Journal of Analytical Toxicology, 2016;40:367–373

doi: 10.1093/jat/bkw028
Article

Article

Evaluation of Postmortem Drug Concentrations

in Bile Compared with Blood and Urine in
Forensic Autopsy Cases
Mariko Tominaga1,2,*, Tomomi Michiue1,2, Shigeki Oritani1,
Takaki Ishikawa1,2, and Hitoshi Maeda1,2
1
Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, Osaka 545-8585,
Japan, and 2Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center
(MLCPI-SC), c/o Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, Osaka 545-8585, Japan
*Author to whom correspondence should be addressed. Email: michi.leg@med.osaka-cu.ac.jp

Abstract
For drug screening and pharmaco-/toxicokinetic analysis, bile as a major drug excretion route in
addition to urine may be used in forensic autopsy cases; however, there are limited published
data on correlations between bile and blood or urine drug concentrations. The present study retro-
spectively investigated drug concentrations in bile, compared with blood and urine concentrations,
reviewing forensic autopsy cases during 6 years (January 2009–December 2014). Drugs were
analyzed using automated gas chromatography–mass spectrometry following solid–liquid phase
extraction. Compared with peripheral blood concentrations, bile concentrations were higher for
most drugs; however, caffeine concentrations were similar. Bile concentrations were mostly lower
than urine concentrations for amphetamines, caffeine and methylephedrine, but were usually similar
to or higher for other drugs. Significant correlations were detected between bile and peripheral blood
concentrations for amphetamines, several cold remedies, phenobarbital, phenothiazine derivatives
and diazepam, as well as between bile and urine concentrations for amphetamines, caffeine, diphen-
hydramine, phenobarbital and promethazine derivatives. These findings suggest that bile can pro-
vide supplemental data useful in routine forensic toxicology, for the spectrum of drugs mentioned
above, as well as for investigating pharmaco-/toxicokinetics and postmortem redistribution when
analyzed in combination with drug concentrations at other sites.

Introduction the intake routes, pharmaco- or toxicokinetics and chemical proper-

In forensic toxicology, various body fluids such as pericardial effusion
and cerebrospinal fluid are used when adequate blood specimens are
not available at autopsy (1–9). Previous studies showed the marked
postmortem redistribution of drugs in the heart and peripheral
blood, which are partly characteristic of individual drugs (10, 11);
thus, it is difficult to estimate blood concentrations during the process
of and at the time of death based on postmortem blood concentrations
(12). With respect to this, simultaneous analyses of blood and other
body fluids provide useful information for investigating the antemor-
tem distribution, postmortem redistribution by diffusion and degrad-
ation, incidental production and contamination, which can depend on
ties of individual drugs and poisons (1–8, 10–20). Urine is a conven-
tional material for drug screening and pharmaco-/toxicokinetic
analysis (21). Several studies have shown that bile as another major
excretion route of a spectrum of drugs and their metabolites may
also be used in forensic autopsy cases (22, 23); however, there are
limited published data on correlations between bile and blood or
urine drug concentrations (24, 25).
Given the observations described above, the present study retro-
spectively investigated drug concentrations in bile, compared with
blood and urine concentrations, reviewing postmortem toxicological
data of serial forensic autopsy cases to examine the potential

