Download Codeine to Morphine Concentration Ratios in Samples

Journal of Analytical Toxicology 2014;38:99 –105
doi:10.1093/jat/bkt099 Advance Access publication December 8, 2013
Article
Codeine to Morphine Concentration Ratios in Samples from Living Subjects and Autopsy
Cases after Incubation
Riikka Mari Berg-Pedersen, Åse Ripel, Ritva Karinen, Merete Vevelstad, Liliana Bachs and Vigdis Vindenes*
Division of Forensic Medicine and Drug Abuse Research, The Norwegian Institute of Public Health, Pb. 4404, Nydalen,
N-0403 Oslo, Norway
*Author to whom correspondence should be addressed. Email: [email protected]
The codeine to morphine concentration ratio is used in forensic toxicology to assess if codeine has been ingested alone or if morphine
and/or heroin have been ingested in addition. In our experience, this
interpretation is more difficult in autopsy cases compared with
samples from living persons, since high morphine concentrations are
observed in cases where only codeine is assumed to have been
ingested. We have investigated if codeine and morphine glucuronides
are subject to cleavage to the same extent in living and autopsy
cases in vitro. We included whole blood samples from eight living
subjects and nine forensic autopsy cases, where only codeine ingestion was suspected. All samples were incubated for 2 weeks at 3788 C
and analyzed for codeine and six codeine metabolites using liquid
chromatography tandem mass spectrometry. A reduction in the
codeine to morphine concentration ratio was found, both in samples
from living subjects (mean 33%, range 22 –50%) and autopsy cases
(mean 37%, range 13 –54%). The increase in the morphine concentrations was greater in the autopsy cases (mean 85%, max 200%)
compared with that of the living cases (mean 51%, max 87%). No
changes were seen for codeine or codeine-6-glucuronide concentrations. The altered ratios might mislead the forensic toxicologist to
suspect morphine or heroin consumption in cases where only
codeine has been ingested.
Introduction
Codeine is an opiate analgesic, predominantly prescribed for the
treatment of moderate pain or as an antitussive agent. In Norway,
codeine is mainly used as an analgesic in combination with paracetamol (acetaminophen) (1). In other countries, codeine is also
available in different combinations, e.g., with acetylsalicylic acid,
ibuprofen, carisoprodol, caffeine, barbiturates or sedative
antihistamines.
The analgesic property of codeine is considered to be due to
the O-demethylation of codeine into morphine by cytochrome
P450 2D6 (CYP2D6), occurring mainly in the liver (2). Lötsch
et al. have shown an overview of the metabolism of codeine into
the different metabolites (3), see Figure 1 for an overview of the
codeine metabolites. Due to genetic polymorphism on the
CYP2D6 gene, the amount of morphine produced from codeine
varies, with somewhere between 0 and 15% of codeine being
metabolized into morphine in the body. The lack of functional
CYP2D6 enzyme activity (‘poor metabolizers’) has been reported
for 7–10% of Caucasians, thereby being completely unable or
only able to convert a small amount of codeine into morphine.
However, another 1–3% of Caucasians have been shown to
possess an allele duplication on the gene encoding CYP2D6
(‘ultrarapid metabolizers’) (4), and are therefore able to metabolize a larger proportion of ingested codeine into morphine (5).
About 90% of the morphine produced from codeine, in the
body, is further metabolized, mainly into morphine-3-glucuronide
(M3G) (45–55%) and morphine-6-glucuronide (M6G) (10–15%)
(6). A minor proportion of morphine is N-demethylated into
normorphine (5).
Codeine-6-glucuronide (C6G) is the major codeine metabolite
(constituting 50–70% of all the metabolites) (7). Due to reports
of analgesic activity by codeine in subjects without morphineformation, it has been suggested that C6G generates the analgesic
activity (8). Codeine is also N-demethylated into norcodeine
(10–15%), via the cytochrome P450 isoenzyme 3A4 (CYP3A4),
and norcodeine is further glucuronidated into norcodeine-6glucuronide (N6G), with a minor proportion being O-demethylated into normorphine (5).
Codeine is widely prescribed in Norway (1) and is frequently
detected in blood samples collected from drivers suspected of
driving under the influence of drugs (9) and from autopsy cases,
analyzed at the Norwegian Institute of Public Health (NIPH). In forensic toxicology, the codeine to morphine concentration ratio,
in blood and urine, is used to evaluate whether codeine has been
ingested (10) or if the findings are due to a consumption of other
opiates (10–13). The interpretation of the codeine to morphine
concentration ratios in autopsy cases are based on knowledge
from the metabolism of codeine to morphine in living subjects.
