Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric analysis of opiates and metabolites in rat urine after inhalation of opium

To examine the urinary excretion of opiates and their metabolites following inhalation exposure of rats to opium, analytical procedures for the simultaneous determination of the compounds in opium, the vapor derived by the volatilization of opium and the urine of rats exposed to the opium vapor were developed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). Seven compounds were determined in the opium, namely morphine, codeine, thebaine, noscapine, papaverine, meconic acid and meconin. All seven were extracted with 2.5% acetic acid solution and subjected to LC-APCI-MS analysis. The separation was performed on an ODS column in acetonitrile-50 mM ammonium formate buffer (pH 3.0) using a linear gradient program and quantitative analysis was carried out in the selected ion monitoring mode ([M+H](+)). For the analysis of the volatilization of opium, the opium (1 g) was added to a glass pipe, which was then heated at 300 degrees C for 20 min. Negative pressure (air flow-rate; 300 ml/min) was used to draw the vapor through a series of glass wool and methanol traps. The total amount of each compound in the vapor was estimated by measurement of the compounds trapped in the glass wool and methanol. Wister rats (n=3) were exposed to the vapor derived from the volatilization system and the urinary amounts (0-72 h) of the six opiates and metabolites including morphine-3-grucronide (M3G) and morphine-6-grucronide (M6G) were measured after solid-phase extraction. The calibration curves for those compounds in the rat urine were linear over the concentration range 10-500 ng/ml. The recoveries for each analyte from the rat urine sample spiked with standard solution were generally greater than 80%, and the relative standard deviation for the analytical procedure was less than 8% with the exception of meconin. After inhalation exposure of rats to opium, M3G (5.45-14.38 micro g), morphine (2.27-4.65 micro g), meconin (0.54-1.85 micro g), codeine (0.54-1.85 micro g), noscapine (0.34-0.40 micro g) and papaverine (0.01-0.04 micro g) were detected in the urine over 72 h. However, only trace levels of thebaine were observed despite it being one of the major alkaloids found in the opium. On the other hand, a relatively large amount of meconin was detected in the vapor and the urine as compared with the opium. It is suggested that the presence of meconin in biological fluids could be indicative of opium ingestion by inhalation.     

Extraction and determination of opium alkaloids in urine samples using dispersive liquid-liquid microextraction followed by high-performance liquid chromatography

A simple, rapid and sensitive method based on dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was used to determine opium alkaloids in urine samples. Some effective parameters on extraction were studied and optimized. Under the optimum conditions, enrichment factors and recoveries for different opiates are in the range of 63.0-104.5 and 31.5-52.2%, respectively. The calibration graphs are linear in the range of 0.50-500 μg L(-1) and limit of detections (LODs) are in the range of 0.2-10 μg L(-1). The relative standard deviations (RSDs) for 200 μg L(-1) of morphine, codeine and thebaine, 5.0 μg L(-1) of papaverine and 10.0 μg L(-1) of noscapine in diluted urine sample are in the range of 2.8-6.1% (n=7). The relative recoveries of urine samples spiked with alkaloids are 84.3-106.0%. The obtained results show that DLLME combined with HPLC-UV is a fast and simple method for the determination of opium alkaloids in urine samples.     

Benzylisoquinoline alkaloid biosynthesis in opium poppy

Opium poppy (Papaver somniferum) is one of the world’s oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.     

Science in drug control: the alkaloid content of afghan opium

Opium samples from Afghanistan were analyzed by HPLC for their content of morphine and three further alkaloids (codeine, thebaine, and papaverine). To our knowledge, this is the largest set of authentic opium samples analyzed in one study until now. The purpose was to assess possible correlations between samples and selected external factors, such as region of origin within Afghanistan, year of harvest, or intra-batch variation. In the investigated samples, a trend towards higher morphine concentrations in opium from the North-Eastern parts of Afghanistan was observed in the period from 2003 to 2005. More than 75% of the samples contained above 10% of morphine, the overall average was 14.4%.     

