Morphine In Biological Samples Research Articles

Cu, I-doped carbon dots as simulated nanozymes for the colorimetric detection of morphine in biological samples

As newly developed synthetic enzymes with exceptional catalytic capabilities and outstanding stability, nanozymes have drawn considerable interest in the realm of sensing. Using a simple hydrothermal process, iodine and copper-doped carbon dots (Cu,I-CDs) with simulated enzymes were fabricated in the current investigation. Cu,I-CDs demonstrate peroxidase-mimicking function together with high catalytic effectiveness due to aforementioned features. This led to generation of a colorimetric sensor for quick and accurate quantitative assessment of morphine (MOR). The outcomes showed the method's usefulness for the colorimetric detection of MOR. The linear range for MOR detection is 0.25–25 μg/mL having a reduced detection limit of 64 ng/mL. This sensor's successful use in the analysis of MOR in biological material is more noteworthy.

On-site quantitation of morphine in urine by fast derivatization and miniature mass spectrometry analysis

Analysis of drugs in urine is particularly useful for screening of public safety issues, monitoring of medicine appliance and detection of drugs of abuse. Tandem mass spectrometry (MS/MS) of morphine usually produces clusters of fragments, hindering accurate quantitation of morphine in biological samples by fast sampling and direct MS/MS method. In this work, we developed a method that enabled determination and quantitation of morphine in urine in 3 min. It consisted of a simple extraction process, a fast dansyl-derivatization process, and direct analysis by a miniature mass spectrometer. MS/MS of derivatized morphine produced a highly abundant product at m/z 285 in positive ion mode, significantly enhancing the sensitivity by more than 10 times. Quantitation of morphine in urine was achieved by in-trap collision-induced dissociation on the miniature mass spectrometer, with the limit of detection of 50 ng/mL and the limit of quantitation of 100 ng/mL. The performance of the method was also evaluated with a standard liquid chromatography-MS method, showing comparable quantitative accuracy. Finally, the method has applied into quantitation of morphine in real urine samples from individuals suspected to have used opioids.

Molecular modeling and experimental study of a new peptide-based microextraction fiber for preconcentrating morphine in urine samples.

Antimicrobial peptides (AMPs) are best known for their bactericidal properties; however, due to their unique and flexible structures, they have also been proposed as potential selective sorbents for specific molecules. In the present study, we aimed to design and produce a new peptide-based microextraction fiber for preconcentrating morphine in urine samples. The binding of morphine to the peptide was first evaluated by computational simulation using the Molecular Operating Environment (MOE) 2015.10 software. A similar study was then performed using DS BIOVIA Materials Studio 2017 v17.1.0.48, which confirmed the results of the simulation carried out with MOE. Afterwards, those results were also confirmed by experimental research. In the experimental evaluation, carbon nanotubes (CNTs) were initially carboxylated with H2SO4/HNO3 (3:1) and then functionalized with the peptide. FTIR analysis, Raman measurements, and SEM imaging were used to confirm that CNT functionalization was successful as well as to check the nanostructure of the fiber. To evaluate the functionality of the fiber, it was inserted into a microtube containing a urine sample that included morphine and then sonicated for 5min at 40°C. Afterwards, the fiber was washed with methanol 20% (H2O/methanol) and the resulting sample was analyzed by HPLC. This procedure was repeated for different concentrations of morphine in the urine sample. The computational and experimental results showed that a morphine concentration as low as 0.25ppb in urine could be adsorbed and detected using the peptide fiber. Therefore, given its semi-selective binding affinity for morphine, this peptide-based fiber can be considered a new approach to the detection of small amounts of morphine in biological samples.

Potential application of amino acids in analytical toxicology

The ability of extraction and preconcentration of small quantities of substances from biological samples is important in analytical sciences, particularly forensic medicine. In the present study, we evaluated the binding potential of amino acids to produce a new solid phase microextraction fiber based on carbon nanotube (CNTs) for extraction and preconcentration of small amount of morphine in urine sample. Raw CNTs were first carboxylated and then functionalized with 3 amino acids including glutamate, arginine, and cysteine. Functionalization was confirmed by FTIR analysis, Raman spectroscopy and SEM imaging. The functionalized CNTs were coated on polypropylene hollow fiber and used for preconcentration. The results of HPLC analysis in isocratic elution mode using acetonitrile-sodium acetate (10:90, v/v; pH 4; 0.01 M) as the mobile phase showed that amino acids are able to adsorb morphine and the prepared fiber could preconcentrate a very low concentration of morphine (0.25 ppb) in urine matrix. In addition, the fiber was successfully used for up to 30 times with no significant loss in the extraction efficiency. Lowest limit of detection (LOD) and limit of quantitation (LOQ) was 0.07 and 0.25, respectively. Also, the lowest and best recovery of the fiber was 87.8% and 139% at LOQ, which belonged to glutamate and arginine, respectively. The fibers based on amino acids can be used for the detection of a small amount of morphine in biological samples, which are not detectable by conventional methods. Simple mechanism of these fibers in preconcentrating morphine makes them a novel candidate for detection of other opiates and drugs of abuses in crime scene investigations and postmortem examinations several days after exposure.

