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1.
Biopharmaceutical production takes place in complex processes which should be thoroughly understood. Therefore, the iConsensus project focuses on developing a monitoring platform integrating several process analytical technology tools for integrated, automated monitoring of the biopharmaceutical process. Water-soluble vitamin monitoring using (microchip) capillary electrophoresis (CE) is part of this platform. This work comprises the development of conventional CE methods as the first part towards integrated vitamin monitoring. The vitamins were divided based on their physical–chemical properties to develop two robust methods. Previously, a method for the analysis of cationic vitamins (pyridoxine, pyridoxal, pyridoxamine, thiamine and nicotinamide) in cell culture medium was developed. This work focused on the development of a micellar electrokinetic chromatography method for anionic and neutral vitamins (riboflavin, d -calcium pantothenate, biotin, folic acid, cyanocobalamin and ascorbic acid). By employing multivariate design of experiments, the background electrolyte (BGE) could be optimised within one experiment testing only 11 BGEs. The optimised BGE conditions were 200 mM borate with 77 mM sodium dodecyl sulphate at a pH of 8.6. Using this BGE, all above-mentioned cationic, anionic and neutral vitamins could be separated in clean samples. In cell culture medium, most anionic and neutral vitamins could be separated. Combining the two methods allows for analysis of cationic, anionic and neutral vitamins in cell culture medium samples. The next step towards integrated vitamin monitoring includes transfer to microchip CE. Due to the lack of fast and reliable methods for vitamin monitoring, the developed capillary methods could be valuable as stand-alone at-line process analytical technology solutions as well.  相似文献   

2.
The rapidly growing, competitive biopharmaceutical market requires tight bioprocess monitoring. An integrated, automated platform for the routine online/at-line monitoring of key factors in the cell culture medium could greatly improve process monitoring. Mono- and disaccharides, as the main energy and carbon source, are one of these key factors. A CE-LIF method was developed for the analysis of several mono- and disaccharides, considering requirements and restrictions for analysis in an integrated, automated monitoring platform, such as the possibility for miniaturization to microchip electrophoresis. Analysis was performed after fluorescent derivatization with 8-aminopyrene-1,3,6-trisulfonic acid. The derivatisation reaction and the separation BGE were optimized using design of experiments. The developed method is applicable to the complex matrix of cell culture medium and proved transferable to microchip electrophoresis.  相似文献   

3.
Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the bioanalytical method of choice to support plate‐based, in vitro early ADME (Absorption, Distribution, Metabolism and Excretion) screens such as metabolic stability (Metstab) assessment. MS/MS method optimization has historically been the bottleneck in this environment, where samples from thousands of discrete compounds are analyzed on a monthly basis, mainly due to the lack of a high‐quality commercially available platform to handle the necessary MS/MS method optimization steps for sample analysis by selected reaction monitoring (SRM) on triple quadrupole mass spectrometers. To address this challenge, we recently developed a highly automated bioanalytical platform by successfully integrating QuickQuan? 2.0, a unique high‐throughput solution featuring MS/MS method optimization by automated infusion, with a customized in‐house software tool in support of a Metstab screen. In this platform, a dual‐column setup running parallel chromatography was also implemented to reduce the bioanalytical cycle time for LC/MS/MS sample analysis. A set of 45 validation compounds was used to demonstrate the speed, quality and reproducibility of MS/MS method optimization, sample analysis, and data processing using this automated platform. Metstab results for the validation compounds in microsomes from multiple species (human, rat, mouse) showed good consistency within each batch, and also between batches conducted on different days. We have achieved and maintained a monthly throughput of 1300 compound assays representing 500 discrete compounds per instrument per month on this platform, and it has been used to generate metabolic stability data for more than 25 000 compounds to date with an overall success rate of more than 95%. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

4.
Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15–120 kDa) within only 15 min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10- to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.  相似文献   

5.
For therapeutic drug monitoring in remote settings, dried blood spots (DBS) are particularly advantageous, as blood sample collection and handling is uncomplicated. The aim of this study was to develop and validate an automated extraction method for the analysis of nevirapine, efavirenz and lopinavir in DBS samples. Automated extraction was performed with methanol : water (70 : 30 v /v ), using a DBS‐MS 500 autosampler coupled to a liquid chromatography tandem mass spectrometry system. The autosampler used digital images of each DBS to position the extraction head, sprayed 10 μl of internal standard onto each DBS and extracted a 4‐mm disc (Ø) from the centre of each spot by unilateral flow using 25‐μl extraction solvent. The analytes were baseline separated on a pentafluorophenyl column and analysed by using electrospray ionization with multiple reaction monitoring in positive polarity mode for nevirapine and lopinavir and in negative mode for efavirenz. The method was linear between 10 and 10 000 ng/ml for all analytes. Automated sample extraction resulted in consistent recoveries (nevirapine: 70 ± 6%, efavirenz: 63 ± 11% and lopinavir: 60 ± 10%) and matrix effects between different donors and concentration levels. Intra‐day and inter‐day accuracy and precision deviations were ≤15%. Manual and automated extractions of DBS samples collected within the framework of an adherence assessment study in rural Tanzania showed good agreements with deviations of less than 10%. Our study highlights that therapeutic drug monitoring samples obtained in the resource‐constrained setting of rural Africa can be reliably determined by automated extraction of DBS. Overall, automatization improved method sensitivity and facilitates analysis of large sample numbers. Copyright © 2017 John Wiley & Sons, Ltd.  相似文献   