Developmental regulation of benzylisoquinoline alkaloid biosynthesis in opium poppy plants and tissue cultures

Summary Opium poppy (Papaver somniferum L.) contains a number of pharmaceutically important alkaloids of the benzylisoquinoline type including morphine, codeine, papaverine, and sanguinarine. Although these alkaloids accumulate to high concentrations in various organs of the intact plant, only the phytoalexin sanguinarine has been found at significant levels in opium poppy cell cultures. Moreover, even sanguinarine biosynthesis is not constitutive in poppy cell suspension cultures, but is typically induced only after treatment with a funga-derived elicitor. The absence of appreciable quantities of alkaloids in dedifferentiated opium poppy cell cultures suggests that benzylisoquinoline alkaloid biosynthesis is developmentally regulated and requires the differentiation of specific tissues. In the 40 yr since opium poppy tissues were first culturedin vitro, a number of reports on the redifferentiation of roots and buds from callus have appeared. A requirement for the presence of specialized laticifer cells has been suggested before certain alkaloids, such as morphine and codeine, can accumulate. Laticifers represent a complex internal secretory system in about 15 plant families and appear to have multiple evolutionary origins. Opium poppy laticifers differentiate from procambial cells and undergo articulation and anastomosis to form a continuous network of elements associated with the phloem throughout much of the intact plant. Latex is the combined cytoplasm of fused laticifer vessels, and contains numerous large alkaloid vesicles in which latex-associated poppy alkaloids are sequestered. The formation of alkaloid vesicles, the subcellular compartmentation of alkaloid biosynthesis, and the tissue-specific localization and control of these processes are important unresolved problems in plant cell biology. Alkaloid biosynthesis in opium poppy is an excellent model system to investigate the developmental regulation and cell biology of complex metabolic pathways, and the relationship between metabolic regulation and cell-type specific differentiation. In this review, we summarize the literature on the roles of cellular differentiation and plant development in alkaloid biosynthesis in opium poppy plants and tissue cultures.     

Capillary zone electrophoresis with diode-array detection for analysis of local anaesthetics and opium alkaloids in urine samples

The present study describes the simultaneous determination of four drugs, two local anaesthetics (lidocaine and bupivacaine) and two opium alkaloids (noscapine and papaverine) by capillary zone electrophoresis (CZE) with solid-phase extraction (SPE) procedure using Oasis HLB cartridges. Their recoveries ranged from 81 to 107% at the target concentrations of 2.0, 5.0 and 8.0 μg mL^?1 in spiked urine samples. Coefficients of variation of the recoveries ranged from 2.1 to 11.3% at these concentrations. The quantitation limits of the method were approximately 300 ng mL^?1 for the different compounds studied. The assay is very specific for these compounds and requires a short sample preparation procedure prior to the electrophoretic analysis.     

Transformation of opium poppy (Papaver somniferum L.) with Agrobacterium rhizogenes MAFF 03-01724

Summary Transformed cultures of opium poppy (Papaver somniferum L.) were established by infecting hypocotyl segments with Agrobacterium rhizogenes MAFF 03-01724. Undifferentiated calli formed on the infected site grew satisfactorily on phytohormone-free solid medium in the dark and produced opine, mikimopine, which could not be detected in a normal culture. Numerous adventitious shoots developed from transformed calli during subculture. The transformed shoots separated individually were cultured on phytohormone-free MS solid medium at 22 ° C under 14 h/day light. They displayed wider leaves and longer internodes than shoots established from seeds or non-transformed root culture. The content of morphinan alkaloids in the cultures and regenerated shoots were quantitatively analyzed by enzyme-linked immunosorbent assay and high performance liquid chromatography. HPLC analysis revealed that non-transformed shoots contained much more codeine (1310 gmg/g dry wt.) than morphine (50 g/g dry wt.), while the transformed shoot cultures did not contain morphine, although the level of morphinan alkaloids in the transformed shoots (213 g morphine equivalents/g fr. wt.) was comparable to that in non-transformed shoots (182 g morphine equivalents/g fr. wt.) by ELISA.Abbreviations MS Murashige-Skoog (Murashige and Skoog 1962) - 1/2 MS half strength MS - HF phytohormone-free - NAA 1-naphthaleneacetic acid - ELISA enzyme-linked immunosorbent assay - HPLC high performance liquid chromatography     

Simultaneous determination of phencyclidine and monohydroxylated metabolites in urine of man by gas chromatography—mass fragmentography with methane chemical ionization

Phencyclidine and monohydroxy metabolites were measured in human urine using gas chromatography—mass fragmentography with methane chemical ionization. Samples were extracted either untreated or following acid hydrolysis, derivatized with heptafluorobutyric anhydride, separated on a 3% SE-30 column and analyzed by mass fragmentography. The assay was sensitive to ca. 0.01 μg/ml for phencyclidine and ca. 0.05 μg/ml for the metabolites. Urine samples from five human subjects enrolled in a methadone maintenance program who had ingested phencyclidine were analyzed. The phencyclidine concentration ranged from 0.3 to 23.7 μg/ml. The concentrations of metabolites ranged from 0 to 1.8 μg/ml. A new monohydroxy metabolite was detected in the samples, but its structure was not fully elucidated. The specificity of the assay was examined.     

