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Evaluation of Postmortem Drug Concentrations in Bile Compared With Blood and Urine in Forensic Autopsy Cases
Evaluation of Postmortem Drug Concentrations in Bile Compared With Blood and Urine in Forensic Autopsy Cases
Evaluation of Postmortem Drug Concentrations in Bile Compared With Blood and Urine in Forensic Autopsy Cases
doi: 10.1093/jat/bkw028
Article
Article
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.
© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com 367
368 Tominaga et al.
usefulness of bile as an alternative to blood and a material for investi- prepared mixture of dichloromethane/isopropanol (78 : 20) ammo-
gating pharmaco-/toxicokinetics and postmortem redistribution. 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
Materials and methods of the extracts were injected into the gas chromatography–mass
Autopsy database spectrometry (GC–MS) system. The recovery of standards ranged
from 60–70% ( phenobarbital) to >95% (codeine) (7, 8).
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- Instrumental conditions
letonization, were retrospectively reviewed, and those with positive Automated GC–MS following solid-/liquid-phase extraction was
toxicological findings in bile were collected to compare the data performed using an Agilent Technologies GC/MS System Model
with those of right heart blood, peripheral blood and urine. These 5975c MSD (column, DB-5MS, 30 m × 0.25 mm i.d., film 0.25 µm;
data analyses as well as sample collections and the analyses described column temperature, 100–325°C; injector temperature, 280°C;
turbocharged carrier gas, He at a flow rate of 48 cm/s; interface tem-
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
Table continues
370 Tominaga et al.
Table I. Continued
Drugs Bile Right heart blood Peripheral external Urine Stomach contents
iliac blood
Cold remedies were also higher than in stomach contents, without markedly high
Bile concentrations of oral caffeine were similar (about ×0.8–1) concentrations related to high stomach concentrations (Table I).
to those in right heart and peripheral iliac venous blood and were Oral chlorpromazine, levomepromazine and promethazine con-
mostly slightly lower (about ×0.7–0.9) than in urine with good centrations in bile were markedly higher (about ×6–45) than those
correlations (Table II). Compared with concentrations in stomach in right heart and peripheral iliac venous blood, partly showing signifi-
contents, bile concentrations were mostly lower (about ×0.5–0.9) cant correlations, which were better for peripheral than right heart
without markedly high concentrations related to high stomach con- blood for most drugs (Table II). Bile concentrations of these drugs
centrations (Table I). were similar to or higher (up to about ×12) than concentrations in
Methylephedrine, chlorpheniramine, dihydrocodeine and di- urine, showing good correlations (Table II). Diazepam concentrations
phenhydramine concentrations in bile were higher than in blood, in bile were also higher (about ×2–4) than in right heart and peripheral
showing various relationships (about ×3–18); bile to blood correla- iliac venous blood, showing significant correlations between bile and
tions of these drugs were excellent for dihydrocodeine in both right peripheral blood concentrations (Table II). No significant relation-
heart and peripheral iliac venous blood, but were better for chlor- ships were detected between bile and blood or urine for midazolam,
pheniramine and diphenhydramine in peripheral iliac venous zolpidem and olanzapine. These bile concentrations were also similar
blood, and for methylephedrine in right heart blood than at the to or higher than in the stomach, without markedly high concentra-
other sites (Table II). Bile diphenhydramine concentrations were tions related to high stomach concentrations (Table I).
higher (about ×3–11) than urine concentrations with a good correl-
ation and were higher (about ×4–6) than in stomach contents with-
Discussion
out markedly high concentrations related to high stomach
concentrations (Tables I and II). Bile concentrations of methylephe- Previous studies demonstrated generally higher drug concentrations in
drine, dihydrocodeine and chlorpheniramine were similar to or bile than in blood with various relationships and suggested the useful-
lower (about ×0.2–2.4) than concentrations in urine; correlations be- ness of bile for screening drug use or abuse (26–31); however, correla-
tween bile and urine concentrations were insignificant (Table II). tions between bile and blood concentrations were investigated for few
These bile concentrations were also similar to or lower than in the drugs (24–26, 29–33). In the present study, bile was collected in most
stomach, without markedly high concentrations related to high cases without cholecystectomy or a gall/bile duct stone(s), including
stomach concentrations (Table I). 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,
Psychotropics as well as in several fatalities involving the possible influence of illicit
Oral phenobarbital mostly showed higher concentrations (about ×2) drugs such as amphetamines. In addition, a spectrum of drug data was
in bile than in the right heart, peripheral iliac venous blood and available for analyses of correlations between bile and blood or urine
urine, with significant correlations (Table II). Bile concentrations concentrations, as described below.
