A specific immunoassay for the determination of morphine and its glucuronides in human blood

The development of specific antisera for immunochemical determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide is described. Morphine was N-demethylated to normorphine and N-alkylated to give N-aminopropyl-normorphine as hapten for antisera against morphine. As haptens for antisera against morphine-3-glucuronide and morphine-6-glucuronide, N-aminopropyl-nor-morphine was glucuronidated in position 3 or 6 respectively. Each of these three haptens were coupled to BSA employing the glutaraldehyde method to obtain three different immunogens. Immunisation of rabbits with these conjugates gave anti-morphine, anti-morphine-3-glucuronide and anti-morphine-6-glucuronide antisera, which were tested in a competitive, heterogeneous radioimmunoassay. Tracers for this radioimmunoassay procedure were synthesised by substitution of morphine and morphine-6-glucuronide in position 2 with ¹²⁵I and indirect iodination of the morphine-3-glucuronide hapten according to the method of Bolton and Hunter. The resulting antisera show very specific reactions with morphine, morphine-3-glucuronide and morphine-6-glucuronide. Cross reactivities of each antiserum with structurally related opiates and opioides are very low. The cross reactivities of the anti-morphine antiserum against morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine were less than 0.3%, the anti-morphine-3-glucuronide antiserum against morphine, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine less than 0.1% and the anti-morphine-6-glucuronide antiserum against morphine, morphine-3-glucuronide, codeine or dihydrocodeine less than 0.1%, against codeine-6-glucuronide less than 2.3%. The determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in blood samples (limit of detection = 3, 1, 0.5 ng/g) of nine cases of fatal heroin overdose with this radioimmunoassay method and the comparison with a GC/MS method is described. Received: 22 December 1997 / Received in revised form: 23 March 1998     

Antibody-mediated clean-up of blood for simultaneous HPLC determination of morphine and morphine glucuronides

For the interpretation of the concentration of morphine in blood samples of heroin consumers information about the concentration of the analgesic active morphine metabolite morphine-6-glucuronide is very important. Thus a simple but specific clean-up procedure based on immuno-affinity chromatography is presented for the extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from whole blood in cases of fatal heroin overdose. The preparation of the immunoabsorber by immobilization of antibodies against morphine-3-BSA and morphine-6-KLH with carbonyldiimidazole-activated trisacrylgel is described. The separation of the extracts is achieved by HPLC using native fluorescence detection. The limits of detection for this method are 10 ng for morphine and morphine glucuronides/g blood. The results for the concentration of morphine and morphine glucuronides in blood from seven cases of heroin overdose are presented. By calculating the quotients for the concentrations of morphine-6-glucuronide/morphine the time elapsed since the last intake of heroin is estimated. Received: 10 November 1996 / Received in revised form: 19 February 1997     

Lack of impairment due to confirmed codeine use prior to a motor vehicle accident: Role of pharmacogenomics

BackgroundWe examined forensic serum toxicology and pharmacogenomics data from a woman on codeine shortly before she caused a motor vehicle accident.MethodsA woman driving erratically collided with a parked car of a highway seriously injuring 2 men working to repair the parked vehicle. The woman tested positive for codeine, acetaminophen and barbital. She had been taking these medications for 20 years due to migraine headache. Serum toxicology and genotype analysis for cytochrome P450, UDP glucuronosyltransferase, and other metabolizing enzymes were measured.ResultsThe woman was tried and convicted of driving under the influence resulting in bodily harm and was sentenced to 6 years. Toxicology results on peripheral blood showed a total and free codeine of 840 and 348 μg/L, respectively, and total morphine of 20 μg/L (17, 3, and 0 μg/L for morphine-3-glucuronide, morphine-6-glucuronide, and free morphine, respectively). She was heterozygous for CYP 2D6 *2/*4 (extensive/poor metabolism) and heterozygous for UGT 2B7 *1/*2 (extensive/ultra-rapid metabolism). The woman was also taking fluoxetine and bupropion which are strong inhibitors of CYP 2D6.ConclusionsBased on her genotype and phenotype and reports by the arresting officer, we suggest that the subject in question was not intoxicated by opiates at the time of her motor vehicle accident and may have been falsely incarcerated.     