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography—mass spectrometry

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography—mass spectrometry (GC—MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester; ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40°C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC—MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.     

Simultaneous determination of in total 17 opium alkaloids and opioids in blood and urine by fast liquid chromatography-diode-array detection-fluorescence detection,after solid-phase extraction

A fast liquid chromatographic method with tandem diode array-fluorescence detection for the simultaneous determination of in total 17 opium alkaloids and opioids is presented. Blank blood and urine samples (1 ml) were spiked with different concentrations of a standard mixture, as well as with the internal standard, butorphanol (2000 ng/ml). After solid-phase extraction, based on weak cation exchange (Bond Elut CBA SPE columns), the extracts were examined by HPLC-DAD-FL. By using a "high-speed" phenyl column (53 x 7.0 mm I.D., particle size 3 microm) eluted with a gradient system (A: water-methanol (90:10, v/v), B: methanol, both containing 25 mM triethylammoniumformate (pH(A) = 4.5)) all compounds could be baseline separated within 12 min. The method was validated and its applicability was demonstrated by the analysis of real-time forensic cases.     

Codeinone reductase isoforms with differential stability,efficiency and product selectivity in opium poppy

Codeinone reductase (COR) catalyzes the reversible NADPH‐dependent reduction of codeinone to codeine as the penultimate step of morphine biosynthesis in opium poppy (Papaver somniferum). It also irreversibly reduces neopinone, which forms by spontaneous isomerization in aqueous solution from codeinone, to neopine. In a parallel pathway involving 3‐O‐demethylated analogs, COR converts morphinone to morphine, and neomorphinone to neomorphine. Similar to neopine, the formation of neomorphine by COR is irreversible. Neopine is a minor substrate for codeine O‐demethylase (CODM), yielding morphine. In the plant, neopine levels are low and neomorphine has not been detected. Silencing of CODM leads to accumulation of upstream metabolites, such as codeine and thebaine, but does not result in a shift towards higher relative concentrations of neopine, suggesting a mechanism in the plant for limiting neopine production. In yeast (Saccharomyces cerevisiae) engineered to produce opiate alkaloids, the catalytic properties of COR lead to accumulation of neopine and neomorphine as major products. An isoform (COR‐B) was isolated from opium poppy chemotype Bea's Choice that showed higher catalytic activity than previously characterized CORs, and it yielded mostly neopine in vitro and in engineered yeast. Five catalytically distinct COR isoforms (COR1.1–1.4 and COR‐B) were used to determine sequence–function relationships that influence product selectivity. Biochemical characterization and site‐directed mutagenesis of native COR isoforms identified four residues (V25, K41, F129 and W279) that affected protein stability, reaction velocity, and product selectivity and output. Improvement of COR performance coupled with an ability to guide pathway flux is necessary to facilitate commercial production of opiate alkaloids in engineered microorganisms.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.     

A rapid high performance liquid chromatographic method for the separation of the alkaloid precursor L-tyrosine and six tetrahydroisoquinoline alkaloids of Papaver somniferum

A high performance liquid chromatographic (HPLC) method was developed for the qualitative and quantitative determination of L-tyrosine and six common tetrahydroisoquinoline alkaloids (papaverine, noscapine, sanguinarine, morphine, codeine and thebaine) of Papaver somniferum. The reversed phase HPLC method yields baseline separation of the alkaloids in 20 min and is achieved using a simple H₂O: MeOH linear gradient. Silanol effects commonly associated with the separation of such strongly basic compounds were minimized by the addition of the amine modifier, triethylamine, to the mobile phase.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Quantitative <Superscript>1</Superscript>H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures

Background  

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (¹H NMR) metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor.