Mário Dias EMCDDA Key Indicator Annual Expert Meeting Lisbon, 18 20 September 1
The National Institute of Legal Medicine and Forensic Sciences (NILMFC) is a public institute, endowed with corporate entity and administrative and financial autonomy, subject to the supervision and guardianship of the Minister of Justice. It is an institution responsible for the co-ordination, guidance and supervision of all the country activities related with forensic expertises and other forensic activities.
65.000 analysis / year GC-MS (8) GC-MS/MS (1) UPLC- MS/MS (3) LC-MS/MS (1) - Post mortem Toxicology - Clinical Toxicology (Emergence; Therapeutic Drug Monitoring) - Human Performance Toxicology (Workplace Drug Testing; Driving)
Information Flow of Special Mortality Registries EMCDDA, 18 20 September 2017
Unnatural / violent deaths Police investigation / hospital information Magistrate of Public Ministry (According police investigation and hospital information) Waive of medico legal autopsy Medico legal autopsy (NILMFC*) Electronic Death Certificate (SICO) Ministry of Health General Mortality Registers Toxicology request (NILMCF*) Cause of death (NILMCF*) Special Mortality Regist (NILMCF*) Electronic Death Certificate (SICO) Ministry of Health General Mortality Registers Autopsy report Magistrate of Public Ministry Ministry of Justice National Focal Point (SICAD) Ministry of Health *NILMCF National Institute of Legal Medicine and Forensic Sciences (Ministry of Justice)
RESULTS
8% Positive cases for DOA - Distribution by Illicit Substance ( n = 7575 ) 17,89% 23,10% 0,80% 0,80% 0,93% 2,54% Morfina 0,80% Codeína 0,40% 2,27% 1,87% 1,74% 6-MAM 8,81% Metadona EDDP 7,74% Benzoilecgona EME Cocaína Cocaetileno 4,81% 0,53% THCCOOH 11-OH-THC THC Anfetamina Efedrina MDA 24,97% MDMA
10 % 8 %
What reality are we seeing through these numbers
Magistrate of Public Ministry: Waive of medico legal autopsy Pathologist: Request or not drug testing Toxicologist: Target substances and cut-off values (Screening); Criteria for identification and limits of detection (Confirmation) Pathologist: Interpretation of results and reference values
TOXICOLOGY ANALYSIS
SAMPLING AND STORAGE Peripheral Blood (femoral) Central Blood (cardiac) Urine Gastric Content Vitreous Humor 10 ml 20-30 ml 30 ml 50 g 1 ml Post Mortem Interval (PMI) < 48 h Samples collected to Polypropylene (PP) tubes Blood samples preserved with Sodium Fluoride Storage at 10 ºC
TARGET SUBSTANCES Prevalence of substances Equipment available (Screening and Confirmation) Budget SCREENING (IMMUNOASSAY) Cut-Off Selectivity Specificity Validated Methods (cut-off) Opiates 50 ng/ml Cocaine 50 ng/ml Cannabis 50 ng/ml Amphetamine 50 ng/ml Methamphetamine 50 ng/ml Benzodiazepines 10 ng/ml
IDENTITY CONFIRMATION OF THE ANALYTES (GC; LC; UPLC) Quadrupole (MS) Triple Quadrupole (MS/MS) Validated Methods (International Standards) CHROMATOGRAPHIC CRITERIA The Relative Retention Time (RRT) of the peak of interest is measured relative to a chromatographic reference compound (CRC): When the CRC is not a stable isotope-labeled analyte, the RRT of the analyte in the Sample shall not differ by more than ± 1% from that of the same analyte in a Positive Spyked Sample or Reference Material analyzed in the same analytical batch. When the CRC is the stable isotope-labeled analyte, the RRT of the analyte in the Sample shall not differ by more than ± 0.5% from that of the same analyte in a Positive Spiked Sample or Reference Material analyzed in the same analytical batch.
IDENTITY CONFIRMATION OF THE ANALYTES (GC; LC; UPLC) Quadrupole (MS) Triple Quadrupole (MS/MS) Validated Methods (International Standards) MASS SPECTROMETRIC IDENTIFICATION CRITERIA MS criteria for identification are based on the presence and relative abundance of a number of ions defined as diagnostic for the analyte during validation of the method. The following identification criteria are applied: When using single-stage MS, at least three (3) diagnostic ions shall be acquired When using multiple-stage MS (e.g. MS/MS), at least two (2) precursor-product ion transitions (i.e. two SRM transitions) shall be monitored. The signal-to-noise (S/N) ratio of all diagnostic ions shall be greater than three to one (3:1)
IDENTITY CONFIRMATION OF THE ANALYTES (GC; LC; UPLC) Quadrupole (MS) Triple Quadrupole (MS/MS) Validated Methods (International Standards) MASS SPECTROMETRIC IDENTIFICATION CRITERIA The Relative Abundance of any of the diagnostic ions shall not differ by more than the amount specified in the following Table from the corresponding Relative Abundances of the same ions acquired from a Spiked Positive Control Sample or Reference Material. Relative Abundance in the reference sample (% of base peak) Maximum tolerance windows for the relative abundance in the sample 50-100 10 (absolute) Relative abundance (% of peak base) 60 95 Example Tolerance window (% of peak base) 50-70 85-105 25-50 20 % (relative) 40 32-48 1-25 5 (absolute) 10 3 5-15 > 0-8
QUALITY CONTROL INTERNAL QUALITY CONTROL (IQC) The preparation of the control samples and the interpretation of results are handled within the laboratory Includes personal, instrumentation, document control, reagent control and corrective actions It s used on daily basis in decision to accept or reject results, and enables the laboratory monitor the quality of its work EXTERNAL QUALITY CONTROL (EQC) The estimation of a test method s accuracy is obtain from the results of analysis of unknown samples sent to different laboratories Permits a comparison of quality between laboratories and it is used to confirm results of IQC
In vivo INTERPRETATION OF RESULTS Post-mortem
INTERPRETATION OF THE RESULTS (REFERENCE VALUES)
INTERPRETATION OF THE RESULTS (REFERENCE VALUES) Sampling (sample type and storage) Specimen collection (post-mortem interval) Stability of the substances Post-mortem redistribution Acute or chronic poisoning Tolerance Metabolism (parent compound and active metabolites) Association of substances (Drug + Drug; Drug + Alcohol) Analytical methods (specificity, sensitivity, LOD and LOQ) Inter-individual variability (pharmacogenetics) Association of substances Age and sex Other pathologies
CONCLUSIONS More uniform analytical approaches will allow the collection of more robust data concerning DRD and provides important contribution to both public safety and public health. To allow the comparability of the results it will be important to elaborate recommendations for operation standards for laboratories involved in DRD toxicological analysis that should include: Sampling and Storage Sample preparation Method validation Core substances Screening methods Confirmation methods Minimum required performance levels for detection and identification