Toxicological Laboratory
History
The first toxicological analyses at the Center were recorded in 1958 when food and pharmaceutical preparations were examined. With advancements in forensics and the increase in the quantities of seized drugs, thin layer chromatography (TLC) was introduced into daily use in 1963 as a simple and selective method for qualitative drug analysis. At about the same time, two methods for the determination of ethanol in biological samples were introduced: the Widmark method and the enzymatic (ADH) method on a UV spectrophotometer. Due to certain practical shortcomings of these methods in the determination of ethanol, in 1969 the first analyses using gas chromatography method (GC-FID) were carried out, which mainly included quantitative analysis of ethanol in blood and urine, and, if needed, to the analysis of illegal drugs. A little later, gas chromatography coupled with mass spectrometry method (GC-MS) was introduced and is still considered one of the most suitable methods for the qualitative determination of drugs.
By moving to its current location in 1997, the Center acquired new modern laboratories that were fully adapted to the needs and requirements of toxicological analyses. The acquisition of new instruments was intensified, computer systems for automatic data processing were introduced, experts were educated in related institutes around the world, and new methods were developed for the quantitative determination of the most common drugs by High Performance Liquid Chromatography (HPLC) and for the qualitative determination of drugs in biological samples by the GC-MS method. As a result of all the above activities, comparative analyses have been conducted since 2002 (initially of heroin and cocaine samples, and later also amphetamines), which combined developed analytical methods upgraded with the specialized expert’s opinion.
Participation in projects financed from the EU pre-accession funds has further modernized the toxicology laboratory, both in terms of equipment and new methods. The most important project was "IPA 2007 - Strengthening Capacities of the Ministry of the Interior to Combat Narcotic Drugs Trafficking and Drug Abuse", through which eleven new instruments were purchased and numerous new methods were developed, the most important of which are methods for chemical profiling of heroin, cocaine and amphetamines and quantitative determination of drugs in biological samples.
Analysis of seized drugs
Qualitative analyses, i.e. the identification of seized drugs and psychotropic substances (in the form of powder, commercial and non-commercial tablets and capsules, liquids, etc.) and the detection of drug traces on objects, for example in syringes and scales, are carried out using Thin-layer Chromatography (TLC), Fourier transform Infrared Spectrophotometry (FTIR) and the associated Gas chromatography-Mass spectrometry (GC-MS) systems. Quantitative analyses, i.e. determination of the purity of sized drugs and psychotropic substances, are carried out using High Performance Liquid Chromatography (HPLC) and gas chromatography whit flame ionization detector methods (GC-FID).
Comparative analysis
Comparative analysis of heroin, cocaine, methamphetamine, MDMA and amphetamine include methods that determine the main components, cutting agents, and characteristic impurities in samples and that compare their content and ratios.
In addition to comparative analysis, chemical profiling is also performed to determine the level of probability that different samples originate from the same source. Chemical profiling of heroin is performed by comparing the ratios of chemical compounds from opium and impurities created during heroin production. Comparative analysis of heroin is performed using FTIR, GC-MS, HPLC, and GC-FID methods.
In the chemical profiling of cocaine, the ratios of trace amounts of organic solvents used in the production of cocaine are compared. Comparative analyses of cocaine are performed using the FTIR, GC-MS, HPLC and HS-GC-FID methods. Chemical profiling of amphetamine, methamphetamine and MDMA is conducted by comparing the proportions and ratios of intermediate products and the impurities formed in the synthesis process of a particular compound. Comparative analyses of amphetamine, methamphetamine and MDMA are carried out using the FTIR, GC-MS and HPLC methods.
Pesticide expertise
Pesticide analysis in plant, powder, liquid and other types of samples and biological samples (for example in gastric contents and liver) includes qualitative and quantitative analysis, i.e., identification and quantification of pesticides. These analyses are carried out using GC-MS and LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) methods.
Drug testing in biological samples
Expert analyses on drugs in biological samples include qualitative and quantitative methods, i.e., the identification and concentration of drugs, psychotropic substances, medicines and its metabolites mostly in blood and urine, but other body tissues and secretions as well. Preliminary analyses of drugs in biological samples are carried out by the enzyme immunological method and then confirmed by GC-MS and LC-MS/MS methods.
Ethanol analysis in biological samples
Ethanol analysis in biological samples includes quantitative analyses of ethanol in biological samples (blood, urine and aqueous/vitreous humor). Analysis of ethanol in biological samples is performed using the HS-GC-FID method (gas chromatography with a headspace injector and a flame ionization detector).