Postmortem Drug Concentrations in Bile
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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) Bile/right heart blood ratio Peripheral external iliac Bile/peripheral blood ratio Urine (x3) Bile/urine ratio
(mean/median) blood (x2) (mean/median) (mean/median)
Methamphetamine y = 2.726 x1 + 1.886 (n = 30, 0.55−52.7 (8.81/3.99) y = 6.573 x2 + 0.851 (n = 19, 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 = 0.592, P < 0.001) r = 0.784, P < 0.0001) r = 0.824, P < 0.0001)
Amphetamine y = 3.856 x1 + 0.41 (n = 30, 0.60−61.0 (8.06/4.42) y = 6.134 x2 + 0.346 (n = 19, 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 = 0.626, P < 0.001) r = 0.880, P < 0.0001) r = 0.572, P < 0.001)
Caffeine y = 0.943 x1 − 0.086 (n = 22, 0.24−3.83 (1.07/0.92) y = 0.861 x2 − 0.275 (n = 13, 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 = 0.865, P < 0.0001) r = 0.856, P < 0.001) r = 0.943, P < 0.0001)
Methylephedrine y = 4.331 x1 + 0.021 (n = 11, 2.68–9.68 (6.71/7.10) y = 1.318 x2 + 0.866 (n = 9, 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 = 0.804, P < 0.01) r = 0.252, P = 0.513) r = 0.146, P = 0.7305)
Chlorpheniramine y = 4.138 x1 + 0.316 (n = 8, 2.32–43.3 (8.96/5.36) y = 8.953 x2 + 0.103 (n = 8, 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 = 0.580, P = 0.1318) r = 0.786, P < 0.05) r = 0.451, P = 0.2619)
Dihydrocodeine y = 10.147 x1 − 0.179 (n = 8, 1.91–14.9 (6.67/6.03) y = 8.8 x2 − 0.127 (n = 5, 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 = 0.993, P < 0.0001) r = 0.989, P < 0.01) r = 0.085, P = 0.8555)
Diphenhydramine y = 3.602 x1 + 1.682 (n = 14, 0.05–66.9 (12.4/6.87) y = 26.207 x2 − 0.092 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 = 0.518, P = 0.0576) (n = 11, r = 0.870, r = 0.850, P < 0.01)
P < 0.001)
Phenobarbital y = 3.673 x1 − 12.353 1.14–5.92 (2.17/1.86) y = 5.164 x2 − 19.584 0.78–7.14 (2.70/2.34) y = 3.978 x3 − 11.318 0.74–11.9 (2.63/1.94)
(n = 28, r = 0.883, (n = 17, r = 0.810, (n = 22, r = 0.509,
P < 0.0001) P < 0.0001) P < 0.02)
Chlorpromazine y = 8.531 x1 + 2.523 (n = 12, 4.65–88.0 (36.5/37.7) y = 6.726 x2 + 2.665 (n = 9, 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 = 0.704, P < 0.02) r = 0.611, P = 0.0805) r = 0.694, P < 0.02)
Levomepromazine y = 7.528 x1 + 1.517 (n = 9, 3.63–224.6 (38.3/15.7) y = 17.971 x2 − 0.766 (n = 8, 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 = 0.557, P = 0.1190) r = 0.792, P < 0.02) r = 0.930, P < 0.0001)
Promethazine y = 3.447 x1 + 1.075 (n = 18, 1.25–73.3 (11.1/6.33) y = 3.097 x2 + 0.526 (n = 14, 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 = 0.781, P = 0.0001) r = 0.951, P < 0.0001) r = 0.960, P < 0.0001)
Midazolam y = 1.07 x1 + 0.416 (n = 11, 0.53–8.75 (3.15/2.16) y = 2.785 x2 + 0.904 (n = 6, 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 = 0.480, P = 0.1349) r = 0.321, P = 0.5350) r = 0.292, P = 0.7081)
Diazepam y = 0.796 x1 + 0.157 (n = 6, 0.49–9.52 (3.84/2.08) y = 3.004 x2 − 0.056 (n = 5, 1.26–7.17 (3.50/2.33) – −
r = 0.194, P = 0.7129) r = 0.931, P < 0.05)
Zolpidem y = 0.878 x1 + 0.257 (n = 9, 0.48–6.15 (2.17/1.89) y = 0.684 x2 + 0.288 (n = 9, 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 = 0.341, P = 0.3695) r = 0.318, P = 0.2783) r = 0.119, P = 0.856)
Olanzapine y = −0.116 x1 + 5.825 (n = 8, 1.95–79.8 (14.7/5.87) y = −0.395 x2 + 7.982 (n = 5, 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 = 0.020, P = 0.9622) r = 0.073, P = 0.9070) r = 0.411, P = 0.4188)
371
372 Tominaga et al.
Drug concentrations in bile were higher than in blood, as previous- were considered, although injected methamphetamine can be distrib-
ly described (26–31), except for oral caffeine, which showed similar uted earlier. It was interesting that bile concentrations were more
concentrations in bile and blood with a good correlation, as previously closely correlated with concentrations in peripheral than in heart
found for pericardial and cerebrospinal fluids (7, 8). Other drugs blood, suggesting greater postmortem interference in heart blood con-
showed various bile-to-blood concentration ratios, ranging from centrations. In contrast, lidocaine, which was often detected in cases
about ×2 to ×45, but significant correlations were detected for amphe- involving critical medical care, showed markedly lower concentrations
tamines, several cold remedies, phenobarbital and phenothiazine in bile than in blood, possibly due to shorter survival before whole-
derivatives. The correlations were mostly better for peripheral than body distribution despite resuscitation and life-supporting measures.
for right heart blood, indicating a closer relationship of bile concentra- Further investigation is needed to assess other factors that might affect
tions with circulating blood concentrations, without evident post- postmortem drug distributions in bile and urine, including the genetic
mortem interference including redistribution and diffusion from background affecting drug metabolism and drug–drug interactions.
stomach contents. These observations suggest the efficacy of bile as In conclusion, the present study demonstrated significant correla-
a supplemental material for investigating these drugs. Different tions between bile and blood or urine concentrations for several drugs,
bile-to-blood concentration ratios among the drugs described above suggesting that bile can provide useful supplemental data in routine
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