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This is to certify that the
thesis entitled

DISTRIBUTION OF METHADONE AND EDDP
IN POSTMORTEM TOXICOLOGY CASES

presented by

Jessica Amanda Jennings

has been accepted towards fulfillment
of the requirements for the
MS.

degree ' Forensic Science

   

 

 

  

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DISTRIBUTION OF METHADONE AND EDDP
IN POSTMORTEM TOXICOLOGY CASES

By

Jessica Amanda Jennings

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE IN FORENSIC SCIENCE
School of Criminal Justice

2005

ABSTRACT

DISTRIBUTION OF METHADONE AND EDDP
IN POSTMORTEM TOXICOLOGY CASES

By
Jessica Amanda Jennings

Methadone, a legal synthetic opioid, has been used for the treatment of heroin and
morphine addiction for over 40 years now in addition to being used as an analgesic. This
drug is less addictive than its opioid counterparts, but it is still abused. Consequently,
methadone accounts for a large portion of drug deaths each year. At the State of
Delaware Office of the Chief Medical Examiner, 100 methadone-related deaths from
September 6, 2001 through March 1, 2005 were analyzed to determine the distribution of
methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine
(EDDP) in these cases. The cases were divided into six groups based on their causes and
manners of deaths. Methadone toxicity is difficult to interpret due to overlapping
concentrations in therapeutic and lethal concentrations. Several factors contribute to this
overlap including tolerance and individual variability in metabolism. Postmortem
redistribution also complicates interpretation. The main specimens that were analyzed in
this study included vitreous humor, peripheral blood (femoral), heart blood, brain, gastric
contents, and liver. Average concentrations of methadone and EDDP were determined
for each specimen in each group. The group in which death was due to methadone
toxicity alone had the highest concentration of methadone for all specimen types with the
exception of liver. In addition, ratios of parent drug to metabolite and ratios of vitreous,
brain, and liver to blood were calculated. These ratios provided valuable information in

some instances, but methadone deaths still remain a challenging category of deaths.

This thesis is dedicated to my Mom and Dad with love and gratitude.

iii

ACKNOWLEDGEMENTS

I would like to first thank Dr. Rebecca Jufer Phipps for all of her guidance and
generosity throughout this research project. She is the one who proposed the project and
also the one who served as my primary advisor. Even though she no longer supervises or
works with me, she continued to help me, which I greatly appreciate. Dr. J ufer has
provided a wealth of knowledge and assistance over the past year. She has taught me
basically everything I know about forensic toxicology. Additionally, I would like to
acknowledge Dr. Jay Siegel who also served as an advisor and committee member. Dr.
Siegel is one of the main reasons I decided to go to Michigan State—due to his
outstanding reputation in forensic science. He has been an inspirational person and role
model ever since I met him. Also serving on my committee is Dr. Sheila Maxwell. I
would like to thank her for her time and concern as well.

I owe a huge thank-you to the staff of the State of Delaware Office of the Chief
Medical Examiner for allowing me to partake in this project using their instruments and
supplies. I would like to especially thank John Marino and Rick Pretzler for their special
efforts on my behalf. Thank you also to my family and friends for all of their love and
support. My parents and sister have always encouraged me and believed in me. I cannot
express my gratitude enough to my parents—for both their emotional and financial
support. I could not have accomplished this goal without them. I owe a warm special
thank-you to my Mom for her countless hours of proofreading. Finally, I would like to
thank the love of my life—my fiance Jeffrey. He and our kitten Gracie have kept me

smiling throughout this entire endeavor.

iv

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vii
LIST OF FIGURES ................................................................................. ix
KEY TO SYMBOLS AND ABBREVIATIONS ................................................ xi
INTRODUCTION ..................................................................................... 1
Aims of this Project ......................................................................... 1
History and Usage of Methadone ......................................................... 2
Metabolism and Excretion ................................................................. 5

Drug Interactions ........................................................................... 9
Pharmacodynamics ......................................................................... 9
Postmortem Redistribution ............................................................... 11
Toxicological Analysis ....................................... , ............................ 13
EXPERIMENTAL ................................................................................. 16
Specimens .................................................................................. 16
Chemicals and Materials .................................................................. 19
Extraction ................................................................................... 19
Instrumentation ............................................................................ 20
Chromatography and Mass Spectrometry .............................................. 21
Quantitation and Acceptability .......................................................... 28
RESULTS AND DISCUSSION .................................................................. 29
Methadone and EDDP Concentrations ................................................. 31

Group 1A .......................................................................... 31

Group 18 .......................................................................... 34

Group 2 ............................................................................. 36

Group 3 ............................................................................ 36

Group 4 ............................................................................ 41

Group 5 ............................................................................ 41

Additional Specimens ............................................................ 43

Summary ........................................................................... 46

Ratios of Methadone to EDDP and Other Matrices to Blood ....................... 52

Group 1A .......................................................................... 53

Group 13 .......................................................................... 53

Group 2 ............................................................................. 56

Group 3 ............................................................................. 56

Group 4 ............................................................................. 56

Group 5 ............................................................................ 60

Additional Specimens ............................................................ 60

Summary ........................................................................... 63

TABLE OF CONTENTS (cont’d).

Central-to-Peripheral Ratios ............................................................. 68
CONCLUSIONS AND FUTURE WORK ...................................................... 70
APPENDIX .......................................................................................... 74
REFERENCES ...................................................................................... 86

vi

LIST OF TABLES

Table 1. Table showing all of the specimens that were analyzed for each case .......... 17

Table 2. Concentrations of vitreous humor, peripheral blood (femoral), heart blood,
brain, gastric contents (total amount), and liver for each case in Group 1A. Averages,
standard deviations, number of positive cases (n), and ranges are also included .......... 32

Table 3. Concentrations of vitreous humor, peripheral blood (femoral), heart blood,
brain, gastric contents (total amount), and liver for each case in Group 1B. Averages,
standard deviations, number of positive cases (n), and ranges are also included .......... 35

Table 4. Concentrations of vitreous humor, peripheral blood (femoral), heart blood,
brain, gastric contents (total amount), and liver for each casein Group 2. Averages,
standard deviations, number of positive cases (n), and ranges are also included .......... 37

Table 5. Concentrations of vitreous humor, peripheral blood (femoral), heart blood,
brain, gastric contents (total amount), and liver for each case in Group 3. Averages,
standard deviations, number of positive cases (n), and ranges are also included .......... 38

Table 6. Concentrations of vitreous humor, peripheral blood (femoral), heart blood,
brain, gastric contents (total amount), and liver for each casein Group 4. Averages,
standard deviations, number of positive cases (11), and ranges are also included .......... 42
Table 7. Concentrations of vitreous humor, peripheral blood (femoral), heart blood,
brain, gastric contents (total amount), and liver for each casein Group 5. Averages,
standard deviations, number of positive cases (It), and ranges are also included .......... 44

Table 8. Concentrations of additional specimens for each case in all six groups. . . . . 45

Table 9. Table summarizing the averages, standard deviations, number of positive cases
(n), and ranges for listed specimens in each group ............................................. 47

Table 10. Ratios for cases in Group 1A. Averages, standard deviations, number of cases
(11), and ranges are also included .................................................................. 54

Table 11. Ratios for cases in Group 18. Averages, standard deviations, number of cases
(It), and ranges are also included .................................................................. 55

Table 12. Ratios for cases in Group 2. Averages, standard deviations, number of cases
(11), and ranges are also included .................................................................. 57

Table 13. Ratios for cases in Group 3. Averages, standard deviations, number of cases
(11), and ranges are also included .................................................................. 58

vii

LIST or TABLES (cont’d).

Table 14. Ratios for cases in Group 4. Averages, standard deviations, number of cases
(n), and ranges are also included .................................................................. 59

Table 15. Ratios for cases in Group 5. Averages, standard deviations, number of cases
(n), and ranges are also included .................................................................. 61

Table 16. Ratios for additional specimens ...................................................... 62

Table 17. Table summarizing the averages, standard deviations, number of cases (n),
and ranges for listed ratios in each group ....................................................... 64

Table 18. Ratios of methadone in heart blood to methadone in peripheral blood
(femoral) for each case in all groups ............................................................. 69

Table A1. Information for all 100 cases, including case number, age, sex, race, brief
history, cause of death, manner of death, group, and other quantitative results ............ 75

viii

LIST OF FIGURES

Figure 1. Chemical structures of methadone and its metabolites—EDDP and EMDP. .. 5
Figure 2. Total ion chromatogram of EDDP and methadone ............................... 22
Figure 3. Ion chromatogram (top) and SIM mass spectrum (bottom) of d3-EDDP ...... 23
Figure 4. Ion chromatogram (top) and SIM mass spectrum (bottom) of EDDP ......... 24
Figure 5. Ion chromatogram (top) and SIM mass spectrum (bottom) of d3-methadone. 25
Figure 6. Ion chromatogram (top) and SIM mass spectrum (bottom) of methadone. . .. 26
Figure 7. Ion chromatogram of ethyl acetate, which was used as a solvent blank ....... 27

Figure 8. Bar graph showing the average concentrations in ng/mL of EDDP and
methadone in vitreous humor for the six groups ................................................ 48

Figure 9. Bar graph showing the average concentrations in ng/mL of EDDP and
methadone in peripheral blood (femoral) for the six groups ................................. 48

Figure 10. Bar graph showing the average concentrations in ng/mL of EDDP and
methadone in heart blood for the six groups .............. ~ ...................................... 49

Figure 11. Bar graph showing the average concentrations in ng/ g of EDDP and
methadone in brain for the six groups ............................................................ 49

Figure 12. Bar graph showing the average totals in mg of EDDP and methadone in
gastric contents for the six groups ............................................................... 50

Figure 13. Bar graph showing the average concentrations in ng/ g of EDDP and
methadone in liver for the six groups ............................................................ 50

Figure 14. Bar graph showing the average ratios of methadone to EDDP in peripheral
blood (femoral) for the six groups ............................................................... 65

Figure 15. Bar graph showing the average ratios of methadone to EDDP in heart blood
for the six groups .................................................................................... 65

Figure 16. Bar graph showing the average ratios of methadone to EDDP in liver for the
six groups ........................................................................................... 66

ix

LIST OF FIGURES (cont’d).

