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At least 217 records · Page 12Linked to original sources

Automated high-performance liquid chromatography of iopentol in human plasma and whole blood using on-line dialysis as sample preparation.

A fully automated high-performance liquid chromatographic method has been developed for the analysis of the radiographic contrast agent iopentol in human plasma and whole blood. This method is based on on-line sample preparation with dialysis followed by pre-column enrichment of the dialysate. The method was compared with a manual sample preparation method based on protein precipitation with tetrahydrofuran. The inter-assay and intra-assay variations and the limits of quantitation were the same for both methods. The on-line combination of dialysis and column-switching high-performance liquid chromatography was shown to be a reliable and time-saving technique for monitoring iopentol in human whole blood and plasma.

Chromatography, High Pressure Liquid↗

The XT-tube extractor: a hollow fiber-based supported liquid membrane extractor for bioanalytical sample preparation.

A new supported liquid membrane extractor for bioanalytical sample preparation is presented. The extractor consists of a polypropylene hollow fiber mounted inside a PTFE tube by means of a cross-connector and a tee-connector. All parts are commercially available, inexpensive, and easily assembled. An organic solvent in the pores of the fiber forms a liquid membrane that separates the sample, which is pumped along the outside of the fiber, from the acceptor phase, which is pumped inside. The length of the hollow fiber may easily be varied to meet different demands on extractive surface and extract volumes. To test the system, the strongly acidic plasticizer/flame retardant metabolite diphenyl phosphate ester (DPhP), with a pKa value of 0.26, was extracted from urine. DPhP was protonated using 4 M hydrochloric acid and extracted into an acceptor phase at pH 9. Thirty extractions were made with the same liquid membrane without any decrease in extraction efficiency and with a relative standard deviation <7%. An analyte concentration enrichment of 5-10 times was achieved in the extraction step, giving a limit of detection (S/N = 3) of 0.014 microg/mL with LC/ESI-MS and 0.18 microg/mL with CE-UV. The effects on extraction efficiency using different sample pH, organic solvents, sample flow rates, and lengths of the fiber were evaluated.

Calibration↗

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform.

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.

Automation↗

Powder dispensing robot for sample preparation.

An automated powder dispensing station capable of transferring milligram quantities (1-100 mg) of powder for sample preparation was developed and integrated into a commercial robotic workstation (Zymark Prelude). The system's performance was optimized with respect to vacuum flow rate and powder transfer tube cross sectional area, and shown to possess excellent powder dispensing accuracy (RSD = < or = 0.1% for target weights < or = 15 mg) and precision (RSD = 3.43%) for a vanillin sample. Using the commercial features of the Zymark Prelude workstation (liquid handling, weighing, and vortexing/mixing) and the custom powder dispensing station, multiple sets of analytical calibration standards were prepared and subsequently analyzed by FIA in order to assess the system's robustness for sample preparation.

Drug Industry↗

[Studies on procedure for sample preparation of ellagic acid in several kinds of foodstuffs].

A sample preparation for ellagic acid in several kinds of foodstuffs, which is included in the List of Existing Food Additives as natural antioxidants in Japan, functioning as an antioxidant, was studied. The solid samples were refluxed with methanol, and then the extract was refined using a solid-phase cartridge. The liquid samples were directly pretreated by solid-phase extraction. On the other hand, test solutions for cooking oils and fats were inapplicable to solid-phase extraction in this work, because the recovery tests from samples spiked with ellagic acid gave low recoveries. Consequently, they were prepared by modified frozen method using methanol or acetonitrile-2-propanol-ethanol (2:1:1). The recoveries from tested foodstuffs spiked with ellagic acid (50 micrograms/g) were 75.2-96.9%. The limits of quantification for ellagic acid were 0.05 microgram/g by the proposed method.

Antioxidants↗

High-resolution magic angle spinning (1)H NMR spectroscopy of intact liver and kidney: optimization of sample preparation procedures and biochemical stability of tissue during spectral acquisition.

