Microwave assisted extraction (MAE) was used to extract drugs of abuse from urine samples for the simultaneous determination of morphine, codeine, 6-acetylmorphine (6AM), cocaine, cocaethylene, benzoylecgonine (BEG), methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) by high performance liquid chromatography with diode array detector (HPLC-DAD). The working wavelengths used were 285 nm for morphine, codeine and 6AM; 233 nm for cocaine, cocaethylene and BEG; and 292 nm for methadone and EDDP. The mobile phase was a gradient of acetonitrile and phosphate buffer at pH 6.5. A full factorial design was used to identify the most suitable extraction conditions as regards pH, solvent and time of extraction. The optimum conditions thus established provided analyte recoveries from 61% to 109%. The detector response was linear over the drug concentration range 0.1-4.0 microg ml(-1) in urine. The within- and between-day precision and accuracy were less than 8% and 6.5%, respectively. The method was applied successfully to samples obtained from Galician hospitals and courts.
Reassortment is one of the main mechanisms of evolution in dsRNA viruses with segmented genomes. It contributes to generate genetic diversity and plays an important role in the emergence and spread of new strains with altered virulence. Natural reassorment has been demonstrated among infectious pancreatic necrosis-like viruses (genus Aquabirnavirus, Birnaviridae). In the present study, coinfections between different viral strains, and genome sequencing by the Sanger and Illumina methods were applied to analyze the frequency of reassortment of this virus in vitro, the possible mechanisms involved, and its effect on virulence. Results have demonstrated that reassortment is a cell-dependent and non-random process, probably through differential expression of the different mRNA classes in the ribosomes of a specific cell, and by specific associations between the components to construct the ribonucleoprotein (RNP) complexes and/or RNP cross-inhibition. However, the precise mechanisms involved, known in other viruses, still remain to be demonstrated in birnaviruses.
High performance liquid chromatography in combination with diode array detection (HPLC-DAD) was used to determine morphine, 6-acetylmorphine, cocaine, benzoylecgonine, cocaethylene, methadone and 2-ethylene-1,5-dimethyl-3,3,-diphenylpyrrolidine in human saliva. For comparison, samples were prepared by either liquid-liquid extraction in Toxitubes A or microwave-assisted extraction (MAE), by mixing 1 ml of saliva with 10 ml of chloroform and operating at 100 degrees C for 10 min. Acetonitrile and 0.02 m phosphate buffer at pH 6.5 were used as mobile phase in HPLC in gradient mode. The detector response was linear over the drug concentration range of 0.05-2.0 microg ml(-1) in human saliva. The analytical method was validated by determining its precision and accuracy (n = 5), which were lower than 5% as relative standard deviation and 6% as relative error. Limits of detection ranged from 10 to 35 ng ml(-1); mean recoveries of drugs were from 53 to 95% with Toxitubes A and from 83 to 100% with MAE at two different concentrations (0.1 and 1.0 microg ml(-1)). The proposed method was applied to 24 saliva samples from individuals poisoned with opiates and/or cocaine.
An analytical method was developed for the simultaneous determination of bromazepam, alprazolam, lorazepam, lormetazepam, diazepam and tetrazepam in vitreous humor, using microwave-assisted extraction (MAE) optimized by a central composite design followed by desirability functions, and high performance liquid chromatography coupled to a photodiode array detector (HPLC-PDA). The optimal conditions were 98 C for the extraction temperature, 10 min for the extraction time, and ethyl acetate (10 mL) as the solvent. The extract was evaporated to dryness, redissolved in 100 mL of mobile phase, and injected (30 mL) into a XBridgeÔ Shield RP18 column (250 Â 4.6 mm id, 5 mm particle size). The mobile phase, composed of acetonitrile and 0.02 M phosphate buffer pH 6, was eluted at a flow rate of 0.8 mL min À1 in gradient mode. The response of the detector was linear in the range of 0.05 to 5 mg mL À1 vitreous humor, the limits of detection ranged between 10 and 13 ng mL À1 , and the coefficients of variation were always lower than 10%. The mean recoveries oscillated between 87.6% for diazepam and 101.2% for lormetazepam. Finally, the MAE/HPLC-PDA method was successfully applied to 16 vitreous humor samples from individuals intoxicated with benzodiazepines, with alprazolam, lormetazepam and diazepam being the most frequently found of these substances.
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