Figure 17. Bar graph showing the average ratios of methadone in vitreous humor to
methadone in peripheral blood (femoral) for the six groups ................................. 66

Figure 18. Bar graph showing the average ratios of methadone in brain to methadone in
peripheral blood (femoral) for the six groups ................................................... 67

Figure 19. Bar graph showing the average ratios of methadone in liver to methadone in
peripheral blood (femoral) for the six groups ................................................... 67

Symbol or Abbreviation

°C

>

/

%

:l:

(i)

®

uL

pm

A

AA
ACS
alpha-1 -AGP
Ante
ASCAD
ASCVD
Bld

C

C/P
CNS
COD
COHb
(cont’d).
COPD
CYP

d3

DI

dL

e. g.
EDDP
EMDP

GC

KEY TO SYMBOLS AND ABBREVIATIONS

Meaning
degrees Celsius

greater than

per

percent

plus or minus

racemic

Registered trademark

microliter

micrometer

accident

Afiican American

American Chemical Society
alpha-l-acid glycoprotein

antemortem

arteriosclerotic coronary artery disease
arteriosclerotic cardiovascular disease
blood

Caucasian

central-to-peripheral

central nervous system

cause of death

carboxyhemoglobin

continued

chronic obstructive pulmonary disease
cytochrome P450

deuterated

deionized/distilled

deciliter

example given

2-ethylidene-1 ,5-dimethyl-3 ,3-diphenylpyrrolidine
2-ethyl-5-methyl-3,3-diphenylpyrroline
female

gram

gas chromatography

xi

Symbol or Abbreviation
GC (in tables)

H

HB

Hep C
Hg

HIV
Hosp
HPLC
HTCVD
HTN

kg

L

m

M
MDN
m8

min

mL

mm
mM
MMT
MOD
Mol. Wt.
MS

n

N

NA

ND

“8

NPD
OCME
P

PB(F em)
PB(Sub)

KEY TO SYMBOLS AND ABBREVIATIONS (cont’d).

Meaning
gastric contents

Hispanic

heart blood

Hepatitis C

mercury

human immunodeficiency virus
hospital

high-performance liquid chromatography
hypertensive cardiovascular disease
hypertension

kilogram

liter

meter

male

methadone

milligram

minute

milliliter

millimeter

millimolar

methadone maintenance treatment
manner of death

molecular weight

mass spectrometry

number of positive cases

natural

not applicable

none detected

nanogram

nitrogen-phosphorus detection
Office of the Chief Medical Examiner
pending

peripheral blood (femoral)
peripheral blood (subclavian)

xii

KEY TO SYMBOLS AND ABBREVIATIONS (cont’d).

Symbol or Abbreviation
pH

pKa

PM/AM

R

RPM

S

S (in Table A1)
SD

SIM

SML

SPE

U

UCR

UCT, Inc.

VH

Meaning
potential of hydrogen

ionization constant
postmortem-to-antemortem
Rectis (Latin for right)
revolutions per minute
Sinister (Latin for left)
suicide

standard deviation

selected ion monitoring
serum methadone level
solid-phase extraction
undetermined

urinary cortisol ratio
United Chemical Technologies, Incorporated
vitreous humor

xiii

INTRODUCTION

Aims of this Project

In Delaware, there have been 100 methadone-related deaths between September
6, 2001 and March 1, 2005. According to the records of the State of Delaware Office of
the Chief Medical Examiner (OCME), there were six deaths in the final four months of
2001. Twenty-three deaths involving methadone toxicity occurred in 2002, 37 in 2003,
and 28 in 2004. In the first three months of 2005, six deaths were associated with
methadone.

Much research has been devoted to methadone-related deaths, but most of the
published articles focus on establishing a lethal methadone concentration range.
However, due to the overlapping concentrations in deaths attributable to methadone
toxicity and those unrelated to it, this is practically impossible. Confounding factors such
as tolerance and postmortem redistribution partially account for this overlap. There has
been little research aimed at evaluating the distribution of parent drug to metabolite in
such cases. One aim of this research project was to determine ratios of methadone to its
main metabolite in Delaware’s methadone cases over the past three and a half years to
see if these would help in classifying such deaths. Since methadone is widely distributed
in tissues such as the liver and kidney, it is possible that these specimens (that are
typically not studied) could provide valuable information. In addition to assessing ratios,
another goal was to determine methadone and metabolite concentrations in other matrices

to determine their usefulness in the evaluation of methadone-related deaths.

History and Usage of Methadone

Methadone is a potent synthetic opioid that produces both circulatory and
respiratory depression. Since it is generally less addictive than heroin and morphine,
methadone (also known as Dolophine®) has been clinically used as an analgesic and as a
legal alternative for opioid addiction through methadone maintenance treatment (MMT)
programs. When used as an analgesic, methadone’s effects only last for four to six hours.
Methadone’s main uses as a painkiller are for chronic pain (most often back pain),
cancer, and terminal illnesses. Compared to heroin, methadone has a longer duration of
action so it effectively reduces cravings and withdrawal symptoms for up to 72 hours.
Additionally, the highs and lows experienced with methadone are less intense than those
resulting from heroin (Inaba and Cohen, 2000).

During World War II, methadone was first synthesized in Germany (Baselt and
Cravey, 1995). Methadone has been used to treat opioid addiction in the United States
since the 19608 when Vincent Dole and Marie Nyswander developed MMT in New York
City (Ali and Quigley, 1999; Inaba and Cohen, 2000). This opioid alternative has been
shown to decrease the amount of illicit drug use, the spread of the human
immunodeficiency virus (HIV) among intravenous drug users, and the amount of
criminal activity. Other advantages of methadone include re-employment, social
functioning and rehabilitation, and an improved quality of life. Methadone is also less
expensive than heroin and other opioids. Moreover, the risks of fatal drug overdose have
declined as a result of methadone maintenance (Ali and Quigley, 1999; Wolff, 1998;

Toombs and Kral, 2005).

Although methadone is less addictive than heroin, it is unfortunately still abused.
There are strict regulations enforced at methadone clinics, but methadone is still sold
illicitly. Additionally, patients will often combine methadone with other drugs such as
benzodiazepines (e. g. alprazolam), heroin, or illicit methadone to enhance the high (Ali
and Quigley, 1999; Inaba and Cohen, 2000). For these reasons, methadone accounts for a
high portion of drug deaths each year (Inaba and Cohen, 2000). Wolf et al. (2004)
reported a remarkable increase in methadone deaths in Palm Beach County, Florida—
from 2 in 1998 to 87 in 2002. Research has identified inadequate dosing as a major cause
for patient noncompliance (Ali and Quigley, 1999). A California study spanning 24
years showed that terminating opioid use takes a long time. Those who are continuing to
abuse opioids in their late 303 are unlikely to ever cease their use (Wolff, 1998).

Finding the optimal methadone dose for a particular patient is crucial to its
success as a maintenance drug. When MMT programs prescribe patients inadequate
doses, they will experience withdrawal symptoms, leading them to return to illicit drugs.
Withdrawal symptoms include depression, irritability, insomnia, nausea, fatigue, and
hot/cold flashes. More severe symptoms include twitching, diarrhea, vomiting, anxiety,
fever, and hypertension. Conversely, signs of overrnedication are drowsiness, miosis
(constriction of the pupil), itching, hypotension, and respiratory depression. When a
patient experiences neither withdrawal symptoms nor overmedication symptoms, he or
she is considered to be in the therapeutic range (Leavitt et al., 2000).

Routine drug monitoring has been recommended as a means to determine this
therapeutic range. This can be done by analyzing serum methadone levels (SMLs). The

level of methadone detected in serum shows what is happening to methadone in the body.

In the study by Leavitt et a1. (2000), there was no correlation between dose and SMLs.
Individual variations in metabolism partially account for this variation. According to this
study, doses of 120-700 mg/day may be more effective than the usual 100 mg/day or less.
Serum methadone levels cannot be used in isolation when determining a person’s optimal
methadone dose. The patient’s symptoms and clinical state must also be monitored for
dosing decisions.

Methadone is available in several forms including liquid, injectable, and tablet.
The preferred method is to dissolve it in juice so that it may be taken at a clinic. If a
patient cannot come into a clinic one day, there are take-home tablets available, but these
often get diverted to the streets (Inaba and Cohen, 2000). Tablets and drinks also run the
risk of being consumed by children when inadvertently left out (DiMaio and DiMaio,
1973). The risk of illicit sale is high with injectable methadone as well (Wolff, 1998). A
typical starting dosage is 10-20 mg daily. With time, this dose may be increased to 40-
100 mg/day with some as high as 180 mg/day (Milroy and Forrest, 2000).

Another problem with methadone is the increased risk of death within the first
two weeks of maintenance treatment. This is often due to the dose being increased too
quickly, resulting in respiratory depression (Karch and Stephens, 2000). In New South
Wales, Caplehom and Drummer (1999) showed that the risk of accidental toxicity during
the first two weeks was 6.7 times that of addicts not in MMT programs. During this time
also, the risk is nearly 98 times higher than that of patients who have been in therapy
longer than two weeks. This data shows that the first two weeks of treatment are
definitely a precarious time. Despite these statistics, it is probable that MMT programs in

New South Wales saved 68 lives in 1994 alone.

Metabolism and Excretion

When administered orally, methadone is absorbed quite rapidly into the body.
The drug is widely distributed in tissues such as the liver, kidney, lungs, and spleen. In
the liver, methadone undergoes biotransformation to the main inactive metabolites 2-
ethylidene-l ,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-
diphenylpyrroline (EMDP) as shown in Figure 1. This process occurs primarily through
mono- and di-N-demethylation. These metabolites are then eliminated by the kidney and
excreted through the bile, urine, and feces. Smaller amounts of methadol and

normethadol are also formed (Jenkins and Cone, 1998; Kerrigan and Goldberger, 2003).

