individual solutions. In such cases, guidelines (SOFT/AAFS, 2006; 
GTFCh, 2009) recommend the standard addition method (SAM), which allows for semi-quantitative 
determination, in which the calibration function is generated directly in the sample matrix. This 
approach takes into consideration the matrix properties of that specific case (including post-mortem 
changes). In this method, the sample to be examined is processed and measured in a completely 
identical manner, first unchanged and then again after the addition of defined amounts of the active 
compound to be determined. The concentration of the compound to be added should correspond to 
the highest expected sample concentration. If the sample quantity is sufficient, several different 
concentrations should be added. The original analyte concentration in the sample can then be 
deduced by linear regression. 

1.2.5. Standards for post-mortem screening and confirmatory analytical methods 

Before 2010 (but still standard practice in many laboratories), analytical techniques relied heavily on 
immunoassay screening analyses and mass spectrometry (MS) for confirmatory analyses using either 
high-performance liquid chromatography or gas chromatography as the separation technique 
(SOFT/AAFS, 2006; Contreras et al., 2006). A Substance Abuse and Mental Health Services 
Administration guidelines consensus panel on ‘Uniform standards and case definitions for classifying 
opioid-related deaths’ noted in 2013 that ‘the most common combination of techniques used for the 
detection of drugs or drug metabolites in urine is immunoassay, followed by confirmation of 
presumptive-positive specimens by gas chromatography-mass spectrometry’(Goldberger et al., 
2013). 

Essentially, there are no major differences between post-mortem forensic toxicology (including DRD 
analysis) and other applications (e.g. human performance forensic toxicology or forensic drug testing 
in live humans) with regard to the quality assurance of analytical strategies and quality control (e.g. 
acceptable deviations of control results, use of deuterated internal standards for MS, ionisation 
methods, interpretation of mass spectra using a minimum number of qualifying ions for each analyte 
in MS). 

18 

 
 
 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Box 3 Technological changes in forensic laboratories 

Available equipment and techniques 

Over the past 10-15 years, conventional techniques such as GC-MS have been supplemented by more 
advanced technology. GC-MS has been a widely used methodology for limited GUS but the application 
is restricted to substances suitable for gas chromatography and electron or chemical ionisation, where 
appropriate after derivatisation (Meyer et al., 2010). 

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) expands significantly the coverage of 
substances, exploiting the polarity and low volatility of many new substances and metabolites. Liquid 
chromatography allows more substances to be analysed in even lower concentrations on liquid rather 
than gaseous carrier-facilitated separation. It relies on pumps to pass a pressurised liquid solvent 
containing the sample mixture through a column filled with a solid adsorbent material. 

High-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) allows 
determination of substances directly through accurate mass. It has become a method of choice for a 
comprehensive screening. 

Ultrahigh-performance liquid chromatography (UHPLC), combined with TOF-MS allows additional selectivity, 
sensitivity and speed resulting from increased chromatographic resolution. Both molecular weight information 
and structural details of unknown analytes are gained by high-resolution high-accuracy mass spectrometry 
(HRMS) enabling accurate-mass determination of ionic species obtained from drugs and their metabolites. 
HRMS and Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) allow measurements at 
extraordinarily high resolution. 

Accurate-mass databases and spectral libraries are used for peak identification, which contain accurate 
mass collision-induced dissociation spectra of more than 2 500, and theoretically calculated accurate 
mass data of several thousands of toxicologically relevant substances (Maurer et al., 2016; Noble et al., 
2018). Assisted by these techniques, forensic toxicologists can keep up with the detection and 
identification of both traditional drugs of abuse as well as NPS. 

Types of screening: comprehensive screening versus immunological screening 

Comprehensive screening means that substances are searched for in a non-targeted manner (GUS). It 
allows for wide-scope screening of many parent substances, metabolites and transformation products 
with an acquisition of accurate-mass full spectrum data. 

By contrast, the established pre-test strategy of immunological screening is always limited to certain 
groups of substances, so it will selectively test for what is expected in the sample. 

Using technologies such as HPLC-TOF-MS, screening (formerly a preliminary indication of a substance) 
and confirmation analysis (for secure identification) partially merge together. Methods for preliminary 
compound identification by liquid chromatography quadrupole time-of-flight mass spectrometry have 
been proposed when reference standards are unavailable (Tyrkkö et al., 2010; Mollerup et al., 2017). 

Quantification 

For quantification, however, reference samples are required for ‘established’ as well as upcoming new 
substances, regardless of the choice of analytical technique. LOD (the lowest quantity of a substance 
that can be detected) and LOQ (the lowest concentration that can be quantified) vary considerably 
depending on various factors, such as the performance of the chosen chromatographic technique and 
mass resolution. 

When immunological and UHPLC-HR-TOF-MS-based screenings have been compared, the latter 
produce a lower number of false-positive results for the main drug groups (such as cannabinoids, 
cocaine, opiates, amphetamines) than occur with immunoassay. Many false-negative immunoassay 
results are a result of higher cut-off concentrations and interference from the matrix, which impede the 
detection of NPS and prescription drugs (Sundström et al., 2015). 

Different extraction and analytical methods are used to monitor different drug classes of interest in 
biosamples. There is consensus that not all controlled drugs or toxins can be routinely screened or 

19 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

tested for in death investigations, and so a negative result does not necessarily exclude a particular 
substance (Byard and Butzbach, 2012). This is a concern particularly when targeted screening is 
relied on, as these methods would probably fail to detect unknown compounds such as NPS. 
However, targeted screening is not an outdated concept. Non-targeted GUS methods for arrays of 
substances are included in current international guidelines (SOFT/AAFS, 2006; GTFCh, 2009) but 
they are far from being generally recommended as a minimum requirement in post-mortem 
investigations. 

Screening methods 
Non-instrumental screening tests (e.g. the analysis of urine at autopsy by test strips) should be 
regarded as a rough indication for the decision to carry out a further toxicological investigation but 
should never justify classification of a death as drug related (SCDAT/AGSA, 2012), nor are 
instrument-based screening tests alone adequate for establishing a cause of death in clinical and 
forensic toxicology (Ceelen et al., 2011; Davis, 2014). 

Screening tests must be appropriate and validated for the type of biological specimens being 
analysed. They may be directed towards a class of drugs, such as opiates, or may be a broad-based 
screen, such as GC-MS (SOFT/AAFS, 2006) and LC-MS (Cooper et al., 2010). If a reported cut-off 
point is used (the threshold below which a result is referred to as negative), the precision of the assay 
around that cut-off point must be demonstrated (SOFT/AAFS, 2006). 

ln line with these recommendations, the UK guidelines state that in most instances in which a 
laboratory is asked to look for drugs in biological specimens, screening tests are employed. More 
recently available techniques that may be available in many laboratories (see above) are not 
mentioned in the guidelines. 

Confirmation methods 
It is recommended that the identity of an analyte is confirmed using a different extract of the same 
specimen as that used for the first test or a second specimen (SOFT/AAFS, 2006). In general, the 
presence of a substance should be verified in more than one specimen if possible (SOFT/AAFS, 
2006). 

Use of an immunoassay system based on another screening method to confirm a previous 
immunoassay is not regarded as acceptable (GTFCh, 2009; Cooper et al., 2010). In addition, the 
detection of an analyte by immunoassay and ‘confirmation’ by gas chromatography with 
propionylation at the N position or gas chromatography FID (flame ionisation detector) does not 
provide sufficient specificity in forensic toxicology (SOFT/AAFS, 2006; Cooper et al., 2010) and is not 
recommended. 

Before about 2010, some guidelines classified (referring to basic equipment for all areas of forensic 
toxicology) flame ionisation nitrogen-phosphorus detectors for gas chromatography and diode-array 
detection (DAD)/ultraviolet/fluorescence detectors for liquid chromatography as alternatives to 
detection by MS (UNDCP, 1995; GTFCh, 2009). Others recommend MS for confirmatory analysis, 
‘where possible and practical’ (Cooper et al., 2010). In recent guidelines, GC-MS for volatile and heat-
stable compounds, as well as HPLC-MS/LC-MS for non-volatile and heat-labile compounds, are 
recommended methods for the confirmatory quantitative determination of DRD-related substance 
groups (opiates/opioids, cocaine/metabolites, methadone/metabolites, amphetamines/metabolites, 
benzodiazepines and ‘z-drugs’, barbiturates (HPLC-DAD is optional for the last four of these) 
(SCDAT/AGSA, 2012). 