Analysis of carboxyhemoglobin (CO-Hb) in biological samples
Carboxyhemoglobin (CO-Hb) testing in biological samples, including quantitative determination of CO-Hb in blood, is carried out using the UV-VIS spectrophotometry method.
Interlaboratory comparisons
Regular participation in interlaboratory comparative tests is the one way to ensure the quality of test results. Therefore, experts and technicians in the field of toxicological expertise participate annually in:
Experts in the field of toxicological expertise regularly participate in the education of police officers for performing drug-related police work. They also participate in the education and the training of employees of other state bodies and institutions regarding drug issues and the emergence of new drugs in Croatia.
They have been involved in the development of the List of Drugs, Psychotropic Substances and Plants from which drugs can be obtained and Substances that can be used for the production of drugs, and its amendments, for many years. They participate in the work of various expert bodies dealing with the issue of drug abuse, of which the following working groups within the National Drug Information System are worth highlighting:
The task of the working groups is to advise the Addiction Service of the Croatian Institute of Public Health on the implementation of the general strategy of the National Drug Information System, on methods of data collection and analysis, on the communication strategy and further development, on current topics and trends, as well as proposing activities and measures and participating in the preparation of documents and professional literature.
In the field of toxicological expertise, there is also the National Contact Point for the Transfer and Analysis of Drug Samples (NCP). The task of the NCP is to send drug samples and exchange data on analyzed samples.
Destruction of seized drugs
Toxicology experts, in collaboration with the Commission for the Destruction of Seized Drugs, are actively involved in both the preparation and execution of the incineration of the seized drugs.
Accredited methods
The methods that are accredited in the flexible area according to the international standard HRN EN ISO/IEC 17025:2017 are:
1. Qualitative/semiquantitative analysis of drugs by TLC method
2. Quantitative analysis of THC by GC-FID method
3. Qualitative analysis of drugs by FTIR method
4. Qualitative analysis of drugs by GC-MS method
5. Quantitative analysis of drugs by HPLC method
6. Quantitative analysis of ethanol in biological samples by HS-GC-FID method
The first toxicological analyses at the Center were recorded in 1958 when food and pharmaceutical preparations were examined. With advancements in forensics and the increase in the quantities of seized drugs, thin layer chromatography (TLC) was introduced into daily use in 1963 as a simple and selective method for qualitative drug analysis. At about the same time, two methods for the determination of ethanol in biological samples were introduced: the Widmark method and the enzymatic (ADH) method on a UV spectrophotometer. Due to certain practical shortcomings of these methods in the determination of ethanol, in 1969 the first analyses using gas chromatography method (GC-FID) were carried out, which mainly included quantitative analysis of ethanol in blood and urine, and, if needed, to the analysis of illegal drugs. A little later, gas chromatography coupled with mass spectrometry method (GC-MS) was introduced and is still considered one of the most suitable methods for the qualitative determination of drugs.
By moving to its current location in 1997, the Center acquired new modern laboratories that were fully adapted to the needs and requirements of toxicological analyses. The acquisition of new instruments was intensified, computer systems for automatic data processing were introduced, experts were educated in related institutes around the world, and new methods were developed for the quantitative determination of the most common drugs by High Performance Liquid Chromatography (HPLC) and for the qualitative determination of drugs in biological samples by the GC-MS method. As a result of all the above activities, comparative analyses have been conducted since 2002 (initially of heroin and cocaine samples, and later also amphetamines), which combined developed analytical methods upgraded with the specialized expert’s opinion.
Participation in projects financed from the EU pre-accession funds has further modernized the toxicology laboratory, both in terms of equipment and new methods. The most important project was "IPA 2007 - Strengthening Capacities of the Ministry of the Interior to Combat Narcotic Drugs Trafficking and Drug Abuse", through which eleven new instruments were purchased and numerous new methods were developed, the most important of which are methods for chemical profiling of heroin, cocaine and amphetamines and quantitative determination of drugs in biological samples.
Analysis of seized drugs
Qualitative analyses, i.e. the identification of seized drugs and psychotropic substances (in the form of powder, commercial and non-commercial tablets and capsules, liquids, etc.) and the detection of drug traces on objects, for example in syringes and scales, are carried out using Thin-layer Chromatography (TLC), Fourier transform Infrared Spectrophotometry (FTIR) and the associated Gas chromatography-Mass spectrometry (GC-MS) systems. Quantitative analyses, i.e. determination of the purity of sized drugs and psychotropic substances, are carried out using High Performance Liquid Chromatography (HPLC) and gas chromatography whit flame ionization detector methods (GC-FID).