  

   
 

Methadone H3O
C21H27NO
/ Mol. Wt.: 309.45
CH3
/
T +
CH
c/
“2 \CH3
EDDP
ConzaN+
Mol. Wt.: 278.41 —'

 

Mol. Wt.: 263.38

Figure 1. Chemical structures of methadone and its metabolites—EDDP and EMDP.

Methadone, a Schedule II narcotic, is typically dispensed as a 50:50 racemic
mixture of the active R-enantiomer and the inactive S-enantiomer (Toombs and Kral,
2005). This basic drug is highly lipophilic with a pKa value between 8.6 and 9.2.
Methadone’s volume of distribution (defined as the dose divided by the concentration at
equilibrium) is 4-5 L/kg (Baselt and Cravey, 1995; Garrido and Troconiz, 1999; Karch
and Stephens, 2000). Since methadone distributes itself extensively throughout the body
(due to its high lipophilicity), it has a long elimination phase. In a study of MMT
patients, 5-50% of the dose was excreted as methadone and 3-25% as EDDP. This
variation is due to factors such as the dose, metabolic rate, and urine pH. When the urine
was acidified, the percentage of unchanged methadone was greater (Baselt and Cravey,
1995)

Methadone has a long elimination half-life that ranges between 15 and 55 hours.
This is the main reason why methadone is so useful as a treatment drug. Since its effects
are long—lasting, patients normally only have to take it once daily. The half-life of
morphine (the primary active metabolite of heroin), on the other hand, is only two to
three hours, so patients go through withdrawal only hours after its use (Baselt, 2004).
The oral bioavailability of methadone has been reported to range between 0.67 and 0.95
(Garrido and Trocc'miz, 1999).

Following oral administration, peak blood concentrations of methadone are
attained approximately four hours after dosing. Four hours after a single 15-mg oral
dose, a peak plasma concentration of 75 ng/mL has been reported. This concentration
slowly declined to 30 ng/mL afier 24 hours with a half-life of 15 hours. Peak

concentrations in brain occur one to two hours after dosing (Jenkins and Cone, 1998). A

study involving nasal administration revealed that peak plasma concentrations occurred
within seven minutes, proving its uptake was very quick (Dale et al., 2002).

Methadone is metabolized by cytochrome P450 (CYP) enzymes in human liver.
Iribame et al. (1996) studied 20 human liver microsomes to determine the involvement of
this family of enzymes in the oxidative N-demethylation of methadone. They determined
that CYP3A4 is, on average, the principal enzyme involved in this metabolism. Out of
nine heterogeneously expressed CYP enzymes, CYP3A4 accounted for 63% of the
catalytic activity. Furthermore, this isoform made up 32% of the total CYP group. The
other CYP isoforrns were CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP2C18, CYP2D6,
CYP3A5, and CYPlEl. Only five of the nine were found to contribute to catalysis—
CYPlAl, CYP2C8, CYP2C18, CYP2D6, and CYP3A4. Data published by Wang and
DeVane (2003) indicate that CYP3A4, CYP2C8, and CYP2D6 are the three prominent
enzymes involved in methadone metabolism. Two other enzymes may also play a role,
and they are CYP2C9 and CYP2C19 (Foster et al., 1999).

The CYP3A4 enzyme is inhibited by several mechanism-based inhibitors such as
gestodene. Other substances such as quinidine and sulfaphenazole did not inhibit
methadone metabolism to the extent of the mechanism-based inhibitors. Inhibition was
only 10% for sulfaphenazole—compared to 60-72%, which was seen with the
mechanism-based inhibitors (Iribame et al., 1996). In another study by Oda and
Kharasch (2001), it was shown that methadone is metabolized by human intestinal
microsomes as well. Methadone undergoes first-pass metabolism in the intestine due to

CYP3A4 activity.

Several other factors are thought to affect the metabolism of methadone. For
example, people who are chronic alcohol abusers metabolize methadone more slowly.
Certain individuals are poor metabolizers because they are deficient in the responsible
enzyme. Vitamins, diet, pregnancy, and physical condition may also influence one’s
metabolism (Leavitt et al., 2000). Studies have shown that methadone concentrations are
increased in users who are infected with HIV (Karch and Stephens, 2000). All such
people are thus potentially at increased risk for accidental overdose of methadone.

Methadone toxicity is difficult to evaluate due to an overlap in therapeutic and
fatal concentrations. One of the main reasons for these coinciding concentrations is
tolerance amongst users. Tolerance is defined as an individual’s decreased response to a
drug following extended use. The body will react to continued drug use in a variety of
ways, leading to tolerance. For example, the metabolism and excretion of the drug may
increase, and nerve cells may become desensitized. Due to these changes, a user must
increase his or her dose to have the same effects. Opioid tolerance occurs quite rapidly
and can develop with sustained use (Inaba and Cohen, 2000).

Research has shown that MMT patients have high tolerance in comparison to
heroin addicts who are not undergoing treatment. Consequently, an MMT patient can
take heroin and survive while a street addict could die from doing so. Even just years
afier MMT programs were established, lack of opioid tolerance contributed to nearly half
of the methadone fatalities (Greene et al., 1974). Tolerance may develop quickly and
extensively, but it can be lost quickly, too. There have been instances in which opioid
addicts go to prison where their tolerance levels become drastically diminished. Upon

being released, they use opioids in the same fashion as they did before going to prison.

Quite often though the results are fatal due to this loss of tolerance (Milroy and Forrest,

2000)

Drug Interactions

Another main factor affecting methadone metabolism is co-medication or drug
interactions. As was previously mentioned, methadone users often combine this drug
with other substances to enhance the high. Certain P450 enzyme-inducing drugs increase
the clearance of methadone. Examples of these drugs include amitriptyline, barbiturates,
phenytoin, and rifampicin. Other drugs such as diazepam and cimetidine do just the
opposite—they inhibit methadone metabolism and therefore account for increased
concentrations of methadone (Oda and Kharasch, 2001). Benzodiazepines such as
alprazolarn, diazepam, and lorazepam are commonly detected in conjunction with
methadone. These, in addition to alcohol, may augment respiratory depression when
used in combination with methadone (Barrett et al., 1996; Rogers et al., 1997). A study
of oxycodone and hydrocodone fatalities revealed that multiple-drug overdoses are seen

with these substances as well (Spiller, 2003).

Pharmacodynamics

Methadone primarily binds to three opioid receptors—mu, kappa, and delta—in
the central nervous system (CNS). Another opioid receptor that is less relevant is the
sigma receptor. These receptors are primarily located in the brain, spinal cord, and
digestive tract (Inaba and Cohen, 2000). Both methadone and morphine are selective to

the mu receptor, although morphine’s affinity is higher. The mu receptor is primarily

responsible for respiratory depression, euphoria, pain relief, blood pressure, pupil
contraction, the gastrointestinal tract, and physical tolerance and dependence. The kappa
receptor, on the other hand, generates dysphoria but is also responsible for pain relief and
sedation (Garrido and Troconiz, 1999; Inaba and Cohen, 2000; Kerrigan and Goldberger;
2003). Studies have suggested that tolerance is partly due to changes in mu-receptor
function (Garrido and Troconiz, 1999).

Cross-tolerance is a type of tolerance that results in a reduced response to an
opioid following treatment with another one. For instance, a person who has built up
tolerance to heroin will also exhibit tolerance to morphine and other opioids. However, it
is important to remember that opioids are selective to certain receptors. Thus, a person
who has acquired a tolerance to an opioid that works at the mu receptor will not have as
much tolerance to a drug that works at a different receptor (Inaba and Cohen, 2000).
Research has indicated that methadone does not induce cross-tolerance as readily as
morphine (Garrido and Troconiz, 1999).

In an analysis of 21 methadone deaths in Milwaukee, Wisconsin, the role of
pharmacogenomics was assessed. In this study, Wong et al. (2003) studied whether or
not CYP2D6 variant alleles were the cause of atypical metabolism, which would lead to
methadone toxicity. They found no significant associations between CYP2D6 mutations
and methadone toxicity. Therefore, the results of genotyping in this study did not provide
any additional information for interpreting the methadone cases and assigning causes and
manners of death. According to their article, methadone metabolism involves CYPl A2,

CYP3A4, and CYP2D6 enzymes, which differs slightly from those previously discussed.

lO

A study aimed at evaluating methadone’s pharrnacokinetics and
pharrnacodynamics involved eight drug-free women who were given a single oral dose.
During a four-day period, their vital signs and CYP3A activity were monitored. As part
of examining their vital signs, the subjects’ eyes were photographed to determine pupil
diameters, which indicate pain intensity. All of the subjects’ pupils were constricted,
which is a common effect of opioids. In addition to measuring CYP3A activity, the
concentrations of alpha-l-acid glycoprotein (alpha-l-AGP) were determined (Boulton et
al., 2001b; Dale et al., 2002).

Methadone readily binds to this glycoprotein, and Boulton et al. (2001b)
concluded that it may function in the first-pass metabolism of R-methadone. While
CYP3A and alpha-l-AGP binding can elucidate the activity of R-methadone, these
factors do not explain the disposition of S-methadone. For these reasons, this
glycoprotein and enzyme may affect the interindividual variability in methadone action.
Research has shown that the urinary cortisol ratio (UCR) may also be a marker for
CYP3A activity. Following oral dosage of methadone, UCR declines, which suggests

that methadone inhibits CYP3A (Boulton et al., 2001a).

Postmortem Redistribution

In addition to tolerance, another phenomenon that complicates interpretation of
methadone toxicity is known as postmortem redistribution. This is the process by which
drugs move between bodily fluids and tissues after death. They essentially diffuse from
areas of higher concentration to areas of lower concentration after cellular membranes

have been disrupted (Cook et al., 2000; Drummer, 2004). Drugs that are highly lipophilic

ll

and have increased concentrations in tissues are more susceptible to redistribution.
Drummer (2004) evaluated 23 different drugs/drug classes based on their extent of
redistribution. The extent was categorized as either low, low to moderate, or moderate.
Of these three, methadone’s extent of redistribution was classified as moderate. This
makes sense considering methadone is a highly lipid soluble drug with a large volume of
distribution.