High-resolution magic angle spinning (MAS) (1)H NMR spectroscopy has been used to investigate the biochemical composition of whole rat renal cortex and liver tissue samples. The effects of a number of sample preparation procedures and experimental variables have been investigated systematically in order to optimize spectral quality and maximize information recovery. These variables include the effects of changing the sample volume in the MAS rotor, snap-freezing the samples, and the effect of organ perfusion with deuterated saline solution prior to MAS NMR analysis. Also, the overall biochemical stability of liver and kidney tissue MAS NMR spectra was investigated under different temperature conditions. We demonstrate improved resolution and line shape of MAS NMR spectra obtained from small spherical tissue volume (12 microl) rotor inserts compared to 65 microl cylindrical samples directly inserted into the MAS rotors. D(2)O saline perfusion of the in situ afferent vascular tree of the tissue immediately postmortem also improves line shape in MAS NMR spectra. Snap-freezing resulted in increased signal intensities from alpha-amino acids (e.g., valine) in tissue together with decreases in renal osmolytes, such as myo-inositol. A decrease in triglyceride levels was observed in renal cortex following stasis on ice and in the MAS rotor (303 K for 4 h). This work indicates that different tissues have differential metabolic stabilities in (1)H MAS NMR experiments and that careful attention to sample preparation is required to minimize artifacts and maintain spectral quality.

Animals↗

Automated sample preparation based on the sequential injection principle. Solid-phase extraction on a molecularly imprinted polymer coupled on-line to high-performance liquid chromatography.

A molecularly imprinted polymer (MIP) prepared using caffeine, as a template, was validated as a selective sorbent for solid-phase extraction (SPE), within an automated on-line sample preparation method. The polymer produced was packed in a polypropylene cartridge, which was incorporated in a flow system prior to the HPLC analytical instrumentation. The principle of sequential injection was utilised for a rapid automated and efficient SPE procedure on the MIP. Samples, buffers, washing and elution solvents were introduced to the extraction cartridge via a peristaltic pump and a multi-position valve, both controlled by appropriate software developed in-house. The method was optimised in terms of flow rates, extraction time and volume. After extraction, the final eluent from the extraction cartridge was directed to the injection loop and was subsequently analysed on HPLC. The overall set-up facilitated unattended operation, operation and improved both mixing fluidics and method development flexibility. This system may be readily built in the laboratory and can be further used as an automated platform for on-line sample preparation.

Automation↗

An automated liquid handling system for polymerase chain reaction sample preparation.

An automated liquid handling system for dispensing liquid samples of sub-microliter volume has been developed. The system has eight nozzles composed of glass capillaries connected to syringe-style pumps. The distance between the nozzles can be changed from 4.5 to 9 mm, which corresponds to the distance between the wells in 96- and 384-well microplates, respectively. The eight nozzles of the system can aspirate and dispense liquid samples in both 96- and 384-well microplates. Sub-microliter volumes of reagents and samples were transferred between 96- and 384-well microplates using the system. This system was successfully used for PCR sample preparation.

Journal Article↗

Effects of sample preparation conditions on biomolecular solid-state NMR lineshapes.

Sample preparation conditions with the 46 kDa enzyme complex of 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, shikimate-3-phosphate (S3P) and glyphosate (GLP) have been examined in an attempt to reduce linewidths in solid-state NMR spectra. The linewidths of 31P resonances associated with enzyme bound S3P and GLP in the lyophilized ternary complex have been reduced to 150 +/- 12 Hz and 125 +/- 7 Hz respectively, by a variety of methods involving additives and freezing techniques.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

High throughput liquid chromatography/mass spectrometry bioanalysis using 96-well disk solid phase extraction plate for the sample preparation.