Specific analytical recommendations for opioids 
In the United States, a National Association of Medical Examiners position paper from 2014 specifies 
recommendations for the investigation, diagnosis and certification of deaths related to opioid drugs: a 
toxicological panel should be comprehensive and include 14 defined opioids, as well as 

20 

 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

benzodiazepines and other potent depressant, stimulant and anti-depressant medications (Davis, 
2014). 

The quantification of codeine and morphine and their major metabolites, particularly morphine-3-
glucuronide (M3G) and morphine-6-glucuronide (M6G) is recommended for the assessment of 
codeine versus morphine- and heroin-related deaths and their survival times, applying codeine to 
morphine and morphine/M3G/M6G concentration ratios (Schanzle et al., 1999; Berg-Pedersen et al., 
2014; Darke and Duflou, 2016). Heroin use is determined by its marker 6-monoacetylmorphine (Darke 
and Duflou, 2016). For this, multi-analyte LC-MS methods have been reported (Frost et al., 2015; 
Frost et al., 2016). 

Enantioselective analysis for methadone (in combination with the metabolite EDDP) has been 
proposed for countries with both racemic methadone and enantiomerically pure R-methadone 
available (Jantos and Skopp, 2013; Rodriguez-Rosas et al., 2007; Holm et al., 2012). 

Earlier reports on the detection of fentanyl in post-mortem specimens described standardised solid-
phase versus liquid-liquid extraction and LC-MS-MS with electrospray source in positive ionisation 
mode for opioids (Coopman et al., 2007; Teske et al., 2007; UNODC, 2017). Recently published 
UNODC recommendations describe the potential problem of the lack of sensitivity of immunochemical 
screening kits of different manufacturers for some fentanyl analogues. GC-MS screening is 
recommended for the common prescription opioids fentanyl, sufentanil, alfentanil and metabolites, 
and liquid chromatography high-resolution high-accuracy mass spectrometry (LC-HRMS) is 
recommended for the screening of a range of opioids/selected fentanyl derivates. GC-MS, LC-MS-MS 
and ultrahigh-performance liquid chromatography high-resolution high-accuracy mass spectrometry 
(UHPLC-HRMS) are recommended for confirmatory and quantitative analysis. The isomer 
differentiation in fentanyl analogues with the same molecular core structure (especially when standard 
reference materials are not available) is reported to be challenging (UNODC, 2017). 

Mass spectrometry-based multi-analyte methods 
In recent years, multi-analyte methods have been refined because they allow a much simpler and 
cheaper identification monitoring of substances of different drug classes in one single body sample 
(Remane, 2010). Multi-analyte methods (including the application of advanced electrostatic traps) 
have repeatedly demonstrated their applicability for post-mortem matrices (Dresen et al., 2010; 
Broecker et al., 2011; Vogliardi et al., 2011; Broecker et al., 2012; Sundström et al., 2013; Montenarh 
et al., 2015; Maurer et al., 2016). Tandem MS has become preferred over single-stage MS (Sauve et 
al., 2012). Drug detection in post-mortem human blood by UPLC-MS and UPLC-MS-MS results in 
higher drug detection rates than GC-MS screening (Rosano et al., 2011) and the LOD in whole blood 
is lower than the lowest therapeutic concentration listed in blood level lists (Montenarh et al., 2015). 
Multi-target screening with electrostatic traps is essentially seen as a complementary method to GC-
MS screening, HPLC-DAD screening and immunoassays. 

However, although ‘multi-target’ screening can be applied to a huge list of substances, as long as 
reference standards, as well as analytical methods, are not available, particularly for a majority of 
NPS and their metabolites, these approaches may still fail to detect some drugs (Favretto et al., 
2013). 

1.2.6. Analytical methods for new psychoactive substances in post-mortem specimens 

A NPS is a new narcotic or psychotropic drug, in pure form or in preparation, that is not controlled by 
the United Nations drug conventions but which may pose a public health threat comparable to that 
posed by substances listed in these conventions (EMCDDA definition according to Council Decision 
2005/387/JHA) (Council of the European Union, 2005). In the past 3 years, death series have been 
reported by EMCCDA risk assessment reports for MDMB-CHMINACA, acryloylfentanyl, 
furanylfentanyl, AB CHMINACA, 5F-MDMBPINACA, 4F-iBF, CUMYL-4CN-BINACA, ADB-

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

CHMINACA, THF-F, carfentanyl, MT45, aPvP, 4,4’-DMAR and 25I-NBOMe (EMCDDA, 2018). 
Although the mortality associated with NPS is still not comparable to that of opiate-related deaths, 
there is uncertainty about the number of undetected cases, and this issue is becoming a more 
significant challenge for post-mortem forensic toxicology. To clarify the chemical structure of an 
unknown drug in a ‘legal high’ seizure, GC-MS, LC-MS-MS, high-resolution MS or TOF-MS can be 
used (Glicksberg et al., 2016). 

Although various human sample matrices are available for testing, urine and blood are the first 
choices for NPS screening. However, many of these drugs, especially unchanged synthetic 
cannabinoids, exist in urine and blood for only a short period. Other matrices such as hair and saliva 
are likely to receive more attention in the future (Namera et al., 2015). 

Current literature on the analytical methodologies that can be applied to these samples is still limited 
and a more thorough validation is often required, including a comparison of the results obtained from 
conventional approaches and from innovative strategies, in order to determine their suitability 
(Mercolini and Protti, 2016). Therefore, the absence of national or international 
recommendations/guidelines — particularly with scope in the post-mortem toxicology field — is not 
unexpected. In 2017, a list of observed NPS in French DRD casework was published as a reference 
for compounds to be included in toxicological analysis in suspected cases (Société Française de 
Toxicologie Analytique, 2017). 

A range of literature reports on validated methods for the analysis of a broad spectrum of NPS in 
urine, whole post-mortem blood, vitreous humour and pericardial fluid (Shanks et al., 2012; Guale et 
al., 2013; Marinetti and Antonides 2013; Pasin et al., 2015; Sundström et al., 2015, Glicksberg et al., 
2016; Margalho et al., 2016; Tynon et al., 2017) (Table 2). Furthermore, the concept of non-targeted 
screening using different approaches for identification of unknown compounds including software 
tools for prediction of molecular structures has been proposed as a valuable tool in forensic cases in 
which intoxication is suspected but no drug is identified by targeted analysis (Mollerup et al., 2017). 

TABLE 2  
Recently published methods for the analysis of NPS in post-mortem specimens (examples) 

Reference 
Shanks et 
al., 2012 

Material 
Post-mortem 
blood 

Guale et al., 
2013 

Post-mortem 
blood and urine 

Whole post-
mortem blood, 
vitreous humour 
and pericardial 
fluid 

Whole blood 

Marinetti 
and 
Antonides, 
2013 

Pasin et al., 
2015 

Glicksberg 
et al., 2016 

Method 
Liquid-liquid 
extraction UHPLC-
MS-MS in positive 
electrospray 
ionisation mode 
LC-TOF-MS after 
solid-phase 
extraction 
Liquid-liquid 
extraction and 
detection by LC-
QTOF-MS 

Liquid-liquid 
extraction followed 
by LC-QTOF-MS 

Urine and blood  Solid-phase 

extraction and LC-
QTOF-MS 

Analyte 
Synthetic cannabinoids JWH-018 and JWH-073  

Newer synthetic ‘Spice/K2’ cannabinoids and 
cathinone ‘bath salt’  

Synthetic cathinones (3,4-
methylenedioxypyrovalerone (MDPV), 3,4-
methylenedioxymethcathinone (methylone), 
pyrovalerone, pentylone, alpha-
pyrrolidinopentiophenone (alpha-PVP) and 
methedrone 
37 NPS including cathinones, hallucinogenic 
phenethylamines and piperazines 

22 synthetic cathinones (methcathinone, 
ethcathinone, pentedrone, buphedrone, 3-
fluoromethcathinone (3-FMC), 4-
fluoromethcathinone (4-FMC), 4-methylethcathinone 
(4-MEC), 4-ethylmethcathinone (4-EMC), 
mephedrone, methedrone, 3,4-
dimethylmethcathinone (3,4-DMMC), ethylone, 
butylone, pentylone, eutylone, methylone, 

22 

 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Margalho et 
al., 2016 

Whole post-
mortem blood, 
vitreous humour 
and pericardial 
fluid 

Mixed-mode solid 
phase extraction, 
followed by 
microwave fast 
derivatisation and 
analysis by GC-MS 
operated in selected 
ion-monitoring mode 

