1. Postmortem Forensic Toxicology
Teri Martin
September 23, 2003

2. Outline Definitions and purpose of postmortem tox
Samples of forensic interest
Handling and storage of samples
Pitfalls in postmortem toxicology
Interpretation of results
Outline
Definitions and purpose of postmortem tox
What is postmortem forensic toxicology? What are the boundaries of postmortem forensic toxicology and the role of the toxicologist in postmortem cases?
Samples of Forensic Interest
What samples are the most relevant to the forensic toxicologist?
Handling and storage of samples
How should postmortem forensic toxicology samples be stored and handled? And why should we care about how postmortem forensic toxicology samples are stored and handled?
Pitfalls in postmortem toxicology
What are some of the most common problems in postmortem forensic toxicology? How do factors such as putrefaction, decomposition and redistribution of drugs after death affect the ability of the forensic toxicologist to draw conclusions?
Interpretation of results
What are the limitations of the toxicologist in reporting results from postmortem blood samples? What are some of the interesting pieces of information that the toxicologist is able to give to the pathologist in order to help them determine cause and circumstances of death - as examples - discuss acute v. chronic ingestion of drugs and identify pathological changes that occur following drug overdose.

3. Postmortem Forensic Toxicology
Qualitative and quantitative analysis of drugs or poisons in biological specimens collected at autopsy
Interpretation of findings in terms of:
Physiological effect at time of death
Behavioural effect at time of death
How might one define postmortem forensic toxicology?
In terms of purpose:
The purpose of postmortem forensic toxicology is to perform qualitative and quantitative analysis for drugs and their metabolites, and poisons such as metals, carbon monoxide, and volatile substances in human fluids and tissues collected after death.
The postmortem forensic toxicologist will then evaluate the role of drugs or poisons as either determining factors or contributory factors in the death of the individual. This includes interpretation as to what the physiological effect at time of death might have been (e.g. acute toxicity such as respiratory depression) and/or what the behavioural effect at the time of death may have been (e.g. impairment leading to drowning, motor vehicle collision or other traumatic cause of death).

4. Quantitative vs. Qualitative
Qualitative analysis – determines the presence or absence of a drug or poison in a submitted sample
Quantitative analysis – determines the amount of drug or poison that is present in the submitted sample
Two important definitions - qualitative versus quantitative analysis.
Qualitative analysis simply determines the presence or absence of a drug or poison in a submitted sample, basically answering the question – was this person exposed to a drug or poison?
Quantitative analysis actually determines the amount of drug or poison that is present in the sample. In other words, you are determining the quantity of drug that is present – this is a memory tool to remember the difference between quantitative and qualitative analysis (determining quantity is quantitative analysis).
Quantitative analysis is always preferable as it is difficult to make a meaningful interpretation based solely on the presence or absence of a drug or poison. However, in some situations qualitative analysis may be all that is meaningful due to the nature of the submitted sample, or qualitative analysis may be a useful starting point in order to direct further work. For example, many initial drug screens are qualitative in their analysis; it is the confirmatory method that is quantitative in its nature.

5. Postmortem Forensic Toxicology
Types of cases:
Suspected drug intoxication cases
Fire deaths
Homicides
Driver and pilot fatalities
Therapeutic drug monitoring
Sudden infant death (SIDS)
These are the types of cases that typically fall into the realm of postmortem forensic toxicology.
Unexplained deaths, with no apparent cause (often suspected to be drug intoxication cases) as well as those that are strongly suspected of being drug intoxication cases.
Fire deaths – measurment of toxic gases such as carbon monoxide and cyanide which may be inhaled during a fire. Furthermore, drugs may be implicated as having incapacitated a victim, thereby preventing their escape from a fire.
Homicides – homicidal poisonings themselves, are rare – but many homicide cases are related to drug use and drug abuse.
Driver and pilot fatalities – where drug impairment may help determine the cause of a crash. Other traumatic causes of death will also require postmortem toxicology analysis (such as drownings, falls)
Therapeutic drug monitoring – for example, determining whether an individual with a seizure disorder has been compliant in their medication use.
SIDS – by definition, SIDS is a diagnosis of exclusion. Therefore, toxicology must be comprehensive in these case to rule out any other cause of death.