Developing a new sensitive solid-phase microextraction fiber based on carbon nanotubes for preconcentration of morphine

The ability of extraction and preconcentration of small amounts of substances from biological samples is important in forensic medicine. In the present study, we produced a new solid-phase microextraction fiber based on carbon nanotube (CNTs) for extraction and preconcentration of small amount of morphine in urine sample. Raw nanofibers were first carboxylated with H2SO4/HNO3 (3:1) and then functionalized with ethylenediamine. Functionalization was confirmed by FTIR and Raman spectroscopy as well as SEM analysis. The functionalized CNTs were coated on a porous polypropylene hollow fiber. A new electrical device was designed and manufactured for adsorption–desorption of the analyte, in which the voltage could be reversed and the analytes could desorb into the washing solution for HPLC analysis. The results showed that the prepared fiber could adsorb a very low concentration of morphine (0.5 ppb) in urine matrix, and was successfully used for up to 50 times with no significant loss in the extraction efficiency. Recovery of the fiber was 77% at 0.5 ppb. The optimum condition for the fiber was 2 min at 1 V of the manufactured electrical catalyst controller. This fiber can be used for the detection of a small amount of morphine in biological samples, which are not detectable by conventional methods. Simple mechanism of this fiber in preconcentrating morphine makes it a novel candidate for detection of other opiates and drugs of abuses in crime scene investigations and postmortem examinations several days after exposure. The prepared electric microextraction device could also efficiently concentrate the morphine in the urine matrix.

Stability of heroin, 6-monoacetylmorphine, and morphine in biological samples and validation of an LC–MS assay for delayed analyses of pharmacokinetic samples in rats

Degradation of heroin to 6-monoacetylmorphine (6-MAM) and then morphine happens rapidly in vivo and in vitro. The rates of heroin and 6-MAM degradation depend on the type of biological samples, and the duration and conditions of storage. In order to optimize conditions for measuring heroin and its metabolites in samples collected for pharmacokinetic studies in rats, we investigated the time course of degradation of heroin, 6-MAM, and morphine in four biological matrices: rat blood, rat brain homogenate, bovine serum, and human plasma under various conditions. Analyte concentrations were measured by LC–MS. The goal was to identify conditions that allow maximum flexibility in scheduling sample collection and analysis, as well as gain more information on the stability of heroin in blood and tissue samples. A solid-phase extraction method with ice-cold solvents, sodium fluoride (NaF) and a low pH (3.0) maintained sample stability. Quality controls were within 94.0–105% of the target value. Variability was 4.0–8.9% for all analytes within the range of 5–200ng/mL for heroin, 5–1000ng/mL for 6-MAM, and 10–200ng/mL for morphine. Heroin degradation to 6-MAM was faster in rat whole blood than in plasma, and faster in rat plasma than in rat brain homogenate. Maintaining NaF at 4mg/mL throughout processing enhanced stability; higher NaF concentrations added to whole blood caused hemolysis. Samples processed through solid phase extraction and stored as dried pellets at 80°C constituted the most stable environment for heroin, and was superior to the storing of samples in solution prior to or after extraction. Nevertheless, post-extraction heroin and 6-MAM levels declined by 6.7–8.3% over one week in rat plasma under these conditions, and by <1–4.7% in bovine serum or human plasma.

Quantitative determination of morphine in biological samples by gas-liquid chromatography and electron-capture detection

A highly sensitive method for the determination of morphine in plasma and brain samples down to 500 pg ml-1 in plasma and 100 pg in 30 mg of brain tissue is described. This sensitivity is in the range of that described for radioactive and radioimmunological tehcniques. The samples (50-500 mu-l) are extracted with toluene-butanol (9:1) at pH 8.9 and then re-extracted into 0.1 N sulphuric acid and back extracted to a toluene-butanol phase at pH 8.9. The pentafluoropropionic anhydride derivatives of morphine and the internal standard nalorphine were quantitatively determined by gas-liquid chromatography with a 3-H electron capture detector.