Comparative analysis
Comparative analysis of heroin, cocaine, methamphetamine, MDMA and amphetamine include methods that determine the main components, cutting agents, and characteristic impurities in samples and that compare their content and ratios.
In addition to comparative analysis, chemical profiling is also performed to determine the level of probability that different samples originate from the same source. Chemical profiling of heroin is performed by comparing the ratios of chemical compounds from opium and impurities created during heroin production. Comparative analysis of heroin is performed using FTIR, GC-MS, HPLC, and GC-FID methods.
In the chemical profiling of cocaine, the ratios of trace amounts of organic solvents used in the production of cocaine are compared. Comparative analyses of cocaine are performed using the FTIR, GC-MS, HPLC and HS-GC-FID methods. Chemical profiling of amphetamine, methamphetamine and MDMA is conducted by comparing the proportions and ratios of intermediate products and the impurities formed in the synthesis process of a particular compound. Comparative analyses of amphetamine, methamphetamine and MDMA are carried out using the FTIR, GC-MS and HPLC methods.
Pesticide expertise
Pesticide analysis in plant, powder, liquid and other types of samples and biological samples (for example in gastric contents and liver) includes qualitative and quantitative analysis, i.e., identification and quantification of pesticides. These analyses are carried out using GC-MS and LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) methods.
Drug testing in biological samples
Expert analyses on drugs in biological samples include qualitative and quantitative methods, i.e., the identification and concentration of drugs, psychotropic substances, medicines and its metabolites mostly in blood and urine, but other body tissues and secretions as well. Preliminary analyses of drugs in biological samples are carried out by the enzyme immunological method and then confirmed by GC-MS and LC-MS/MS methods.
Ethanol analysis in biological samples
Ethanol analysis in biological samples includes quantitative analyses of ethanol in biological samples (blood, urine and aqueous/vitreous humor). Analysis of ethanol in biological samples is performed using the HS-GC-FID method (gas chromatography with a headspace injector and a flame ionization detector).
Analysis of carboxyhemoglobin (CO-Hb) in biological samples
Carboxyhemoglobin (CO-Hb) testing in biological samples, including quantitative determination of CO-Hb in blood, is carried out using the UV-VIS spectrophotometry method.
Interlaboratory comparisons
Regular participation in interlaboratory comparative tests is the one way to ensure the quality of test results. Therefore, experts and technicians in the field of toxicological expertise participate annually in:
- two UNODC ICE (United Nations Office on Drugs and Crime International Collaborative Exercises) interlaboratory tests for the qualitative and quantitative determination of drugs in sized substances and in two tests for the qualitative and quantitative determination of drugs in biological samples,
- ENFSI proficiency testing for the identification and quantification of drugs in seized materials,
- two Forensic Testing Programs "Referenz Institut für Bioanalytik" for quantitative determination of ethanol in blood.
Experts in the field of toxicological expertise regularly participate in the education of police officers for performing drug-related police work. They also participate in the education and the training of employees of other state bodies and institutions regarding drug issues and the emergence of new drugs in Croatia.
They have been involved in the development of the List of Drugs, Psychotropic Substances and Plants from which drugs can be obtained and Substances that can be used for the production of drugs, and its amendments, for many years. They participate in the work of various expert bodies dealing with the issue of drug abuse, of which the following working groups within the National Drug Information System are worth highlighting:
- Drug-Related Deaths and Mortality among Addicts
- Drug Crime Data
- Early Warning System for the appearance of a new psychoactive substance in Croatia.
The task of the working groups is to advise the Addiction Service of the Croatian Institute of Public Health on the implementation of the general strategy of the National Drug Information System, on methods of data collection and analysis, on the communication strategy and further development, on current topics and trends, as well as proposing activities and measures and participating in the preparation of documents and professional literature.
In the field of toxicological expertise, there is also the National Contact Point for the Transfer and Analysis of Drug Samples (NCP). The task of the NCP is to send drug samples and exchange data on analyzed samples.
Destruction of seized drugs
Toxicology experts, in collaboration with the Commission for the Destruction of Seized Drugs, are actively involved in both the preparation and execution of the incineration of the seized drugs.
Accredited methods
The methods that are accredited in the flexible area according to the international standard HRN EN ISO/IEC 17025:2017 are:
1. Qualitative/semiquantitative analysis of drugs by TLC method
2. Quantitative analysis of THC by GC-FID method
3. Qualitative analysis of drugs by FTIR method
4. Qualitative analysis of drugs by GC-MS method
5. Quantitative analysis of drugs by HPLC method
6. Quantitative analysis of ethanol in biological samples by HS-GC-FID method