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368
usefulness of bile as an alternative to blood and a material for investi-
gating pharmaco-/toxicokinetics and postmortem redistribution.
Materials and methods
Autopsy database
All forensic autopsy cases (n = 1,003) during a period of 6 years
(January 2009–December 2014), excluding those where adequate
specimens were not available due to advanced decomposition or ske-
letonization, were retrospectively reviewed, and those with positive
toxicological findings in bile were collected to compare the data
with those of right heart blood, peripheral blood and urine. These
data analyses as well as sample collections and the analyses described
below were performed within the framework of the routine medicole-
gal casework following the autopsy guidelines (2009) and ethics
guidelines (2003) of the Japanese Society of Legal Medicine, approved
by the institutional ethics committee.
Analytical procedures
Autopsy material
Besides heart blood and peripheral external iliac blood, bile and urine,
as well as the stomach contents, were routinely collected at autopsy
and analyzed in parallel. Peripheral blood (∼5 – 25 mL) was drawn
using an aseptic syringe from the external iliac vein. Bile (∼5 – 15 mL)
and urine (∼5 – 25 mL) were drawn using an aseptic syringe after
opening the abdominal cavity. These specimens were subsequently
stored at −20°C until analysis.
Chemicals and reagents
Bond Elut Certify columns were provided by Agilent Technologies
(Santa Clara, CA, USA). Deionized pure water was obtained using a
Milli-Q Purification System (Millipore, Bedford, MA, USA). Other
chemicals and reagents, including standard drugs and internal
standards, as listed below, were of the highest purity commercially
available (7, 8).
Sample preparation
Midazolam, diazepam, olanzapine and zolpidem were extracted using
liquid/liquid phase extraction: 50 µL of internal standard ( prazepam)
solution and 1 mL of saturated sodium bicarbonate solution were
added to 1 mL of each sample and vortexed; after 0.5 g of sodium
chloride was added and vortexed, 3 mL of toluene was added for
extraction by shaking for 30 min; the solvent was separated by centri-
fugation. Other drugs and chemicals were extracted using a Gilson
ASPEC XL-274 automated SPE solid-/liquid-phase extraction instru-
ment (Middleton, WI, USA) as follows. To 0.5 mL of each sample,
50 µL of solution containing the following internal standards
was added: allobarbital for phenobarbital; 3-phenylpropylamine for
amphetamine and methamphetamine; and cocaine for caffeine, chlor-
pheniramine, chlorpromazine, dihydrocodeine, diphenhydramine,
methylephedrine, levomepromazine, lidocaine and promethazine
(7, 8). The pH of the sample was adjusted to ∼7.0 by adding 6 mL
of 0.1 M potassium phosphate buffer pH 6.0. The mixture was mildly
mixed, centrifuged at 2,500 × g for 5 min, poured into HF-Bond Elut
Certify columns (acetaminophen; Autoprep EDS-1) and gently
aspirated. Columns had been previously conditioned with 2 mL of
methanol and 2 mL of 0.1 M potassium phosphate buffer pH 6.0.
After applying the samples, the columns were then successively
washed with 1 mL of buffer, 1 mL of 1 M acetic acid solution and
1 mL of methanol. Finally, analytes were eluted with 3 mL of a freshly
Tominaga et al.
prepared mixture of dichloromethane/isopropanol (78 : 20) ammo-
nium hydroxide. The eluates were collected and evaporated to dryness
under a gentle stream of nitrogen at room temperature. The residues
were reconstituted with 100 µL of ethyl acetate and 1 µL aliquots
of the extracts were injected into the gas chromatography–mass
spectrometry (GC–MS) system. The recovery of standards ranged
from 60–70% ( phenobarbital) to >95% (codeine) (7, 8).
Instrumental conditions
Automated GC–MS following solid-/liquid-phase extraction was
performed using an Agilent Technologies GC/MS System Model
5975c MSD (column, DB-5MS, 30 m × 0.25 mm i.d., film 0.25 µm;
column temperature, 100–325°C; injector temperature, 280°C;
turbocharged carrier gas, He at a flow rate of 48 cm/s; interface tem-
perature, 300°C). Analytical precision ranged from 9.6% ( phenobar-
bital) to 16.1% (midazolam) (7, 8).
Statistics
Regression equation analysis was used to study the relationship between
pairs of parameters for drug concentrations. These analyses were carried
out using StatView (Version 5.0; SAS Institute, Inc., Cary, NC, USA).
Results
Drugs detected in bile
Bile was collected in 949 cases (94.6%); adequate bile samples were not
collectable in 54 cases, which mostly included post-cholecystectomy
cases (n = 44; 81.5%), as well as some cases of decomposing gallblad-
der (n = 3; 6.8%), gall/bile duct stone(s) (n = 3; 6.8%) and insufficient
gallbladder contents (n = 4; 7.4%). In bile samples of 160 cases
(16.8%), 46 drugs including metabolites were detected (Table I).
Among these cases, the following drugs were identified in bile of
14 cases where adequate blood specimens were not available because
of severe bodily injury/damage or decomposition, with regard to the
documented cause of death: alprazolam, olanzapine and prometha-
zine in a fatal drug abuser, and chlorpromazine, phenobarbital
and promethazine in a chronic alcohol abuser, as well as amphetamine
(n = 3), chlorpromazine (n = 1), dihydrocodeine (n = 1), methampheta-
mine (n = 4), phenobarbital (n = 2) and theophylline (n = 1) in other
cases (blunt injury, n = 6; drowning, n = 1; hyperthermia, n = 1;
natural death, n = 4). Among these, drugs that were detected in bile
and blood or urine in more than five cases were used for the following
statistical analysis (Table II), excluding lidocaine used for intubation
in critical medical care, because of diverse and partly very low bile
concentrations, as well as diminished vitality after administration.
Data on the stomach contents were used to evaluate the influence on
bile drug concentrations.
Amphetamines
Oral or injected methamphetamine, as well as amphetamine, usually
as the possible metabolite, mostly showed about 2–10 times higher
concentrations in bile than in right heart and peripheral iliac venous
blood, as estimated using mean and median ratios, with moderate
to excellent correlations (Table II). Bile concentrations were lower
(about ×1/4–1/2) than urine concentrations, showing excellent corre-
lations, but were slightly higher (about ×1.2–2) than in stomach con-
tents without markedly high concentrations related to high stomach
concentrations (Tables I and II). Greater deviations of these site-to-site
ratios were seen in low drug concentration ranges, and also for the
drugs described below.
Postmortem Drug Concentrations in Bile 369