Tynon et al., 
2017 

Whole blood 

Separate extraction 
procedures followed 
by LC-MS-MS 

methylenedioxypyrovalerone (MDPV), 4-
methylpyrrolidinobutiophenone (MPBP), 3,4-
methylenedioxypyrrolidinobutiophenone (MDPBP), 
α-pyrolidinopentiphenone (α-PVP), pyrovalerone, 
and naphyrone) 
D-cathine (D-norpseudoehedrine), ephedrine, 
methcathinone, 1-(4-methoxyphenyl)-propan-2-
amine (PMA), mephedrone, methedrone, 2,5-
dimethoxy-4-methylamphetamine (DOM), 4-bromo-
2,5-dimethoxyamphetamine (DOB), 2,5-
dimethoxyphenethylamine (2C-H), 4-bromo-2,5-
dimethoxyphenethylamine (2C-B), 4-iodo-2,5-
dimethoxyphenethylamine (2C-I), 2-[2,5-dimethoxy-
4-(ethylthio)phenyl]ethanamine (2C-T-2), 2,5-
dimethoxy-4-isopropylthiophenethylamine (2C-T-4) 
and 2-[2,5-dimethoxy-4-
(propylthio)phenyl]ethanamine (2C-T-7), 
Diverse synthetic cannabinoid drugs, targeting 
arylindole compounds as well as the emerging 
aminocarbonyl/carboxamide (NACA) compounds 

Note: LC-QTOF-MS, quadrupole time-of-flight liquid chromatography mass spectrometry. 

Although strongly recommended, the sharing of reference materials and expertise (e.g. between 
laboratories in the same country, or between laboratories analysing seized substances or biological 
samples, or between laboratories in the same international network) is challenging, especially 
considering the number and rapid evolution of the substances that are detected on the market. The 
exchange of analytical data is promoted by databases such as the European Database on new drugs 
(EDND) under the framework of the EMCDDA Early Warning System (EMCDDA, 2007). The 
detection of use of NPS is a particular analytical challenge where identification of metabolites and/or 
the parent molecule in biofluids can be difficult because of the low concentrations encountered for the 
more potent substances and the lack of knowledge about many of them (Guillou, 2017). 

1.2.7. Interpretation and reporting of findings in forensic toxicology 

The interpretation of concentrations of post-mortem drugs of abuse requires correlation with medical 
history (prescriptions), scene investigation (drug paraphernalia), seized substances (transdermal 
patches, pills in or outside vials and autopsy findings) (SOFT/AAFS, 2006; GTFCh, 2009; Davis, 
2014). 

If death is attributed to any drug or combination of drugs (whether as cause or contributing factor), the 
certifier should list all the responsible substances by generic name in the autopsy report and on the 
death certificate (Davis, 2014). In the toxicology report, applied methods should be described, 
including the substances included, their LOQ and cut-off values (obviously this is not appropriate for 
reported non-targeted screening results, but international guidance in such cases is missing). No 
quantitative value from a non-specific immunological or other initial testing procedure should be 
reported, unless the procedure has been appropriately validated through parallel studies with a 
reference quantitative method (SOFT/AAFS, 2006). 

Consideration needs to be given to potential interactions of pharmacokinetic and pharmacodynamic 
variables when making a cause of death determination (Davis, 2014). In some cases, a drug can 
cause death even at a concentration below a reported lethal range. Conversely, the presence of a 
drug concentration within the reported lethal range does not necessarily make the drug the cause of 
death. Published tables of therapeutic, toxic and lethal concentrations can be highly misleading 
(Musshoff et al., 2004; Kennedy, 2010; Launiainen and Ojanpära, 2013). Tolerance accounts for 
some part of the overlap between therapeutic and lethal concentrations of opioid analgesics (Ferner, 
2008), but there is no reliable quantifiable measure of drug tolerance before or after death (with hair 

23 

 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

analysis only providing an indication of tolerance) (Davis, 2014). With regard to NPS, there are often 
no defined concentration ranges associated with (especially emerging) NPS that would correspond to 
degrees of toxicity and expected outcomes. However, toxicological significance scores have been 
proposed that consider a range of different sources of evidence to assign a level of significance (low, 
medium, high or unclassified) that indicates the likely role of a substance in contributing to or causing 
death (Elliott et al., 2017). 

Box 4 Pitfalls in the interpretation of substance concentrations in post-mortem specimens 

Drug concentrations measured in post-mortem samples cannot be used to reliably calculate the precise quantity 
of medication consumed (Davis, 2014). A drug level can be elevated exclusively because of post-mortem 
redistribution (exchange between tissues leading to changes in drug blood levels in the post-mortem period) 
(Cooper et al., 2010). This is particularly relevant for centrally acting drugs with large volumes of distribution (the 
theoretical volume that would be necessary to contain the total amount of an administered drug at the same 
concentration that is observed in the blood plasma) (Flanagan, 2012-2013). The interpretation of solid tissue 
concentrations of drugs is complicated by the fact that drugs may distribute unequally throughout body tissues 
because of variations in blood flow, bio-accumulation and other factors (Davis, 2014). Reference concentrations 
in different tissues related to concentrations in post-mortem blood have rarely been reported (Skov et al., 2015). 
Relevant technical literature should be consulted (Flanagan et al., 2008; Moffat et al., 2011; Suzuki and 
Watanabe, 2011; Kintz, 2012; Baselt, 2017). Tissue levels may be affected by the post-mortem interval, direct 
(e.g. stomach) and indirect (bladder diffusion) contamination (GTFCh, 2009), refrigeration before autopsy and the 
position of the body (Pounder et al., 1996). Endogenous metabolism (e.g. production of gamma-hydroxybutyrate, 
or GHB) and decomposition involving both autolysis and putrefaction may increase or reduce drug and 
metabolite levels in post-mortem specimens (e.g. microbial production or loss of alcohol) (GTFCh, 2009; Skopp, 
2010) depending on the tissue/organ in question (GTFCh, 2009; Lafreniere and Watterson, 2009; Wyman et al., 
2011) and storage conditions (Holmgren et al., 2004). Such effects may be minimised by the prompt refrigeration 
of the body and by performing the autopsy quickly (Flanagan, 2012-2013). 

Conclusions should never be based solely on the drug level detected but should also include an in-
depth review of the death scene or clinical scenario and drug characteristics (Patel, 2012); however, 
interpretation of analytical results is often limited by the inadequate information provided in a 
particular case (Skopp, 2010). 

With regard to deaths involving opiates, the presence of 6-acetylmorphine (6-AM) rather than heroin 
is sufficient to ascribe intoxication to heroin. It is important that the presence of 6-AM is confirmed by 
toxicological investigations, as heroin fatalities frequently cannot be distinguished from morphine 
intoxication by the scene investigation (Gill et al., 2016). In the absence of 6-AM, heroin use can be 
reasonably inferred by other means (Davis, 2014). A morphine-to-codeine ratio of greater than 1 may 
be considered evidence of heroin use (Jones and Holmgren, 2011). 

1.2.8. Certification of drug-related deaths 

In accordance with World Health Organization (WHO) guidelines and death certification legislation, 
US guidelines recommend that toxicological results should be included on the death certificate only in 
cases of a pathological contribution to death (Gill and Stajíc, 2012). With regard to the assignment of 
the cause of death on the death certificate, listing the generic names of all chemical agents 
considered responsible for causing death is recommended (Department of Health and Human 
Services, Centers for Disease Control and Prevention, National Centre for Health Statistics, 2003). 
The recommended approach applies to drugs present in concentrations sufficient to have caused 
death or contributed to death in a given case. Predisposing physical conditions that might have 
predisposed the person to a fatal outcome being neither necessary nor sufficient to cause death 
should be listed in the death certificate as ‘other significant conditions’ (Davis, 2014). 

24 

 
 
 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

1.3. Discussion 

The scoping study found that although European national or multi-national guidelines identified for 
forensic toxicology post-mortem investigations — including aspects for DRDs — are in general 
accordance with the guidelines of international professional societies, there is no agreed guideline 
from the EU Member States in this field. This results in some differences across countries with regard 
to the current analytical strategies and practices, which in turn affect the sensitivity of the toxicological 
investigations of suspected DRD cases. This limits the comparability of the data available, and this 
should therefore be reflected in the analysis and presentation of the data on DRDs at a European 
level. The scoping study identifies the various kinds of differences and elements that can influence the 
results of a toxicology investigation. It provides a description of specific weaknesses and difficulties 
with regard to the sensitivity and comparability of the data. These are detailed below, along with 
suggestions for ways forward and recommendations. A high level of specialisation is required for the 
investigation of post-mortem material from suspected DRD cases and evaluation of the findings. 
While the complexity of post-mortem analysis is high, the market for these analytical services is 
relatively limited, compared with markets for highly standardised urine-based doping tests or 
workplace testing, and post-mortem analyses are frequently restricted to official public bodies and 
state laboratories. 