6. Samples of Forensic Interest

7. Issues in Specimen Collection
Selection
Multiple, varied sites of collection
Collection
Appropriate method of collection
Adequate volumes for analysis
Storage and handling
Important to ensure analytical results are accurate and interpretations are sound
In postmortem forensic toxicology, the onus for specimen collection lies entirely on the pathologist performing the autopsy. Since it is out of the toxicologist’s hands, we can only hope that the pathologist has properly selected the most appropriate samples for analysis and collected them using good technique.
In terms of selection, in the most general sense, the pathologist should collect samples from a variety of sources (example blood, urine, liver, stomach contents, hair) and should collect them in sufficient quantity for analysis. We will further discuss which samples are the best samples for analysis and ultimately for interpretation of findings in postmortem cases. For example, we’ll talk about how some blood sampling sites are preferable over others.
Method of collection is also important. Has the pathologist performed a full autopsy or only an external autopsy? In the case of an external autopsy, there is a danger that the pathologist, in attempting to collect a heart blood sample may actually be sampling pericardial fluid. For interpretive purposes the ideal toxicological blood sample is a specimen that is obtained from a ligated vessel immediately after death (in other words, the vein should be selected, clamped off and then sampled. In reality, most autopsy samples do fall short of this ideal.
Another issue is adequacy of collection – comprehensive drug testing may require 20 – 30 mL of blood, so the pathologist should ensure they have collected enough sample to meet the needs of the investigation.
Storage and handling are also important aspects of specimen collection. We’ll talk about some of the conditions under which samples for toxicology are stored and some of the criteria under which samples should be stored.

8. Typical autopsy specimens
Blood
Urine
Stomach contents
Bile
Liver
Hair
Vitreous humor

9. Blood Antemortem  ideal blood sample
Postmortem blood is not truly “blood”
Anatomical site of collection at autopsy should be noted
The best possible blood sample, even in postmortem forensic toxicology is an antemortem blood sample. In the case of postmortem forensic toxicology, this antemortem blood sample would ideally be a blood sample that is collected just prior to the expiration of the patient. Occasionally these samples are available - for example, in cases where the deceased was rushed to hospital and where blood was collected in the hospital emergency room.
What we are usually faced with, however, is an analysis in postmortem blood samples. Something to keep in mind is that postmortem blood is not truly blood. It may look like blood - but in fact it is not - it is a fluid, from the vasculature that is collected after death. Postmortem blood however has already begun to break down and hemolyze and is not the same as an antemortem blood sample.
As will become clear through discussions of the limitations and pitfalls of postmortem forensic toxicology, the anatomical site of collection at autopsy should be noted and MUST be accurate (for an appropriate interpretation of the blood drug concentrations).
Example: “heart blood” collected from a patient who was an organ donor and whose heart was taken for transplantation purposes in hospital.

10. Central sites Peripheral sites Other sites Heart Femoral Iliac
Subclavian
Other sites
Head blood
Hematoma blood
Subclavian
Heart
Iliac
Femoral
Examples of central blood collection sites and peripheral blood collection sites. Ideally, blood should be collected from both a central site, such as from the chambers of the heart – in addition to one or more samples being collected from a peripheral site. The most common peripheral sites of blood collection are femoral, iliac, and subclavian (emphasis on femoral). [No, students do not need to know exact location of these veins in the body, this picture is for reference only.]
Other sites of blood sample collection:
“Head blood” – rarely seen in adults, but is not an uncommon site of selection in babies, especially since in infants it is difficult to obtain blood from the peripheral sites and blood volume of the heart is small.
Hematoma blood – a hematoma is a blood clot, formed extravascularly (outside the circulating blood system). Because it is outside the blood system, any drugs or poisons that are contained in the hematoma are actually protected from metabolism. This can be important in providing information as to what drugs were present in the system at the time of hematoma formation.
Analysis of hematoma blood has has proven especially useful with alcohol…

11. Hematoma Extravascular blood clot Protected from metabolism
Analysis will indicate what drugs were present in the blood at the time of formation

