Evaluation of a method for simultaneous quantification of codeine, ethylmorphine and morphine in blood

doi:10.1016/0379-0738(91)90210-A

Forensic Science International

Volume 51, Issue 1, October 1991, Pages 105-110

https://doi.org/10.1016/0379-0738(91)90210-A Get rights and content

Abstract

Codeine, ethylmorphine and morphine are the most commonly detected opiates in forensic blood samples in Norway. A method for the simultaneous quantification of these opiates utilizing solid phase extraction and gas chromatography-mass spectrometry has been evaluated. The detection limits were 0.026 μmol/l for codeine, 0.025 μmol/l for ethylmorphine and 0.032 μmol/l for morphine (corresponding to 7.8, 7.8 and 9.1 μg/l, respectively). The analytical variations at concentrations of 1.0 μmol/l codeine, 1.0 μmol/l ethylmorphine and 0.5 μmol/l morphine were less than 5%.

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Gas chromatographic-mass spectrometric determination of morphine, codeine and 6-monoacetylmorphine in blood extracted by solid phase
J. Chromatogr.
(1989)

There are more references available in the full text version of this article.

Cited by (32)

Petechial hemorrhages, ethanol, and opioids in victims from intoxication
2022, Forensic Science International
Citation Excerpt :
Screening for other drugs was performed using high-performance liquid chromatography with mass spectrometry detection [26]. Confirmatory analyses were carried out by gas chromatography with mass spectrometry (GC–MS) or LC–MS [27–29]. We considered heroin present if morphine and 6-monoacetylmorphine (6-MAM) had been found.
Petechial hemorrhages are of interest to forensic pathologists because of their association with pressure on the neck. This study shows the associations between ethanol, opioids in blood and the risk of petechiae in conjunctivae and eye lids of 865 medico-legally examined victims from intoxication, 112 (12.9 %) with petechiae. Livor mortis on the front, face down body position, higher body weight, and younger age of the victims were independently associated with higher risk of petechiae. These variables were used for adjustment in the logistic regression analyzes. We found associations between ethanol, opioids, and the risk of petechiae when analyzed simultaneously. The association between ethanol and the risk of petechiae differed in opioid negative and positive victims (interaction, p = 0.028). In the opioid negative group, the association was J-formed, victims with low to medium level ethanol having lower risk (OR = 0.77) than those without ethanol or opioids, whereas high ethanol level gave a 4-fold higher risk (OR = 3.97). In the opioid positive group, the J-formed pattern was reversed. Victims with low to medium level ethanol had more than 4 times higher risk (OR = 4.65), whereas high level ethanol gave a slightly elevated risk (OR = 1.34) only compared to no ethanol or opioids. The results suggest that ethanol and opioids have a complex association with the risk of petechiae independent of livor mortis, initial body position, body weight, and age in victims from intoxication. Of practical value for the post-mortem examination is that the pathologist must consider both the ethanol level and the presence of opioids when judging the significance of petechiae in the eye regions.
Differences in combinations and concentrations of drugs of abuse in fatal intoxication and driving under the influence cases
2017, Forensic Science International
In toxicology, international classification systems focus on single intoxicants as the cause of death. It is, however, well known that very few drug related deaths are caused by a single substance and that information concerning the drug concentrations as well as the combinations of drugs are essential in order to ascertain the cause of death. The aim of the study was to assess whether those prone to fatal intoxications differ significantly from chronic drug users – in terms of demographics and drug exposure patterns.
Fatal psychoactive drug intoxications in Norway during 2012, where a forensic autopsy including toxicological analysis were performed, were included. Analytical findings in blood were compared with concentrations in blood from apprehended drivers under the influence of drugs and ethanol (DUID) during the same time period. The opioid and benzodiazepine concentrations were assessed as morphine and diazepam equivalents, respectively, in order to compare concentrations across the different groups.
A total of 194 autopsy cases and 4811 DUID cases were included. Opioids were detected in around 90% of the drug intoxication cases, but in only 16% of the DUID cases. The number of substances detected in fatal intoxications was 4.9 compared to 2.6 in the DUID cases. The total opioid concentrations were significantly higher in the fatal intoxication cases compared to DUID cases (229 ng/mL versus 56.9 ng/mL morphine equivalents, respectively). Benzodiazepines were detected in 90% of the fatal cases. Only one fatal opioid mono-intoxication was found; a case with a very high methadone concentration (1238 ng/mL).
Mono-intoxication with heroin was not seen in any of the fatal intoxications in Norway, and single drug intoxications were rare (1.5%). Fatal intoxications were caused by a combination of drugs with significantly more substances as well as higher total drug concentrations among the fatal cases compared to the DUID cases. The combination of opioids and benzodiazepines seemed to represent an increased risk of death.