National expert panel recommendations tend to allow some degree of flexibility, taking account of the 
fact that minimum standards may put pressure on and be challenging for laboratories with a lack of 
adequate equipment and/or limited resources for reference standards for constantly emerging new 
substances appearing on the market. 

The absence of specific European guidelines on forensic toxicology for DRD investigations is a barrier 
to the desired harmonisation of monitoring DRDs. Technical guidelines tend to be limited in their 
statements on minimum standards (as they are cautious about not excluding laboratories). Guidelines 
also fast become outdated in times of significant technical advances. It is probable that general 
technical instructions for the determination of non-opioid NPS are more likely to be developed in the 
area of human performance toxicology rather than the post-mortem area. Nevertheless, specific 
expert recommendations at the European level are needed for the inclusion of opioids in analytical 
strategies, as this is the most important substance group in DRDs. 

Furthermore, specific medico-legal recommendations for decision-making on the ordering of 
toxicological examinations following any type of post-mortem examination would have a promising 
impact on the harmonisation of DRD monitoring. This should include guidance for cases with an 
equivocal cause for death identified at autopsy or with a potentially natural cause of death, whenever 
a drug may have precipitated or contributed to the death by an additive mechanism. 

For laboratories specialising in the forensic evidence of NPS, the availability of reference materials 
(seized substances or reference standards) is essential. This would require the establishment of 
cooperation between forensic/police laboratories and medico-legal laboratories for the analysis of 
biological samples, because in many countries these are institutionally separated. Reference 
standards for the quantitative determination of emerging NPS are often available only — if at all — 
with a considerable time delay (up to years) from the relevant industrial suppliers. However, they are 
produced under rigorous quality assurance standards. The use of confiscated samples for reference 
purposes may be a compromise. However, it requires information exchange on where and when high-
purity samples are available. It should be noted that these substances may not be legally marketable 
and can be exchanged only in a legal grey area in many countries. 

25 

 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Part 2: Survey of practices — mapping the ‘typical’ or ‘standard’ 
toxicology practices in place in each country 

2.1. Objective 

The objective of this expert survey was to analyse how the international and national guidance with 
regard to the post-mortem investigation of suspected DRD cases are translated into practice in 
Europe (including minimum requirements, recommendations by national expert associations 
regarding sampling, processing, confirmatory testing, GUS, technical specifications and reporting — 
prioritising cases of polydrug and mixed poisonings) with special reference to NPS. 

2.2. Methods 

The identification of experts for the survey was carried out using the following strategies: 

  nomination by national focal points and national experts for DRDs from the EMCDDA working 

group; 

  direct  communication  to  state/public  institutes  for  forensic  medicine  and  forensic  toxicology 

departments in universities; 

  direct communication with authors of on-topic publications; 
  national professional associations. 

In total, 118 experts at 95 European laboratories were identified, to whom an online questionnaire 
(using SurveyMonkey) containing 54 items was sent. 

The questionnaire contained questions on the following topics: 

  whether or not the participating laboratory undertakes analyses of biological samples in cases 

of DRDs; 

  national  or  laboratory  guidelines  regarding  general  principles  of  post-mortem  toxicological 

 
 
 

 

 

 

 

 
 

analysis; 
laboratory performance (the number of suspected DRD cases handled per year); 
the laboratory’s work as a share of the total national post-mortem analyses; 
the  analytical  strategy  used  (pre-test/confirmation;  detection/quantification  of  substances 
causing death/contributing/additional substances); 
the use of pre-tests by immunological screening/others; substances included in pre-tests and 
since when these have been included; 
the  technical  equipment  available  for  quantification  of  substances,  including  a  retrospective 
timeline indicating how long they have been used for; 
coverage  of  substance  groups  and  examples  for  specific  analytes/special  drugs  such  as 
mescaline, phencyclidine, psilocybin/selected pharmaceutical medicines; 
coverage  of  NPS, 
benzodiazepines, piperazines, phenethylamines, cocaine derivatives; 
standards for toxicological reports (what is included/not included); 
the  level  of  satisfaction  regarding  the  exchange  of  case-related  information  between 
institutions (police/hospitals/forensic pathologists) and forensic laboratories; 

including  synthetic  cannabinoids,  synthetic  cathinones,  opioids, 

  potential hindrances to daily work and analytical advancements; 
  an awareness of the laboratory’s role as gate-keeper for statistical cause of death registration; 
  suggestions for improving the analytical strategy of suspected DRDs. 

26 

 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

2.3. Results 

2.3.1 Characteristics of participating laboratories 

Two thirds (or 63 out of 95) of laboratories from 27 EU Member States, Norway, Turkey and 
Switzerland responded. Of these, 95 % reported that they perform analyses from biological samples 
in cases of DRDs, 5 % (n = 3: 2 in Malta and 1 in the United Kingdom) did not. Accordingly, these 
three laboratories were excluded from the final analysis, as was another laboratory (in Hungary) 
owing to problems with data transmission. Czechia had a particularly high number of laboratories 
identified and a very high rate of response. Some of the answers came from laboratories that reported 
a relatively low annual number of DRD analyses. It was assumed that the national standards of DRD 
analysis in Czechia are likely to be represented by laboratories with larger numbers of cases, and 
therefore five Czech laboratories were excluded from the final analysis to reduce the potential for 
participant bias in the analysis as a result of the over-representation of Czech laboratories. A total of 
54 laboratories were used for the final analysis (Table 3). 

TABLE 3  
Survey response: laboratories per country 

Country 

Austria 

Belgium 

Bulgaria 

Croatia 

Cyprus 

Czechia 

Denmark 

Estonia 

Finland 

France 

Germany 

Greece 

Hungary 

Ireland 

Italy 

Latvia 

Lithuania 

Luxembourg 

Included in final analysis (n) 

1 

2 

1 

1 

1 

6 (of 11 laboratories investigating DRD) 

3 

1 

1 

4 

6 

1 

0 (1 laboratory investigating DRD but problems 

with data transmission) 

1 

3 

1 

1 

1 

Malta 

0 (2 laboratories responded, but were not 

investigating DRDs)  

Netherlands 

Norway 

Poland 

Portugal 

Romania 

1 

2 

3 

1 

0 

27 

 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Slovakia 

Spain 

Sweden 

Switzerland 

Turkey 

1 

3 

1 

3 

1 

United Kingdom 

3 (4 laboratories responded, one of which was not 

Total 

investigating DRDs) 

54 

With regard to laboratory capacity, 12 % of the laboratories reported that they conduct post-mortem 
analyses on more than 2 500 cases per year. A total of 13 % reported fewer than 100 cases. Most of 
the other laboratories carry out fewer than 500 analyses per year (43 %), with results for a further 7 % 
of laboratories being unknown. With regard to the analysis of suspected DRD cases, 20 % of 
laboratories reported conducting analyses on more than 250 cases per year and 30 % reported 
conducting analyses on between 51 and 250 cases. The biggest group, 43 %, responded that they 
work on fewer than 50 cases per year. This indicates that drug analyses are not a focus of activity in 
many laboratories; even in post-mortem analyses they are usually a secondary activity. 

In response to the question on an estimation of their share of nationwide DRD analyses, 35 % of the 
participating laboratories indicated that they perform 1-10 % of all cases in the country, 29 % reported 
that they carry out about 11-50 % of cases, 12 % reported that they carry out 51-95 % of cases and 
another 12 % reported that they are doing all or nearly all national toxicological investigations for their 
country (12 % unknown). 

In this respect, the study was able to fully reflect the standard in six EU Member States on the basis 
of the answers of the single responding laboratory that investigates 100 % of the national share of 
DRDs. These countries are Estonia, Finland, Latvia, Lithuania, Portugal and Sweden. For another six 
countries (Croatia, Cyprus, Ireland, the Netherlands, Slovakia, Turkey), the one responding laboratory 
was highly representative of a large number of cases investigated in that country (> 50 % of national 
share). 

