Soluble manganese(IV) as a chemiluminescence reagent for the determination of opiate alkaloids, indoles and analytes of forensic Interest

We present the results of our investigations into the use of soluble manganese(IV) as a chemiluminescence reagent, which include a significantly faster method of preparation and a study on the effect of formaldehyde and orthophosphoric acid concentration on signal intensity. Chemiluminescence detection was applied to the determination of 16 analytes, including opiate alkaloids, indoles and analytes of forensic interest, using flow injection analysis methodology. The soluble manganese(IV) reagent was less selective than either acidic potassium permanganate or tris(2,2′-bipyridyl)ruthenium(III) and therefore provided a more universal chemiluminescence detection system for HPLC. A broad spectral distribution with a maximum at 730 ± 5 nm was observed for the reaction between the soluble manganese(IV) and a range of analytes, as well as the background emission from the reaction with the formaldehyde enhancer. This spectral distribution matches that reported for chemiluminescence reactions with acidic potassium permanganate, where a manganese(II) emitting species was elucidated. This provides further evidence that the emission evoked in reactions with soluble manganese(IV) also emanates from a manganese(II) species, and not bimolecular singlet oxygen as suggested by previous authors.

Determination of opiate alkaloids in process liquors using capillary electrophoresis

This paper describes the determination of opiate alkaloids (morphine, codeine, oripavine and thebaine) in industrial process liquors using capillary zone electrophoresis with UV-absorption detection at 214 nm. A study of cyclodextrin type and concentration revealed that the addition of 30 mM hydroxypropyl-β-cyclodextrin to the electrolyte solution (100 mM Tris adjusted to pH 2.8) was suitable to resolve the four analytes of interest. Typical analysis time was 12 min and the limit of detection for each alkaloid was 2.5 × 10⁻⁶ M. The results for the proposed methodology were in good agreement with those of a conventional HPLC procedure. Under the same conditions, short-end injection was used to reduce the effective separation length from 41.5 to 8.5 cm, which allowed the determination of morphine and thebaine in process liquors within 2.5 min.

An improved process for the N-demethylation of opiate alkaloids using an iron(II) catalyst in acetate buffer

An improved process to N-demethylate opiate alkaloids utilising a solution of the ferrous porphyrin, tetrasodium 5,10,15,20-tetra(4-sulfophenyl)porphyrinatoiron(II) [=Fe(II)-TPPS (8)], in acetate buffer is described. This method provided the corresponding N-demethylated opiates in good yield with high reproducibility.

Preliminary evaluation of dual acidic potassium permanganate and tris(2,2`-bipyridyl)ruthenium(II) chemiluminescence detection for the HPLC determination of Papaver somniferum alkaloids

This paper describes a dual chemiluminescence reagent for the determination of the opiate alkaloids morphine, codeine, oripavine, and thebaine in Papaver somniferum extracts. Detection was achieved using a mixture of acidic potassium permanganate and tris(2,2′-bipyridyl)ruthenium(ii), where the former acted as both the oxidant for the latter and as a chemiluminescence reagent in its own right. The analytes were separated on a C₈ column using ion-pairing HPLC. The application of the mixed reagent detection compared favourably with results obtained using standard HPLC methodology. Detection limits for the alkaloids were 10^-6, 5 × 10^-7, 3 × 10^-6, and 2 × 10^-6 mol L^-1 for morphine, codeine, oripavine, and thebaine, respectively.

Rapid determination of papaver somniferum alkaloids in process streams using monolithic column high-performance liquid chromatography with chemiluminescence detection

We have combined high-performance liquid chromatography (HPLC) separations using a monolithic column with acidic potassium permanganate and tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence detection in a rapid and highly sensitive method to monitor the process of extracting opiate alkaloids from Papaver somniferum. Due to the high flow rates allowed with the monolithic column and the inherent selectivity of the chemiluminescence reactions, the four predominant alkaloids – morphine, codeine, oripavine and thebaine – were determined in less than 2 min. The results obtained with numerous process samples compared favourable with those of the standard HPLC methodology. Limits of detection were 1 × 10⁻¹⁰ M, 5 × 10⁻¹⁰ M, 5 × 10⁻¹⁰ M and 1 × 10⁻⁹ M, for morphine, codeine, oripavine and thebaine, respectively.

The use of CE-electrochemiluminescence with ionic liquid for the determination of bioactive constituents in Chinese traditional medicine

CE/tris(2,2-bipyridyl) ruthenium(ll) (Ru(bpy)(3)(2+)) electrochemiluminescence (ECL), CEECL, with an ionic liquid (IL) detection system was established for the determination of bioactive constituents in Chinese traditional medicine opium poppy which contain large amounts of coexistent substances. A minimal sample pretreatment which involves a one-step extraction approach avoids both sample loss and environmental pollution. As the nearby hydroxyl groups in some alkaloid such as morphine may react with borate to form complexes and IL, as a high-conductivity additive in running buffer, could cause an enhanced field-amplified effect of electrokinetic injection. Running buffer containing 25 mM borax-8 mM 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF(4)) IL (pH 9.18) was used which resulted in significant changes in separation selectivity and obvious enhancement in ECL intensities for those alkaloids with similar structures. Sensitive detection could be achieved when the distance between the Pt working electrode and the outlet of separation capillary was set at 150 mu m and the stainless steel cannula was fixed approximately 1 cm away from the outlet of the capillary. Quantitative analysis of four alkaloids was achieved at a detection voltage of 1.2 V and a separation voltage of 15 kV in less than 7 min.