Kronstrand et al. showed a maximum codeine to morphine
concentration ratio in blood from living subjects of 32 (ranging
24– 49), after the ingestion of 100 mg codeine, with a ratio of 2
after 23 h (14). Quiding et al. reported that in plasma the morphine concentration is 2 –3% of the codeine concentration,
when measured simultaneously (15).
Postmortem changes will affect drug concentrations, due to
the redistribution of the drug along a concentration gradient,
from tissues like lungs, heart or liver into the blood, and also due
to the formation and breakdown of drugs (16). In the case of
morphine, it has been shown that morphine glucuronides may
be hydrolyzed postmortem, releasing free morphine. As a consequence, the postmortem concentrations of morphine are not
necessarily representative of the perimortem blood concentration levels (17, 18). It is still unclear if the previous also holds
true for codeine and C6G.
Changes in the concentration ratios of codeine to morphine
may also take place after blood samples have been collected, due
to in vitro changes in the vial. It is not known if codeine and
morphine glucuronides are subject to the same extent of cleavage in authentic samples from living persons compared with
autopsy cases.
From our routine cases, we have experienced that the interpretation of codeine and morphine concentrations is more difficult in autopsy cases compared with samples from living
# The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Codeine metabolism in humans. Adapted by permission from Macmillan Publishers Ltd, Lötsch et al. (3).
persons. In several autopsy cases, we have found high morphine
concentrations, and thus low codeine to morphine ratios indicating additional ingestion of morphine, although other information
in these cases have indicated ingestion of codeine alone. This is
not a challenge in the samples from living persons, but to our
knowledge, no studies have investigated these differences. The
aim of this study was therefore to investigate if changes could
occur in vitro, after sampling, in the concentrations of six
codeine metabolites, using samples collected both from living
subjects and from forensic autopsy cases, after being incubated
at a high temperature to create a condition that may facilitate
the cleavage of the glucuronides and allowing time for in vitro
changes to occur in the metabolite concentrations. Our purpose
is to investigate whether the cleavage of morphine glucuronides
is more extensive than the cleavage of codeine glucuronides,
thus changing the codeine to morphine ratio in the samples.
Experimental
Samples
All samples utilized, in the presented study, are authentic whole
blood samples, collected over a 6-year period, and analyzed at the
NIPH. The institute receives venous whole blood samples from
100 Berg-Pedersen et al.
living subjects suspected of being under the influence of drugs.
Postmortem whole blood samples are received from forensic autopsies, sampled from the femoral vein. All samples are routinely
screened for a standard selection of drugs of abuse, and all of the
positive results are confirmed by a second analytical method.
Blood from living subjects were collected into 5 mL Vacutainerw
tubes, containing 20 mg sodium fluoride (NaF; a preservative) and
143 IU of heparin (BD Vacutainer Systems, Belliver Industrial
Estate, Plymouth, UK). Postmortem blood samples from autopsies
were collected into 20 mL Sterilinew tubes (Bibby Sterilin,
Staffordshire, UK), containing 0.3 mL 67% (w/v) potassium
fluoride (KF) solution (also a preservative).
Eight samples from living subjects and nine samples from
autopsy cases, containing codeine and morphine, were selected,
for this study, using a specific set of criteria. In order to investigate if different changes might take place at varying concentration levels, samples containing both moderate and high codeine
concentrations were used.
Criteria for selecting the samples
To be able to investigate codeine cases specifically, only cases
containing low morphine concentrations were included. Samples
containing both codeine and morphine, in whole blood, were
included by applying the following criteria:
Cases containing ‘high’ codeine concentrations:
Four postmortem samples were selected with codeine
.1.0 mg/L and morphine ,0.1 mg/L. Among the living cases,
however, only three samples were found in the database containing codeine .1.0 mg/L and low morphine concentrations.
C6G varied from 3 to 10% (morphine:0.0043/0.043/0.43 mg/L;
M3G: 0.032/0.32/3.2 mg/L; M6G: 0.0032/0.032/0.32 mg/L; codeine: 0.0045/0.045/0.45 mg/L; K6G: 0.0071/0.071/0.71 mg/L).
Norcodeine and normorphine had a day-to-day RSD between 6
and 23% (norcodeine: 0.0020/0.020/0.20 mg/L; normorphine:
0.0019/0.019/0.19 mg/L). Intraday relative standard deviations,
for all of the compounds, were ,12%, at the same concentration
levels. Changes in concentrations, after reanalysis, between +20%
are considered to be within the normal analytical variation.