12. Hematoma case example
A 26 year old man was found dead at the bottom of a staircase. Death was due to physical injuries.
Question as to alcohol use prior to fall down stairs
No urine available at autopsy
Alcohol not detected in femoral blood
Alcohol in hematoma blood  150 mg/100 mL
The deceased had been drinking prior to receiving the head trauma.
The deceased had survived for several hours after the injury.
For example…
These results indicate that the deceased had been drinking prior to receiving the head trauma. Since ethanol is detected in the hematoma, but not in the femoral blood, you can also conclude that this individual survived for a substantial period of time AFTER receiving the head trauma but BEFORE death occurred. In the case of alcohol, the rate of elimination of alcohol from the blood is very well know and I can predict that this individual would have had to survive between 7 and 15 hours after hematoma formation in order to clear the femoral blood of alcohol.
Caution: Hematomas may not form immediately upon receiving an injury. Therefore this hematoma alcohol concentration will not necessarily indicate the BAC at the time of the accident….more appropriate to state the hematoma alcohol concentratoin indicates the BAC at the time of hematoma formation.

13. Hematoma
Caution: There may be a delay between the incident which resulted in hematoma and the actual formation of the hematoma
Therefore, this alcohol concentration does not necessarily indicate the BAC at the time of the fall down the stairs.

14. Urine Produced by the kidneys Blood filtered by the kidneys
Stored in the bladder until voided
Qualitative - the presence of a drug in the urine of an individual indicates that some time prior to death the drug or poison was present in the blood of the individual
Urine is produced by the kidneys as a result of blood filtration. Along with other wastes, drugs and their metabolites are filtered by the kidneys and are contained in urine. Urine, is stored in the bladder until voided. It may sound weird to think of it his way – but urine, since it is stored in the bladder is actually “outside of the body” – and it is no longer subjected to metabolism.
Since urine is stored for a period of time before it is expelled, the presence of a drug in the urine does not necessarily mean the drug was present in the blood at the time of death. It simply means that some time prior to death the drug or poison was present in the blood.
In this way, urine analysis – in isolation of blood analysis is of limited value.

15. Stomach contents Visual examination may reveal tablets
Drugs that have been orally ingested may be detected in stomach contents
Caution: drugs administered by other routes may also diffuse into stomach contents from the blood
Generally qualitative:
Stomach contents are not homogeneous
Only a portion of stomach contents collected (unmixed?)
Useful for directing further analysis
A portion of the stomach contents are typically collected at autopsy. Stomach contents may contain unabsorbed poisons, tablets, capsules, caplets which may be intact and visible. These can be removed from the stomach contents, photographed, and identified.
Drugs in the stomach contents do not necessarily indicate oral ingestion!
Many weak bases are subject to secretion into the gastric contents due to the pH gradient between plasma and the stomach. Ion trapping in the stomach produces very high concentrations of these agents, even after intravenous or other non-oral routes of administration. Thus the finding of even appreciable amounts of a basic drug in the gastric contents is not a reliable indication of oral ingestion.
Can you quantify stomach contents to determine if a person has taken a drug overdose?
Stomach contents are not homogenous in their nature.
Correct procedure of collecting stomach contents is for the pathologist to ligate the stomach, remove it from the body, remove all stomach contents, mix thoroughly and provide a small quantity to the toxicology lab – again – this is rarely, if ever, done.
If stomach contents were collected properly, quantitation may be valid and may be useful…however, in light of the fact that stomach contents are typically a scooped, non-homogenous sample, it is not acceptable practice to quantify the contents and make any valid interpretation from the results.

16. Case Example A 26 year old woman is found dead in bed
Numerous medications in her home:
Amitriptyline, Oxycodone, Morphine, Paroxetine, Diphenhydramine, Pseudoephedrine, Phenobarbital, Codeine, Temazepam, Diazepam
Only 3 mL of blood collected at autopsy
Qualitative analysis of stomach contents:
Amitriptyline: detected
Nortriptyline: detected
Quantitation can now be performed in blood
Point out that 3 mL of blood is not a lot of blood for analyses – in fact, probably only enough blood to perform one or two quantitative analyses.