Time-dependent postmortem redistribution of morphine and its metabolites in blood and alternative matrices—application of CT-guided biopsy sampling

Interpretation of postmortem morphine concentrations in forensic toxicology provides several pitfalls such as missing information on tolerance, analyte stability, or postmortem redistribution (PMR). Recently, it had been shown that computed tomography (CT)-guided collection of biopsies using a robotic arm (virtobot) provides a valuable strategy for systematic studies on time-dependent PMR. Using this technique, time-dependent PMR of morphine and its metabolites was investigated in 12 cases. At admission to the institute (t1), femoral and heart blood (right ventricle) as well as biopsies from the right lung, the right kidney, liver, spleen, and muscle tissue were collected. At autopsy approximately 24 h later (t2), samples from the same body regions were collected again. Additionally, gastric contents, urine, brain tissue, and heart blood from the left ventricle was collected. Morphine, normorphine, hydromorphone, morphine-3-glucuronide, morphine-6-glucuronide, and morphine-sulfate were quantified with LC-MS/MS. In femoral blood, significant increase of morphine concentrations was observed, although ultimately not relevant for forensic interpretation. In the alternative matrices, increases as well as decreases were observed without a clear trend. The morphine metabolites did not exhibit relevant concentration changes. Investigation of underlying redistribution mechanisms indicated that concentration change (i.e., increase) of morphine in femoral blood rather resulted from diffusion processes than from release of morphine from its conjugates. Concentration changes in heart blood might have been caused by redistribution from lung tissue or gastric content. This study also proved that CT-guided collection of biopsies using a virtobot arm is an invaluable tool for future studies on PMR redistribution of other substance groups.

     

Postmortem distribution pattern of morphine and morphine glucuronides in heroin overdose

The postmortem distribution of morphine and its metabolites was investigated in four cases of heroin overdose to evaluate some of the factors that influence intravasal blood concentrations. Variables included were the chemical stability of morphine conjugates, hemoconcentration, incomplete distribution of the drug and diffusion processes. Blood samples from different sampling sites including the aorta, the infra- and suprarenal portion of the inferior vena cava, the superior vena cava, the femoral and subclavian veins, and the right and left ventricles were examined for morphine, morphine-3-glucuronide and morphine-6-glucuronide, hematocrit and water content. Drug concentrations were determined by HPLC based on the native fluorescence of the analytes. Morphine glucuronides proved to be stable for a time period of 72 h. The water content ranged from 65 to 83% and hematocrit values from 25 to 75%, and were seen as contributory factors to the dramatic differences observed for drug concentrations from different sampling sites. The differences could neither be attributed to incomplete distribution during life-time nor to a diffusion process following the different distribution volumes of morphine and its conjugates. A definite relationship between the ratio of the molar concentrations of morphine and its glucuronides, as assessed in pharmacokinetical studies after morphine dosing, could not be established. For a better understanding more cases and changes over time and tissue concentrations should be analysed.     

Analytical confirmation of lethal heroin overdose by the use of liquid chromatography methods

BACKGROUND/AIM: Heroin is diacetylated morphine. Its ability to induce euphoria has led to its frequent abuse, giving rise to psychological and physical dependence. It has a short half-life, of approximately 2-6 min. In the brain, heroin undergoes deacetylation to 6-monoacetylmorphine (6-MAM) and morphine. Detection of 6-acetylmorphine in the urine is indicative of heroin use. The aim of this study was to compare sensitivity and reliability of two analytical methods, a multicolumn liquid chromatography system with UV scanning detector (HPLC-UV) and liquid chromatography-mass spectrometry detection (LC-MS) in opiate determining in post mortem material. METHODS: Post mortem samples (blood, urine and vitreous humor) were analyzed by liquid chromatography with UV and MS detection. The samples were prepared by liquid-liquid extraction with mixture chloroform-isopropanol (9:1). Separation was performed on C8 column with mobile phase composed of 55% acetonitrile-glacial acetic acid (99:1) and 45% 20 mM ammonium acetate. RESULTS: The analysis of blood samples, urine, and eye liquid by the use of multicolumn HPLC-UV method confirmed the presence of morphine in the samples of blood and urine, codeine only in urine, and 6-MAM in the samples of urine and eye liquid. Using LC-MS method morphine was confirmed in all of the samples, while codeine was confirmed in urine and in the sample of eye liquid. In the samples of eye liquid and urine 6-MAM was confirmed. CONCLUSION: For determination of opiates in post mortem material LC-MS technique is more sensitive and reliable as compared to multicolumn liquid chromatography.     