A hybrid FIA/HPLC system incorporating monolithic column chromatography

We have combined the generation of solvent gradients using milliGAT pumps, chromatographic separations with monolithic columns and chemiluminescence detection in an instrument manifold that approaches the automation and separation efficiency of HPLC, whilst maintaining the positive attributes of flow injection analysis (FIA), such as manifold versatility, speed of analysis and portability. As preliminary demonstrations of this hybrid FIA/HPLC system, we have determined six opiate alkaloids (morphine, pseudomorphine, codeine, oripavine, ethylmorphine and thebaine) and four biogenic amines (vanilmandelic acid, serotonin, 5-hydroxyindole-3-acetic acid and homovanillic acid) in human urine, using tris(2,2′-bipyridyl)ruthenium(III) and acidic potassium permanganate chemiluminescence detection.

A screening test for heroin based on sequential injection analysis with dual-reagent chemiluminescence detection

A sequential injection analysis procedure with dual-reagent chemiluminescence detection was applied to the screening of street drug seizure samples for the presence of heroin. The chemiluminescence reagents (acidic potassium permanganate and tris(2,2′-bipyridine)ruthenium(III)) were aspirated from either side of a sample aliquot that was sufficiently large to prevent interdispersion of the reagent zones, and therefore two different chemical reactions could be performed simultaneously at either end of the sample zone. The presence of heroin in seizure samples was indicated by a strong response with the tris(2,2′-bipyridine)ruthenium(III) reagent and confirmed by a significant increase in the response with the permanganate reagent when the sample was treated with sodium hydroxide to hydrolyse the heroin to morphine. Nicomorphine (a morphine-derived pharmaceutical) was synthesised and tested under the same conditions. The responses with the permanganate reagent were similar to those for heroin, which supports the proposed chemical basis for the test. However, the responses with tris(2,2′-bipyridine)ruthenium(III) were far lower for nicomorphine than heroin (approximately 5-fold for the samples that had not been hydrolysed).

Enhancing the chemiluminescence determination of biologically and forensically important molecules

This thesis describes a systematic approach to the design, evaluation and optimisation of novel flow-cells for liquid-phase chemiluminescence detection. Various approaches were employed for improving the sensitivity of several analytically important reactions utilised for the determination of biologically significant thiols and disulfides as well as forensically important opiate alkaloids.

The determination of bioactives using multidimensional separation and detection systems

This project was focused on the development of platform technology for the separation and detection of complex samples. The samples investigated included roasted coffee beans, opiate alkaloids and amino acids.

Ethanol as an alternative to formaldehyde for the enhancement of manganese(IV) chemiluminescence detection.

Previous applications of manganese(IV) as a chemiluminescence reagent have required the use of formaldehyde to enhance the emission intensity to analytically useful levels. However, this known human carcinogen (by inhalation) is not ideal for routine application. A wide range of alternative enhancers have been examined but to date none have been found to provide the dramatic increase in chemiluminescence intensities obtained using formaldehyde. Herein, we demonstrate that ethanol offers a simple, safe and inexpensive alternative to the use of formaldehyde for manganese(IV) chemiluminescence detection, without compromising signal intensity or sensitivity. For example, chemiluminescence signals for opiate alkaloids using 50-100% ethanol were 0.8-1.6-fold those using 2M formaldehyde. This innocuous alternative enhancer is shown to be a particularly effective for the direct detection of thiols and disulfides by manganese(IV) chemiluminescence, which we have applied to a simple HPLC procedure to determine a series of biomarkers of oxidative stress.

Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.

A novel SNP in COMT is associated with alcohol dependence but not opiate or nicotine dependence: a case control study

Background It is well established that COMT is a strong candidate gene for substance use disorder and schizophrenia. Recently we identified two SNPs in COMT (rs4680 and rs165774) that are associated with schizophrenia in an Australian cohort. Individuals with schizophrenia were more than twice as likely to carry the GG genotype compared to the AA genotype for both the rs165774 and rs4680 SNPs. Association of both rs4680 and rs165774 with substance dependence, a common comorbidity of schizophrenia has not been investigated. Methods To determine whether COMT is important in substance dependence, rs165774 and rs4680 were genotyped and haplotyped in patients with nicotine, alcohol and opiate dependence. Results The rs165774 SNP was associated with alcohol dependence. However, it was not associated with nicotine or opiate dependence. Individuals with alcohol dependence were more than twice as likely to carry the GG or AG genotypes compared to the AA genotype, indicating a dominant mode of inheritance. The rs4680 SNP showed a weak association with alcohol dependence at the allele level that did not reach significance at the genotype level but it was not associated with nicotine or opiate dependence. Analysis of rs165774/rs4680 haplotypes also revealed association with alcohol dependence with the G/G haplotype being almost 1.5 times more common in alcohol-dependent cases. Conclusions Our study provides further support for the importance of the COMT in alcohol dependence in addition to schizophrenia. It is possible that the rs165774 SNP, in combination with rs4680, results in a common molecular variant of COMT that contributes to schizophrenia and alcohol dependence susceptibility. This is potentially important for future studies of comorbidity. As our participant numbers are limited our observations should be viewed with caution until they are independently replicated.