6.
《Electrophoresis》2018,39(14):1754-1762
A novel and fully automated sequential injection analysis manifold coupled to a capillary electrophoresis apparatus with amperometric detection, is described. The sequential injection manifold was isolated from the high voltage by inserting an air plug into the circuit. Small buffer reservoirs were used to avoid the need to pump fresh buffer to the interface during the electrophoretic separation. No decoupling device was used to mitigate the interference from the high voltage electric field, instead the potential shift induced by the separation voltage, was accounted for. The new hydrodynamic injection method presented is based on the overpressure created in the circuit when a pinch valve is closed for a predetermined time. The injection method yields RSD values of peak height and area below 2.55 and 1.82%, respectively, at different durations of valve closure (n = 5). The capillary and working electrode alignment was achieved by adapting a commercial available capillary union. When the electrode was replaced, the alignment method proved to be very reliable, yielding RSD values of peak height and area lower than 2.64 and 2.08%, respectively (n = 8). Using this system with a gold microelectrode, dopamine, and epinephrine could be quantified within the concentration range of 1–500 μM and detected at a concentration of 0.3 μM. The methods here presented could be applied for the development of new capillary electrophoresis systems with amperometric detection and/or to the design of fully automated systems for online process monitoring purposes.  相似文献   

7.
Permanent monitoring of waterborne pathogens is important for securing the hygiene of water. Enumerating bacteria in water at low concentrations and minute quantities demands rapid and efficient enrichment methods in order to improve the signal-to-noise ratio of subsequent determination methods. In this work an automated cross-flow microfiltration (CFM) system is presented which is usable in the field to concentrate large volumes of environmental water for analytical purposes. It was designed as a rapid enrichment apparatus achieving high recovery and high concentration factors. The efficiency of the CFM system was studied for E. coli spiked in a 10-L tap water sample. By this technique, a 10-L water sample was concentrated by a factor of 200 in 15 min. The high and consistent recovery of 91.3 ± 5.4% living cells in the concentration range 0.01 and 100 cfu mL−1 is suitable for rapid enumeration of bacteria in water.  相似文献   

8.
In the present study, an ultra-sensitive and highly reproducible novel SERS-based capillary platform was developed and utilized for the trace detection of tetrahydrocannabinol (THC). The approach combines the advantages of microwave-assisted nanoparticle synthesis, plasmonics and capillary forces. By employing a microwave-assisted preparation method, glass capillaries were reproducibly coated with silver nanoparticles in a batch fabrication process that required a processing time of 3 min without needing to use any pre-surface modifications or add surfactants. The coated capillaries exhibited an excellent SERS activity with a high reproducibility and enabled the detection of low concentrations of target molecules. At the same time, only a small amount of analyte and a short and simple incubation process was required. The developed platform was applied to the spectroscopic characterization of tetrahydrocannabinol (THC) and its identification at concentration levels down to 1 nM. Thus, a highly efficient detection system for practical applications, e.g., in drug monitoring/detection, is introduced, which can be fabricated at low cost by using microwave-assisted batch synthesis techniques.  相似文献   

9.
《Electrophoresis》2017,38(13-14):1764-1770
Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on‐demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary‐ and chip‐based methods offering low consumable costs (<0.1$) circumventing cost‐intensive microfluidic chip fabrication, short process times (5 min per analysis) and high sensitivity (4 ng/μL dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2–4 μL in volume, a semi‐contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non‐contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR‐grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi‐circular cross section of adaptable width (∼200–600 μm) and height (∼30–80 μm) as well as a typical length of 15–55 mm. Layout of such liquid structures is adaptable on‐demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label‐free DNA ladder (100–1000 bp) at 100 V/cm via in‐line staining and laser induced fluorescent end‐point detection using an automated prototype.  相似文献   

10.
A fully integrated and automated electromembrane extraction LC-MS (EME-LC-MS) system has been developed and characterized. Hyphenation of a flow–flow EME probe to LC-MS was accomplished by using an in-built 10-port switching valve of the LC-MS system. The 10-port switching valve decoupled the high pressure of the UHPLC-system from the low pressure required for operation of the EME-probe by automated switching between a sample extraction/analysis and a sample load position. In the sample load position the extracted analytes were loaded into a HPLC sample loop. By switching the valve to the sample extraction/analysis position the setup allowed simultaneous analysis of previously loaded analytes while extracting a new sample. Performance of the system was characterized with respect to precision and linearity (RSD < 2.5%, R2: 0.998) and the setup was applied for studying the in-vitro metabolism of methadone by rat liver microsomes. As the metabolic reaction proceeded, methadone and its metabolites were extracted and analyzed in parallel by LC-MS using either isocratic or gradient elution. Compared to a conventional in-vitro metabolism analysis based on protein precipitation followed by LC-MS analysis the fully automated EME-LC-MS system offers a significant time saving and in addition demonstrates increased sensitivity as the analytes were automatically enriched during the extraction process. The experiment revealed 6 to 16 times higher S/N ratios of the EME-LC-MS method compared to protein precipitation followed by LC-MS and thus concomitantly lower LOD and LOQ. The setup integrates a complete analytical workflow of rapid extraction, enrichment, separation and detection of analytes in a fully automated manner. These attributes make the developed system a powerful alternative approach for a wide range of analytical applications.  相似文献   

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