If the gastrointestinal tract contains large amounts of unabsorbed drug, this could
leak to surrounding tissues and fluids. Analyses of heart blood in the central region and
peripheral blood (such as that acquired from the femoral vein) have shown that the latter
is less subject to contamination. Peripheral blood (femoral) is thus the preferred
specimen for toxicological analysis the majority of the time. Due to the perplexing factor
of postmortem redistribution, it is important for toxicologists to know the approximate
timing of specimen collection relative to the time of death (Cook et al., 2000).

A case at the OCME in Baltimore, Maryland prompted Levine et al. (1995) to
study 15 methadone cases to evaluate site dependence of this drug. The methadone
concentrations in this initial case were 2.4 mg/L in heart blood and 0.8 mg/L in
subclavian blood. In addition to testing heart blood, an alternative sample such as
subclavian blood was analyzed for each case. They then compared the ratios of these
samples and found variation of concentrations. Only four of the 15 cases had results
within 20% of each other. From their study, they could not conclude that one sample
would always have a higher concentration than another or that one sample was

preferential to another.

12

Milroy and Forrest (2000) also examined postmortem site dependence and drug
redistribution of methadone. Of their 111 cases, gastric contents were analyzed for 94 of
them. Furthermore, multiple site sampling was performed for 26 of the cases. This is the
process of collecting peripheral blood in both the arm and leg and comparing their
methadone concentrations. They found extreme unpredictable variation in concentrations

from the different sites, suggesting redistribution.

Toxicological Analysis

A variety of acceptable toxicological analysis schemes for methadone-have been
reported in the literature. In addition to the method of gas chromatography/mass
spectrometry (GC/MS) utilized herein, hi gh-performance liquid chromatography (HPLC)
has been used as well as gas chromatography with nitrogen-phosphorus detection (GC-
NPD). The most common type of extraction used for methadone analysis is solid-phase,
although liquid-liquid extraction has been documented as well (Levine et al., 1995; Rio et
al., 1987). When methadone toxicity is suspected, pathologists nearly always perform a
full autopsy and collect a combination of the following samples: heart blood, peripheral
blood, vitreous humor, brain, liver, kidney, gastric contents, urine, and bile (Drummer,
2004). Of these samples, blood, liver, and urine are the most common specimens
analyzed in drug-suspected cases. Due to postmortem redistribution, it is important for
medical examiners to collect two different blood specimens (one from the central region
and one from a peripheral site) so that their concentrations may be compared. In addition
to redistribution, there are other difficulties of working with postmortem samples,

especially in cases of decomposition. These oily samples are useful for drug screening,

13

but they are not favorable for quantitative analysis. In instances of severe decomposition,
skeletal muscle is oftentimes the only available specimen for collection (Drummer,
2004)

Blood is definitely a useful indicator of drug toxicity. Since metabolism primarily
occurs in the liver, results from this tissue can be valuable as well. Analysis of vitreous
humor has provided the detection of drugs, although this type of testing is especially
useful for the quantitation of ethanol and other volatiles. Since many drugs act on the
brain, toxicologists have analyzed this tissue for drug concentrations. However, these
results are often difficult to interpret due to the uneven distribution of drugs there.

Gastric contents function mainly for determining time and type of drug administration
(Drummer, 2004).

Karch and Stephens (2000) reviewed 38 methadone-related deaths at the San
Francisco Medical Examiner’s Office. The average concentration of methadone in blood
for these cases was 957 ng/mL, and that for EDDP was 253 ng/mL. The mean ratio of
parent drug to EDDP was 13.6 with a range of 0.572 to 60. Diazepam was the most
common drug detected in addition to methadone. They also reported that methadone
concentrations were increased in HIV patients.

Baselt and Cravey (1995) report methadone concentrations in fatalities for various
specimens. The average concentrations in blood and brain were 1.0 mg/L and 1.0 mg/kg,
respectively, and that in kidney was 2.9 mg/kg. Liver was the tissue with the highest
concentration of methadone at 3.8 mg/kg. Other reported methadone concentrations
ranged from 0.18-3.99 mg/L for deaths caused or related to drug toxicity. In this same

study, the range for deaths not related to methadone toxicity was 018-303 mg/L

14

(Gagajewski and Apple, 2003). For deaths attributable to methadone overdosage in a
separate study, the listed range was 0.114-1.939 mg/L. For those due to trauma, the
range was 0.072-2.7 mg/L. The maximum concentration here is much higher than that

from the former group, and again there is an overlap (Wolf et al., 2004).

15

EXPERIMENTAL

Specimens

The cases included in this study were all methadone-positive postmortem cases
received at the State of Delaware OCME during a three and a half year period—from
September 2001 through the beginning of March 2005. All specimens collected during
autopsy (with the exception of bile and urine) were analyzed for methadone and its
primary metabolite (EDDP). When available, the following specimens were analyzed:
vitreous humor, peripheral blood (femoral), heart blood, brain, gastric contents, liver, and
kidney. In some instances, antemortem blood or serum specimens were also available for
analysis. Table 1 shows all of the specimens that were analyzed for each of the 100
cases. Following collection at autopsy, specimens were placed into refrigerated storage
at approximately 5°C. Specimens were later transferred to a fi'eezer for long-term storage
(approximately -10 to -12°C).

One milliliter of blood or vitreous humor was required for analysis. Solid tissue
specimens such as brain, liver, and kidney were homogenized with deionized/distilled
(DI) water to prepare a 1:4 tissue homogenate for analysis. All gastric contents were
initially prepared at a 1:25 dilution. If the initial results fell outside of the linear range of
the calibration curve, specimens were reanalyzed at an appropriate dilution. All dilutions

were prepared with DI water.

16

Table 1. Table showing all of the specimens that were analyzed for each case.

1
2
3
4
5
6
7
8
9
10
ll
12
l3
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43

Mbhbbbh
C‘OOOQO‘MA

 

H8
H8
Blood
Fluid
HB
Blood
HB
Ante Blood
118
Blood
em
HB
HB
HB
HB
HB Liver
HB
Aorta Blood
em HB
em
HB
HB
Ante Blood
HB
HB
em

Liver
Liver
Liver
Liver
B Liver
HB Liver
P em Sub
em HB B Liver
HB Liver
em Liver
em Liver Sub
P em HB Liver
em HB B Liver
em HB B Liver
em HB B Liver
P em HB GC
B Liver
HB Liver
em Liver
P Fem HB
em HB Liver

17

Table l (cont'd).

P em HB Liver
P Fem
P em HB B Liver
HB GC Liver P Sub
118 B GC Liver P Sub
HB ’ Liver P Sub
P Fem HB B GC Liver
P Fem HB Brain
HB B Liver
P Sub
HB
VH
em HB B Liver
P Fem HB
em HB B ' Liver
Fern HB Liver
Fem HB B GC Liver
em HB B ' Liver
HB Brain, Liver Blood
HB
Ante B Ante Serum
P em HB B Liver
P Fem HB B Liver
P em HB B Liver
P em 113
P em HB Liver
Fem Sub
P em HB B ° GC Liver
P em HB GC Liver
P em [-13 Liver
Ante B Ante Serum
P Fem HB Brain
Fem [-18 B Liver
P em HB Liver
HB
P em HB B Liver
em [-13 Liver
Liver
em HB Liver
B P Sub
P em HB Liver
P em HB Liver
P em HB B Liver
P em HB B Liver
em HB Liver
em Sub
P em HB B GC Liver
P em l-IB Liver
l-IB Liver Ante B
HB

18

Ante Serum

 

Chemicals and Materials

(i)-Methadone, (i)-Methadone-d3, EDDP, and EDDP-d3 standards were
purchased from Cerilliant® (Round Rock, Texas). The following reagents were required
as well: methanol, sodium phosphate monobasic, sodium phosphate dibasic, glacial
acetic acid, methylene chloride, isopropanol, concentrated ammonium hydroxide, and
ethyl acetate (absolute). All reagents were ACS Grade and purchased from Fisher
Scientific (Pittsburgh, Pennsylvania). Solid-phase extraction columns (ZSDAU020) were

purchased from United Chemical Technologies (UCT), Inc. (Bristol, Pennsylvania).

Extraction

For each run, a matrix blank and six calibrators containing both methadone and
EDDP were prepared (using calibrated pipettes) in properly labeled 16 x 125 mm test
tubes. The calibrators were prepared in drug-free whole blood and ranged in
concentration from 25 ng/mL to 1000 ng/mL. Two whole blood controls were prepared
at concentrations of 100 and 500 ng/mL. Case specimens were prepared in l-mL
aliquots. Solid tissue specimens were prepared in duplicate, and one aliquot was spiked
with 500 ng of methadone and 500 ng of EDDP. The spiked tissue specimens were
prepared so the method of standard addition could be used to ensure that there were no
significant matrix effects on the quantitation of methadone and EDDP in solid tissue
specimens (Andollo, 1998). Internal standard containing both d3-methadone and d3-
EDDP (100 ng each) was added to all calibrators, controls, and case specimens. All

samples were then mixed with 4 mL DI water and 2 mL phosphate buffer (100 mM, pH

19

6.0) and vortex-mixed for 15 seconds. The samples then sat at room temperature for 5
minutes followed by centrifugation at 3000 RPM for 10 minutes.

Solid-phase extraction of methadone and EDDP was performed with combination
reverse phase/cation exchange columns using a method adopted from UCT, Inc. (Moore,
1994). The SPE columns were conditioned with 3 mL methanol, 3 mL DI water, and 2
mL phosphate buffer. The supernatant of each sample was then applied to a column,
allowing gravity flow to pull it through. The columns were washed with 3 mL DI water,
1.25 mL 100 mM acetic acid, and 3 mL methanol, respectively. Following this, the
columns were dried for 5 minutes under vacuum (>10 inches Hg).

The analytes were eluted with 3 mL Basic Drug Elution Solvent (containing
methylene chloride/isopropanol/ammonium hydroxide—78/20/2) into properly labeled 5-
mL conical centrifuge tubes. Extracts were evaporated to dryness at approximately 35°C
under nitrogen in an adjustable temperature evaporator and then reconstituted in 50 uL
ethyl acetate. Final extracts were transferred to autosarnpler vials with reduced volume

inserts, and a l-uL aliquot was injected into the gas chromatograph for analysis.

Instrumentation

Analysis of methadone and EDDP was performed on a Hewlett Packard (HP)
model 6890 gas chromatograph (GC) with an Agilent 5973 Network Mass Selective
(MS) Detector (Wilmington, Delaware). An I-IPS-MS column (30 m length x 250 um
diameter x 0.25 pm film thickness) was used for separation. The GC was operated in the
constant pressure mode; the pressure was variable to maintain a constant retention time

for methadone. The GC oven temperature program started at 90°C where it was held for

20

1 minute, then ramped to 200°C at 25°C/min, increased and then ramped to a final
temperature of 300°C at 20°C/min where it was held for 2 minutes for a total run time of
12.40 minutes. The GC inlet mode was pulsed splitless with an inlet temperature of
260°C. The purge flow was 50 mL/min with a purge time of 1.00 minute. Ultrapure
helium was the carrier gas.

The mass spectrometer was operated in the selected ion monitoring (SIM) mode.
The transfer line temperature was set to 280°C. The ions monitored for d3-EDDP and
EDDP were 276, 277, 278, 279, 280, and 281. The ions monitored for d3-methadone and

methadone were 223, 226, 294, 297, 309, and 312.

Chromatography and Mass Spectrometry

Figure 2 is a total ion chromatogram of EDDP and methadone, showing that
EDDP elutes first. Figures 3-6 are ion chromatograms and corresponding SIM mass
spectra of d3-EDDP, EDDP, d3-methadone, and methadone, respectively. These figures
illustrate the peaks and ions used to identify and quantitate the analytes for each case. As
Figures 3 and 4 show, the retention time for d3-EDDP was 8.47 min, and that for EDDP
was 8.49 min. The retention time for d3-methadone was 9.00 min, and that for
methadone was 9.02 min (Figures 5 and 6). The deuterated internal standards (which
contain three deuterium atoms in place of three hydrogen atoms) were identified by ions
that were three atomic mass units heavier than those seen with EDDP and methadone.
Ethyl acetate was used as a solvent blank between standards, controls, and case
specimens where necessary. An ion chromatogram of ethyl acetate is also shown as

Figure 7. There was no carryover present in any of the blanks.

21

3200000 ' EDDP
3000000
2800000 : ,
2600000;
2400000 I
2200000 ”
2000000
1800000
1600000 . I
1400000 1
1200000 1
1000000
800000

600000 1 1

400000 I

200000 , I

0 WI 6.00 "7.00 . 8.00 ' 0.00 010.000”11.00”MW—“1’220'13'~
Time

Abundance

I Methadone

 

 

Figure 2. Total ion chromatogram of EDDP and methadone.

22

180000
160000
140000 8.47
120000
100000 I
80000 1
60000 II
40000 1
20000 '

0 l 7.40 7.60 7.80 8I00 8.20 8.40 8.60 8.80 9.00 9.20340326160“
Time

Abundance

 

 

70000 ' 279 I
60000 , I
50000 ‘ r J d3-EDDP
40000; .
30000 I
20000‘ I
10000I " a I i

I I
1... ~_..,_v . _- . *‘I‘Hf' 4. 1-7-” 72.. _.-_.__._.- _. -Vfi ._¢ .2;

0 ”268270272 277?? 276 278 280 282284 "286' 288 2'90"
m/z

Abundance

 

.
I
I I

Figure 3. Ion chromatogram (top) and SIM mass spectrum (bottom) of d3-EDDP.

23

1 800000
8.49
1400000 I

Abundance

1000000 ‘ I

EDDP

600000 I
I E

200000 II
0 7.40 7.60 7.80 8I00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60

Time

 

 

277

700000j 276 1

600000 ‘
500000 ‘ EDDP

400000

Abundance

300000‘

200000 ‘ 273

I

I

I

I

I

I

I

I I

I

1 I I : i

L. - t-.-LT__LLI-I. , 1 ,1 1-, -- , . . 2_TH_

0 268 270 272 274 276 278 280 282 284 286 288 290
m/z

 

100000 I

 

Figure 4. Ion chromatogram (top) and SIM mass spectrum (bottom) of EDDP.

24

Abundance

Abundance

r
I

46000
38000
30000
22000
14000.

6000 .

 

32000
28000:
24000:
20000:
16000,
12000‘
8000.
4000,
0

 

072,322.-.. .3. 2; _.:_..._:.I_._._.. _.

I
I4
‘ *W'I . *H‘rfi'fih-fi‘r—rfvr'r‘m

"I220 230 240 280IM26000270‘T280 290' 300

9.00

I d3-Methadone

I“
1 I

80608807003089.1101 9609801000
Time

80 8.0 8.20 8.40

226

I
I

dg-Methadone

I
I
I

I 312
II .
I I

f,-.-y-Tw—v—v—y 1 v—vw—f—vv-v—T—wv-

I

I

I

I I
I

I

I

 

310
m/z

Figure 5. Ion chromatogram (top) and SIM mass spectrum (bottom) of d3-methadone.

25

._ w» .L-_.LJ

"'—'_"I -fiuvwl ..A -—V ,2.

700000
600000 9'02

500000 I Methadone
400000 ‘

300000 I

200000 I

100000 I

Abundance

 

 

Time

36000I 223 I
32000;: : I
28000I ‘

24000?
20000:
16000:
12000 _ .
8000 i

4000 i I I I

~7—y—v—v—v~—Y-w—v-- . _-~—.——.—--~w—y .-. v v—r-~—.—~—v—-—v—y— v-v

(I 220'"230m"240""2§0 260” 270 280FI290"V300""310”V""'

m/z

Abundance

I

I

I
I Methadone I
I I
I I
I

309

I
I

 

Figure 6. Ion chromatogram (top) and SIM mass spectrum (bottom) of methadone.

26

07.80 8.00 8.20 8.40I8.60 8.80 9.00 920 9.40 I9I60m980"10.00””

 

 

 

 

220000 I I
200000‘
180000' I

9 160000 I I Ethyl Acetate

2 140000“

4:; 120000I ’ .*

3 100000 ‘ I I

< 80000I : I I I I I I
60000 I I I I I I I I I L I
400001 I I I I I I I I I' I I
20000‘ I I I I I I I II ;I I ‘

6.00 7.00 8.00 9.00 10. 0 11.00 12.00
Time

Figure 7. Ion chromatogram of ethyl acetate, which was used as a solvent blank.

27

Quantitation and Acceptability

A multi-point linear calibration curve containing seven points was constructed for
each separate analytical batch. Each calibration curve had a linear correlation coefficient
of 0.985 or better. All data files were quantitated, and calculations were based on the
internal standardization method. The internal standards were detected in all samples.
The limits of quantitation for methadone and EDDP were 25 ng/mL and 50 ng/mL,
respectively. Quantitative results for all calibrators were within i20% of their expected
values. Qualifier ion ratios were also evaluated. The ratios of positive findings were
required to be within 20% of the ion area ratios of a calibrator of similar concentration.

All quantitative results for the controls were also within i20% of their target values.

28

RESULTS AND DISCUSSION

This study included 100 methadone-related deaths that were identified during a
three and a half year period—September 6, 2001 through March 1, 2005—in the small
state of Delaware. Of the total, 61 were male and 39 were female. Eighty-one percent
were Caucasian, 18% were African American, and only one person was Hispanic. The
average age of the decedents was 41 years old (with a range of 16-65). These statistics
are very similar to those reported from other states.

In a study by Wolf et a]. (2004) of 125 methadone-related deaths in Palm Beach
County, Florida, 98 (78%) were males. The average age was 39 years old, and all but
one of their decedents was Caucasian. Similarly, in Hennepin County, Minnesota, over
90% of the methadone decedents from 1992 to 2002 were Caucasian, nearly 80% were
male, and the average age was 45 years old (Gagajewski and Apple, 2003). These same
figures were seen in the past as well. A Harris County, Texas study from 1987 to 1992
revealed that 77% of the methadone decedents were male, 85% were Caucasian, and the
median age was 35 years old (Barrett et al., 1996).

For this study, investigative reports were reviewed for case histories including any
known methadone usage, and toxicology reports were examined to determine additional
positive drug findings. Additional drugs were detected in approximately 70% of the 95
completed cases. Eighteen decedents were positive for ethanol. Eight decedents had
alprazolam in their systems, nine had diazepam, and 12 had nordiazepam (the major
metabolite of diazepam). Alprazolam and diazepam are benzodiazepine tranquilizers that

are primarily used to treat anxiety disorders. Cocaine was detected in ten cases, and

29

benzoylecgonine and ecgonine methyl ester (metabolites of cocaine) were found in 23%
and 20% of the 100 cases examined, respectively.
Autopsy reports were also reviewed for the causes and manners of death in each

case. The manner of death in over half the cases was accident, which accounted for 54
cases. Of the remaining 46 cases, 26 were classified as natural deaths, seven were
classified as suicides, four were undetermined, and nine are still pending. From this
information, the cases were categorized into groups as follows:

Group 1A) Drug-related deaths in which death was due to methadone

only;

Group 1B) Drug-related deaths in which death was due to methadone in

combination with other drugs;

Group 2) Drug-related deaths in which methadone was not a contributing

factor;

Group 3) Deaths in which methadone was an incidental finding (e. g.

deaths due to trauma);

Group 4) Natural deaths that were aggravated by methadone; and

Group 5) Undetermined or pending cases.
Of the 100 cases, 13 were placed in Group 1A, 27 in Group 13, 6 in Group 2, 33 in
Group 3, 9 in Group 4, and 12 in Group 5. Table A1, which can be found in the
Appendix, is a compilation of all this information. As the table shows, there were several
frequent findings. These include a history of heroin, cocaine, alcohol, and/or prescription

medication abuse, a history of depression and/or suicide attempts, back pain, Hepatitis C,

30

cirrhosis, HIV, and pneumonia. Karch and Stephens (2000) listed similar findings such

as cirrhosis, pneumonia, and HIV.