Kinetic spectrophotometric method for the determination of morphine in biological samples

In this paper a simple, selective and inexpensive kinetic method was developed for the determination of morphine based on its inhibitory effect on the Janus green–bromate system in sulfuric acid media. The reaction was monitored spectrophotometrically at 618 nm by a fixed time method. The effect of different parameters such as concentration of reactants and temperature on the rate of reaction was investigated and optimum conditions were obtained. The calibration curve was linear in the concentration range 0.07–7.98 mg L −1 of morphine, and detection limit of the method was 3.0 × 10 −2 mg L −1. The relative standard deviation for five determinations of 3.74 mg L −1 of morphine was 0.57%. Finally, the proposed method was successfully applied to the determination of morphine in human urine and serum as real samples.

Determination of morphine in the plasma of addicts in using Zeolite Y extraction following high-performance liquid chromatography

Background The measurement of morphine in biological samples has become a routine in many clinical and forensic toxicology laboratories. A high-performance liquid chromatography (HPLC) method was developed for the determination of morphine in plasma. Methods Samples were extracted using Zeolite Y column followed by reversed phase HPLC with fluorescence detection. This method was based on an ex-calibration procedure and was linear between 20 and 200 ng/ml of morphine. Blood from 10 male opiate addicts were obtained from Rosbeh Hospital. All of the male smoked opiate (heroin, opium) and cigarettes. Results The mean total level 5 h after the last abuse was 152.4 ng/ml and 37.6 ng/ml at 10–15 h. The method was reliable for morphine determination in blood even after 5 half-lives after the last abuse. Conclusions This method is simple and rapid and may be useful for routine monitoring of plasma morphine concentration.

Determination of morphine in urine by solid-phase immunoextraction and high-performance liquid chromatography with electrochemical detection

The analysis of morphine in biological fluids is of vital interest in monitoring opiate abuse and in drug abuse research. Although methods for analysis of morphine and its metabolites are well established, studies are still being carried out to improve sample preparation procedures as well as detection levels of morphine in biological samples. In this study, morphine-specific immunosorbents were developed to concentrate morphine prior to HPLC analysis. Urine (0.1 ml) was diluted 10-fold with phosphate-buffered saline, pH 7.4 (PBS), loaded onto a solid-phase immunoextraction column and washed with 15 ml PBS followed by elution with 2 ml of elution buffer (40% ethanol in PBS, pH 4). The eluted fraction was analysed for morphine by HPLC–electrochemical detection using a cyanopropyl (CN) analytical column with 25% acetonitrile in phosphate buffer–sodium lauryl sulphate, pH 2.4 as the mobile phase. Duration of the extraction procedure was approximately 40 min. Calibration graphs were linear from 100 ngml−1 to 500 ngml−1 in urine. The inter-assay R.S.D. was <10% and the recovery of morphine from urine was >98%. Immunocolumns demonstrated remarkably high specificity towards morphine showing minimal binding with other opiate metabolites such as codeine, normorphine, norcodeine, morphine-3-glucuronide, morphine-6-glucuronide.

High-performance liquid chromatographic determination of morphine in biological samples: An overview of separation methods and detection techniques

High-performance liquid chromatography with electrochemical detection at present suits most of the needs of toxicologists for the determination of morphine and some related compounds in biological samples, although fluorescence detection is still a useful alternative. Chemiluminescence detection may be promising, but needs further optimization of its coupling with HPLC to give the best performances. Morphine detection by absorbance spectrophotometry does not seem to allow the degree of sensitivity and selectivity from matrix interferences that is required in most instances. However, this approach is useful when morphine congeners undetectable by alternative means (i.e., heroin and morphine-3-glucoronide) are to be determined or when a general toxicological screening is required.

Liquid chromatography with pre-column dansyl derivatisation and fluorimetric detection applied to the assay of morphine in biological samples

A simple method employing pre-column dansylation and liquid chromatography is proposed for a very sensitive and specific assay of morphine in biological samples. Nalorphine is used as an internal standard. The detection limit is 0.2 picomol of injected morphine. In the assay of human sera spiked with 150 nmol/1, the intra- and inter-assay coefficients of variation were 3.7% ( n  10) and 4.5% ( n  10), respectively. No interferences were observed from more than 70 opiate and non-opiate drugs. Urine, plasma and total blood were assayed, using different extraction methods, with negligible interference from coextractives.

