Evaluation of an on-site test device for the heroin metabolite 6-acetylmorphine in urine.

Detection of heroin use is an important task in clinical drug testing and can be best performed by using 6-acetylmorphine as the target analyte. This study was performed to evaluate an on-site test for 6-acetylmorphine screening in urine with an assigned cut-off limit at 10 ng/mL. The reference method was a forensic accredited liquid chromatography-tandem mass spectrometry method. The study confirmed that negative controls and negative authentic specimen resulted in negative readings. Low cross-reactivity was recorded from other potential interfering opioids. Prepared standards and commercial calibrators demonstrated that the cutoff level of the test was lower than the assigned value and rather 2 ng/mL. A study using authentic specimens from patients on substitution treatment with methadone, morphine, and buprenorphine confirmed that the real cut-off level was 2 ng/mL. Using this value as cutoff limit the sensitivity and specificity of the test was 100%.

Metabolites of Heroin in Several Different Post-mortem Matrices.

In some forensic autopsies blood is not available, and other matrices are sampled for toxicological analysis. The aims of the present study were to examine whether heroin metabolites can be detected in different post-mortem matrices, and investigate whether analyses in other matrices can give useful information about concentrations in peripheral blood. Effects of ethanol on the metabolism and distribution of heroin metabolites were also investigated. We included 45 forensic autopsies where morphine was detected in peripheral blood, concomitantly with 6-acetylmorphine (6-AM) detected in any matrix. Samples were collected from peripheral blood, cardiac blood, pericardial fluid, psoas muscle, lateral vastus muscle, vitreous humor and urine. Opioid analysis included 6-AM, morphine, codeine, and morphine glucuronides. The 6-AM was most often detected in urine (n = 39) and vitreous humor (n = 38). The median morphine concentration ratio relative to peripheral blood was 1.3 (range 0-3.6) for cardiac blood, 1.4 (range 0.07-5.3) for pericardial fluid, 1.2 (range 0-19.2) for psoas muscle, 1.1 (range 0-1.7) for lateral vastus muscle and 0.4 (range 0.2-3.2) for vitreous humor. The number of 6-AM positive cases was significantly higher (P = 0.03) in the ethanol positive group (n = 6; 86%) compared to the ethanol negative group (n = 14; 37%) in peripheral blood. The distribution of heroin metabolites to the different matrices was not significantly different between the ethanol positive and the ethanol negative group. This study shows that toxicological analyses of several matrices could be useful in heroin-related deaths. Urine and vitreous humor are superior for detection of 6-AM, while concentrations of morphine could be assessed from peripheral or cardiac blood, pericardial fluid, psoas muscle and lateral vastus muscle.

A morphine conjugate vaccine attenuates the behavioral effects of morphine in rats.

Vaccines for opioid dependence may provide a treatment that would reduce or slow the distribution of the drug to brain, thus reducing the drug's reinforcing effects. We tested whether a conjugate vaccine against morphine (keyhole limpet hemocyanin-6-succinylmorphine; KLH-6-SM) administered to rats would produce antibodies and show specificity for morphine or other heroin metabolites. The functional effects of the vaccine were tested with antinociceptive and conditioned place preference (CPP) tests. Rats were either vaccinated with KLH-6-SM and received two boosts 3 and 16 weeks later or served as controls and received KLH alone. Anti-morphine antibodies were produced in vaccinated rats; levels increased and were sustained at moderate levels through 24 weeks. Antibody binding was inhibited by free morphine and other heroin metabolites as demonstrated by competitive inhibition ELISA. Vaccinated rats showed reduced morphine CPP, tested during weeks 4 to 6, and decreased antinociceptive responses to morphine, tested at week 7. Brain morphine levels, assessed using gas-chromatography coupled to mass spectrometry (GC-MS) on samples obtained at 26 weeks, were significantly lower in vaccinated rats. This suggests that morphine entry into the brain was reduced or slowed. These results provide support for KLH-6-SM as a candidate vaccine for opioid dependence.

CE-MS analysis of heroin and its basic impurities using a charged polymer-protected gold nanoparticle-coated capillary.