Methadone and EDDP Concentrations

All available specimens from each case were analyzed. Following GC/MS
analysis, the methadone and EDDP concentrations were determined. These results have
been categorized in tables according to the various groups so that trends in concentrations
could be evaluated. In all subsequent tables, any blank spaces indicate that a given
specimen was either not analyzed (due to an insufficient quantity) or not received. The
average, standard deviation (SD), number of positive cases (It), and range is included for
each category as well. An asterisk next to a case number indicates that additional
specimens such as antemortem blood were analyzed.
Group [A

The concentrations for Group 1A (drug deaths attributable to methadone only) are
shown on the next page in Table 2. As the table shows, the average concentration of
methadone in vitreous humor was 260 ng/mL (n = 10). EDDP was detected in vitreous
humor in only one case (Case 82) in this group. This case also happened to have the
highest methadone concentration detected in this specimen (840 ng/mL) amongst all six
groups. This analyte was only detected in one other vitreous sample from Group 1B.
The peripheral blood (femoral) concentrations ranged from 330-1800 ng/mL for
methadone with an average of 950 ng/mL (n = 9; SD = 510). The average concentration
of EDDP in peripheral blood was 110 ng/mL with a range of 50-220 ng/mL (n = 8; SD =

54).

31

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32

In a study of 125 methadone-related deaths in Palm Beach County, Florida, the
deaths were grouped as follows: methadone toxicity, combined drug toxicity, other
drugs, natural causes, and trauma. These groupings are very similar to those chosen for
this project. In deaths attributable to methadone toxicity alone, the mean concentration of
methadone in peripheral blood was 559 ng/mL with a range of 1 14-1939 ng/mL (Wolf et
al., 2004). The average in this study for Group 1A was significantly higher. In another
study by Milroy and Forrest (2000), the average concentration of methadone for a
category in which methadone was the only drug involved was 584 ng/mL, which is again
less than that seen in this project. The case with the highest reported methadone
concentration in this group was Case 68. Referring back to Table A1, it is interesting to
see that this was the only case in this group in which the manner of death was suicide
(intentional overdose). All others were accidents.

In heart blood, the mean concentration of EDDP was 96 ng/mL (n = 6). That for
methadone was 590 ng/mL (n = 12). The case with the maximum methadone
concentration was again Case 68. EDDP was not detected in brain for any of the cases.
The average concentration of methadone in this tissue was 1800 ng/ g (n = 8). The
highest concentration was not seen with Case 68 this time but with Case 82 (Caucasian
female on methadone for chronic back pain).

The average total amount of methadone in the gastric contents was 2.3 mg (n = 7).
The highest total amount (7.2 mg) was seen for Case 50, which was deemed an accident.
Gastric contents can be particularly useful to establish route of administration or a
minimum dose administered. However, one must ensure that the entire gastric contents is

collected and that it is homogenized prior to analysis to obtain a reliable result. The

33

concentration range for liver was 310-970 ng/ g for EDDP (n = 7; mean = 680; SD = 250)
and 1500-5600 ng/g for methadone (n = 8; average = 4000; SD = 1400). Case 76 had the
highest concentration of methadone, and this was another person who was taking
methadone for back pain. He could have been taking the drug for an extended time,
which would cause it to accumulate in his body.

Group 13

Table 3 provides concentrations of the various specimens for cases in Group 18
(multiple drug deaths involving methadone). The mean concentration of methadone in
vitreous was 160 ng/mL (n = 16). The average concentration of methadone in peripheral
blood was 640 ng/mL (n = 16; SD = 370; range = 170—1500). The average for EDDP was
180 ng/mL (n = 10; SD = 130; range = 64-450). Referring back to the study by Wolf et
a1. (2004), the average concentration of methadone in deaths due to combined drug
toxicity was 411 ng/mL. Again, the average for this study is higher. Case 49 had the
concentration of 1500 ng/mL, which was the maximum for this group. It is worthy to
note that this person was HIV positive. As was already mentioned, research has indicated
that methadone concentrations tend to be high in individuals infected with HIV (Karch
and Stephens, 2000).

The mean EDDP concentration in heart blood was 190 ng/mL (n = 1 1). For
methadone, the average was 530 ng/mL (n = 20). The average concentration of
methadone in brain was 1300 ng/ g (n = 13). The maximum concentration of 2900 ng/ g
was seen with a person who was on methadone up to three times a day for chronic back
pain (Case 34). This case also had the highest total amount of methadone in the gastric

contents (5.9 mg). The average was 0.80 mg (n = 14). The average concentration of

34

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35

EDDP in liver was 530 ng/ g (n = 15; SD = 300; range = 220-1500). That for methadone
was 3600 ng/g (n = 16; SD = 2000; range = 990-9200). The decedent with the maximum
methadone concentration here (Case 91) was known to go to the methadone clinic daily
for heroin abuse.
Group 2

Group 2 consists of drug deaths in which methadone was not contributory. Table
4 contains concentrations for cases in this group. The mean concentration of methadone
in vitreous was 110 ng/mL (n = 3). Peripheral blood had an average methadone
concentration of 410 ng/mL (n = 3; SD = 290; range = 82-600). Averages for heart blood
were as follows: EDDP—60 ng/mL (n = 2) and methadone—480 ng/mL (n = 4).

Methadone was detected in the brain at an average amount of 1000 ng/ g (n = 3).
The average total amount of EDDP in gastric contents was 0.017 mg (n = 2), and that for
methadone was 1.0 mg (n = 3). Liver had an average EDDP concentration of 570 ng/ g
with a range of 400-780 ng/g (n = 3; SD = 190). The mean methadone concentration in
liver was 5000 ng/g with a range of 2800-7900 ng/g (n = 3; SD = 2600). The majority of
the concentrations in this group (with the exception of liver) were lower than those from
Groups 1A and 18.
Group 3

This group, which was the largest of all, contains deaths in which methadone was
an incidental finding. Table 5 displays results from this group. The average methadone
concentration in vitreous was 180 ng/mL (n = 16). EDDP was detected in peripheral
blood at a mean concentration of 100 ng/mL (n = 6; SD = 31; range = 63-150).

Methadone was detected at an average concentration of 630 ng/mL (n = 12; SD = 370;

36

 

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range = 120-1400). The average EDDP amount in heart blood was 80 ng/mL (n = 12)
while that for methadone was 560 ng/mL (n = 23). The average concentration of
methadone detected in brain was 1700 ng/ g (n = 10). For gastric, the mean total amount
of methadone was 1.4 mg (n = 9), and that for EDDP was 0.089 mg (n = 2).

The average liver concentration was 760 ng/g for EDDP (n = 10; SD = 490; range
= 240—1600) and 5700 ng/g for methadone (n = 10; SD = 4800; range = 2300-19000).
This maximum concentration of methadone in liver (19000 ng/g) was seen with Case 48.
This was the greatest liver concentration amongst all the groups. It is very interesting to
note that the decedent here was an African American female (34 years old) who was
pregnant and taking methadone for heroin and cocaine addiction. The cause of death in
this case was ruptured ectopic pregnancy—not drug toxicity. Even though this was the
maximum concentration of methadone detected in liver for this project, studies have
actually shown increased clearance rates of methadone in pregnant women (particularly
during the second and third trimesters) (N anovskaya et al., 2004).

A study by Wong et a1. (2003) involved a female decedent who was six months
pregnant. This woman had a known history of drug and alcohol abuse and had been
taking methadone and amitriptyline (an antidepressant). The methadone levels in this
case were elevated due to deficient CYP2D6 metabolism, resulting from a mutation of
this enzyme. Perhaps genetic predisposition causing poor drug metabolism is the
explanation for the high methadone levels seen with Case 48 in this study. Another
explanation could be that the woman had acquired tolerance to methadone after possible
prolonged use. This case clearly illustrates the problem of overlapping concentrations in

methadone-related deaths.

40

Group 4

Group 4 consists of natural deaths that were aggravated by methadone. Table 6
contains information on concentrations for this group. The average concentrations of
EDDP and methadone in peripheral blood were 100 ng/mL (n = 4; SD = 45; range = 60-
140) and 400 ng/mL (n = 7; SD = 150; range = 160-630), respectively. The mean values
in heart blood were 73 ng/mL for EDDP (n = 5) and 410 ng/mL for methadone (n = 6).
The average methadone concentration in vitreous was 120 ng/mL (n = 7) and in brain
was 1000 ng/ g (n = 6). The average total EDDP content in stomach contents was 0.13
mg (n = 3). The mean total methadone content was 1.5 mg (n = 5).

Liver EDDP concentrations ranged from 240-1000 ng/ g with an average of 560
ng/ g (n = 6; SD = 290). Methadone was detected at a mean concentration of 2700 ng/ g (n
= 6; SD = 1500; range = 1400-5600). The maximum liver concentration (Case 80) was
more than double that of any other reported amount in this group. This person died from
bronchopneumonia, which was aggravated by methadone. The presence of pneumonia in
such cases ofien indicates methadone ingestion or complications of intravenous opioid
use (Ropero-Miller and Winecker, 2004; Karch and Stephens, 2000).
Group 5

The final group contains the 12 cases that are either undetermined or pending and
could thus not be grouped in one of the main groups. The average vitreous concentration
for methadone was 87 ng/mL (n = 8). That for brain was 1100 ng/ g (n = 8). Peripheral
blood had a mean EDDP concentration of 91 ng/mL (n = 5) and a mean methadone
concentration of 310 ng/mL (n = 9). The averages in heart blood were 110 ng/mL for

EDDP (n = 4) and 300 ng/mL for methadone (n = 9). The average total amount of

41

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42

methadone in the gastric contents was 0.71 mg (n = 6), which was the smallest average
seen for all the groups. The mean liver concentration was 670 ng/ g for EDDP (n = 6) and
2900 ng/ g for methadone (n = 9). The decedent with the highest concentration of 6800
ng/ g (Case 99) had an extensive history of prescription medication abuse, which is a
common finding among methadone users. All of this information for Group 5 can be
found on the following page in Table 7.