Table I. Drugs Detected in Bile, Right Heart Blood, Peripheral External Iliac Blood, Urine and Stomach Contents

Drugs Bile Right heart blood Peripheral external Urine Stomach contents
iliac blood

n Concentration n Concentration n Concentration n Concentration n Concentration

(mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL)
mean/median mean/median mean/median mean/median mean/median

7-Aminoflunitrazepam 4 0.21–1.38 4 0.008–0.59 3 0.11–1.34 4 0.009–2.94 4 0.07–13.4

0.74/0.69 0.18/0.05 0.63/0.48 1.38/1.27 3.85/0.98
Acetaminophen 3 1.1–30.8 3 0.3–19.8 2 0.47, 3.18 2 15.8, 35.7 2 0.43, 230.2
10.9/1.1 6.8/0.33 1.83/– 25.7/– 115.3/–
Alprazolam 1 1.12 0 – 0 – 0 – 0 –
Amitriptyline 4 0.001–22.3 4 0.001–0.59 2 0.001, 0.15 2 0.61, 1.13 3 0.36–1.97
7.38/3.64 0.24/0.18 0.076/– 0.87/– 1.10/0.96
Amphetamine 39 0.016–5.8 30 0.006–1.2 19 0.006–1.05 30 0.02–23.7 34 0.01–3.30
1.16/0.65 0.38/0.11 0.18/0.077 6.1/2.8 2.35/0.37
Bromazepam 3 0.02–1.90 3 0.01–0.47 2 0.01, 0.02 1 0.31 3 0.05–1.10
0.67/0.08 0.17/0.02 0.016/– 0.45/0.21
Caffeine 24 0.09–11.8 22 0.09–6.96 13 0.38–5.82 16 0.12–5.92 19 0.07–13.6
1.43/0.78 1.65/0.99 2.20/1.69 1.69/1.31 3.47/2.60
Carbamazepine 1 4.68 1 2.68 0 – 1 0.52 1 1.71
Chlorpheniramine 11 0.01–1.59 8 0.002–0.29 8 0.001–0.20 8 0.037–4.54 8 0.018–2.14
0.48/0.36 0.096/0.069 0.066/0.054 0.93/0.159 0.51/0.25
Chlorpromazine 17 0.021–51.6 12 0.005–1.79 9 0.007–1.49 12 0.007–6.82 16 0.008–116.4
0.62/1.36 0.28/0.06 0.35/0.06 1.10/0.19 17.5/2.32
Clomipramine 2 0.97, 3.84 2 0.15, 0.35 2 0.20, 0.35 2 0.12, 1.77 2 0.60, 1.00
2.41/– 0.25/– 0.21/– 0.95/– 0.80/–
Desalkylflurazepam 4 0.13–0.83 4 0.05–0.19 4 0.06–0.17 3 0.06 2 0.06, 0.18
0.33/0.17 0.09/0.08 0.09/0.06 0.06/0.06 0.12/–
Desipramine 1 0.04 1 0.02 1 0.04 1 0.71 1 0.04
Diazepam 8 0.03–0.79 6 0.008–0.20 5 0.009–0.261 3 0.007–0.024 6 0.03–3.40