The first application of charged polymer-protected gold nanoparticles (Au NPs) as semi-permanent capillary coating in CE-MS was presented. Poly(diallyldimethylammonium chloride) (PDDA) was the only reducing and stabilizing agent for Au NPs preparation. Stable and repeatable coating with good tolerance to 0.1 M HCl, methanol, and ACN was obtained via a simple rinsing procedure. Au NPs enhanced the coating stability toward flushing by methanol, improved the run-to-run and capillary-to-capillary repeatabilities, and improved the separation efficiency of heroin and its basic impurities for tracing geographical origins of illicit samples. Baseline resolution of eight heroin-related alkaloids was achieved on the PDDA-protected Au NPs-coated capillary under the optimum conditions: 120 mM ammonium acetate (pH 5.2) with addition of 13% methanol, separation temperature 20 degrees C, applied voltage -20 kV, and capillary effective length 60.0 cm. CE-MS analysis with run-to-run RSDs (n=5) of migration time in the range of 0.43-0.62% and RSDs (n=5) of peak area in the range of 1.49-4.68% was obtained. The established CE-MS method would offer sensitive detection and confident identification of heroin and related compounds and provide an alternative to LC-MS and GC-MS for illicit drug control.

Oral fluid as an alternative matrix to monitor opiate and cocaine use in substance-abuse treatment patients.

Interest in oral fluid as an alternative matrix for monitoring drug use is due to its ease-of-collection and non-invasiveness; however, limited data are available on the disposition of drugs into oral fluid. The objective of this research was to provide data on the presence and concentrations of heroin, cocaine and multiple metabolites in oral fluid after illicit opioid and cocaine use. Thrice weekly oral fluid specimens (N=403) from 16 pregnant opiate-dependent women were obtained with the Salivette oral fluid collection device. Evidence of heroin (N=62) and cocaine (N=130) use was detected in oral fluid by LC-APCI-MS/MS. 6-Acetylmorphine (6-AM), heroin and morphine were the major opiates detected, with median concentrations of 5.2, 2.3, and 7.5 microg/L, respectively. Cocaine and benzoylecgonine (BE) had median concentrations of 6.4 and 3.4 microg/L. Application of the Substance Abuse Mental Health Services Administration (SAMHSA) recommended cutoffs for morphine and codeine (40 microg/L), 6-AM (4 microg/L) and cocaine and BE (8 microg/L), yielded 28 opiate- and 50 cocaine-positive specimens. Oral fluid is a promising alternative matrix to monitor opiate and cocaine use in drug testing programs. These data guide interpretation of oral fluid test results and evaluate currently proposed SAMHSA oral fluid testing cutoffs.

La inyección de drogas en Bogotá: una amenaza creciente / Drug injection in Bogotá: a growing threat

Este proyecto hace parte de la Fase II del estudio mundial sobre Uso de Drogas Inyectadas y Riesgos para la salud de la Organización Mundial de la Salud. Esta fase de la investigación debía proveer información acerca de la naturaleza actual, extensión e implicaciones del uso intravenoso de drogas, así como una mejor comprensión de los factores socio-culturales que podrían estar influenciando la toma de riesgos. Se entrevistaron 410 personas en la ciudad de Bogotá, Colombia, (inyectores, ex-inyectores, expendedores y no inyectores). Las personas fueron contactadas inicialmente en su ambiente natural y posteriormente en recintos privados para la realización de grupos focales. Los resultados sugieren que el uso intravenoso de drogas es más frecuente de lo que se pensaba; los riesgos del policonsumo de drogas, del uso intravenoso de éstas, de la actividad sexual desprotegida, evidencian que la población de inyectores es un foco importante para el desarrollo de epidemias como el VIH y la Hepatitis B y C. La realidad del uso intravenoso de drogas es un problema que ha permanecido "oculto" y las consecuencias para la salud de los inyectores no han sido del todo evidentes hasta ahora. El potencial de una difusión continuada de la práctica entre redes de consumidores en la ciudad y las consecuencias que de ello se derivarían, deberán ser contempladas por quienes construyen políticas, ofrecen servicios y diseñan intervenciones

This Project is part of Phase II of the WHO Injected Drug Use and Health Risks worldwide study. The objective was to obtain information on the current nature, extension and implications of injected drug use, in addition to gaining a better understanding of the socio-cultural factors that could influence risk taking. 410 people were interviewed in a sample drawn from Bogóta, Colombia (injectors, former injectors, dealers and non-injectors). SubjBcts were initially contacted in their natural environment and met, subsequently, on private premises for focal groups. Results show that drug injection is more common than previously assumed. Risk behaviours associated with multidrug use, injecting these, and unsafe sex suggest that this population of injectors is a significant source for epidemics such as HIV and hepatitis B and C. The reality of intravenous drug use has been "hidden" and its consequences on the health of those who inject have not been apparent upto now. The potential for a continual spread of such a practice among user networks in the city and the repercussion deriving from this must be taken into consideration by policymakers in implementing services and drawing up interventions

Reversible parkinsonism following heroin pyrolysate inhalation is associated with tetrahydrobiopterin deficiency.