Additional Specimens

As Table 8 shows, additional specimens were available for approximately a
quarter of the cases. Such samples included peripheral blood (subclavian), kidney,
antemortem blood and serum, blood (type not indicated), aorta blood, and cavity fluid.
At the Delaware OCME, subclavian blood is typically collected during inspections when
a full autopsy is not performed (whereas heart blood is collected during autopsies). Of
particular interest to this project are concentrations in kidney since this is yet another type
of tissue. Unfortunately though, this specimen was only available for five cases.

For Case 33, the concentration of methadone in kidney was 1100 ng/ g. For this
same case, methadone was found at 1300 ng/ g in brain and 4200 ng/ g in liver. Similarly,
for Case 95, the kidney concentration for methadone was 1300 ng/g, which closely
compares to the amount of 1400 ng/ g in brain. The concentration in liver was 5500 ng/ g.
Case 99 also had comparable findings. The amount of methadone in kidney was 2000
ng/g. That in brain was 1900 ng/g, while that in liver was 6800 ng/ g. These three
specific cases suggest that methadone concentrations are similar in brain and kidney.

Liver, on the other hand, tends to have the highest concentration of all the samples.

43

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Summary

Table 9 is included to summarize all the averages, standard deviations, and ranges
for each of the specimens in each of the groups. Additionally, bar graphs have been
incorporated to show the average concentrations of the main specimen types in each of
the six groups (Figures 8-13). Both this table and these figures illustrate that the average
methadone concentration was highest in Group 1A for all specimens except liver. Group
3 had the greatest concentration, and this is also the group that contained the value
of 19000 ng/ g (the pregnant woman). Even after eliminating this value from the average,
it is still 4200 ng/ g, which would be the second highest average after Group 2. Even with
all the variables affecting methadone concentrations and metabolism, one would expect
to see the greatest concentrations of methadone in each of the samples in Group 1A since
this is the group in which death was due solely to methadone toxicity.

While viewing the bar graph for vitreous humor, one can see that EDDP is not
readily detected in this matrix. Ziminski et al. (1984) studied methadone, barbiturates,
and morphine in vitreous humor, blood, and several tissues. Their study showed that
water-soluble drugs are more likely to diffiise from the blood to the vitreous. Drugs must
also have adequate lipid solubility and must not be significantly affected by protein
binding. This suggests that EDDP does not have these characteristics since it was only
detected in two cases. The group with the second highest methadone average in vitreous
was Group 3—the group in which methadone was an incidental finding.

Figure 9 graphically shows the results for femoral blood. As was already
mentioned, the average is greatest for Group 1A. When comparing the bar graphs for all

the blood and tissue samples, it is clear that the average for femoral blood stands out most

46

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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47

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Figure 9. Bar graph showing the average concentrations in ng/mL of EDDP and
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48

 

 

 
 
  

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Figure 11. Bar graph showing the average concentrations in ng/ g of EDDP and
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49

 

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50

as being the greatest. For the other sample types, the averages are closer in range, so the
bar lines are closer in height. For peripheral blood, the group with the second highest
average was 1B (multiple drug deaths involving methadone), which again would be
expected. Group 3 was very close though; there was only a 10 ng/mL difference between
Groups 1B and 3. Methadone’s metabolite had the highest average in Group 1B, and all
the others were very similar in concentration.

The heart blood graph (Figure 10) reveals that concentrations in this specimen are
close in range amongst the various groups (mainly Groups 1A, 1B, 2, and 3). Group 1B
has the largest EDDP concentration again. Brain is similar to vitreous in that EDDP is
not normally present. Figure 11 illustrates that Group 1A has the greatest concentration
of methadone in brain, followed by Group 3. The figure for gastric contents is interesting
as well. The average total for Group 1A is far greater than those for the other groups.
Again, this would be expected since these are deaths of known methadone toxicity.
Surprisingly though, all groups registered having EDDP except Group 1B. This is
probably just coincidental since this compound is not readily present in gastric contents.

The bar graph for liver concentrations is the most baffling of the six. The reason
for this is that Group 3 has the maximum average followed by Group 2, and that is not
what would be expected. It is rather difficult to assess these results without a more
detailed history for each case including how long each person had been on methadone.
This information would help determine a person’s potential tolerance level at the time of
death. People who are poor metabolizers would have increased concentrations of
methadone. From the provided histories, it is hard to determine which individuals, if any,

had slower metabolisms. Thus, no additional conclusions could be made from this data.

51

Looking back to the cases with kidney samples, one can see that the concentration
of methadone in liver is between three and four times the concentration in brain or
kidney. This data compares closely to published data containing methadone
concentrations in brain and liver. It is important to note though that such data is scarce.
In an article by Bastos and Galante (1976) on traumatic deaths, they report median blood
and brain concentrations of 0. 13 mg/ 100 mL. A median liver concentration of 0.53
mg/lOO mL is also given. From these averages, one can see that the median liver

concentration is four times that of brain.

Ratios of Methadone to EDDP and Other Matrices to Blood

Most published data on methadone-related deaths focus on determining a lethal
concentration range. There are also many articles devoted to the pharmacodynamics and
pharmacokinetics of this drug. The specimen commonly analyzed in these studies is
blood (most often peripheral). A purpose of this study was to determine if other sample
types provide valuable information for the interpretation of methadone deaths. Another
focus was to study whether or not ratios of parent drug to metabolite and ratios of other
matrices to peripheral blood would help in classifying methadone deaths. When possible,
ratios of methadone to EDDP were calculated for peripheral blood (femoral), heart blood,
and liver. Additionally, ratios of methadone in vitreous, brain, and liver to methadone in
femoral blood were also calculated. These results are displayed in tables for each of the

groups. Averages, standard deviations, and ranges are included.

52

Group [A

For methadone intoxication deaths (Table 10), the average methadone-to-EDDP
ratio was 11 for peripheral blood (n = 8), 8.9 for heart blood (11 = 6), and 7.0 for liver (n =
7). The average ratio of vitreous to peripheral blood for methadone was 0.27 (n = 9).
The corresponding ratio for brain was 2.2 (n = 7), and that for liver was 5.7 (n = 7).
Ropero-Miller and Winecker (2004) report liver-to-central ratios of 6.2 and 7.5 for two
different groups of methadone-related deaths. These are in the same broad range as the
average for this study. Perhaps the reason for their increased ratio is that they used
central blood for the calculation rather than peripheral blood. Another explanation could
be a different sampling site in the liver.

The maximum ratio of methadone to EDDP in peripheral blood was 24 and in
liver was 10. These were seen with Case 74. The maximum in heart blood was 18 (Case
68). Both of these high ratios can be partially explained by information obtained from
the investigative reports. Case 74 involved a 23-year-old Caucasian male who recently
purchased methadone tablets from an illicit source. If this was one of his initial
exposures to methadone, then it would likely result in acute overdose. The second case
was a suicide, so intentional overdose would justify the high ratio.

Group IB

Table 11 contains ratios for Group 1B. The mean methadone-to-metabolite ratio
was 5.1 for peripheral blood (n = 10), 6.1 for heart blood (11 = 11), and 7.7 for liver (11 =
15). Case 7 had the highest ratio in heart blood, and perhaps this was due to a recent
switch fi'om a morphine-based medication to methadone. Methadone is not cross-tolerant

to morphine tolerance, which could help explain the ratio (Garrido and Troconiz, 1999).

53

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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55

The average ratio of vitreous to femoral blood was 0.30 (n = 12). The average ratio of
brain to femoral blood was 2.6 (n = 10), and that for liver was 7.3 (n = 11).
Group 2

Ratios for Group 2 are listed in Table 12. The mean methadone-to-EDDP ratio
was 12 for heart blood (n = 2), and 8.8 for liver (n = 3). The average ratio of vitreous to
peripheral blood was 0.46 (n = 3), that for brain was 4.2 (n = 3), and that for liver was 36
(n = 3). The reason for this extremely high liver-to-blood ratio is due to Case 87, which
has an individual ratio of 96. This decedent was in early stages of decomposition, which
could have affected the concentrations. This case is one that demonstrates why it is
important to know the history and circumstances surrounding death before interpreting
the drug findings.
Group 3

Table 13 consists of information for Group 3. In peripheral blood, the average
ratio of methadone to EDDP was 9.3 (n = 6). The ratio in heart blood was 9.5 (n = 12)
and in liver was 8.9 (n = 10). All ratios are fairly close in range here. Moreover, the
maximum in each of these three groups was seen with Case 84—a Caucasian male (44
years old) with a history of asthma, Hepatitis C, and heavy smoking. The average ratio
of vitreous to peripheral blood was 0.26 (n = 10). The brain-to-blood ratio was 2.3 (n =
6), and the liver-to-blood ratio was 12 (n = 7).
Group 4

The average ratio of methadone to EDDP in peripheral blood for Group 4 was 4.3
(n = 4). This data can be found in Table 14. The ratio in heart blood was 5.7 (n = 4) and

in liver was 5.2 (n = 6). The mean ratio of vitreous to peripheral blood was 0.30 (n = 6).

56

 

 

 

 

 

 

 

 

 

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59

 

That for brain was 3.0 (n = 6), and that for liver was 7.7 (n = 6). The maximum ratio of
liver to blood in this group was 13 (Case 80). This was the person who died from
bronchopneumonia (aggravated by drugs).