0.18/0.11 0.08/0.07 0.09/0.08 0.02/0.018 0.70/0.19
Dihydrocodeine 10 0.06–1.3 8 0.006–0.614 5 0.04–0.65 7 0.006–3.47 8 0.02–4.31
0.41/0.21 0.12/0.052 0.19/0.065 1.43/1.13 0.74/0.26
Diphenhydramine 15 0.001–14.5 14 0.001–2.16 11 0.001–0.50 9 0.002–4.46 12 0.004–149.9
3.13/0.35 0.43/0.052 0.11/0.058 0.89/0.055 15.2/0.19
Estazolam 3 0.09–0.74 2 0.10, 0.18 2 0.09, 0.11 2 0.01, 007 3 0.001–4.31
0.34/0.19 0.14/– 0.10/– 0.04/– 1.46/0.08
Flunitrazepam 3 0.003–4.42 2 0.29, 0.42 2 0.65, 0.96 2 0.19, 0.82 3 0.96–25.1
0.46/3.93 0.36/– 0.80/– 0.50/– 11.6/8.8
Flurazepam 3 0.06–0.12 2 0.003, 0.04 1 0.002 2 0.007, 0.008 2 0.13, 16.4
0.09/0.09 0.02/– – 0.008/– 8.25/–
Imipramine 2 0.77, 1.15 2 0.05, 0.13 2 0.05, 0.12 2 0.22, 0.83 1 0.64
0.96/– 0.09/– 0.08/– 0.53/–
Isoniazide 1 0.96 1 10.5 1 11.6 1 57.2 1 0.62
Lamotrigine 1 6.94 1 0.16 1 0.28 1 1.85 1 9.98
Levomepromazine 11 0.043–12.9 9 0.008–0.91 8 0.009–0.60 10 0.009–20.9 9 0.006–423.0
3.5/2.6 0.36/0.35 0.27/0.37 3.12/1.27 45.0/0.47
Lidocaine 55 0.004–2.72 49 0.001–3.41 37 0.001–2.21 20 0.007–2.74 42 0.003–7.40
0.39/0.07 0.42/0.31 0.22/0.08 0.50/0.13 1.33/0.57
Marprotiline 1 34.7 1 3.12 0 – 1 5.11 1 27.8
Metoclopramide 1 0.3 1 0.03 1 0.03 1 0.45 0 –
Methamphetamine 36 0.005–17.9 30 0.01–4.52 19 0.001–2.07 28 0.005–136.44 34 0.06–10.22
4.02/1.74 0.75/0.38 0.47/0.27 21.2/6.76 3.25/2.07
Methylephedrine 12 0.05–5.25 9 0.03–0.75 11 0.006–1.12 8 0.321–16.93 34 0.05–11.35
1.17/0.75 0.21/0.11 0.28/0.09 4.75/2.49 2.84/0.76
Mexiletine 1 2.71 0 – 1 0.48 1 14.6 1 4.68
Mianserin 2 1, 20.7 2 0.11, 0.54 1 0.13 2 0.11, 0.23 1 0.38
10.9/– 0.33/– 0.17/–
Midazolam 11 0.031–3.4 11 0.012–1.74 6 0.005–0.43 4 0.046–3.22 10 0.032–3.21
0.96/0.29 0.49/0.56 0.30/0.21 0.50/0.13 1.06/0.75
Mirtazapine 4 0.66–3.82 3 0.01–0.03 4 0.02–0.15 3 0.01–0.74 4 0.08–1.20
1.75/1.26 0.02/0.03 0.07/0.04 0.36/0.34 0.48/0.26

Table continues
370 Tominaga et al.