The total load of drugs influence the degree of intoxication and the total concentration level must be considered, including the total number of substances. Our findings imply that international statistics regarding an opioid being the main intoxicant should have a shift in focus towards combinations of drugs (especially opioids and benzodiazepines) as a major risk factor for fatal drug overdoses.
Toxicological findings in suspected drug-impaired drivers in Norway — Trends during 1990–2015
2017, Forensic Science International
Citation Excerpt :
UHPLC-MS/MS screening for gamma-hydroxybutyrate (GHB) and pregabaline was introduced in 2010 [14]; earlier, these drugs were only analysed in cases where such use was suspected. In the 1990s, confirmation of positive screening results was performed by GC with EC- or MS-detection [15–18]. From 2001, confirmation of amphetamines and cannabinoids continued using GC–MS, whereas other compounds were confirmed by LC–MS [12] or UHPLC-MS/MS [19,20].
This study describes trends in drug use among drivers suspected of driving under the influence of drugs, apprehended by the police in Norway during 1990–2015. Chromatographically determined toxicological findings in blood samples were retrospectively investigated. Drug findings above defined cut-off concentrations were considered positive; hence making the annual prevalence comparable during the 26 years studied.
Blood samples from 112,348 drivers were included, of which 63% were positive for drugs; 43% had combined drug with alcohol or other drugs. In total, 87% of the drug-positive drivers were men, and a higher proportion of them were positive for illicit drugs compared to the women. Benzodiazepines and related drugs were found in 57% of the drug-positive drivers, stimulants in 51%, cannabis (tetrahydrocannabinol, THC) in 34%, and opioids in 18%. The types of benzodiazepines and opioids changed over time. The age distribution also changed; the proportion of drug-positive drivers above 40 years of age increased for all drug classes. The annual number of suspected drug-impaired drivers increased by 122% from 1990 to 1999, and by 54% from 2000 to 2015; the annual number of drug-positive samples increased by 260% from 1990 to 1999, and by 60% from 2000 to 2015. During 2000–2015, an increasing prevalence of amphetamines was found among suspected drug-impaired drivers above age 30; the highest rate of increase was observed among those at or above age 40. In the same period, the prevalence of benzodiazepines and related drugs decreased among all age groups, whereas the prevalence of THC increased; the highest prevalence and rate of increase were among suspected drug-impaired drivers under the age of 30. The results from this study indicate a slight change in the types of drugs used by drivers in Norway.
Pharmacokinetic interactions between ethanol and heroin: A study on post-mortem cases
2014, Forensic Science International
Citation Excerpt :
The analytical cut-off values for this method were 0.01 mg/L for 6-MAM and free morphine, and 0.02 mg/L for M3G and M6G. The confirmation analysis of 6-MAM in urine was until August 2007 performed using GC–MS, modified from the previously published method for blood samples mentioned above [26], and from August 2007 using the previously published UPLC–MS–MS method, also mentioned above [28]. In the present study, all analytical results at and above the cut-off level were included, while the results below cut-off were set to zero.
Ethanol and heroin are both depressant drugs on the central nervous system, and combined use is known to be dangerous due to pharmacodynamic interactions, leading to an even higher risk of respiratory depression and death. In addition, previous studies have suggested a pharmacokinetic interaction between ethanol and the metabolism of heroin. The aim of the present study was to investigate if there was a pharmacokinetic interaction between heroin and ethanol, by comparing concentrations of heroin metabolites in cases with and without ethanol, as detected in blood samples collected from a large material of forensic autopsy cases.
The material consisted of 1583 forensic autopsy cases, all containing 6-monoacetylmorphine (6-MAM), as evidence of heroin intake, in either blood or urine samples, from the time period between the 1st of January 2000 and the 31st of December 2012. Due to the high risk of post-mortem ethanol formation in cases revealing blood ethanol concentrations between 0.1 and 0.3‰, these cases were excluded from the study, along with cases where the analysis for ethanol was missing. After this exclusion of cases, the material (n = 1474) was divided into two groups; one group where ethanol was not detected in blood (n = 1160), and another group where ethanol was detected in blood at or above the concentration of 0.4‰ (n = 314). Furthermore, the material was also divided into two other subgroups; one group where 6-MAM was detected in blood samples, indicating a very recent intake of heroin, and another group where 6-MAM was detected in the urine, but not in blood, indicating a less recent heroin intake.
The concentration ratios of morphine/6-MAM, morphine-3-glucuronide (M3G)/morphine, and morphine-6-glucuronide (M6G)/morphine in blood samples, were all significantly lower in the ethanol positive cases compared with that of the ethanol negative cases. For the subgroup of cases revealing a very recent intake of heroin (n = 645), only the morphine/6-MAM ratio was significantly lower in the ethanol positive cases than in the ethanol negative cases. For the subgroup of cases with a less recent heroin intake (n = 817), lower M3G/morphine and M6G/morphine ratios were found among the ethanol positive cases.
The results indicate that ethanol inhibits two steps in the heroin metabolism; the hydrolysis of 6-MAM to morphine, and the glucuronidation of morphine to M3G and M6G. This pharmacokinetic interaction could further complicate the outcome after combined use of heroin and ethanol, in addition to the already well-known pharmacodynamic interactions.
Long-term stability of morphine, codeine, and 6-acetylmorphine in real-life whole blood samples, stored at -20°C
2014, Forensic Science International
Citation Excerpt :
Post mortem blood samples were collected in 20 mL Steriline® tubes (Bibby Sterilin, Staffordshire, UK), containing 0.3 mL of 67% (w/v) potassium fluoride solution (a final concentration of 1%), as a preservative. The confirmation analyses for morphine, codeine, and 6-AM, at the time of both the initial- and the reanalysis, were performed using a gas chromatography–mass spectrometry (GC–MS) method [20]. The analytical cut-off values for this method were 0.009 mg/L for morphine, 0.009 mg/L for codeine and 0.010 mg/L for 6-AM.
Stability of drugs during storage is important in forensic toxicology. For the analytes detected after intake of heroin (6-acetylmorphine (6-AM), morphine and codeine), long-time stability in real life whole blood samples are studied in only a small number of cases.
Whole blood post mortem (n = 37) and whole blood samples from living persons (n = 22) containing morphine and codeine as well as 6-AM in blood or urine were selected. All cases represented intake of heroin. All samples contained fluoride and were initially analysed and stored in normal conditions (−20 °C) for 4–9 years. All samples were then reanalysed using the same analytical methods and the results were compared.
For samples from living persons, the median change in concentration was −3.7% for morphine and −5.3% for codeine. For post mortem samples, the median change in concentration was −12% for morphine and −11% for codeine. Both for samples from living persons and post mortem samples, the decrease in the concentrations from the original analysis to reanalysis were statistically significant for morphine and codeine. Regarding 6-AM, all living samples were negative at reanalysis. For post mortem samples, four cases still tested positive for 6-AM at reanalysis with a median change in the concentrations of −81%. There was no significant change in the morphine to codeine concentration ratios neither for living nor post mortem samples.
This study showed that in real life whole blood samples, the concentrations of morphine and codeine are relatively stable during long-term storage at −20 °C. 6-AM on the other hand, shows a considerable decrease in concentrations that is important to consider when interpreting results from reanalyses of forensic cases.
Morphine to codeine concentration ratio in blood and urine as a marker of illicit heroin use in forensic autopsy samples
2012, Forensic Science International
Citation Excerpt :
From May 2009 a specific screening method by UPLC–MS-MS [11], with a cut-off level of 0.006 mg/L for both free morphine and codeine, has been used. The positive screening findings of free morphine and codeine were confirmed and quantified with GC–MS, modified from previously published method [27]. From May 2009, findings were confirmed/quantified using an UPLC–MS-MS method modified from previously used method [9,10].
A morphine to codeine ratio greater than unity (M/C > 1) has been suggested as an indicator of heroin use in living individuals. The aim of this study was to examine the morphine to codeine ratio in a large population (N = 2438) of forensically examined autopsy cases positive for 6-monoacetylmorphine (6-MAM) and/or morphine in blood and/or urine. Blood and urine concentrations of 6-MAM, morphine and codeine were examined using GC–MS and LC–MS/MS methods. In 6-MAM positive samples, the M/C ratio was greater than unity in 98% (N = 917) of the blood samples and 96% (N = 665) of the urine samples. Stratification of 6-MAM negative cases by M/C above or below unity revealed similarities in morphine and codeine concentrations in cases where M/C > 1 and 6-MAM positive cases. Median blood and urine morphine concentrations were 8–10 times greater than codeine for both groups. Similarly to 6-MAM positive cases, 25–44 year-old men prevailed in the M/C > 1 group. In comparison to cases where M/C ≤ 1, the M/C ratio was a hundred times higher in both 6-MAM positive and M/C > 1 cases. The range of morphine concentration between the lowest and the highest quintile of codeine in M/C > 1 cases was similar to that in 6-MAM positive cases. This range was much higher than for M/C ≤ 1 cases. Moreover, linear regression analyses, adjusted for age and gender, revealed a strong positive association between morphine and codeine in 6-MAM positive and M/C > 1 cases. The M/C ratio appeared to be a good marker of heroin use in post-mortem cases. Both blood and urine M/C > 1 can be used to separate heroin users from other cases positive for morphine and codeine.

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Evaluation of a method for simultaneous quantification of codeine, ethylmorphine and morphine in blood

Abstract

J. Chromatogr.