Cases containing ‘moderate’ codeine concentrations:
Results
Five samples were selected from each category (living and
postmortem) with codeine between 0.2 and 0.6 mg/L and
morphine ,0.03 mg/L.
All cases containing the heroin metabolite 6-acetylmorphine
(6-AM), in blood or urine, were excluded.
Methods
After the initial routine analyses at the NIPH, all of the samples
were stored in a freezer at 2208C. The samples chosen for this
study were thawed and allocated into two vials; one sample for
analysis the following day, and the other for incubation at 378C
for 14 days before reanalysis. Analyses for codeine, morphine,
M3G, M6G, C6G, norcodeine and normorphine were performed
by using a previously published LC–MS-MS method (19).
Limits of detection (LOD) for morphine, M3G, M6G and normorphine were 0.00049, 0.0065, 0.00060 and 0.00081 mg/L,
and limits of quantification (LOQ) 0.0012, 0.019, 0.0014 and
0.0016 mg/L, respectively. LOD for codeine, C6G, and norcodeine
were 0.0015, 0.0010 and 0.00086 mg/L. LOQ was 0.0030, 0.0048
and 0.0017 mg/L for codeine, C6G and norcodeine, respectively.
Day-to-day relative standard deviations (RSD), determined at
three concentration levels, for morphine, M3G, M6G, codeine and
Samples from living subjects
The concentrations detected for codeine and its metabolites are
presented in Table I. The changes, given in percentage, from the
initial concentrations are also shown. Five samples, with codeine
in the range of 0.2 –0.6 mg/L, were analyzed, but for samples
containing codeine .1.0 mg/L, with low morphine concentrations, only three samples were found.
Only minimal changes were observed for the codeine concentrations after incubation, from a reduction of 5% to an increase
of 13% (mean 5%), which is within the range considered to be
caused by analytical variation. For morphine, the concentrations
increased between 33 and 87% (mean 51%).
For most of the metabolites, no changes in the concentrations
were found after incubation (the results were within +20%,
which is within the analytical variation), but an increase in normorphine concentrations were seen in all the cases, from 43 to
116% (mean 78%).
Autopsy samples
The concentrations of codeine and its metabolites are presented
in Table II. The changes from the initial concentrations, given in
percentage, are also shown.
Table I
The concentrations of codeine and codeine metabolites (mg/L) in samples collected from living subjects on Day 1 and after 14 days of incubation, and the change in concentrations given in percentage
Study day
Subject 1
1
14
Subject 2
1
14
Subject 3
1
14
Subject 4
1
14
Subject 5
1
14
Subject 6
1
14
Subject 7
1
14
Subject 8
1
14
Mean
change in %
Morphine
(mg/L)
%
change
M3G
(mg/L)
%
change
M6G
(mg/L)
%
change
Normorphine
(mg/L)
0.010
0.019
87
0.21
0.18
214
0.046
0.041
210
0.073
0.14
92
0.52
0.49
0.014
0.019
37
0.21
0.19
211
0.033
0.033
0
0.060
0.11
78
0.014
0.023
61
0.31
0.27
213
0.052
0.051
22
0.046
0.086
0.019
0.029
50
0.31
0.29
27
0.053
0.049
28
0.013
0.020
46
0.09
0.08
217
0.015
0.015
0.037
0.053
46
0.31
0.28
210
0.303
0.404
33
2.31
2.48
8
0.018
0.028
51
51%
0.18
0.17
27
11%
C6G
(mg/L)
%
change
Norcodeine
(mg/L)
%
change
6
1.38
1.30
26
0.099
0.089
210
0.49
0.47
24
0.80
0.81
2
0.049
0.044
210
86
0.38
0.40
6
1.80
1.84
2
0.066
0.055
217
0.083
0.14
63
0.44
0.42
24
1.15
1.12
23
0.049
0.047
25
0
0.020
0.032
56
0.41
0.39
25
0.52
0.50
24
0.053
0.039
226
0.050
0.049
21
0.070
0.13
89
1.02
1.06
4
1.31
1.32
1
0.13
0.13