Table I. Continued

Drugs Bile Right heart blood Peripheral external Urine Stomach contents
iliac blood

n Concentration n Concentration n Concentration n Concentration n Concentration

(mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL)
mean/median mean/median mean/median mean/median mean/median

Olanzapine 9 1.13–12.2 8 0.15–1.99 5 0.10–1.66 6 0.45–6.4 8 0.20–4.24

5.64/5.00 0.91/0.83 0.68/0.52 2.23/1.27 2.10/1.57
Paroxietine 1 0.02 1 0.03 1 0.007 1 0.05 1 0.26
Pentobarbital 2 0.56, 18.9 2 0.14, 14.7 0 – 0 – 2 0.06/13338
9.72/– 7.41/– 6669/–
Phenobarbital 31 0.12–242.4 28 0.08–52.8 17 1.66–33.9 22 0.04–20.5 29 0.05–71.2
22.9/12.4 9.93/7.25 9.73/6.56 9.59/8.63 13.9/7.64
Phentermine 1 0.013 1 0.05 1 0.02 1 0.01 1 0.021
Phenytoin 1 43.9 1 8.15 1 7.01 1 5.21 1 4.83
Promethazine 23 0.001–14.3 18 0.002–3.44 14 0.002–4.01 15 0.008–14.7 21 0.007–31.5
2.82/1.29 0.46/0.12 0.62/0.15 2.06/0.41 4.74/1.12
Propofol 3 0.18–20.3 3 0.08–0.12 3 0.12–0.19 2 0.18, 23.9 3 0.015–0.39
7.19/1.13 0.11/0.11 0.15/0.15 12.1/– 0.21/0.23
Secobarbital 4 0.29–47.2 4 0.08–2.76 1 2.22 4 0.08–3.69 4 0.07–3.78
16.1/8.45 1.36/1.29 – 2.30/2.71 1.82/1.71
Theophylline 1 0.07 0 – 0 – 1 0.36 1 0.09
Thiamylal 4 0.24–70.2 4 0.04–15.5 1 8.73 2 10.7, 21.0 3 1–26.4
29.8/24.4 7.76/7.76 15.9/– 13.6/13.5
Triazolam 1 0.17 0 – 0 – 0 – 1 1.33
Zolpidem 9 0.002–0.99 9 0.004–0.55 9 0.004–0.57 5 0.06–0.57 8 0.03–15.5
0.33/0.27 0.15/0.11 0.14/0.08 0.19/0.06 3.74/1.38
Zopiclone 1 3.35 1 0.38 1 0.02 1 1.99 1 1.4

Cold remedies
Bile concentrations of oral caffeine were similar (about ×0.8–1)
to those in right heart and peripheral iliac venous blood and were
mostly slightly lower (about ×0.7–0.9) than in urine with good
correlations (Table II). Compared with concentrations in stomach
contents, bile concentrations were mostly lower (about ×0.5–0.9)
without markedly high concentrations related to high stomach con-
centrations (Table I).
Methylephedrine, chlorpheniramine, dihydrocodeine and di-
phenhydramine concentrations in bile were higher than in blood,
showing various relationships (about ×3–18); bile to blood correla-
tions of these drugs were excellent for dihydrocodeine in both right
heart and peripheral iliac venous blood, but were better for chlor-
pheniramine and diphenhydramine in peripheral iliac venous
blood, and for methylephedrine in right heart blood than at the
other sites (Table II). Bile diphenhydramine concentrations were
higher (about ×3–11) than urine concentrations with a good correl-
ation and were higher (about ×4–6) than in stomach contents with-
out markedly high concentrations related to high stomach
concentrations (Tables I and II). Bile concentrations of methylephe-
drine, dihydrocodeine and chlorpheniramine were similar to or
lower (about ×0.2–2.4) than concentrations in urine; correlations be-
tween bile and urine concentrations were insignificant (Table II).
These bile concentrations were also similar to or lower than in the
stomach, without markedly high concentrations related to high
stomach concentrations (Table I).
Psychotropics
Oral phenobarbital mostly showed higher concentrations (about ×2)
in bile than in the right heart, peripheral iliac venous blood and
urine, with significant correlations (Table II). Bile concentrations
were also higher than in stomach contents, without markedly high
concentrations related to high stomach concentrations (Table I).
Oral chlorpromazine, levomepromazine and promethazine con-
centrations in bile were markedly higher (about ×6–45) than those
in right heart and peripheral iliac venous blood, partly showing signifi-
cant correlations, which were better for peripheral than right heart
blood for most drugs (Table II). Bile concentrations of these drugs
were similar to or higher (up to about ×12) than concentrations in
urine, showing good correlations (Table II). Diazepam concentrations
in bile were also higher (about ×2–4) than in right heart and peripheral
iliac venous blood, showing significant correlations between bile and
peripheral blood concentrations (Table II). No significant relation-
ships were detected between bile and blood or urine for midazolam,
zolpidem and olanzapine. These bile concentrations were also similar
to or higher than in the stomach, without markedly high concentra-
tions related to high stomach concentrations (Table I).
Discussion
Previous studies demonstrated generally higher drug concentrations in
bile than in blood with various relationships and suggested the useful-
ness of bile for screening drug use or abuse (26–31); however, correla-
tions between bile and blood concentrations were investigated for few
drugs (24–26, 29–33). In the present study, bile was collected in most
cases without cholecystectomy or a gall/bile duct stone(s), including
cases where adequate blood specimens were not available due to the
severe bodily injury/damage or decomposition, providing useful infor-
mation in a case where the cause of death was attributed to drug abuse,
as well as in several fatalities involving the possible influence of illicit
drugs such as amphetamines. In addition, a spectrum of drug data was
available for analyses of correlations between bile and blood or urine
concentrations, as described below.