17. Liver Drug metabolism occurs in the liver
Both parent compounds and metabolites may be present in higher concentrations in the liver than in the blood  ease of detection
Limitation is that drugs are not uniformly distributed throughout the liver  confounds interpretation
A sample of liver (not the whole liver) is also typically collected at autopsy and submitted to the tox lab.
Liver is the main drug metabolizing organ. As such, most drugs and poisons that enter the blood stream will travel to the liver for biotransformation. Both parent compounds (the drug that is ingested) and their metabolites may not only be present in the liver, but may also be present in higher concentrations than in the blood – this may help to identify drugs that are present in quantities that are below detection limits in the blood.
Quantitative analysis of liver can be performed, however it is very difficult to make an interpretation with any certainty from liver analysis. Drugs are not uniformly distributed throughout the liver and therefore, where you sample the liver for drug analysis may have an effect on the quantity of drug that is identified.

18. Bile Digestive secretion Continuously produced by the liver
Stored in the gallbladder
Qualitative - the presence of a drug in the bile of an individual indicates that sometime prior to death, the individual was exposed to the drug
Bile is another sample that is often collected at autopsy. Bile is a digestive secretion that is continously produced by the liver and stored in the gallbladder. [Not the policy of the CFS tox lab to analyze bile samples]; however some labs do perform analysis in bile and some labs also quantify the amount of drug in bile (although I would caution that bile rarely has a quantitiative value). Since it is produced by the liver, it is similar in respect to the liver samples in that concentrations of drugs in the bile are usually greater than concentrations in the blood. Therefore the duration of detection of a drug may be increased in the bile compared to the blood.
As with any quantitative analysis, however, the presence of a drug in the bile – simply indicates that sometime prior to death the individual was exposed to the drug.

19. Vitreous humor
Fluid that occupies the space between the lens and the retina of the eye.
Sequestered from putrefaction, charring and trauma, microorganisms.
Useful in cases where decomposition is advanced, body is exhumed or in fire deaths
Limitation is blood:vitreous ratio may not be known
Vitreous humor is in a protected position behind the lens of the eye. Because of this protected position, it is isolated from putrefactive processes, from charring and from trauma – for example, the vitreous humor can be obtained intact even if a corpse has been extensively burned or damaged. Blood is very susceptible to postmortem changes (more on this later) – however the vitreous fluid is less susceptible to these effects, particularly because it is likely to be free from microorganisms.
Particularly useful for determining alcohol concentrations and may be useful for determining some drug concentrations - depending on whether or not the blood:vitreous ratio of the drug is known.

20. Hair Recent specimen of interest Metabolism does not occur in hair
Can provide a historical record of drug or poison exposure
Pros and cons of hair analysis still being uncovered  racial variability?
Hair analysis – has, in recent years, become a new interest in postmortem forensic toxicology. Drugs and poisons are stable in hair – that is, they are not subject to metabolism or other non-enzymatic degradation.
Since hair grows at a predictable rate (generally 1 cm/month) – hair can be used to provide a historical record of drug or poison exposure. Procedure is to chop a hair sample into 1 cm increments and analyze them separately to “track” drug exposure over a long period of time (as long as the hair will allow, basically).
Pros and cons of hair analysis are still being uncovered – for example there have been studies that have shown racial variability in the binding of drugs to hair. It is a developing area of research in forensics right now [not something that we at the CFS do]

21. Case Example 30 year old woman, previously in good health
Poklis, A Abstract SOFT, Dearborn, Michigan.
30 year old woman, previously in good health
Nausea, vomiting, diarrhea, rash, fever
Weakness in hands and feet  Guillian Barre?
Hospitalized with hypotension, seizures
Misplaced laboratory result  Arsenic!
Sequential hair analysis for arsenic showed chronic arsenic poisoning over 8 month period
A case of homicide by chronic arsenic poisoning. The victim was a 30 year old mother of two who was in excellent health until 8 months prior to her death when she developed an apparent viral syndrome characterized by persistent nausea, vomiting and diarrhea with low grade fever and rash. Within 2 weeks she developed a symmetrical parasthesia and weakness in both her hands and feet. These symptoms progressed and resulted in a diagnosis of Guillian Barre syndrome. During the following 2 months she had several episodes of severe g.I. distress. Two weeks prior to her death she was hospitalized for mental confusion, hypotension and seizures. Her condition improved until she had dinner with her husband after which she developed severe gastrointestinal distress. Her condition then gradually deteriorated until death.
A misplaced laboratory result found after her death indicated an arsenic concentration of 2.1 mg/100 mL.

