This tutorial provides an overview on a new technique for sample preparation, microextraction by packed sorbent (MEPS). Not only the automation process by MEPS is the advantage but also the much smaller volumes of the samples, solvents and dead volumes in the system. Other significant advantages such as the speed and the simplicity of the sample preparation process are provided. In this tutorial the main concepts of MEPS will be elucidated. Different practical aspects in MEPS are addressed. The factors affecting MEPS performance will be discussed. The application of MEPS in clinical and pre-clinical studies for quantification of drugs and metabolites in blood, plasma and urine will be provided. A comparison between MEPS and other extraction techniques such as SPE, LLE, SPME and SBSE will be discussed. (C) 2011 Elsevier B.V. All rights reserved.
Microextraction by packed sorbent (MEPS) is a new technique for sample preparation that can be connected on-line with liquid chromatography (LC) or gas chromatography (GC) systems without any modifications. This article describes the use of MEPS in clinical and preclinical studies to quantify different drugs in whole blood samples. MEPS was used to determine cyclophosphamide in mouse blood from preclinical g studies using 20 mu L of blood samples. The interday accuracies and 0 precisions ranged from 107-109% and from 2.0-7.0%, respectively. The determination of four immunosuppressive drugs in human blood by MEPS and liquid chromatography-mass spectrometry (LC-MS) is described. The method showed a good selectivity and sensitivity. The calibration curves for everolimus, sirolimus, and tacrolimus ranged from 0.5 to 50 ng/mL and for cyclosporine from 3.0 to 1500 ng/mL. Intraday precisions for the studied immunosuppressive drugs were 2.0-11.7% and interday precision ranged from 5.1 to 13.7% (CV).
The importance of sample preparation methods as the first stage in bioanalysis is described. In this article, the sample preparation concept and strategies will be discussed, along with the requirements for good sample preparation. The most widely used sample preparation methods in the pharmaceutical industry are presented; for example, the need for same-day rotation of results from large numbers of biological samples in pharmaceutical industry makes high throughput bioanalysis more essential. In this article, high-throughput sample preparation techniques are presented; examples are given of the extraction and concentration of analytes from biological matrices, including protein precipitation, solid-phase extraction, liquid-liquid extraction and microextraction-related techniques. Finally, the potential role of selective extraction methods, including molecular imprinted phases, is considered.
To improve the efficacy of the dendritic cell vaccine Alv B DC from Alv B, the PD-L1 expression in cancer cells was attempted to be reduced through a transfection with a custom designed siRNA. Before transfecting the dendritic cells, the siRNA functionality had to be tested through flow cytometry, that resulted in negative results and therefore led to a RT-qPCR protocol that indicated that the siRNA was functional. Protocols for the two methods were developed and a cell line expressing PD-L1 was set up as a tool for testing. The final goal of testing the effects in Alv B DC was never performed due compromising time but the positive result from the PCR provides a promising start to further testing.
Background: The triggers of the acute local inflammatory response to peritoneal dialysis (PD) fluid exposure remain unknown. In the present study, we investigated the effects of neurogenic inflammation and mast cell degranulation on water and solute transport in experimental PD. Methods: Single 2-hour dwells in rats with PD catheters were studied. Histamine and the neuropeptides substance P and calcitonin gene-related peptide (CGRP) were measured in PD fluid samples by ELISA. Radiolabeled albumin (I-125 and I-131 respectively) was used as an intraperitoneal (IP) and intravascular tracer. Glucose and urea concentrations were measured in plasma and PD fluid. The effects of varying the volume and osmolarity of a lactate-buffered PD fluid were compared and related to the effects of pharmacologic intervention. Results: Application of 20 mL 3.9% glucose PD fluid induced an IP histamine release during the first 30 minutes, blockable by the mast cell stabilizer doxantrazole and the substance P neurokinin-1 receptor (NK1R)-blocker spantide. Histamine release was also inhibited at a reduced PD volume (14 mL), but was not affected by normalizing the PD fluid osmolarity. Blockade of NK1R also reduced plasma albumin leakage to the peritoneal cavity. Inhibition of CGRP receptors by CGRP8-37 improved osmotic (transcapillary) and net ultrafiltration and reduced the dialysate urea concentration. Neuropeptide release was not clearly related to activation of the TrpV1 receptor, the classic trigger of neurogenic inflammation. Conclusions: Neuropeptide release exaggerated albumin loss and reduced ultrafiltration in this rat PD model. Intervention aimed at the neuropeptide action substantially improved PD efficiency.