Table II. Correlations of Drug Concentrations Between Urine (y) and Right Heart Blood (x1), Peripheral External Iliac Venous Blood (x2) or Urine (x3)
Drugs Right heart blood (x1)
Methamphetamine y = 2.726 x1 + 1.886 (n = 30,
r = 0.592, P < 0.001)
Amphetamine y = 3.856 x1 + 0.41 (n = 30,
r = 0.626, P < 0.001)
Caffeine y = 0.943 x1 − 0.086 (n = 22,
r = 0.865, P < 0.0001)
Methylephedrine y = 4.331 x1 + 0.021 (n = 11,
r = 0.804, P < 0.01)
Chlorpheniramine y = 4.138 x1 + 0.316 (n = 8,
r = 0.580, P = 0.1318)
Dihydrocodeine y = 10.147 x1 − 0.179 (n = 8,
r = 0.993, P < 0.0001)
Diphenhydramine y = 3.602 x1 + 1.682 (n = 14,
r = 0.518, P = 0.0576)
Phenobarbital y = 3.673 x1 − 12.353
(n = 28, r = 0.883,
P < 0.0001)
Chlorpromazine y = 8.531 x1 + 2.523 (n = 12,
r = 0.704, P < 0.02)
Levomepromazine y = 7.528 x1 + 1.517 (n = 9,
r = 0.557, P = 0.1190)
Promethazine y = 3.447 x1 + 1.075 (n = 18,
r = 0.781, P = 0.0001)
Midazolam y = 1.07 x1 + 0.416 (n = 11,
r = 0.480, P = 0.1349)
Diazepam y = 0.796 x1 + 0.157 (n = 6,
r = 0.194, P = 0.7129)
Zolpidem y = 0.878 x1 + 0.257 (n = 9,
r = 0.341, P = 0.3695)
Olanzapine y = −0.116 x1 + 5.825 (n = 8,
r = 0.020, P = 0.9622)
Bile/right heart blood ratio
(mean/median)
0.55−52.7 (8.81/3.99)
0.60−61.0 (8.06/4.42)
0.24−3.83 (1.07/0.92)
2.68–9.68 (6.71/7.10)
2.32–43.3 (8.96/5.36)
1.91–14.9 (6.67/6.03)
0.05–66.9 (12.4/6.87)
1.14–5.92 (2.17/1.86)
4.65–88.0 (36.5/37.7)
3.63–224.6 (38.3/15.7)
1.25–73.3 (11.1/6.33)
0.53–8.75 (3.15/2.16)
0.49–9.52 (3.84/2.08)
0.48–6.15 (2.17/1.89)
1.95–79.8 (14.7/5.87)
Peripheral external iliac
blood (x2)
y = 6.573 x2 + 0.851 (n = 19,
r = 0.784, P < 0.0001)
y = 6.134 x2 + 0.346 (n = 19,
r = 0.880, P < 0.0001)
y = 0.861 x2 − 0.275 (n = 13,
r = 0.856, P < 0.001)
y = 1.318 x2 + 0.866 (n = 9,
r = 0.252, P = 0.513)
y = 8.953 x2 + 0.103 (n = 8,
r = 0.786, P < 0.05)
y = 8.8 x2 − 0.127 (n = 5,
r = 0.989, P < 0.01)
y = 26.207 x2 − 0.092
(n = 11, r = 0.870,
P < 0.001)
y = 5.164 x2 − 19.584
(n = 17, r = 0.810,
P < 0.0001)
y = 6.726 x2 + 2.665 (n = 9,
r = 0.611, P = 0.0805)
y = 17.971 x2 − 0.766 (n = 8,
r = 0.792, P < 0.02)
y = 3.097 x2 + 0.526 (n = 14,
r = 0.951, P < 0.0001)
y = 2.785 x2 + 0.904 (n = 6,
r = 0.321, P = 0.5350)
y = 3.004 x2 − 0.056 (n = 5,
r = 0.931, P < 0.05)
y = 0.684 x2 + 0.288 (n = 9,
r = 0.318, P = 0.2783)
y = −0.395 x2 + 7.982 (n = 5,
r = 0.073, P = 0.9070)
Postmortem Drug Concentrations in Bile
Bile/peripheral blood ratio Urine (x3) Bile/urine ratio
(mean/median) (mean/median)
0.72−47.9 (1.85/9.05) y = 0.144 x3 + 0.844 (n = 28, 0.015−2.01 (0.36/0.27)
r = 0.824, P < 0.0001)
0.86−35.1 (10.6/8.46) y = 0.111 x3 + 0.402 (n = 30, 0.02−2.79 (0.52/0.26)
r = 0.572, P < 0.001)
0.17−2.26 (0.85/0.68) y = 1.558 x3 − 0.932 (n = 16, 0.14–2.67 (0.85/0.69)
r = 0.943, P < 0.0001)
1.75–36.9 (11.9/8.80) y = 0.04 x3 + 1.093 (n = 8, 0.04–2.51 (0.57/0.23)
r = 0.146, P = 0.7305)
2.90–34.2 (10.3/5.24) y = 0.223 x3 + 0.595 (n = 8, 0.29–4.74 (1.85/0.63)
r = 0.451, P = 0.2619)
1.82–10.6 (5.55/2.65) y = −0.114 x3 + 1.049 (n = 7, 0.029–9.17 (1.66/0.57)
r = 0.085, P = 0.8555)
0.43–65.2 (18.1/8.25) y = 2.274 x3 + 0.879 (n = 9, 0.18–47.0 (11.3/3.07)
r = 0.850, P < 0.01)
0.78–7.14 (2.70/2.34) y = 3.978 x3 − 11.318 0.74–11.9 (2.63/1.94)
(n = 22, r = 0.509,
P < 0.02)
3.37–166.8 (45.9/36.8) y = 2.14 x3 + 2.374 (n = 12, 1.19–63.6 (11.9/5.34)
r = 0.694, P < 0.02)
4.74–39.0 (16.8/13.2) y = 0.477 x3 + 1.069 (n = 10, 0.39–16.5 (2.99/1.40)
r = 0.930, P < 0.0001)
1.5–20.7 (7.64/6.96) y = 0.888 x3 + 0.55 (n = 15, 0.44–9.71 (2.39/1.87)
r = 0.960, P < 0.0001)
2.02–17.4 (8.31/6.16) y = 0.126 x3 + 0.513 (n = 4, 0.27–7.35 (3.65/3.49)
r = 0.292, P = 0.7081)
1.26–7.17 (3.50/2.33) – −
0.52–6.30 (2.52/2.04) y = 0.065 x3 + 0.287 (n = 5, 0.38–7.81 (3.45/2.86)
r = 0.119, P = 0.856)
4.63–121.0 (32.1/11.6) y = −0.687 x3 + 8.338 (n = 6, 0.52–27.2 (7.25/3.67)
r = 0.411, P = 0.4188)
371
372
Drug concentrations in bile were higher than in blood, as previous-
ly described (26–31), except for oral caffeine, which showed similar
concentrations in bile and blood with a good correlation, as previously
found for pericardial and cerebrospinal fluids (7, 8). Other drugs
showed various bile-to-blood concentration ratios, ranging from
about ×2 to ×45, but significant correlations were detected for amphe-
tamines, several cold remedies, phenobarbital and phenothiazine
derivatives. The correlations were mostly better for peripheral than
for right heart blood, indicating a closer relationship of bile concentra-
tions with circulating blood concentrations, without evident post-
mortem interference including redistribution and diffusion from
stomach contents. These observations suggest the efficacy of bile as
a supplemental material for investigating these drugs. Different
bile-to-blood concentration ratios among the drugs described above
may depend on individual pharmacokinetics. Poor bile–blood correla-
tions for other psychotropic drugs, such as several benzodiazepines,
zolpidem and olanzapine, may predominantly be attributed to greater
influences of the genetic background of complex drug metabolism in
the liver, including degradation and conjugation (28); further investi-
gation is needed, including the analysis of metabolites and conjugates
in bile.
The relationship between bile and urine concentrations also
differed by drug, possibly depending on different elimination pro-
cesses; bile concentrations were mostly lower than urine concentra-
tions for amphetamines, caffeine and methylephedrine, reflecting
their major elimination process in urine, but were usually similar
to or higher for other drugs. In addition, there were significant
correlations between bile and urine concentrations for amphetamines,
caffeine, diphenhydramine, phenobarbital and phenothiazine deriva-
tives; however, correlations were insignificant for other psychotropic
drugs. These findings suggest that bile-to-urine concentration ratios
also depend on pharmacokinetics involving the genetic background
and the elimination process of individual drugs without evident post-
mortem interference. These observations suggest that bile is useful for
instrumental toxicological screening of therapeutic and abused drugs,