22. Non-biological submissions
Used to direct analysis of biologicals
May indicate the nature of substances that may have been ingested, inhaled or injected
Examples:
Containers found at the scene
Syringes
Unidentified tablets or liquids
Case example of the veterinarian who was found seated in a chair in his office, intravenous infusion machine attached to empty I.v. bag, and needle in left antecubital fossa. As a veterinarian, this individual had access to a number of drugs – analysis of the I.v. bag showed it to contain phenobarbital and isopropanol. Blood analysis then revealed a fatal blood concentration of phenobarbital and also a potentially fatal level of isopropanol (although it was likely simply meant to be the vehicle for the phenobarbital).

23. Autopsy specimens of limited value
Pleural fluid
Chest cavity blood
Gutter blood
Samples taken after embalming
Samples taken after transfusion in hospital
Pleural fluid – fluid taken from the space surrounding the lungs.
Chest cavity blood - trauma to the chest, heart is no longer intact, basically a “scooped” sample.
Gutter blood - blood that is scooped from the gutter of the autopsy table in cases of severely decomposed bodies (bodies that are decomposed to the point of liquefaction). May also be collected by irresponsible pathologists in non-decomposed cases (a slice and drain technique)
Specimens collected after embalming - process may dilute or destroy any drugs that are present. An alcohol of some sort is usually used in the embalming process (typically methanol) - most drugs are soluble in methanol and therefore, you are essentially washing the aviculture clean of any drug as you embalm. Obviously, alcohol analysis is not probative due to possible presence of methanol or ethanol in the embalming fluid.
Samples taken after treatment with intravenous fluids usually renders postmortem blood and tissues devoid of detectable drugs.
It goes without saying that if you don’t know what a sample is – you can’t make any sort of valid interpretation of the toxicological analysis of the sample. Spleen squeezings and esophogeal scrapings, would quite reasonably fall into this category.
“Spleen squeezings”
“Esophageal scrapings”

24. Chest Cavity Fluid Not readily definable
Most likely to be collected if:
Traumatic injury to the chest
Advanced decomposition
A “contaminated” blood sample, chest cavity fluid may contain fluids from stomach, heart, lungs etc.

25. Samples taken after embalming
Methanol is a typical component of embalming fluid
Most drugs are soluble in methanol
Embalming process will essentially “wash” the vasculature and tissues
Qualitative analysis can be performed on body tissues

26. Case Example
A 72 year old woman, given meperidine to control pain following surgery, later died in hospital. The woman was in poor health and it is possible that death was due to natural causes. However, coroner requests toxicology to rule out inappropriate meperidine levels.
BUT:
Body had been embalmed
Liver and spleen submitted

27. Storage and Handling

28. Proper specimen handling
Identification of samples
Continuity
Contents
Specimens delivered to lab without delay
Specimens should be analyzed as soon as possible
Storage areas should be secure

29. Storage and Handling Not feasible to analyze specimens immediately
Sample should be in well-sealed container
Sample containers must be sterile
Use of preservatives and anti-coagulants
Refrigeration vs. Freezing
Both inhibit bacterial action; esp. freezing
Freezing results in  prep time
Freeze-thaw cycle may promote breakdown
Glass tubes will break during the freeze-thaw cycle

30. Storage of Samples Preservative Anti-coagulants Sodium fluoride
Sodium citrate
Potassium oxalate
EDTA
Heparin
Not imperative for postmortem blood samples
Anticoagulants are not really necessary in postmortem blood samples since the blood is hemolyzed!
But any changes that have occurred before the sample is put into the proper container cannot be reversed.

31. Determining analyses Case history Experience of the toxicologist
Medical history
Autopsy findings
Symptomatology
Experience of the toxicologist
Amount of specimen available
Nature of specimens available
Policies of the organization
Case History
Were drugs found at the scene? What drug did the individual have access to?
Can prescription drugs be accounted for (in terms of when prescription was filled versus when death occurred)
Was the patient treated - did they receive medication?
Example: Cocaine in a child! - Later found out to have been used as a local anesthetic in the hospital during resuscitation attempt and insertion of a nasal tube.
Symptomatology - was anything witnessed? Seizures? Delirium? Another example: Opioid overdoses - typical for deceased person to have been witnessed to have been snoring heavily at some point before death.
Autopsy findings - if there is an anatomical cause of death then the number of analyses may be limited or there are pathological changes that can implicate drug involvement in the cause of death.
Policies of the organization - example SIDS - full toxicology screen.