2.3.2. Collection of test material for toxicological analysis 

Our hypothesis was that there may be variations in how evidence for post-mortem toxicological 
analysis is gathered. Recent developments in autopsies and post-mortem toxicology techniques may 
have opened up new options for securing specimens from the human corpse. 

In 35 laboratories (65 %), samples for toxicological analyses are always collected from a complete 
autopsy. However, in 12 countries, some laboratories (see Table 4) reported the use of ‘needle 
autopsies’ (minimally invasive procedures for sampling body fluids that waive the need for a full-scale 
autopsy). The extent to which these procedures are routinely used in individual countries remains 
unclear. In Hamburg, Germany, it was reported that about 40-60 % of all DRD toxicological analyses 
are based on blood and urine from ‘needle autopsies’, but in several other countries the use of this 
approach was indicated to be rare. It is likely that there is a negative correlation between such 
minimally invasive sampling and full-scale legal autopsies in suspected DRD cases. 

28 

 
 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

TABLE 4  
Laboratories reporting using specimens from ‘needle autopsies’ 

Country 

City 

Belgium 

Liège 

Bulgaria 

Sofia 

Czechia 

Prague 

Needle 
autopsy? 

Comment 

Yes 

Yes 

Yes 

France 

Strasbourg 

Yes 

Paris 

Yes 

Grenoble 

Yes 

Versailles 

Yes 

Germany 

Hamburg 

Yes 

40-60 % of the DRD cases 

Munich 

Yes 

Very few 

Dresden 

Yes 

Italy 

Rome 

Yes 

Luxembourg 

Luxembourg 

Yes 

Netherlands 

The Hague 

Yes 

Norway 

Oslo 

Poland 

Lublin 

Turkey 

Istanbul 

United 
Kingdom 

London 

Yes 

Yes 

Yes 

Yes 

Very few cases 

Blood samples occasionally 

Glasgow 

Yes 

Rarely, but it does happen with certain 
pathologists in certain circumstances 

The specimens reported as being used in pre-tests (multiple choices) were urine (95 %), serum 
(39 %), whole blood (50 %) and sometimes other matrices (21 %). Other matrices mentioned included 
cerebrospinal fluid, vitreous humour, bile, liver, stomach contents, pericardial fluid, muscle and kidney 
tissues. Apart from cerebrospinal fluid and vitreous humour, other matrices have a major role in cases 
in which decomposition of the corpse has occurred. 

2.3.3. Laboratory equipment 

Laboratories in the EU are regularly equipped with — and therefore are supposedly using — modern 
chromatography equipment, which is usually coupled with MS (Figure 1). Thin-layer chromatography, 
one of the oldest methods, is still used in 26 % of the laboratories. Various chromatographic 
techniques for separating analytes coupled with different detectors (‘GC coupled’ (without GC-MS) 

29 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

and ‘HPLC coupled’) are available in about half of the laboratories. Headspace-FID, which uses a 
special technique for applying samples in a gaseous state to a column for separation and subsequent 
analysis in a FID, is commonly available (81 %). The equipment most commonly found in laboratories 
is GC-MS units (87 %); however, liquid chromatography-tandem mass spectrometry units (LC-MS-
MS) are also widespread (78 %). Over the past 10-15 years, a shift from GC-MS to LC-MS-MS has 
been observed due to advances in instrument technology, fuelled by the polarity and low volatility of 
many new relevant substances and metabolites. 

HPLC-TOF-MS, which enables elemental composition to be determined directly, has become the 
method of choice for GUS in an increasing number of laboratories in recent years and was found in 
50 % of the institutions surveyed. The most advanced technical equipment is provided by FTICR-MS, 
LC-HRMS and UHPLC-HRMS devices, but only two participants in the study had them available. 
High-resolution mass spectrometry (HRMS) allows mass measurement with high resolution and very 
high accuracy, while UHPLC, combined with TOF-MS allows additional selectivity, sensitivity and 
speed resulting from increased chromatographic resolution. 

FIGURE 1  
Laboratory equipment for analytical oriented chromatography and mass spectrometry 
(percentage of laboratories equipped) 

100

80

60

40

20

0

81 

87 

78 

59 

43 

26 

35 

33 

50 

4 

Laboratories equipped with GC-MS and HS-FID, the most common types of equipment, have 
generally had these available for more than 10 years. The introduction of HPLC-TOF equipment, now 
reported in half of laboratories, is more recent and has occurred in the past decade, often in the past 
5 years. Technical upgrading with LC and GC tandem MS is also a recent development in the majority 
of laboratories now equipped with it (Figure 2). 

30 

 
 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

FIGURE 2  
Time of implementation of new techniques/methods (percentage of laboratories, unknown 
excluded) 

100.0

80.0

60.0

40.0

20.0

0.0

GCMS

HS-FID

HS-MS

LCMSMS

GCMSMS

HPLC TOF

> 10 years

5-10 years

2-5 years

Previous 2 years

2.3.4. Analytical strategy 

All the responding laboratories reported that the objective of their analytical strategy in cases of 
poisonings is the detection and quantification of the drug or drugs that was/were a direct cause of 
death. However, 81.5 % of laboratories confirmed that the analysis for further (additional/concomitant) 
drugs/substances, which are not relevant to death, is not the objective of their strategy. This means 
that in almost 20 % of the surveyed laboratories the completeness of the confirmation of all detectable 
substances is not a priority. It can also mean that at least 20 % of the laboratories will decide step by 
step which substance indications in pre-tests will be followed up and which will not. As a result, there 
may be substances that remain unconfirmed and thus unreported. 

It is routinely an option to limit analytics in certain cases. In addition, a laboratory might decide not to 
determine certain substance groups (in the NPS groups) at all, since the effort required for the 
validation of appropriate methods involved seems too high or the appropriate techniques may not be 
readily available. The laboratories reported experiencing a variety of limitations with respect to the 
toxicological analyses in DRD cases. One third (33 %) reported budgetary reasons resulting in 
limitations, while a similar proportion (35 %) mentioned shortcomings regarding their laboratory 
equipment. The most frequent problem reported was the insufficient availability of reference 
standards (43 %). 

The laboratories were asked if they apply conventional pre-tests (screening tests). Immunochemical 
and chromatographic tests were specified in the question, but there was also an option to provide 
free-text information on further test principles. A conventional laboratory strategy, which has been 
available for several decades, is the application of immunoassays, which enable a two-stage 
analytical approach. If a substance is detected in pre-tests, the findings are confirmed by a 
confirmatory test. This currently still common practice was affirmed by 70 % of the laboratories. 
Almost all reported that they had been using these tests for more than 10 years, with only one 
laboratory reporting their introduction 5-10 years ago. Some laboratories reported immunochemical 
tests being carried out outside the laboratory by forensic pathologists or coroners in order to have an 
indicative result at autopsy stage. 

By contrast, the question on chromatographic pre-tests may not have been sufficiently precise. 
Almost all the laboratories confirmed their use; however, it is possible that failing to draw a distinction 

31 

 
 
 
TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

between chromatographic pre-tests and the use of current high-performance chromatographic 
analyses for ‘comprehensive screenings’ may have posed a problem. These comprehensive 
screening analyses should not be regarded as a conventional ‘pre-test’, as they rather have the 
character of a qualitative confirmation, even if quantification is a further step. In fact, many 
laboratories use different chromatographic systems for screening and quantification/confirmation of 
positive immunoassay results depending on the analyte. 

Figure 3 shows how often specific substance groups are tested for by those laboratories that 
indicated their use of immunological screening or other pre- tests. If a specific substance group is not 
tested for, this may mean that without GUS even in further confirmation analysis this substance group 
will not be found, even if it is contained in the sample. 

Natural cannabinoids, cocaine and opiates are nearly always included in routine screening (one 
laboratory used immunological screening for natural cannabinoids only and further relied on GUS). 
Only a few laboratories do not pre-test for amphetamines (5 %) and benzodiazepines (10 %). 3,4-
Methylenedioxymethamphetamine (MDMA) screening by immunoassay is performed in 74 % of the 
laboratories. The use of methadone (66 %) and buprenorphine (58 %) screening is limited and may 
depend on the probability of occurrence according to substitution treatment practices in individual 
Member States. 

There are other tests that are rarely used, such as those for lysergic acid diethylamide (LSD), but LSD 
is not relevant for determining the cause of death in poisonings. Fentanyl and oxycodone are rarely 
included in a pre-test, although they may have a role in the cause of death. For substances GHB, 
screening tests have not been available for very long. It should be noted that many laboratories that 
affirm that they use immunological screenings for these groups of substances have established GUS 
procedures at the same time, so that a double strategy enhances the probability of detection. 