Group 5

The various ratios were determined for Group 5 as well. The average ratio of
parent drug to metabolite was 4.3 for peripheral blood (n = 5), 3.5 for heart blood (n = 4),
and 6.8 for liver (11 = 6). It is interesting to note that the maximum ratio in liver was from
Case 88, which was an exhumed body. The body was exhumed due to suspicion of
poisoning, which surfaced after burial. The mean vitreous-to-blood ratio was 0.28 (n =
7). The average ratio of brain to blood was 3.2 (n = 6), and the average ratio of liver to
blood was 8.9 (n = 7). This information is listed in Table 15.

Additional Specimens

When possible, ratios were determined for additional specimens as well (Table
16). The ratio of methadone to EDDP was calculated for peripheral blood (subclavian),
kidney, antemortem blood and serum, blood, and aorta blood. These ratios were low for
kidney and antemortem blood and serum (compared to those seen with the typical
specimens). Additionally, subclavian-to-femoral ratios were determined for methadone
as well as kidney-to-blood ratios.

In this study, antemortem blood was only available in five cases. Antemortem
serum was also available in three of these cases. The work of Cook et a1. (2000) showed
that postmortem-to-antemortem (PM/AM) ratios are typically similar to central-to-
peripheral (C/P) ratios. When the C/P ratio is high for a given drug, it is likely that the

PM/AM ratio will also be high. In 11 cases they examined, the average PM/AM ratio

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62

was 1.4. In this present study, Case 99 was the only case in which a PM/AM ratio could
be calculated. The PM/AM ratio for this case was 1.3, which compares closely with the
average reported by Cook et al. The C/P ratio for Case 99 was 1.6, showing that the
PM/AM ratio is also similar to this value.

Summary

Table 17 is provided to summarize all the average ratios for all the different
specimens and groups. Bar graphs have been included as graphical representations of
this data as well (Figures 14-19). Figure 14 shows that the mean methadone-to-EDDP
ratio was the greatest in Group 1A, which is again what was expected. Group 3 had the
second highest ratio. In heart blood, however, the maximum ratio was seen in Group 2.
This confirms that peripheral blood is the preferred specimen for analysis.

The ratios of parent drug to metabolite in liver were very similar amongst the six
groups. For the ratios of alternate matrices to blood, Group 2 had the maximum average
ratio for each. This is, however, due to the fact that Case 87 was in this group. Since
there were only three cases, this one case significantly skewed the ratios. If this ratio of
96 for liver-to-blood (Case 87) was eliminated from the average, the average ratio would

drop from 36 to 6.3. This value would place Group 2 in the same range as the other

groups.

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Figure 14. Bar graph showing the average ratios of methadone to EDDP in peripheral
blood (femoral) for the six groups.

 

 

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for the six groups.

65

 

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66

 

 

 

 

 

 

 

 

 

 

 

 

Average Ratio

 

 

 

 

1A 13 2 3 4
Group

Figure 18. Bar graph showing the average ratios of methadone in brain to methadone in
peripheral blood (femoral) for the six groups.

 

 

 

 

 

 

 

 

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Group

Figure 19. Bar graph showing the average ratios of methadone in liver to methadone in
peripheral blood (femoral) for the six groups.

67

 

Central-to-Peripheral Ratios

Since postmortem redistribution is a recognized phenomenon with methadone, its
role was considered in this project. The central blood in this study was heart blood, and
femoral blood was used for the peripheral blood. The ratio of heart blood to peripheral
blood was calculated for each case when possible. An average of all the cases as one big
group was also determined (Table 18). The average was 1.3 with a range of 054-95 (n =
47; SD = 1.3). The greatest C/P ratio of all the cases was 9.5 (Case 87). This high ratio
is not surprising considering this was a decomposed body so there was ample time for the
drugs to move between the tissues and blood. When specimen collection occurs more
than 72 hours following death, peripheral blood is preferred for analysis as it is less
subject to contamination and redistribution (Wong et al., 2003).

The average C/P ratio from this research project correlates strongly with C/P
ratios reported by Ropero-Miller and Winecker (2004). For methadone-related deaths,
the Cl? ratio was 1.5 for Group 1 (tolerant users) and 1.8 for Group 2 (non-tolerant
users). Another source sites a study in which the average C/P ratio was 1.1 (Wong et al.,
2003). From published data by Levine et a1. (1995), the average heart blood-to-altemate
blood ratio for 15 methadone cases was calculated to be 1.3—the same exact ratio seen in
this study. In the study by Levine, the alternate blood was subclavian for nine of the
cases, pericardial for three of the cases, inferior vena cava for two of the cases, and
femoral for just one of the cases. The mean C/P ratio from this research project closely
agrees with values from the literature. Methadone is a highly lipophilic drug, so it will

readily diffuse from higher concentrations to lower concentrations, redistributing in the

body (Drummer, 2004).

68

Table 18. Ratios of methadone in heart blood to
methadone in peripheral blood (femoral) for each case in all groups.

em)
G Case Ratio for MDN
1A 50 0.54
65 0.93
66 0.73
68 0.83
74 0.67
76 0.91
82 0.56
89 1.4
l .5
30 1.5
32 0.80
34 0.96
41 0.85
43 0.85
49 0.87
51 1.2
53 1.6
73 0.54
83 1.2
86 0.82
91 1.6
57 .80
72 1.1
87 9.5
33 1.4
37 1.4
39
45 2.7
48 1.6
58 1.1
64
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78
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93 .7

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63 1.0
67 1.4
80 1.4
79 .65
92 0.70
94 1.6
95 1.1
97 1.0
98 1.2
99 1.6

1.3
47
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69

CONCLUSIONS AND FUTURE WORK

In conclusion, this project has demonstrated the value of analyzing alternative
specimens such as brain and liver for methadone—related deaths to determine the
distribution of this drug and EDDP. The usefulness of calculating ratios of parent drug to
metabolite and concentrations in other matrices has also been assessed. Methadone is a
highly lipophilic drug with a high volume of distribution, so it is extensively distributed
throughout the body following administration. Afier dividing the 100 cases into six
different groups based on methadone’s contribution to death, the mean concentration of
methadone and its metabolite were determined for each specimen in each group.
Additionally, blood concentrations were related to concentrations in alternative matrices.

Group 1A (drug deaths in which death was attributable to methadone only) had
the highest concentration for all specimens except liver. The maximum methadone
concentration in liver (19000 ng/ g) was actually seen with a case in Group 3 (deaths in
which methadone was an incidental finding). Liver concentrations were greater than
blood concentrations in all cases. The liver-to-blood ratios ranged from 2.7 to 96 among
all groups and were, on average, between five and ten. Brain concentrations were greater
than corresponding blood concentrations in all cases. Brain-to-blood ratios ranged from
1.4 to 8.5 among all groups and were, on average, between two and four. The vitreous-
to-blood ratios were less than or equal to one in all cases. The maximum mean
methadone-to-EDDP ratio in peripheral blood was observed for Group 1A (average = 1 1;

n = 8). No noticeable patterns were observed for these ratios in the other specimens.

70

It is important to note that Baselt and Cravey (1995) report fatal methadone
concentrations of 1.0 mg/L in blood, 1.0 mg/kg in brain, and 3.8 mg/kg in liver. This
would equate to a brain-to-blood ratio of 1.0 and a liver-to-blood ratio of 3.8. These
results are from a study of only 10 methadone cases and are reported in Disposition of
Toxic Drugs and Chemicals in Man (a popular reference material among forensic
toxicologists). The average brain-to-blood ratios from this research project are all greater
than one. Thus, the results from this study of 100 methadone cases could provide an
additional reference guide to toxicologists concerning the distribution of methadone in
tissues.

Factors such as tolerance and individual differences in metabolism make
methadone deaths difficult to interpret due to overlapping therapeutic and toxic
concentrations. Drug interactions also complicate interpretation, and additional drug
findings are common among methadone deaths. Users will often take other drugs such as
benzodiazepines to increase the effects of their highs. These drugs are known to increase
respiratory depression when taken with methadone. Individuals who have acquired some
tolerance to heroin or morphine can still overdose on methadone since methadone
accumulates in the body and does not induce tolerance as readily as other opioids
(Garrido and Troconiz, 1999).

Another problem with interpretation of methadone-related deaths is the
phenomenon of postmortem redistribution. The extent of redistribution was evaluated in
this study by determining heart blood-to-peripheral blood ratios. The average C/P ratio
was 1.3 (n = 47), which is very close to reported values for methadone (Levine et al.,

1995; Ropero-Miller and Winecker, 2004). One case in this study had a C/P ratio of 9.5,

71

which was not unexpected since the decedent was in early stages of decomposition. In
other words, there was a long time interval between death and specimen collection in
which redistribution occurred.

The findings fi‘om this study re-emphasize the importance of obtaining additional
information when evaluating the role of methadone in death. There was a considerable
overlap of concentrations and ratios between the various groups. Consequently, blood
concentrations in isolation are not always useful in interpreting deaths involving
methadone. If a blood concentration were ever questionable, then analyzing tissues
would help the toxicologist assess the distribution of methadone throughout the body. If
the concentrations in these alternative specimens were within the ranges reported in this
study, then the toxicologist would be able to better evaluate the blood concentrations.

This research is particularly useful for pathologists and toxicologists in
methadone cases where the cause and manner of death are uncertain. This project is not
suggesting that additional specimens such as brain and gastric contents be routinely
analyzed for methadone cases as this would be ineffective and time-consuming. When
applicable though, analysis of alternative specimens may aid in evaluating methadone-
related deaths. These cases, however, still remain challenging when little information is
known (e. g. dosing history).

Future work could include more in-depth evaluation of drug interactions for each
of the specific cases. Additionally, it would be interesting to attempt to retrieve
additional case information fi'om methadone clinics in the state. In several cases where
high concentrations were unexplainable, it would be valuable to learn of the decedent’s

dosing history and how long he or she had been taking methadone. This information

72

would help in assessing the person’s probable tolerance. Future work might also involve
comparing these postmortem methadone concentrations with concentrations obtained
from “Driving Under the Influence” cases in which the methadone users are alive.

Comparing concentrations and ratios across these two groups might prove educational.

73

APPENDIX

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88

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