0
0.426
0.470
10
0.36
0.52
43
2.83
2.86
1
5.00
4.69
26
0.26
0.22
217
0.040
0.038
25
5%
0.10
0.21
%
change
116
78%
Codeine
(mg/L)
2.13
2.41
%
change
13
5%
2.66
2.64
21
3%
0.33
0.34
2
11%
Codeine to Morphine Concentration Ratios after Incubation 101
Table II
The concentrations of codeine and codeine metabolites (mg/L) in samples collected from postmortem cases on Day 1 and after 14 days of incubation, and the change in concentrations given in percentage
Study day
Subject 9
1
14
Subject 10
1
14
Subject 11
1
14
Subject 12
1
14
Subject 13
1
14
Subject 14
1
14
Subject 15
1
14
Subject 16
1
14
Subject 17
1
14
Mean
change in %
Morphine
(mg/L)
%
change
M3G
(mg/L)
0.020
0.060
200
0.093
0.099
0.027
0.039
45
0.026
0.035
M6G
(mg/L)
%
change
Normorphine
(mg/L)
%
change
Codeine
(mg/L)
7
0.015
0.017
11
0.061
0.130
116
0.26
0.38
50
0.44
0.66
49
0.016
0.024
55
0.13
0.12
28
0.017
0.017
4
0.018
0.032
74
0.37
0.36
22
0.24
0.25
5
0.014
0.017
19
37
0.046
0.027
242
0.0090
0.0077
215
0.051
0.068
35
0.42
0.42
0
0.22
0.23
4
0.031
0.030
23
0.016
0.027
66
0.035
0.033
27
0.0085
0.0076
210
0.041
0.054
32
0.42
0.48
14
0.25
0.29
19
0.032
0.043
37
0.019
0.026
37
0.046
0.041
210
0.0053
0.0060
13
0.018
0.025
38
0.25
0.24
26
0.36
0.39
8
0.012
0.014
13
0.082
0.090
10
0.032
0.020
238
0.0061
0.0055
211
0.010
0.013
28
2.42
2.34
23
0.11
0.10
29
0.094
0.072
224
0.008
0.021
152
0.021
0.025
21
0.0063
0.0089
40
0.045
0.088
93
1.24
1.82
47
0.89
1.22
37
0.39
0.51
31
0.018
0.024
31
0.077
0.056
226
0.013
0.014
2
0.029
0.039
33
2.10
1.55
226
0.91
0.73
219
0.21
0.18
214
0.14
0.40
185
85%
1.40
1.12
%
change
220
20%
0.34
0.36
4
12%
1.21
1.84
The changes in concentrations of codeine after incubation
ranged from a reduction of 26% to an increase of 50% (mean
19%). For morphine, the concentrations increased between 10
and 200% (mean 85%).
As observed for the living cases, an increase in normorphine
concentrations were also seen in all the autopsy cases, from 28
to 116% (mean 57%).
Changes in codeine to morphine concentration ratios
The changes in codeine to morphine concentration ratios after
14 days of incubation are shown in Table III, and a reduction in
the ratios were seen for all the samples. The mean reduction in
the codeine to morphine concentration ratio after incubation
for samples from living subjects was 33% (ranging 22 –50%). The
mean reduction in the codeine to morphine concentration ratio
in the postmortem samples after incubation was 37% (ranging
13 –54%).
Discussion
The study reveals that the codeine to morphine concentration
ratios in blood were reduced after incubation, both in samples
collected from living subjects and from autopsies. Morphine and
normorphine concentrations increased in both categories. The
codeine and C6G concentrations were not altered after incubation. Why changes are seen in the concentrations of morphine
and its metabolites, and not codeine and its metabolites, is not
known, but this fact is very important to be aware of during
interpretation of such cases.
The interindividual variation in the codeine to morphine
ratios may be due to interindividual variation in vivo, such as
102 Berg-Pedersen et al.
52
57%
%
change
3.37
4.20
25
19%
C6G
(mg/L)
5.68
6.85
%
change
21
19%
Norcodeine
(mg/L)
%
change
2.07
2.64
27
25%
Table III
The codeine to morphine ratios on Day 1 and after incubation at 378C for 14 days, in samples
collected from living subjects (L) and in postmortem samples (PM) collected from autopsy cases
Subject
no.