A patient who inhaled heroin vapour (chasing the dragon), resulting in temporary parkinsonism, is described. A reversible deficiency of tetrahydrobiopterin, causing altered dopamine metabolism, is demonstrated as the metabolic basis of this aspect of the encephalopathy.

Pharmacological characterization of dihydromorphine, 6-acetyldihydromorphine and dihydroheroin analgesia and their differentiation from morphine.

The present study examined the pharmacology of dihydromorphine, 6-acetyldihydromorphine and dihydroheroin (3,6-diacetyldihydromorphine). Like morphine, dihydromorphine and its acetylated derivatives all were highly selective mu-opioids in receptor binding assays. All the compounds were potent mu-selective analgesics, as shown by their sensitivity towards the mu-selective opioid receptor antagonists naloxonazine and beta-funaltrexamine. However, the actions of dihydromorphine and its analogs were readily distinguished from those of morphine, differences that were surprising in view of the very limited structural differences among them that consisted of only the reduction of the 7,8-double bond. Like heroin and morphine-6beta-glucuronide, the analgesic actions of dihydromorphine and its two acetylated derivatives were antagonized by 3-O-methylnaltrexone at a dose that was inactive against morphine analgesia. Antisense mapping also distinguished between morphine and the dihydromorphine compounds. Antisense oligodeoxynucleotides targeting exon 2 of the cloned MOR-1 gene decreased dihydromorphine analgesia and that of its acetylated derivatives, but not morphine analgesia. Conversely, the exon 1 antisense that effectively lowered morphine analgesia was inactive against dihydromorphine and its analogs. Finally, dihydromorphine and its analogs retained their analgesic activity in a mouse model of morphine tolerance, consistent with incomplete cross-tolerance. Together, these findings imply that the mu-opioid receptor mechanisms mediating the analgesic actions of dihydromorphine and its acetylated analogs are distinct from morphine and more similar to those of heroin and morphine-6beta-glucuronide.

Comparison of four derivatizing reagents for 6-acetylmorphine GC-MS analysis.

The propionyl, trimethylsilyl, trifluroacetyl, and heptafluoroacyl derivatives of 6-acetylmorphine (6-AM) were evaluated with respect to optimal method performance, derivative stability, and methods characterization for use in gas chromatographic-mass spectrometric (GC-MS) analysis with electron ionization mode and selected ion monitoring. The most common potential interferences and compatibility with other derivatives when used on the same GC-MS were determined for the derivatizing reagents. The propionyl, trimethylsilyl, and trifluroacetyl derivatives produced adequate stability, accuracy, and precision for the method. The 6-AM derivatization with commercially available propionic anhydride generated a relatively small amount of 6-AM-propionyl derivative from the free morphine present in a specimen. The trimethylsilyl derivative obtained by the reaction with MSTFA did not require incubation, was the easiest to prepare, and had the highest potential for use on an automated sample-preparation device. An important advantage of derivatization with MSTFA is elimination of the possibility of heroin decomposition to 6-AM that is due to incubation at elevated temperature.

[Economic evaluation in a trial of medically controlled prescription of narcotics to dependent users (PROVE)]. / Gesundheitsökonomische Bewertung der Versuche für eine ärztliche Verschreibung von Betäubungsmitteln (PROVE).

In the 1994-1996 trial of medically controlled prescription of narcotics to dependent users, 800 places were ascribed to heroin substitutes and another 200 for methadone and morphine substitutes. The trial was evaluated with the aid of an accompanying research. Among the results demonstrated in the evaluation was an improvement of the health of the participants. The economic assessment was drawn from observations of health effects within a sub-sample of 142 participants from four centers. In a retrospective statistical survey, for each acute illness which could be influenced through the trial, the number of diagnoses was recorded in the first and thirteenth month after study entry. Also, based on a number of representative cases for each of these acute illnesses, the resource use, i.e. the types and numbers of medical products and services rendered to the patients, was recorded. The results showed a clear decline in depressive episodes, skin diseases, digestive system disorders as well as epileptic attacks and intoxication. Treatment costs could be reduced from a total of CHF 94875.--to CHF 21,998.--/month or from CHF 22.27 to CHF 5.15/patient per day. The improvement of somatic and psychic health due to the medically controlled prescription of narcotics resulted in a benefit of CHF 17.11/person per day.