FIGURE 3  
Substance groups that are at present included in immunological screening (percentage of 
laboratories with positive answer) 

Opiates
Cocaine
Cannabinoids
Amphetamines
Benzodiazepine
Ecstasy
Methadone
Barbiturates
Buprenorphine
Trycyclic antidepressant
LSD
Fentanyl
GHB
Oxycodone
Tramadol
Zolpidem

37 

26 

18 

16 

13 
13 

3 

97 
97 
97 

95 

89 

74 

66 

60 

58 

0

10

20

30

40

50

60

70

80

90

100

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

The vast majority of laboratories have not changed their immunoassay pre-testing programme in 
recent years; individual substance groups were reported as having been added to the programme by 
less than 10 % of the participants (Figure 4). 

FIGURE 4  
Substance groups included in immunological screening in the past 5 years (percentage of 
laboratories with positive answer) 

Substance groups recently included in immuno-screening in last 5 years 

Opiates
Cocaine
Cannabinoids
Amphetamines
Benzodiazepine
Ecstasy
Methadone
Barbiturates
Buprenorphine
Trycyclic antidepressant
LSD
Fentanyl
GHB
Oxycodone
Tramadol
Zolpidem

8 
8 
8 
8 

8 
8 

2 

4 

5 

5 

5 
5 
5 

3 

4 

0 

0

5

10
% 

15

20

Nearly all laboratories reported that they routinely confirm positive screening test results, with only 
one laboratory indicating that this decision was made on a case-by-case basis (a laboratory with < 10 
DRD cases per year). 

2.3.5. Analytical strategy: classification 

The results indicate that there are four different analytical strategies (Table 5), mainly due to the 
differences in the technical equipment and thus the established methods used in the laboratories: 

1.  A comprehensive screening in the strictest sense of a GUS strategy is applied for analysis in 

2. 

suspicious DRD cases in 26 % of all laboratories (n = 14). These laboratories prefer a 
systematic screening for all substances that can be detected on the available devices, 
regardless of the case. They use chromatographic multi-target methods applied on LC-MS-
MS, HPLC-TOF-MS, UHPLC-HRMS or FTICR-MS. These devices enable the qualitative 
detection of thousands of substances in a first run, followed by confirmatory analyses with 
quantitative measurement, using essentially the same methods. 
In 12 out of the 14 laboratories, the routine screening procedure was performed with a Q-
TOF-MS and the quantification with GC-MS, HPLC or LC-MS-MS, depending on the 
analyte/substance, the case and the circumstances of the death. These 12 laboratories 
reported using GUS systematically in all cases. However, 3 out of 12 laboratories stated that 
they still make case-dependent decisions (in addition to a primary GUS), so that it is not 
always a rigid system. A case dependency could refer to sample properties, to the 
documented survival time and other factors. The remaining 2 of the 14 laboratories with high-

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

quality equipment essentially use case-dependent analysis strategies instead of a standard 
GUS. 

3.  Mixed methods: the questionnaire form did not allow the laboratories to differentiate their 

strategy for every typical situation in suspected DRDs. On the basis of their report on pre-test 
screening, GUS and type of available devices, however, it can be concluded that about 43 % 
of the laboratories (n = 23) use advanced technologies such as LC-MS-MS, HPLC-TOF-MS 
and UHPLC-HRMS for screening followed by quantification, but they still combine it with 
conventional immunoassays. This situation is typical for laboratories that are not (or not yet) 
in a position to be certain that their GUS analysis makes immunoassays completely 
dispensable. 

4.  Alternative options for the laboratories are to have a limited GUS coupled with either 

chromatographic methods such as GC-MS or with immunological screening methods. This 
strategy is combined with analytical decisions depending on the available case information 
and may proceed with individual selective analyses with different methods. Limited GUS 
combined with immunoassays was reported by 9 % (n = 5) of the laboratories. 

A conventional two-step strategy, relying on immunoassays as the first step, was reported by 17 % 
(n = 9) of the laboratories. 

Approximately 6 % (n = 3) of the laboratories could not be unambiguously classified on the basis of 
their data. 

TABLE 5  
Classification of analytical strategies for screening and confirmation in the event of a 
suspected DRD 

Classification 

Group 

n 

% 

1 

2 

3 

4 

5 

Comprehensive screening with multi-target chromatography screening, including LC-
MS-MS, HPLC-TOF-MS, UHPLC-HRMS or FTICR-MS 

14 

26 

Mixed methods: immunological screening AND multi-target chromatography 
screening, including LC-MS-MS, HPLC-TOF-MS or UHPLC-HRMS 

23 

43 

Primarily targeted immunological screening or screening based on GC-MS/HS-FID, 
confirmed by coupled chromatography/MS methods 

5 

9 

Primarily targeted immunological screening, confirmed by coupled 
chromatography/MS methods 

Others 

Total 

9 

17 

3 

6 

54 

100 

2.3.6. Special analytical considerations and reporting 

Two thirds (67 %) of laboratories confirmed that they send substances to a specialised laboratory 
when they are not able to analyse them themselves (28 % responded ‘no’; the situation was unknown 
for 5 % of laboratories). However, the expectation that laboratories with limited technological 
equipment/methods will send samples to other laboratories more frequently than laboratories with 
advanced technology was not confirmed. Rather, fewer than half of the laboratories in the first group 
reported being able to send samples to a specialist laboratory, compared with three quarters in the 
other group, which reported having more advanced technology available (Figure 5). 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

FIGURE 5  
‘When questions arise about substances you cannot analyse in your laboratory, do you have 
the opportunity to send samples to a specialised laboratory?’ (%) 

Laboratories with conventional 
technology (n=14)  

Laboratories with advanced 
technology (n=37) 

7 

47 

Yes

No

Unknown

46 

5 

21 

74 

Yes

No

Unknown

Almost all of the participating laboratories (93 %) indicated that they performed a quantitative 
determination of the major drug(s) that caused death. Eighty-seven per cent also performed a 
quantitative determination of additional drugs that might have contributed to death, but only 65 % 
reported undertaking a quantitative determination of additional findings (e.g. natural cannabinoids). 
Therefore, there are still some laboratories that base their key findings, at least in part, on qualitative 
evidence. A clear correlation with the analytical methodology used was not found for this item. 
However, all laboratories equipped with HPLC-TOF-MS quantified both the primary causes of death 
and the additional potential contributors to the cause of death. 

Quantitative results are included in the final report by only 71 % of laboratories. Two laboratories 
never or ‘rarely’ give quantitative statements. An interpretation of the results is not regularly given by 
the toxicologist in the final report: 77 % of the laboratories with advanced technologies, but only 33 % 
of the laboratories with conventional methods, provide this aid to interpretation. 

Incidental or additional findings that are rated as being irrelevant to the cause of death are rarely 
listed in the report. Even in the case of laboratories with advanced technologies, only 57 % include 
this sort of information, meaning that these substances may not be included in any further processing 
for registration/codification of cause of death or epidemiological surveillance. 

2.3.7. Substance group coverage 

Opiates, cocaine, amphetamines, methamphetamine, MDA, MDMA, MDEA and the widely used 
prescription benzodiazepines are practically fully covered by any routine analytical strategy being 
reported by more than 95 % of all laboratories who provided a response for this item. The coverage of 
natural cannabinoids (93 %) was slightly lower (Figure 6). This was also the case for the prescription 
opioids, methadone and tramadol (93 % each), as well as for the anaesthetic ketamine (94 %). Drugs 
that are routinely determined slightly less often are the prescription opioids buprenorphine and 
fentanyl, the anticonvulsant pregabalin and ‘z-drugs’ (including the hypnotics zopiclone, zolpidem, 
zaleplon), with coverage rates of around 70 %. Finally, routine testing for the psychoactive 
neurotransmitter drug GHB/GBL, volatiles, gases such as butane and propane and ‘poppers’(alkyl 
nitrites, tested for by only one laboratory) are even less common (Figures 7 and 8). 