32. Pitfalls in Postmortem Forensic Toxicology

33. Decomposition Autolysis Putrefaction
The breakdown of cellular material by enzymes
Putrefaction
A septic/infectious process
The destruction of soft tissues by the action of bacteria and enzymes
Traumatic deaths may demonstrate  putrefaction
The first and most visible problem facing postmortem forensic toxicology is the process of decomposition. It is not uncommon in postmortem forensic toxicology for a body to lie decomposing for several days (if not longer) before discovery (contrast with criminal events which are often immediately identified).
The process of decomposition comprises two main stages - the first is autolysis, which occurs when the cellular material of the body begins to break down due to enzymatic action.
The second stage - which is of far more importance to the forensic toxicologist is putrefaction. Putrefaction is an infectious process, meaning that bacteria are involved - it occurs when soft tissues are destroyed by both the actions of bacteria and enzymes.
The bacteria - commensal bacteria such as those found in the respiratory tract and gastrointestinal tract - invade the surrounding tissues.
The speed at which putrefaction takes place increases if death is due to a septic condition or if injuries to the body surface have occurred (provides a portal for entry for bacteria (e.g. traumatic injury).

34. Decomposition Fewer samples available for collection
Quality of samples is diminished
Putrefaction produces alcohols
Ethanol
Isopropanol
Acetaldehyde
n-propanol
Fewer samples available for collection - liquefication means fewer intact vessels from which to sample
Quality of samples is diminished - “dirty” samples; interfering compounds during GC analysis

35. Postmortem redistribution
A phenomenon whereby increased concentrations of some drugs are observed in postmortem samples and/or site dependent differences in drug concentrations may be observed
Typically central blood samples are more prone to postmortem changes (will have greater drug concentrations than peripheral blood samples)

36. Possible mechanisms of postmortem redistribution
Diffusion from specific tissue sites of higher concentration (e.g. liver, myocardium, lung) to central vessels in close proximity
Diffusion of unabsorbed drug in the stomach to the heart and inferior vena cava
Diffusion of drugs from the trachea, associated with agonal aspiration of vomitus
Postmortem redistribution typically results in elevated cardiac blood concentrations.
Little is known about which mechanism or mechanisms are primarily responsible for increases in drug concentrations in cardiac blood. Possible explanations include:
1. Postmortem diffusion of drugs from surrounding tissues. LUNGS - have the richest supply of blood vessels in the body and drugs can be sequestered in the pulmonary tissues at high concentrations antemortem. These drugs may be redistributed rapidly into pulmonary venous blood and then into the left cardiac chamber.
2. Postmortem diffusion of drugs from the stomach, containing large amounts of drug.
3. Postmortem diffusion of drugs through the trachea associated with agonal aspiration of vomitus or medical treatment.
Postural changes may play a role; movement of blood may be facilitated.

37. Case Example 37 year old man found dead in his home
Cause of death identified at autopsy as asphyxia due to choking; white pasty material lodged in throat
Heart blood
Morphine: ng/mL
Amitriptyline: 0.36 mg/dL
Femoral blood
Morphine: 442 ng/mL
Amitriptyline: 0.01 mg/dL
Examination of esophageal and tracheal contents revealed presence of both morphine and amitriptyline

38. Susceptible Drugs
Drugs most commonly associated with postmortem redistribution:
are chemically basic
have large volumes of distribution
Susceptible drugs are chemically basic in nature and with large volumes of distribution.

39. Volume of distribution
Review from last lecture:
Volume of distribution is the amount of drug in the whole body (compared to the amount of drug in the blood)
If a drug has a large volume of distribution, it is stored in other fluids and tissues in the body

40. Susceptible Drugs Tricyclic antidepressants Antihistamines
Amitriptyline
Nortriptyline
Imipramine
Desipramine
Antihistamines
Diphenhydramine
Narcotic Analgesics
Codeine
Oxycodone
Propoxyphene
Doxepin
Digoxin
Susceptible drugs are chemically basic in nature and with large volumes of distribution.