Human liver carboxylesterase hCE-1: binding specificity for cocaine, heroin, and their metabolites and analogs.

Purified human liver carboxylesterase (hCE-1) catalyzes the hydrolysis of cocaine to form benzoylecgonine, the deacetylation of heroin to form 6-acetylmorphine, and the ethanol-dependent transesterification of cocaine to form cocaethylene. In this study, the binding affinities of cocaine, cocaine metabolites and analogs, heroin, morphine, and 6-acetylmorphine for hCE-1 were evaluated by measuring their kinetic inhibition constants with 4-methylumbelliferyl acetate in a rapid spectrophotometric assay. The naturally occurring (R)-(-)-cocaine isomer displayed the highest affinity of all cocaine and heroin analogs or metabolites. The pseudo- or allopseudococaine isomers of cocaine exhibited lower affinity indicating that binding to the enzyme is stereoselective. The methyl ester, benzoyl, and N-methyl groups of cocaine play important roles in binding because removal of these groups increased K(i) values substantially. Compounds containing a variety of hydrophobic substitutions at the benzoyl group of cocaine bound to the enzyme with high affinity. The high K(i) value obtained for cocaethylene relative to cocaine is consistent with weaker binding to the esterase and a longer elimination half-life reported for the metabolite. The spectrophotometric competitive inhibition assay used here represents an effective method to identify drug or environmental esters metabolized by carboxylesterases like hCE-1.

Pyrolysis products of heroin.

Heating of heroin hydrochloride or of heroin at 250 degrees C led to extensive degradation. Major components of the pyrolysate were identified as heroin, 6-acetylmorphine, N,6-diacetylnormorphine, and N-acetylnorheroin by comparison of mass spectra and 13C- and 1H-nuclear magnetic resonance (NMR) spectra with those of authentic compounds. There was evidence for degradation of the piperidino moiety and the structure 3,4-diacetoxyphenanthrene was proposed for a minor product.

Chemistry and pharmacology of homologs of 6-acetyl-and 3,6-diacetylmorphine.

3,6-Diformyl- and 3,6-dipropanoylmorphine and 6-formyl- and 6-propanoylmorphine were prepared to obtain longer acting, heroin-like compounds. The 6-acylated compounds were more potent than heroin subcutaneously and were orally effective, and their duration of action was at least two to three times greater than that of heroin in monkey species.

Preparation and analgesic activity of 3,6-diacetylnormorphine and 6-acetylnormorphine.

3,6-Diacetylnormorphine (norheroin) and 6-acetylnormorphine have been prepared in excellent yield through the 3,N-bis(tert-butoxycarbonyl) derivative of normorphine via acetylation and selective removal of protecting groups. This general procedure would be applicable to the preparation of various 3,6-diesters or 6-monoesters of normorphine. The analgesic potency of norheroin was found to be the same as that of 6-acetylnormorphine, about 0.05 that of heroin. The onset, peak, and duration of action of these compounds were nearly identical and comparable with morphine.

GLC determination of heroin and its metabolites in human urine.

Heroin and its metabolites, 6-monoacetylmorphine, morphine, and normorphine, were determined in human urine with a GLC procedure. Heroin was extracted with chloroform at pH 4.5 and chromatographed at a temperature programmed from 200-250 degrees by 8 degrees/min. 6-Monoacetylmorphine and morphine were extracted with ethylene dichloride containing 30% isopropanol at pH 8.5, and normorphine was extracted at pH 10.4 wtih the same solvent. The extract was derivatized with trimethylsilylimidazole and chromatographed at 230 degrees for the determination of 6-monoacetylmorphine and morphine and at 220 degrees for normorphine and morphine.

Rapid and sensitive gas chromatographic determination of diacetylmorphine and its metabolite monoacetylmorphine in blood using a nitrogen detector.

A quantitative gas chromatographic method for the determination of plasma concentrations of diacetylmorphine and its metabolite monacetylmorphine using an alkali flane detector (nitrogen detector) is described. Plasma samples (pH 9.0) containing ethylmorphine acetate as internal standard are extracted with benzene. The dried benzene extracts are analysed as their corresponding acetylated derivatives following treatment with trifluoroacetic anhydride-benzent (1:5). The nitrogen detector permits quantitation of narcotic levels down to 100ng/ml with detection as low as 20 ng/ml. The higher sensitivity and selectivity of the nitrogen detector are compared to those obtained in flame ionization detection. Species differences in the rate of conversion of diacetylmorphine to monacetylmorphine in vitro in blood are also presented.