吗啡代谢产物的药理作用

吗啡主要在肝脏与葡糖苷酸结合并产生两种代谢产物：吗啡-3-葡糖苷酸(M3G)和吗啡-6-葡糖苷酸(M6G)。M6G能结合阿片受体，动物实验显示其镇痛作用比吗啡强，不良反应较轻微。M3G与阿片受体亲和力较低，没有镇痛作用，且动物实验表明M3G能对抗吗啡和M6G的镇痛作用，可能参与吗啡耐受的形成。本文综述了吗啡的代谢过程及其代谢产物的药理作用。     

GC-MS detection and tentative characterization of two noscapine metabolites in human urine and their potential as markers for opium and illicit heroin use

Urine samples for cases of drug abuse are regularly screened by gas chromatography-mass spectrometry (GC-MS) after enzyme hydrolysis, liquid-liquid extraction, and trimethylsilyl (TMS) derivatization. During such routine analysis, two compounds (labeled as X1 and X2) were detected in urine samples of opium and illicit heroin users as their TMS derivatives, which led us to tentatively characterize the compounds. It was found that the compounds were noscapine metabolites as determined by GC-MS in both electron-impact ionization (EI) and chemical ionization modes. Together with the analysis of fragment ions in the EI mass spectra, the X1 and X2 compounds were further assigned as 6,7-dihydroxynoscapine and its desmethyl product at either position 6′ or 7′, respectively. The two tentatively characterized compounds were found to be useful as markers for excluding medicinal morphine use and for refuting claims of codeine use or Golden-Seal preparation use often made by heroin addicts to evade conviction. By addition of the above two metabolites, the arsenal of markers for opium and illicit heroin use has now been strengthened.     

Identifying cases of heroin toxicity where 6-acetylmorphine (6-AM) is not detected by toxicological analyses

Purpose

Heroin has a half-life of 2–6 min and is metabolized too quickly to be detected in autopsy samples. The presence of 6-acetylmophine (6-AM) in urine, blood, or other samples is convincing evidence of heroin use by a decedent, but 6-AM itself has a half-life of 6–25 min before it is hydrolyzed to morphine, so 6-AM may not be present in sufficient concentration to detect in postmortem samples. Codeine is often present in heroin preparations as an impurity and is not a metabolite of heroin. Studies report that a ratio of morphine to codeine greater than one indicates heroin use. We hypothesize that the ratio of morphine to codeine in our decedents abusing drugs intravenously will be no different in individuals with 6-AM present than in individuals where no 6-AM is detected, and we report our study of this hypothesis.

Methods

All accidental deaths investigated by the Jefferson County Coroner/Medical Examiner Office from 2010 to 2013 with morphine detected in blood samples collected at autopsy were reviewed. Five deaths where trauma caused or contributed to death were excluded from the review. The presence or absence of 6-AM and the concentrations of morphine and codeine were recorded for each case. The ratio of morphine to codeine was calculated for all decedents. Any individual in whom no morphine or codeine was detected in a postmortem sample was excluded from further study. Absence or presence of drug paraphernalia or evidence of intravascular (IV) drug use was documented in each case to identify IV drug users. The proportion of the IV drug users with and without 6-AM present in a postmortem sample was compared to the M/C ratio for the individuals.

Results

Of the 230 deaths included in the analysis, 103 IV drug users with quantifiable morphine and codeine in a postmortem sample were identified allowing for calculation of an M/C ratio. In these IV drug users, the M/C ratio was greater than 1 in 98 % of decedents. When controlling for the absence or presence of 6-AM there was no statistically significant difference in the proportion of IV drug users when compared to non IV drug users with an M/C ratio of greater than 1 (p = 1.000).