Determination of morphine in biological samples by gas chromatography—mass spectrometry: Evidence for persistent tissue binding in rats twenty-two days post-withdrawal

Combined gas chromatography—mass spectrometry with capillary and packed column gas chromatography and a deuterium-labelled internal standard was used to determine morphine in biological specimens from rats 22 days after abrupt withdrawal. Morphine was extracted from urine and body organs at pH 9 and the pentafluoropropionyl derivatives were made for analysis by gas chromatography—mass spectrometry. The stationary phase was OV-17 and the mass spectrometer was focused on m/z 414 for morphine and m/z 417 for the internal standard, [NC 2H 3]morphine. With fused-silica capillary columns, the sensitivity of the assay was increased about ten-fold over packed columns. Urinary excretion of total morphine (free + conjugated) was 22 ng/h (range 11–51 ng/h, n = 8) at 22 days post-withdrawal. Free morphine was mainly detected in the lung (1.8–6.5 ng/g, n = 7), kidney (1.5–4.0 ng/g, n = 7) and liver (1.8–4.6 ng/g, n = 4). Traces of morphine were also detected in brain of some rats. Treatment with the opiate antagonist naltrexone, 10 mg/kg on four consecutive days before death, failed to change the urinary excretion pattern or the concentration of free morphine in body organs. The biological significance of the residual morphine, if any, remains to be determined.

High-performance liquid chromatography detection of morphine by fluorescence after post-column derivatisation

A reversed-phase high-performance liquid chromatographic method for the fluormetric determination of morphine in biological samples has been developed. Column effluent is mixed with alkaline potassium ferricyanide to produce the flurorescent dimer pseudomorphine. The method provides higher chromatographic specificity and sensitivity than conventional high-performance liquid chromatography. Morphine can be detected at levels of 10 ng on column.

The Distribution of Morphine in the Brain in Fatal Cases Due to the Intravenous Administration of Heroin

Heroin (3,6-diacetylmorphine) is deacetylated in the human body to 6-mono-acetylmorphine and finally to morphine. These metabolites are responsible for the major pharmacologic effects of heroin (I,2). Since the conversion of heroin to morphine is rapid, it is that metabolite that has been measured in tissues following administration. In deaths due to heroin overdose, the tissue distribution of morphine has been studied (3),but the concentrations in brain regions have scldom been reported. The most extensive study of brain morphine concentrations following the intravenous administration of heroin was that of Richards et al. (4), who provided blood:brain correlations in 50 cases. This study did not, however, utilize any particular section of the brain. Animal studies have indicated a close relationship between the concentration of morphine in the brain and the degree of analgesia (5,6). In these studies, entire rat brains were homogenized and aliquots analyzed, thus yielding no information on distribution within the brain. The investigation of the fine distribution of morphine has been hindered by the lack of sensitivity of analytical procedures for morphine. In 1970 Spector and Parker (7) reported the production of antibodies to morphine using 3-0 carboxymethoxy-morphine-bovine serum albumin. Spector and Parker employed this morphine antibody in quantitative determination of morphine by a competitive binding assay using the ability of morphine to inhibit binding of dihydromorphine3H to the antimorphine antibody. The radioimmunoassay for morphine has a sensitivity of less than I ng morphine added and a specificity sufficient to detect and quantitate morphine in biological samples without extraction of the drug. The sensitivity and specificity of morphine radioimmunoassay has been applied to the study of morphine distribution in postmortem human tissues by Coubis and Kaul (8). Spiehler et al. have compared results for quantitative determination of morphine in urine, postmortem blood, bile, brain, and lung by radioimmunoassay, enzyme immunoassay, and spectrofluorometry (9). From our preliminary study (9). it appears that morphine radioimmunoassay possesses the necessary sensitivity for determination of morphine distribution in the brain.

Simultaneous determination of codeine and morphine in biological samples by gas chromatography with electron capture detection.

A sensitive gas chromatographic method for the simultaneous determination of codeine and morphine in plasma and brain samples is described. The method involves solvent extraction of the compounds from plasma, derivatization with pentafluoropropionic anhydride and subsequent separation on a 3% OV-17 column. The quantification is performed with electron capture detection. The sensitivity of the method (0.75 ng of morphine and 7.5 ng of codeine in a sample) makes it especially useful for pharmacokinetic investigations. The method was successfully applied to determine the time course of codeine and its metabolite morphine after intravenous administration of codeine to the rat.