Even on request, many of these substances are not tested for in a considerable proportion of 
laboratories (e.g. testing for buprenorphine either routinely or on request is not available in 9 % of the 
laboratories; for fentanyl the figure is 4 %, for tilidin it is 46 %, for gases it is 26 %, for volatiles it is 
39 %, for ‘poppers’ it is 41 %, for pregabaline it is 33 % and for GHB it is 13 %). 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

FIGURE 6  
Analytical strategy and capacity for established substance group: coverage in routine versus 
‘on request’ testing (percentage of laboratories) 

Routinely 

97 

100

83 

96 

98 

98 

96 

93 

56 

7 

2 

On request 

%

80

60

40

20

0

%

100
80
60
40
20
0

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

FIGURE 7  
Analytical strategy and capacity for some exemplary opioids and other substances: coverage 
in routine versus ‘on request’ testing (percentage of laboratories) 

Routinely 

93 

98 

74 

74 

94 

78 

65 

37 

35 

100

93 

%

80

60

40

20

0

On request 

%

100

80

60

40

20

0

22 

17 

6 

6 

17 

17 

2 

4 

52 

Regardless of the case in question, laboratories with a routine GUS analyse more systematically for 
substances or groups of substances. The difference between buprenorphine and fentanyl is 
particularly striking: whereas 92 % and 100 %, respectively, of the 12 laboratories with routine GUS 
examine these opioids systematically in each sample, the remainder do so in only 69 % and 67 % of 
laboratories, respectively. Even with pregabalin, GHB and ‘z-drugs’, the methodological coverage for 
these substances is considerably lower in laboratories without comprehensive GUS (Figure 8). 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

FIGURE 8  
‘Routine analytical strategy covers…’: laboratories with (n = 12) versus laboratories without 
(n = 42) routine GUS 

Laboratories with routine GUS 

100

92 

100 

92 

100 

100 

100 

100 

80

60

40

20

0

33 

92 

75 

50 

Laboratories without routine GUS 

100

93 

90 

90 

98 

95 

69 

67 

71 

62 

38 

31 

80

60

40

20

0

2.3.8. Collaboration/information exchange 

When asked about their level of satisfaction regarding the exchange of case-related information 
between institutions (police/hospitals/forensic pathologists) and their laboratory, only 50 % of the 
respondents stated that they were ‘satisfied’ or ‘very satisfied’, while 21 % of respondents expressed 
dissatisfaction (Figure 9). 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

FIGURE 9  
Level of satisfaction regarding the exchange of case-related information between 
institutions 

4 

13 

37 

17 

29 

Very satisfied

Satisfied

OK

Dissatisfied

Very dissatisfied

Over half (54 %) of the laboratories were aware that the results of their toxicology reports are used to 
code causes of death (27 % not sure, 19 % not aware). However, a smaller proportion of participants 
knew whether the toxicological findings they produced were sent to a General Mortality Register or a 
comparable institution for the coding of causes of death according to the International Classification of 
Diseases, Tenth Edition criteria (35 % ‘yes’; 33 % ‘not sure’; 26 % ‘no’; 6 % unknown). 

2.3.9. Coverage of new psychoactive substances 

Among NSP, synthetic cathinones and phenethylamines show the highest coverage in routine 
diagnostics, in 81 % and 74 % of the laboratories, respectively (Figure 10). Synthetic cannabinoids 
and piperazines follow, with 65 % each. Synthetic opioids (e.g. new fentanyls), benzodiazepine and, 
in particular, cocaine derivatives are less systematically investigated routinely. 

FIGURE 10  
Analytical coverage of NPS in EU Member State laboratories 

%

100

80

60

40

20

0

In laboratories with comprehensive screening and mixed methods, almost all NPS groups are 
examined more systematically and independently of case information than in laboratories with a 
strategy based on more conventional methods (Figure 11). 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

FIGURE 11  
Analytical coverage of NPS according to laboratory classification in Table 5 (laboratories with 
advanced multi-target methods = groups 1 and 2; laboratories with conventional methods = groups 3 
and 4) (percentage of laboratories with positive answer) 

Laboratories using conventional methods 

60 

47 

53 

40 

27 

27 

Laboratories using advanced multi-target methods 

90 

72 

74 

82 

74 

59 

26 

%

100

80

60

40

20

0

%

100

80

60

40

20

0

In response to a question about the capacity for analysis of specific substances that exemplify the 
NPS substance groups included in the previous questions, lower coverage rates were reported than 
indicated for the groups as a whole. Most of the individual substances had reported coverage rates of 
between 40 % and 70 % (Table 6). 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

TABLE 6  
‘What kind of substances does your analytical strategy usually cover?’ (examples) 

Substance group 

Substance example 

Synthetic cannabinoids 

Synthetic cathinones 

Designer opioids 

Designer benzodiazepines 

Piperazines 

Phenethylamines 
Synthetic derivative of cocaine 

Hallucinogens 

JWH-18 
AKB48 Apinaca 
Mephedrone 
Methylone 
Pentedrone 
O-Desmethyltramadol  
3-Methyl-fentanyl 
Acryloylfentanyl 
Flubromazepam 
Clonazolam 
mCPP 
BZP 
2CB 
Dimethocaine 
3-pFBT 
Phencyclidine 

Mescaline 

Psilocybin 

Coverage? ‘Yes’ 
(% of laboratories) 
59.3 
37.0 
81.5 
70.4 
50.0 
64.8 
44.4 
27.8 
40.7 
40.7 
68.5 
63.0 
70.4 
14.8 
9.3 
63.0 

46.3 

44.4 

2.3.10. Suggestions for improvements 

When asked for suggestions to improve the analysis strategy for suspected cases of DRD, several 
participants mentioned the following points: 

  networking/communication  with  other  stakeholders  (police,  justice,  health  system/clinical 

doctors); 
cooperation with forensic pathologists; 
standardisation of sample collection; 

 
 
  with regard to technical and operational needs: equipment, methods needed; 
 
funding for method development and research; 
  guidelines for post-mortem toxicological analyses; 
  guidelines for coding cause of death on death certificate. 

Box 5 Limitations of the survey 

The survey gives a rough overview of the state of the forensic post-mortem toxicology field among EU 
Member States, Norway and Turkey as well as Switzerland. The data collection covered almost exclusively 
public institutions such as university, state or police laboratories, but it seems likely that post-mortem 
analyses are performed predominantly in the public sector. The representativeness of the results may be 
questionable, in particular in large EU Member States such as Spain, France, Italy and the United Kingdom, 
where post-mortem analyses are shared among quite a large number of laboratories. 

Detailed survey questions were generally limited to broad substance groupings, which were considered as a 
group. It was not feasible within the scope of this survey to collect details on specific analytical methods for 
particular substances of interest or to gather analytical threshold levels (LOD, LOQ); indeed, the different 
detection methods reported already reveal dimensional differences in sensitivity. The present study could not 
describe distinct detailed timelines (e.g. for the past decade), in terms of the development of new methods on 
new devices for individual laboratories. Hence, a breakdown of new trends in the incidence of certain DRD 
substances in relation to technical innovations in the laboratories in place remains preliminary at the moment. 

Finally, the survey provided evidence, but did not query the causes, for the apparent dissatisfaction regarding 
inter-sectoral communication (between coroners/forensic pathologists, police, health institutions). Future work 
should analyse in more detail whether, in individual countries/between individual regional actors, the 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

exchange of information as provided by regulations remains insufficient or whether there are fundamental 
obstacles, such as privacy policy and interface issues (e.g. between public authorities, university research 
laboratories or independent laboratories). 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

3. Discussion and conclusions 

3.1 Interpreting drug-related death prevalence data against the background of 
toxicology standards and capacity in European countries 

The implementation of new methods and technology in the laboratories yields more results for 
substances that are missed by traditional analytical strategies. These include new substances that 
may cause harm owing to their chemical and pharmacological properties and/or to their high potency, 
which may cause hazardous health effects at very low concentrations. Changing from one method or 
standard to another will influence trends. 

The principle ‘the more you search, the more you find’ clearly has an impact on DRD monitoring at 
regional or country level. This has been shown for Sweden, where the number of detected fentanyl 
cases doubled when there was a switch from the earlier occasion-related testing for drugs to 
comprehensive (‘full’) screening on every forensically investigated death in September 2011 (Leifman, 
2016). The implementation of new technology allowed for lower LODs in a centralised forensic 
laboratory for post-mortem specimens from all over the country. A technical upgrade of laboratory 
methods — which could be synchronised at a national level — offers the opportunity for the 
retrospective analysis of stored samples. Such local studies could provide insight into the shift of 
analytical (and monitoring) sensitivity for some opioids and more particularly for NPS, for example. 