Conclusion

The M/C ratio in IV drug users, if greater than 1, is seen in deaths due to heroin toxicity where 6-AM is detected in a postmortem sample. This study provides evidence that a M/C ratio greater than one in an IV drug user is evidence of a death due to heroin toxicity even if 6-AM is not detected in the blood. Using the M/C ratio, in addition to scene and autopsy findings, provides sufficient evidence to show heroin is the source of the morphine and codeine. Listing heroin as a cause or contributing factor in deaths with evidence of IV drug abuse and where the M/C ratio exceeds 1 will improve identification of heroin fatalities, which will allow better allocation of resources for public health initiatives.

     

Diskurs zu Morphinbefunden im Autopsieblut nach palliativer Schmerzbehandlung

The finding of morphine and morphine glucuronide concentrations in post-mortem blood samples assists to certify deaths associated with narcotics. However, the forensic interpretation of the analytical results affords profound knowledge, especially in cases after palliative pain treatment. This paper gives a comprehensive review about the pharmacokinetics of morphine and its glucuronides collected from experimental and clinical data. The hallmark of morphine kinetics is the large interindividual variation for all parameters. The advantages and disadvantages of the different analytical methods are given and the importance of the direct determination of the pharmacologically active metabolite morphine-6-glucuronide is stressed. Special aspects of post-mortem blood samples, sampling techniques and the particular blood provenience, factors affecting the drug concentrations such as the ante-mortem haemodynamic status as well as arterio-venous differences, blood inhomogeneity, possible bacterial deconjugation of the glucuronides in cardiac blood samples as well as the relevance of unequal distribution in blood and plasma for interpretation are explained. Drug stability does not seem to be affected in peripheral blood (femoral vein) and post-mortem diffusion of the analytes must not be considered to be crucial for evaluating morphine concentrations and its metabolites.     

Pharmacokinetics of heroin and its metabolites in vitreous humor and blood in a living pig model

Vitreous humor (VH) is an alternative matrix for drug analysis in forensic toxicology. However, little is known about the distribution of xenobiotics, such as opioids, into VH in living organisms. The aim of this study was to simultaneously measure heroin and metabolite concentrations in blood and VH after injection of heroin in a living pig model. Six pigs were under non-opioid anesthesia during the surgical operation and experiment. Ocular microdialysis was used to acquire dialysate from VH, and a venous catheter was used for blood sampling. Twenty milligrams of heroin was injected intravenously with subsequent sampling of blood and dialysate for 6 h. The samples were analyzed by ultra-performance liquid chromatography–tandem mass spectrometry. Heroin was not detected in VH; 6-monoacetylmorphine (6-MAM) and morphine were first detected in VH after 60 min. The morphine concentration in VH thereafter increased throughout the experimental period. For 6-MAM, C _max was reached after 230 min in VH. In blood, 6-MAM reached C _max after 0.5 min, with a subsequent biphasic elimination phase. The blood and VH 6-MAM concentrations reached equilibrium after 2 h. In blood, morphine reached C _max after 4.3 min, with a subsequent slower elimination than 6-MAM. The blood and VH morphine concentrations were in equilibrium about 6 h after injection of heroin. In conclusion, both 6-MAM and morphine showed slow transport into VH; detection of 6-MAM in VH did not necessarily reflect a recent intake of heroin. Because postmortem changes are expected to be small in VH, these experimental results could assist the interpretation of heroin deaths.     

Concentrations of morphine and codeine in urine of heroin abusers

We have measured concentrations of morphine, codeine and 6-monoacetylmorphine in urine of people admitted to the Los Angeles County + University of Southern California Medical Center. Of 60 patients positive for morphine and/or codeine, 10 were judged to be heroin abusers based on positive results for 6-monoacetylmorphine, a specific metabolite of heroin. In nine of these ten patients, 0.028-39.4 microg/ml free codeine and 0.070-307 microg/ml total codeine were detected along with 1.74-218 microg/ml of free morphine and 11.2-2870 microg/ml of total morphine; the morphine-to-codeine ratios were 3.65-228 and 2.27-207 for the free forms and total amounts of these opiates, respectively. In the one patient who was negative for codeine, the concentrations of free and total morphine were 0.114 and 2.22 microg/ml, respectively. Based on our data and literature data available, the following criteria are proposed for judging heroin use from the results of urinalysis, especially when no 6-monoacetylmorphine is detected: (1) a detectable amount of free morphine exists and the concentration of total morphine is higher than 10 microg/ml; (2) a detectable amount of codeine exists; and (3) the morphine-to-codeine ratio is higher than 2 for both the free forms and total amounts of these opiates.     

HPLC with laser-induced native fluorescence detection for morphine and morphine glucuronides from blood after immunoaffinity extraction

A new immunoaffinity solid phase extraction of morphine and its phase II metabolites, morphine-3-ß-D-glucuronide and morphine-6-ß-D-glucuronide is described. An immunoadsorber was applied which was created for the first time by the immobilisation of specific antibodies (polyclonal, host: rabbit) by the sol-gel method. The extraction method in combination with high performance liquid chromatography-fluorescence determination has been validated and shown to be applicable to blood samples of heroin victims in a low concentration range. Blood extracts were essentially free of interfering matrix components when compared to C₈-extracts. Additionally, a novel, sensitive and selective detection system for wavelength-resolved analysis of laser-induced fluorescence coupled to HPLC was developed. The analytes were excited with a frequency tripled Ti:Sa laser (=244 nm quasi cw). The total emission spectrum was recorded with a detection system consisting of an imaging spectrograph and a back-illuminated CCD camera. This technique of detection, combined with an extended optical path (at least 6 mm could be illuminated by the laser), resulted in an optimal fluorescence intensity of the analytes. The method permitted the analysis of morphine, morphine-3-ß-D-glucuronide and morphine-6-ß-D-glucuronide in a low concentration range and could be applied to a complex matrix such as postmortem blood samples because analyte peaks could be discriminated from matrix peaks by their characteristic emission spectra.     

Drug abuse in Denmark (Jutland and Funen)

Summary Two thousand fifty-three samples submitted to forensic chemical analysis during the period 1982–1987 were characterized according to their content of narcotics and the year of seizure. Heroin and cannabis dominated the material, but a significant decrease in the number of heroin samples was observed in 1987. Amphetamine, on the contrary, hardly seen during the period 1982–1984, accounted for half of the narcotics submitted in 1987. The misuse of morphine, especially in the form of poppy capsules, was a problem for some years, whereas cocaine has not yet been a drug of abuse in Denmark. The mean concentrations of the heroin and the amphetamine samples were 30% and 44%, respectively, both for retail drugs and the entire material.     

Cross-reactivity of the CEDIA buprenorphine assay with opiates: an Austrian phenomenon?

When testing the Microgenics CEDIA assay for immunological buprenorphine analysis, cross-reactivity between the buprenorphine reagents and opiates was observed at concentrations higher than 120 mg/l morphine, 320 mg/l methadone, 30 mg/l codeine, 60 mg/l dihydrocodeine and 520 mg/l morphine-3-glucuronide. The cross-reactivity with morphine has the greatest impact on routine screening as opiate maintenance therapy in Austria is also performed with slow-release oral morphine. The use of a second cutoff value of 30 g/l for urine samples that are (immunologically) positive for opiates is therefore suggested, compared to the cutoff value of 5 g/l proposed by the manufacturer.     

Death of a female addict due to heroin and cocaine overdoses: a case report with multiparameter evaluation

This study undertook a multiparameter evaluation of the death of a 21-year-old woman known to be an abuser of heroin and cocaine. The toxicological analysis of multiple postmortem specimens such as blood and hair was carried out using liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS). The blood specimens of the deceased showed the presence of opium components such as morphine and its glucuronides together with cocaine and benzoylecgonine. The detected xenobiotic levels probably explained the cause of her death resulting from combined action of unintentional illicit drug overdose. By analysis of four 2-cm long hair segments, a heroin-cocaine addiction for at least 8 months antemortem was able to be documented; the presence of 6-monoacetylmorphine (6-MAM), cocaine, and benzoylecgonine was demonstrated. The histopathological findings of lesions of the internal organs of the deceased were consistent with long heroin and cocaine abuse. The use of multiple parameters, such as blood and hair segments as matrices and drug metabolites such as 6-MAM, morphine, glucuronides, and benzoylecgonine as target compounds, gave a well-defined outline of her death.     

Four nonfatal and six fatal cases of opiate use: utility of morphine, its metabolites, and their ratios in blood specimens

Four nonfatal and six fatal cases of opiate use are presented with careful toxicological analysis. Levels of morphine (M), 6-monoacetylmorphine (6-MAM), morphine-6-glucuronide (M6G), and morphine-3-glucuronide (M3G) in blood specimens were measured by the sophisticated method of liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS-MS). Fatal cases were characterized by much higher levels of free M than the nonfatal cases; this caused lower ratios of M6G/M and M3G/M in the fatal cases when compared with the nonfatal cases. Among the six fatal cases, the M6G/M ratios were especially low in four cases, in which rapid deaths were estimated. The present data are compared with data previously reported by other groups, and we discuss the utility of the levels of M, M6G, and M3G in blood and their ratios for estimating the antemortem status of each individual.     

GC-MS detection and characterization of thebaine as a urinary marker of opium use

The differentiation between legal and illegal opiate product use through the detection of marker compounds in urine is one of the most important subjects in forensic toxicology. Thebaine, an opium alkaloid, can be detected by gas chromatography-mass spectrometry (GC-MS) and has been suggested as a good marker of poppy seed use in differentiation from that of illicit heroin. In the present study, thebaine was targeted as a urinary marker of opium use because it is a major constituent of opium. Although thebaine lacks the conventional functional group, it was found to form a trimethylsilyl (TMS) derivative. This has made it possible to simultaneously detect thebaine together with morphine and codeine in the same urine sample to confirm opium use, thus saving time and cost of analysis while using less material. The TMS derivatization was found to enhance the stability and chromatographic properties of thebaine and to increase the sensitivity of its detection about 50-fold in comparison with that of the underivatized compound. Using the present method, thebaine was analyzed for a number of actual urine samples obtained from users of opium, illicit heroin, or codeine preparation. The results showed that thebaine is a useful opium-use marker subject to its oral administration.     

Direct quantification of morphine glucuronides and free morphine in urine by liquid chromatography–tandem mass spectrometry

A simple and robust liquid chromatographic-tandem mass spectrometric method was developed and validated for the direct quantification of the structural isomers morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), and morphine in urine. Method development showed that dilution of the urine samples was mandatory to obtain reproducible chromatography and that the use of deuterated internal standards was needed to compensate for ion suppression. Sample preparation consisted of a 20-fold dilution of urine into the internal standard solution. Chromatography was performed on a C₈-column using gradient conditions. The mobile phase consisted of water/methanol, both containing 0.1 % acetic acid and 1 mM ammonium acetate. Calibration curves were constructed between 0.05 and 2 μg/ml. Validation data showed biases ranging from 1.5 to 5.2 % for M3G, −2.7 to −5.9 % for M6G, and 3.7 to 7.1 % for morphine. Imprecision never exceeded 5.2, 5.9, and 5.7 % for M3G, M6G, and morphine, respectively. The applicability of the method was checked by the analysis of 20 urine samples that were analyzed concurrently with a routine gas chromatography-mass spectrometry method. Results showed good correlation between the methods with a correlation coefficient of approximately 0.95.     

Changes in place of origin of heroin seized in Denmark from 1981 to 1986

Summary Hundred thirty-eight samples of heroin weighing more than 0.1g seized between 1981 and 1986 were characterized according to their contents of opium alkaloids, adulterants, and diluents together with their form and color. The chemical fingerprint was used to establish a change in the heroin during the period. As compared to the first few years covered by the survey, a predominant number of the samples at the end of the period were in the base form and contained the opium alkaloids papaverine and noscapine. In particular, the concentration of noscapine as related to the heroin content of each sample had increased considerably, indicating Pakistan or Iran as being the places of origin of most of the heroin seized in Denmark at the end of the period.