41. Example: Digoxin
p. 60, Principles of Forensic Toxicology
A 33 year old white female is admitted to hospital after taking 60 digoxin tablets
An antemortem blood sample collected 1 hour prior to her death indicates a blood digoxin level of 18 ng/mL
Heart blood digoxin concentration obtained at autopsy is 36 ng/mL

42. Example: Digoxin
Postmortem increase in blood digoxin concentrations is suspected to be due to the release of the drug from the myocardium
Postmortem levels > Antemortem levels
Heart blood levels > Femoral blood levels

43. Postmortem redistribution
Coping with the problem of postmortem redistribution:
Analysis of both central blood and peripheral blood in cases where postmortem redistribution may be a factor
Compilation of tables to determine average and range of postmortem redistribution factors for drugs

44. Incomplete Distribution
Site dependent differences in drug levels due to differential distribution of drugs at death
Has been noted in rapid iv drug deaths
Example:
Intravenous injection of morphine between the toes
Fatal amount of drug reaches the brain
Full distribution of the morphine throughout the body has not occurred
Femoral concentration > Heart concentration
Site dependent differences may be due to incomplete distribution as opposed to postmortem redistribution
The difference between this and postmortem redistribution (in my mind) is that there is a differential distribution in the drugs at the time of death.

45. Drug Stability
Knowledge of a drug’s stability is necessary to facilitate interpretation of concentrations
Breakdown of drugs may occur after death and during storage via non-enzymatic mechanisms
Cocaine  Benzoylecgonine (Hydrolysis)
LSD  degradation due to light sensitivity
Others ?
Cocaine will hydrolyze spontaneously especially under alkaline conditions (e.g. blood) to benzoylecgonine. Mechanism is the action of plasma cholinesterase. This process occurs both in vivo and in vitro, which further complicates interpretation. Fluoride and refrigeration help to prevent the conversion to benzoylecgonine.

46. Example: Bupropion
Bupropion, an antidepressant, was identified and confirmed during a GC drug screen
Blood analyzed using a quantitative analysis:
Bupropion  not detected
Review of the literature:
Laizure and DeVane, Ther. Drug. Monit.
“Bupropion showed a log linear degradation that was both temperature and pH dependent…”

47. Evaporation of volatiles
Ethanol
Carbon monoxide
Cyanide
Toluene
Other alcohols

48. Example: Carbon Monoxide
Ocak et al J. Analytical Toxicology. 9:
Effects of storage conditions on stability of CO
No significant change in % CO saturation in capped samples stored at room temperature or 4oC
Significant losses in % CO saturation in uncapped samples stored at room temperature and at 4oC
Mechanism for loss  diffusion

49. Interpretation

50. Interpretation Therapeutic, toxic or fatal? How do you know?
Compare measured blood concentrations with concentrations reported in the literature:
Clinical pharmacology studies
Incidental drug findings
Plasma  blood
Consider case history:
Symptoms observed by witnesses?
Tolerance of the individual to the drug
Therapeutic and toxic blood ranges have been established for many drugs.

51. Blood:plasma ratios
Knowledge of the blood:plasma ratio can be very important when applying information from clinical studies to postmortem forensic tox
Cocaine, blood:plasma ratio is 1.0
Phenytoin, blood:plasma ratio is 0.4
Ketamine, blood:plasma ratio is 1.7
Hydroxychloroquine, blood:plasma ratio is 7.2
Phenytoin is a drug that is used to control seizures.
Hydroxychloroquine (Plaquenil) is used in the treatment of malaria, lupus and rheumatoid arthritis. Why would this blood to plasma ratio be so high? Question the class – what does blood have that plasma does not? Red and white blood cells – hydroxychloroquine must bind to one of these components in order to have such a discrepant blood:plasma ratio (over 7x as much drug in a whole blood sample as in a plasma sample).
Phenytoin actually has less drug in a whole blood sample than in a plasma sample. Why might this be? Binding to plasma proteins results in more drug in the plasma portion than in the whole blood cell portion…in essence, the blood cells in such a situation are actually acting to “dilute” the drug concentration. This also happens with alcohol – which is such a simple molecule that it affiliates with the watery portion of the blood (plasma or serum).

52. Example: THC
Six healthy male volunteers recruited for a study of the pharmacokinetics of THC in humans
Smoked a “high-dose” THC cigarette
15 minutes after cessation of smoking, plasma THC concentrations averaged 94.8 ng/mL
The plasma:blood ratio for THC is 1.8
Plasma contains 1.8x as much THC as whole blood
The results of this study correspond to a blood THC concentration averaging 53 ng/mL

53. Importance of History: Tolerance
Drug concentrations in non-drug related deaths may overlap with reported drug concentrations in fatal drug intoxications
Methadone example:
Naïve users - deaths due to methadone are associated with blood levels > 0.02 mg/100 mL
Patients on methadone maintenance – peak blood concentrations may range up to 0.09 mg/100 mL
Tolerance is a state of decreased responsiveness to a drug that accompanies long term exposure to the same agent or to a closely related agent.
The toxicity of methadone is highly dependent on an individual’s tolerance to the drug. In naïve users, fatalities due to methadone have been associated with postmortem blood concentrations of 0.02 mg/100 mL and greater (Clark et al., J. Clin. Forensic Med., 2: ). However, abusers of the drug, or patients receiving methadone on maintenance programs may develop tolerance to high blood concentrations. The administration of a single daily oral dose of mg methadone to 5 methadone maintenance subjects resulted in peak blood concentrations ranging mg/100 mL (Inturrisi and Verebely, Clin. Pharmacol. Ther., 13: ) (converted from plasma using a plasma:blood ratio of 1.3)(Moffat et al. (Eds.) Clarke’s Isolation and Identification of Drugs, 2nd ed.).
Tolerance to a drug can complicate interpretation to the point that you may not be able to answer the question as to whether or not the drug caused death in the individual. Example:

54. Interpretation Acute vs. Chronic Ingestion: Can you tell?
Parent:metabolite drug concentration ratio may be of assistance in differentiating between acute and chronic ingestion of a drug

55. Example: Amitriptyline
Case 1
Amitriptyline: 0.4 mg%
Nortriptyline: 0.02 mg%
Parent >> Metabolite
Suggestive of acute overdose and rapid death
Case 2
Amitriptyline: 0.04 mg%
Nortriptyline: 0.08 mg%
Parent < Metabolite
Slow death and/or chronic administration

56. Interpretation
Metabolites are produced when drugs are biotransformed (converted) into other chemicals, more easily excreted from the body
Metabolite drug concentrations may be the more useful measure of exposure or toxicity
Metabolism is a biological process that alters the chemical form of a substance. The interaction of a drug or chemical with the body’s enzyme systems results in the formation of one or more metabolites. The purpose of metabolism is to detoxify exogenous substances and render them more polar and readily excretable.

57. Metabolites: Exposure
The parent compound may be a prodrug or may have a shorter t1/2 than the metabolite:
Clorazepate  nordiazepam
Flurazepam  N-desalkylflurazepam
Heroin  morphine
A prodrug is an inactive substance that has an active metabolite.
Flurazepam is a benzodiazepine with hypnotic efficacy. The half life of flurazepam is 1-3 hours (review of what a half-life is?), however, the half life for the metabolite N-1_desalkylflurazepam is between hours. The difference in detectability here is hours (for the parent) versus days-weeks (for the metabolite). N-desalkylflurazepam is an active metabolite with relative activity about 10x that of the parent drug.
Heroin – t½ is only 2-6 minutes long…whereas the half-life of morphine is 2-3 hours. Heroin is rapidly converted to morphine (its active metabolite) in the body.

58. Metabolites: Toxicity
The metabolite may have  toxicity over the parent compound:
Acetaminophen  N-Acetylbenzoquinoneimine
Meperidine  normeperidine
Methanol  formic acid
Ethylene glycol  oxalic acid  calcium oxalate
Methanol – industrial solvent, major toxic effects associated with methanol exposure are marked acidosis and blindness. Methanol is metabolized in the body to formaldehyde which is further converted to formic acid – formic acid contributes to the production of acidosis and to blindness by destruction of the retina and optic nerve degeneration.