The survey reported here showed that HPLC-TOF mass spectrometers have been used in some 
laboratories in Helsinki, Finland, Copenhagen, Denmark, and Frankfurt, Germany, for more than 10 
years; in laboratories responding from other countries, the investment in this technology was made 
less than 10 years ago. Laboratories in Nicosia, Cyprus, Berlin, Germany and Birmingham, United 
Kingdom, recently put the technology into operation (less than 2, years ago). In many other 
laboratories, the technical upgrade to an HPLC-TOF mass spectrometer took place 2-5 years ago. 
Among these laboratories, those in Istanbul, Turkey, Oslo and Trondheim, Norway, and Vilnius, 
Lithuania, are in the position of handling a majority or nearly all DRD cases in their countries. 
Therefore, in these countries, a trend analysis of deaths related to synthetic opioids and NPS in the 4-
year period from 2013 to 2016 could be a promising way to analyse a potential monitoring artefact. 
Importantly, in the other countries, where the responding laboratories handle only part of all DRD 
cases, generalisation of the findings to the country level should be avoided. 

However, interpreting trends will be challenging and consideration will need to be given to a range of 
potentially confounding factors and interactions, including those between changes in patterns of use 
and the influence of various regulatory and health policy measures on both use of substances and the 
availability of the equipment for new toxicological investigations and the extent to which these are 
implemented. The pattern of polydrug poisonings will require an in-depth analysis in these 
observational studies, as some illicit substances that were previously hidden but that accompanied 
other, easier to detect, substances may become visible. 

3.2. Conclusions 

In Europe, the majority of DRDs are registered with a ‘known toxicology’ in the EMCDDA Statistical 
Bulletin, but there is only fragmentary knowledge of limitations and regional or national differences in 
the analytical capacity of forensic toxicology laboratories in terms of routine substance screening. 
While rapidly evolving technologies have resulted in an increasing number and range of toxicological 
assessment methods being available, the diversity of substances that are potentially involved in 
deaths means that there is not a single method that can cover them all (at least with adequate LODs). 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Importantly, in many countries in Europe, post-mortem forensic toxicology is restricted to a single or 
small number of laboratories, run by public authorities. Hence, any change in standard operating 
procedures in a single laboratory can potentially have a major influence on DRD prevalence data. 

Over the past 10 years, a comprehensive screening approach using advanced HPLC-TOF-MS, 
FTICR-MS, GC-MS-MS or LC-MS-MS has become increasingly available in forensic laboratories. 
Such screenings do not cover all possible substances but have become the method of choice for 
analytes with forensic relevance in very low concentrations in biological specimens. 

As there is currently limited experience in the post-mortem analysis of NPS-related poisoning cases, 
universal analytical recommendations are very difficult to make in this field. In addition, the lack of 
reference standards leads to great restraint in the reporting of results, even where qualitative 
determinations are possible. 

The survey conducted with a sample of 54 forensic laboratories in Europe showed that the majority of 
the responding laboratories have shifted to multi-target screening methods, including advanced 
technologies and instrumentation. A strict GUS that assesses the broadest spectrum of substances 
currently possible for every incoming case is meanwhile applied in just over one quarter of the 
participating laboratories. Conventional targeted methods such as immunochemical screening are still 
common as a first step but are used selectively. 

There are still challenges for the reliable determination of highly potent opioids and NPS in 
laboratories without systematic GUS strategies, which, as indicated, are still not adopted in many 
countries. Particularly in polydrug poisonings, prescription opioids and other NPS may go 
undetected/unreported, although they may have caused or contributed to death; the same is true for 
the coverage of certain non-opioid prescription drugs such as pregabalin. Some other substances that 
may be misused and result in sudden death, for example solvents and alkyl nitrites (‘poppers’), are 
also challenging to detect in routine sampling and analysis owing to their volatility, and, for the 
majority of laboratories, their detection needs case-dependent decisions. 

In the case of an NPS-related death, a laboratory without adequate equipment for comprehensive 
screening will struggle to identify cause of death, and further analytical decisions will critically depend 
on the quality of the case information. Nonetheless, even if police information actually gives specific 
indications on the use of NPS before death, in many cases it will be necessary to send specimens to 
a specialised laboratory. However, this study indicates that, for the survey respondents, this option is 
not always available, particularly for those laboratories without advanced technical equipment. In 
addition, the potential to send specimens away for analysis may be limited owing to sample volumes, 
storage and transport conditions (and funding). 

3.2.1. Consequences and priorities for public health 

One of the important conclusions of this review is that specific medico-legal recommendations for 
decision-making on ordering toxicological examinations after autopsies would be valuable. Such 
recommendations should include guidance for ordering and conducting examinations on all cases of 
young/middle age unexpected deaths or with equivocal cause of death identified at autopsy or with a 
potential natural cause of death whenever a drug may have precipitated or contributed to death by an 
additive mechanism. Importantly, it has been demonstrated that the threshold level for toxicological 
investigations has a considerable impact on DRD monitoring and on the quality of substance-related 
data available. Therefore, guidelines should be instrumental in helping to harmonise these threshold 
levels. 

The report suggests that national guidelines for forensic toxicological laboratory analysis should be 
reviewed, addressing in particular the following questions: ‘Do laboratories analysing biological 
samples have sufficient facilities, competence and training to contribute to reporting of data relevant 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

for public health?’, ‘Is the codification of the toxicological evidence of NPS in deaths sufficiently clear 
for General Mortality Registries under WHO rules?’ and ‘Is the integration of the laboratories in early 
warning networks sufficiently ensured?’. 

In the context of rapidly changing laboratory techniques, it is also important to highlight that guidelines 
often take into account the average technical laboratory instrumentation available at the time they are 
developed, and thus they should be updated regularly to reflect developments in instruments and 
analytical approaches. Another important conclusion of this work is that there is a need for an update 
of national/European/professional society reference documents to include a recommendation that a 
comprehensive screening strategy based on advanced analytical technology is adopted. That will be 
useful for DRD casework but also for the epidemiological monitoring of substance use. 

For the epidemiological surveillance of NPS, the challenge is that at least qualitative results (i.e. the 
detection and identification) are reported. Bearing this minimum requirement in mind, owing to the 
specific challenges associated with the large and rapidly growing number of NPS, it is likely that only 
selected or centralised laboratories in relatively large Member States will be able to become 
specialised in analyses for these from biological samples. Indeed, there are special requirements in 
post-mortem analysis and difficulties in their interpretation, which challenge laboratories with relatively 
few DRD cases per year. 

Therefore, experience and expertise in the determination of hazardous NPS in specialised 
laboratories should be shared (e.g. by facilitating further updates of mass spectral libraries). This 
knowledge sharing could entail in particular police laboratories analysing substances from seizures, in 
collaboration with the laboratories analysing biological samples. Furthermore, this sharing, within 
countries, could stimulate further European/international collaboration, through a strategically 
strengthened exchange of information across the relevant EU organisations, the EMCDDA, and the 
forensic and toxicology organisations (e.g. to provide online access to mass spectra of emerging 
NPS). 

Beyond qualitative analysis, it is important to note that reference standards necessary for the 
quantitative determination of emerging NPS are available from industrial sources but only after a 
considerable time delay and at significant cost. To address this issue, the use of high-purity 
confiscated samples as a reference may solve the problem, but, evidently, this requires the exchange 
of information about where such samples are available. In this context, the problem arises because 
the substances are not marketable by law and, consequently, situations can vary across countries. 
The situation is indeed complicated by the fact that, while in some countries the process is well 
specified by legal provisions, in others the exchange among laboratories constitutes a legal grey area. 
In this context, an EU-wide inventory of how to deal with legal grey areas while exchanging seized 
materials for analytical purposes may increase the security and success of action taken by forensic 
laboratories. Furthermore, the question of improving national and international data exchange 
between laboratories on available new substances should be prioritised. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

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analytical toxicology, John Wiley & Sons Ltd, Chichester. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Frost, J., Lokken, T. N., Brede, W. R., Hegstad, S., Nordrum, I. S. and Slordal, L. (2015), ‘A 
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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

femoral blood in heroin-related poisoning deaths’, Legal Medicine (Tokyo, Japan) 13, pp. 171-173. 

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194. 

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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Noble, C., Weihe Dalsgaard, P., Stybe Johansen, S. and Linnet, K. (2018), ‘Application of a screening 
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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

Shanks, K. G., Dahn, T. and Terrell, A. R. (2012), ‘Detection of JWH-018 and JWH-073 by UPLC-
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TECHNICAL REPORT I An analysis of post-mortem toxicology practices in drug-related death cases in Europe 

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