Although commonly used synthetic vascular grafts perform satisfactorily in large caliber blood vessels, they are prone to thrombosis in small diameter vessels. Therefore, small vessels might benefit from tissue engineered vascular grafts. This study evaluated bacterial cellulose (BC) as a potential biomaterial for biosynthetic blood vessels. We implanted the dorsal skinfold chambers in three groups of Syrian golden hamsters with BC (experimental group), polyglycolic acid, or expanded polytetrafluorethylane (control groups). Following implantation, we used intravital fluorescence microscopy, histology, and immunochemistry to analyze the biocompatibility, neovascularization, and incorporation of each material over a time period of 2 weeks. Biocompatibility was good in all groups, as indicated by the absence of leukocyte acti-vation upon implantation. All groups displayed angiogenic response in the host tissue, but that response was highest in the polyglycolic acid group. Histology revealed vascularized granulation tissue Surrounding all three biomaterials, with many proliferating cells and a lack of apoptotic cell death 2 weeks after implantation. In conclusion, BC offers good biocompatibility and material incorporation compared with commonly used materials in vascular surgery. Thus, BC represents a promising new biomaterial for tissue engineering of vascular grafts. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: 140-149, 2010
The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.
Background: Microextraction by packed sorbent (MEPS) is used as an online sample-preparation method. The determination of local anesthetics lidocaine, ropivacaine and bupivacaine directly in human blood was performed using MEPS online with LC-MS/MS. Results: The range of the calibration curves in whole blood was 10-10000 nmol/l. The lower limit of quantification was set to 10.0 nmol/l. The accuracy of the quality control samples ranged from 85 to 97%. The interday precision of the studied analytes was within the range 1-5%. The regression correlation coefficient (r(2)) was over 0.995 for all runs. The present method is rapid, reliable and robust and may be used for therapeutic drug monitoring of studied analytes in whole blood. Conclusion: This assay allows the analysis of drugs in human blood directly. Sample preparation is simple and automated. The assay reduced the handling time and the cost, and could handle small volumes of whole blood samples (25 mu l).
The pulp and paper industry is entering a new era. Pulp mills will be transformed to biorefineries that produce not only pulp, but also biofuels and novel products from lignocellulose. This thesis addresses problems connected with the industrial transition to environmental-friendly technologies and the implementation of the biorefinery concept.
Peroxide bleaching and enhanced recirculation of process water may lead to increased problems with oxalate scaling. Enzymatic elimination of the oxalate problem could be the ultimate industrial solution. The activities of oxalate oxidase, oxalate decarboxylase and a novel oxalate-degrading enzyme provided by Novozymes have been tested in industrial bleaching filtrates. Chemical characterization of the filtrates was used in combination with multivariate data analysis to identify potential enzyme inhibitors. A method based on oxalate oxidase was developed to determine the levels of oxalic acid in process water.
The precursors of oxalic acid formed during bleaching of pulp have been reassessed. New experimental data indicate that alkaline oxidative degradation of dissolved carbohydrates is the main source of oxalic acid. These findings are contradictory to previous hypotheses, which have been focused on lignin. Xylan was more important than lignin as precursor of oxalic acid under peroxide-bleaching conditions. Hot-water extraction of hemicelluloses from softwood mechanical pulp prior to the peroxide-bleaching stage reduced the formation of oxalic acid by one third.
Lignocellulosic materials were characterized chemically with regard to their suitability as feedstocks in biorefineries producing bioethanol. Four agricultural and agro-industrial residues were investigated; cassava stalks, peanut shells, rice hulls, and sugarcane bagasse. Pretreated sugarcane bagasse was the material that was most susceptible to hydrolysis by cellulolytic enzymes. Waste fiber sludges from three pulp mills were characterized. The waste fiber sludge with the lowest content of lignin was hydrolyzed most efficiently by the enzymes. Oligomeric xylan fragments were isolated as by-products from a waste fiber sludge. Hydrolysis of the waste fiber sludges resulted in solid residues with improved fuel properties. The waste fibers were found to be suitable as a feedstock for the production of biofuels in a pulp mill-based biorefinery.
Several bacterial species are capable to use perchlorate and/or chlorate as an alternative electron acceptor in absence of oxygen. Microbial respiration of oxochlorates is important for biotreatment of effluent from industries where oxochlorates are produced or handled. One of these species, the Gram-negative Ideonella dechloratans, is able to reduce chlorate but not perchlorate. Two soluble enzymes, chlorate reductase and chlorite dismutase, participate in the conversion of chlorate into chloride and molecular oxygen. The present study deals with the electron transport from the membrane-bound components to the periplasmic chlorate reductase. Soluble c cytochromes were investigated for their ability to serve as electron donors to chlorate reductase. The results show that a 6 kDa c cytochrome serves as electron donor for chlorate reductase. This cytochrome also serves as electron donor for a terminal oxidase in the reduction of oxygen that is produced in the course of chlorate respiration. A gene encoding a soluble c cytochrome was found in close proximity to the gene cluster for chlorate reduction. This gene was cloned and expressed heterologously, and the resulting protein was investigated as a candidate electron donor for chlorate reductase. Electron transfer from this protein could not be demonstrated, suggesting that the gene product does not serve as immediate electron donor for chlorate reductase.