Living/
postmortem
cases
Codeine/
morphine ratio
Day 1
Codeine/
morphine ratio
Day 14
Change in
ratio Days
1 – 14
Change in
ratio (%)
1
2
3
4
5
6
7
8
L
L
L
L
L
L
L
L
Mean
PM
PM
PM
PM
PM
PM
PM
PM
PM
Mean
52
36
26
23
30
28
9
117
40
13
14
16
26
13
30
149
117
24
45
26
25
17
15
20
20
7
87
27
6
9
12
18
9
26
87
66
11
27
226
211
29
28
210
28
22
230
213
27
25
24
28
24
24
262
251
213
218
250
231
235
235
233
229
222
226
233
254
236
225
231
231
213
242
244
254
237
9
10
11
12
13
14
15
16
17
differences in the genotype encoding enzymes metabolizing
codeine to morphine, and also different time periods between
last codeine ingestion and sampling. Frost et al. performed CYP2D6
genotyping in samples from deceased subjects to investigate
if genotyping could be applied to predict the morphine to
codeine concentration ratios in postmortem toxicological specimens (20). He concluded that investigations on codeine-related
deaths should include quantification of morphine and morphine
metabolites, together with a detailed case history, in order to
achieve a comprehensive interpretation of postmortem codeine
findings, and that CYP2D6 genotyping may be of interest in
cases with unexpectedly high or low ratios. In our study, the
samples were not genotyped, as it was not considered relevant.
The lack of genotyping would, according to the previous conclusion, only influence the interpretation of the concentrations
measured on Day 1, and not influence the changes observed in
the vials in vitro nor during incubation.
Previously published data indicate that codeine and morphine
may exhibit postmortem redistribution, but the results are
inconsistent, particularly for morphine (16, 21–26). The increased
morphine concentrations observed in our study are likely due to
the cleavage of morphine glucuronides. A slightly larger reduction was seen for M3G concentrations, compared with M6G concentrations, but due to the small number of cases investigated,
this could be only within the range of analytical variation.
Romberg and Lee have however compared the hydrolysis rates
of M3G with M6G, and showed that the enzymatic hydrolysis
rate of M6G constituted 25% of that of M3G (27). They found
that the hydrolysis of M3G and M6G was complete within 24 h.
Two other studies have investigated the stability of the morphine
glucuronides in vitro, under different storage conditions (17,
18). Carroll et al. concluded that hydrolysis, during specimen
storage, can generate free morphine from M3G; possibly resulting in erroneous conclusions when certifying narcotic deaths (18).
Skopp et al. found that morphine and its glucuronides, in both
blood and plasma, are stable at 48C for the observation period;
however, in postmortem blood, the analytes are stable only
when stored at 2208C. Thus, it is likely that M3G is more prone
to hydrolysis than M6G, but the reason for this fact remains
unknown. Because the concentrations of M3G are several times
higher than both that of M6G and morphine, the contribution of
morphine from M3G might be expected to result in a significant
increase in the morphine concentration, but it is however important to have in mind that the higher concentrations of the
morphine-glucuronides are due to a lower volume of distribution compared with morphine.
Our study reveals that both codeine and C6G concentrations
remain stable during incubation, and this is an important finding.
Using incubation to study changes taking place after sampling
constitutes a worst case scenario. In forensic toxicology cases, it
is important to be able to reveal what might happen in such
samples, since the conclusion might have serious consequences
for the sample donor. The reason for codeine and C6G appearing
more stable than morphine and morphine glucuronides is
unknown, but one explanation may be an inherent stability in
the chemical structure of the substance. No previous studies
seem to have investigated this hypothesis. For M6G, C6G and
norcodeine, opposing changes in the concentrations were
observed, in both samples from living subjects and from autopsy
cases; however, the changes were within the range of normal
analytical variation of +20%.
No differences in the codeine concentrations were observed
between samples with ‘high’ and ‘moderate’ codeine concentrations, after incubation. But due to the limited number of cases
included, a conclusion cannot be drawn as to whether such differences may or may not take place. Because the changes observed
appear to be due to in vitro changes in the morphine concentrations, and not in the codeine concentrations, larger differences in
the codeine to morphine concentration ratio might have been
expected in cases with higher original morphine concentrations.
It is well known that due to genetic polymorphism on the
CYP2D6 gene, the amount of morphine produced from codeine
varies. About 1 –3% of Caucasians have an allele duplication
resulting in ‘ultrarapid metabolizers’ (4), and larger proportion
of the ingested codeine can be metabolized into morphine (5).
In such cases, the codeine to morphine ratio might thus be
lower than expected as a consequence of ingestion of only
codeine, and there might be a risk that intake of morphine and/
or heroin is suspected.
An increase in the normorphine concentrations are seen for
all the samples from both living and autopsy cases. We have not
found any literature that can explain this increase. This finding
does however reveal that this metabolite might not be a good indicator for the morphine concentrations at the time of sampling
from living persons, or at the time of death. The postmortem
changes in normorphine concentrations are also unknown.
The samples included in this study were selected using a specific set of criteria, with the intent of making it more likely that
the concentrations of codeine and morphine detected were due
to the ingestion of codeine alone. The ingestion of morphine or
heroin cannot be ruled out completely, but the criteria, including low morphine and high codeine concentrations, should
make it less likely. Cases revealing 6-AM, in blood or urine, were
excluded.
Finding cases suitable for this study was a challenge. Although
codeine is frequently used in Norway, ingestion of codeine only,
is very rare in these types of cases, and the sample size is therefore small. Samples from living persons or from autopsies are
however obligated to study such changes, since processes that
have taken place after death lead the different level of glucuronides and matrix effects might influence the drug concentrations in vitro to a different extent in samples from different
persons. Spiked samples can thus not be used to examine this.
Due to the low number of cases, statistical analyses have not
been performed. The aim of the study was, however, to investigate if changes could occur in vitro after sampling; and even
with our low number of cases, we were able to show such
changes to take place. It is therefore likely that even greater
changes would have been observed if the sample size was larger.
For the overall cases from the living persons and the autopsy
cases, we did not see major differences between the groups.
Looking at the range for the different parameters that we have
investigated, we found large changes in the autopsy group. Our
impression prior to this study was that the changes in the
codeine to morphine ratio were most pronounced in the autopsy
cases, and the findings from this study could only to some extent
confirm this assumption. One of the autopsy cases did however
show a 200% increase in the morphine concentration after incubation, and if such an increase is found in cases where a larger
amount of morphine has been formed from codeine, this might
lead to difficulties regarding interpretation. The changes observed in routine autopsy cases received at our lab might also be
due to changes that have taken place in the body before sampling, and further studies are needed to investigate this.
Tolliver et al. examined the relationship between ante- and
postmortem morphine and codeine concentrations in whole
blood (23). Their study indicated that several factors, such as
metabolism and postmortem interval, may affect the postmortem drug concentrations in an unpredictable manner. The
Codeine to Morphine Concentration Ratios after Incubation 103
majority of the cases, where postmortem concentrations
exceeded that of the antemortem concentrations, could not
simply be explained by metabolism alone, but, in addition, by the
postmortem release of drugs. The previous may be further exacerbated by the increase in postmortem intervals and by the
sampling of a pooled mixture of blood. In our study, the influence of postmortem redistribution and the metabolism of
codeine were negligible as it had already taken place before sampling. The results reveal that changes also may take place after
sampling, during transportation to the analysis laboratory, under
freezing and thawing, and before analysis. Such in vitro changes,
occurring in the vials, must be caused by formation from other
metabolites or by degradation. Incubation is not a realistic scenario for the handling of such routine samples, but this has been
used to illustrate what might happen with the different codeine
metabolites in vitro. It is important to emphasize that this study
has not investigated the stability of the drug concentrations
during storage. The concentration ratios selected in this study
do not represent cases where there have been difficulties with
the interpretation of the findings, with respect to codeine ingestion. But the knowledge from this small study provides important information that can be applied on other cases, where such a
conclusion is challenging.
Conclusion
The codeine to morphine concentration ratio in blood depends
on interindividual variation in the metabolism of codeine to morphine, on postmortem redistribution, and on in vitro changes
occurring after sampling. In this study, the changes that may
take place in vitro, following sampling have been investigated
after incubation, using a worst case scenario to be able to illustrate what might take place. The increase in the morphine concentrations, followed by the reduction in codeine to morphine
ratios, is seen both in samples collected from autopsies and
from living samples. Increased concentrations of normorphine
were also found, while minor changes were seen for the other
metabolites.
The number of samples studied is small, and compared with
what might be seen in such cases, we have only included
samples with low morphine concentrations. Larger changes in
the codeine to morphine ratios are therefore likely to be found,
especially in cases from ‘ultrarapid metabolizer’ at the CYP2D6,
where higher morphine concentrations are present, and
changes in the morphine concentration to a larger extent will
contribute to lowering the codeine to morphine ratio. This must
be kept in mind when interpreting opiate cases. The mechanisms underlying the differences we have found for codeine and
morphine are still unknown, and also if codeine and morphine
have different properties regarding postmortem changes. Even
though this is an in vitro study, the changes observed is likely to
be part of the postmortem redistribution process, and are
changes that might take place in the body, after death. Further
investigations are warranted.
Acknowledgment
Thanks to Stine Marie Haavig for critical reading of the
manuscript.
104 Berg-Pedersen et al.
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Codeine to Morphine Concentration Ratios after Incubation 105