POSTMORTEM TOXICOLOGY

Farmasi Forensik
• To determine whether alcohol, drugs, or other poisons may have caused or
contributed to the death of person
• Clinical assays need to be modified to give acceptable results with the unique
fluids and tissues
• Whole blood contributes a large number of endogeneous compounds that are
at much lower concentrations (fatty acids, cholesterol, sterols)
 REQUEST, RECEIPT, AND STORAGE

• Tanggung jawab lab untuk memastikan klien (coroner, dokter forensic, pengacara,
pathologist) jenis dan jumlah specimen yang dibutukan untuk uji toksikologi
postmortem , serta pengawet apa yang harus digunakan (bila perlu)
• Setidaknya 1 tube darah utuh yang ditambah 1% sodium fluoride harus disediakan
untuk uji etanol dan kokain
• Isi lambung dan jaringan tidak diberikan pengawet
• Daftar permintaan bertujuan untuk:
• Identifikasi korban. Memberikan informasi demografik dan sejarah kasus (circumstance of death,
relevant medical history, autopsy finding)
• Identikasi specimen spesifik yang diberikan
• Memberikan ruang identifikasi pada uji yang diperlukan
• Identifikasi orang yang memberikan data dan menjaga chain-of-custody
• Memberikan petunjuk dan informasi untuk pengemasan dan transportasi spesimen
 SPECIMEN TYPES (1)

• May be numerous or limited : depend on the case history and preferences of the
submitter
• Recent death: blood, vitreous humor, at least one organ tissue (liver), gastric
contests
• Decomposed cases: muscle, hair, bone
• Toxicology testing usually limited to those where there is appropriate database
available
• Proper collection and preservation of postmortem specimens is critical: why?
 SPECIMEN TYPES (2)

• Blood:
• sampling by syringe and large-gauge needle
• Postmortem blood concentrations of many drugs vary from site to site  collection site must be chose wisely
• Label must be appropriate and detail
• Vitreous humor (volume +/- 3 mL)
• Postmortem confirmation of the ingestion of ethanol
• Measurement of drugs: e.g. digoxin, cocaine, morphine
• Disadvantage: volume is small, little information on the concentration expected after therapeutic doses for most drugs
 SPECIMEN TYPES (3)

• Urine:
• Comprises more than 99% water, contains relatively few endogenous substances interfering with chromatography or
immunoassay tests
• Disadvantage:
• Urine is only available in about 50% of deaths
• Many drugs are metabolized extensively that the parent drug is not detected in urine
• Urinary concentrations of most drugs are difficult to interpret  Why?
• Liver:
• Most important tissue to be collected and analysed in post-mortem toxicology
• Large amount of tissue available, ease of collection, relative ease sample preparation
• Relatively large database of liver drug concentrations in literature
 SPECIMEN TYPES (4)

• Liver (continued)
• Concentrations of many basic drugs are also higher in the liver compared to blood  detection easier
• Concentration of drugs in the liver after death  relatively stable
• Stomach Contents:
• After over-dosage, drug concentrations in the stomach may be quite high, even after the majority of the drug has passed into the
small intestine
• Analysis is uncomplicated by metabolism
• Drugs that may be difficult to detect in the blood (due to extensive distribution) : detected readily in stomach
 SPECIMEN TYPES (5)

• Stomach content (continued):

• Disadvantage: c
• omposition varies from a thin watery fluid to semi-solid,
• stomach contents are rarely homogeneous (difficult to measure accurately the representative concentration of
drug in the volume of stomach contents revealed)
• Total stomach contents often not sent to the lab  accuracy of dose estimation can be questioned
• Gastric drug concentrations should neverbe interpreted on the ame basis as those for blood
• Other fluids,tissues, and organs
• Nasal swab
• Antemortem specimen
 SCOPE OF TESTING

• Common practice: search for the common drugs of abuse, prescription and
non-prescription drugs, followed, by specific analyses as indicated by the case
history
 SPECIMEN PREPARATION &
EXTRACTION

• 1st stage: separation of drug/compound of interest from biological matrix

(except from urine and other non-viscous fluid specimen)
• Volumetric measurement with positive displacement pipette designed for
viscous sample or gravimetric sampling
• Use of standard glass pipettes is discouraged  inaccurate
• LLE or SPE  appropriate procedures to extract drugs from urine/blood
• Clotted blood may be homogenized in water or buffer prior to analysis
• Extraction of drugs from tissue  tissue matrix must be broken down
(homogenization, acid/alkaline hydrolysis, or enzyme digeston)
• Use of protein-precipitation reagents (Barium chloride, zinc sulfate, tungstic
acid) discouraged for quantitative work  significant portion of the analyte
may be co-precipitated with the coagulated protein
 SCREENING & DETECTION

• Postmortem toxicological analysis starts with a drug screen

• Drug screen cannot be a single test, most commonly is an open-ended panel of
tests designed to detect the maximum number of substances of toxicological
interest
• Targeted testing sometimes justified where the case history strongly indicates
a specific substance is involved  still there’s problem with it. What is it?
• Drug screen consists of a panel of immunoassay tests and headspace analysis
for alcohol & other volatiles, combined with one or more broad-based GC or
HPLC .
 FORENSIC IDENTIFICATION &
CONFIRMATION

• Principle: forensic identification of an analyte requires the use of two

techniques that employ different physical and chemical principles (SOFT/AAFS
Guidelines Committee 2002)
• Advantage: supportive in arriving positive result
• Identification of a drug by the use of two similar methods, such as 2 different
immunoassay  not acceptable (even though tests may employ different
endpoint reactins)
 QUANTIFICATION OF DRUGS AND
OTHER TOXINS

• Usually quantified using GC or HPLC based on the total peak area

• SIM GC-MS (Selected ion monitoring): more specific & sensitive
• Important in postmortem work  endogeneous lipids and putrefactive products can
produce significant interference
• Major considerations:
• Reproducibility & robustness of the extraction procedure
• Choice of an appropriate calibration method
 CALIBRATION METHODS

• Choice of an appropriate calibration method is critical to obtaining reliable results.

• Single-point calibrations : unacceptable for postmortem toxicology
• Unless it can be shown that the calibration is table & linear over the dsired range
• A minimum of three calibration points is usually recommended, although 5 or more are preferred (depending on
the linearity)
• An appropriate internal standard is considered essential to minimize matrix effect and correct other variable
• If analyte concentration lies outside that range: it may either reported as greater than or less than the alibration
range
• OR additional calibration range/calibrato/control could be run to validate
• Standard curve should not be extrapolated and if an accurate result is required  specimen should be diluted and
re-analysed