especially for several drugs that showed higher concentrations in bile
than in urine. In addition, further autopsy data analyses involving
multiple-site drug distributions, including pericardial effusion,
cerebrospinal fluid and bone marrow aspirate, in consideration of
individual site characteristics and relationships, can provide useful
information for the investigation of antemortem pharmaco-/toxicoki-
netics and postmortem redistribution, including the survival time after
administration for the equilibration of drug concentrations in blood,
tissue and body fluids (6–8).
Major limitations of the present study were: (i) limited numbers of
cases for several psychotropic drugs, which showed poor bile–blood
or bile–urine correlations, (ii) a lack of drug metabolite data including
conjugates of these drugs in bile and urine, (iii) few fatal intoxication
cases for toxicokinetic analysis, (iv) a partial lack of blood/urine data
for statistical analysis, and (v) unpredictable postmortem interference
at each site, because of a retrospective review of routine postmortem
toxicological data using GC–MS. However, the present data demon-
strated significant bile–blood and/or bile–urine correlations of several
drug concentrations, for which substantial equilibrium and the stabil-
ity of postmortem distribution were suggested; bile may be a useful
supplementary material for postmortem analysis of these drugs, al-
though quantitative interpretation should be reserved for other
drugs. In these cases, hours-long survival after drug intake,
independent of the route, may have contributed to systemic drug
distributions including bile and urine, when therapeutic blood drug
concentrations with relevant stomach concentrations in most cases
Tominaga et al.
were considered, although injected methamphetamine can be distrib-
uted earlier. It was interesting that bile concentrations were more
closely correlated with concentrations in peripheral than in heart
blood, suggesting greater postmortem interference in heart blood con-
centrations. In contrast, lidocaine, which was often detected in cases
involving critical medical care, showed markedly lower concentrations
in bile than in blood, possibly due to shorter survival before whole-
body distribution despite resuscitation and life-supporting measures.
Further investigation is needed to assess other factors that might affect
postmortem drug distributions in bile and urine, including the genetic
background affecting drug metabolism and drug–drug interactions.
In conclusion, the present study demonstrated significant correla-
tions between bile and blood or urine concentrations for several drugs,
suggesting that bile can provide useful supplemental data in routine
forensic toxicology including instrumental screening for such drugs,
as well as for investigating pharmaco-/toxicokinetics and postmortem
redistribution, when analyzed in combination with drug concentra-
tions at other sites. Further investigation is needed for other drugs.
Conflict of interest
None declared.
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