The 96 well solid phase extraction (SPE) operated by robot in the LC/MS/MS bioanalysis offered rapid sample preparation for drugs and metabolites in biological matrices, based on simultaneous extraction of 96 samples. The use of a disk as sorbent in the 96 well plate further improved the performance of SPE and allowed for small elution volumes, making it possible to 'dilute and shoot" after SPE elution. In this study, a 96 well plate (Empore) was developed, characterized and optimized for several pharmaceutical compounds. In addition, a robot (MultiProbe) was modified to automate the 96 well plate operation. Examples were given to illustrate the major differences of using 96 well disk plate SPE in the method development as compared to the traditional SPE. This technology has been successfully used to support many clinical studies. Typically, a batch of 96 samples were prepared in 1-1.5 hours unattended (except for the replacement of a collection plate). Considerable savings in disposable supplies were also noted.

Anti-Asthmatic Agents↗

Improved matrix-assisted laser desorption/ionisation sample preparation of a partially depolymerised cellulose derivative by continuous spray deposition and interfacing with size-exclusion chromatography.

Continuous spray deposition (CSD) of aqueous solutions of partially depolymerised methyl cellulose was found to improve matrix-assisted laser desorption/ionisation (MALDI) sample preparation. One feature was that the sensitivity in MALDI time-of-flight mass spectrometry increased up to an order of magnitude compared with the standard sample preparation method. Another feature was that CSD provided targets for MALDI with homogeneously distributed analyte. This resulted in a more even signal intensity and a higher reproducibility than in the standard method. High-mass discrimination was more pronounced in CSD than in the standard method. Size-exclusion chromatography with aqueous eluent was coupled online to CSD onto matrix-precoated foils. The suitability for determination of the molar mass distribution of methyl cellulose was investigated.

Biopolymers↗

Sample preparation project for the subcellular proteome of mouse liver.

Organelle proteome has become one of the most important fields of proteomics, and the subcellular fractionation with high purity and yield has always been a challenge for cell biologists and also for the Human Liver Proteome Project (HLPP). The liver of a C57BL/6J mouse was chosen as the model to find the optimum method for subcellular preparation. The method we selected could obtain the multiple fractions including plasma membrane, mitochondria, nucleus, ER, and cytosol from a single homogenate. With the same procedure, it is for the first time that the preparation method of frozen homogenized livers was compared with that of the fresh livers and frozen livers. We systematically evaluated the purity, efficiency, and integrity by protein yield, immunoblotting, and transmission electron microscopy. Taken together, the method of multiple fractions from a single tissue is effective enough for subcellular fractionation of mouse liver. We give a selective sample preparation method for frozen homogenized livers, for rare clinical samples, which cannot easily be used for subcellular separation immediately. But the frozen livers are not recommended for organelles isolation. This result is especially useful for sample preparation of human liver for subcellular fractionation of HLPP.

Animals↗

Miniaturized sample preparation and separation methods for environmental and drug analyses.

Miniaturized extraction and separation media have been successfully developed from precisely controlled technologies. In this article, recent developments in these high performance analytical methods, such as miniaturized sample preparation methods and the coupling of these techniques with microscale separation systems, have been reviewed, along with some applications to environmental and biological analysis. The advantage of the miniaturization is not only for the environmental compatibility but also for the developments of the high performance analytical systems. Down-sizing also makes it possible to investigate and introduce various compounds and materials as novel media (such as tailor-made materials and devices) in separation science. As a typical example of the novel miniaturized sample preparation system, the applications of fibrous materials for microcolumn liquid-phase separation methods are described.

Environmental Monitoring↗

Development of an automated sample preparation module for environmental monitoring of biowarfare agents.

An automated sample preparation module, based upon sequential injection analysis (SIA), has been developed for use within an autonomous pathogen detection system. The SIA system interfaced aerosol sampling with multiplexed microsphere immunoassay-flow cytometric detection. Metering and sequestering of microspheres using SIA was found to be reproducible and reliable, over 24-h periods of autonomous operation. Four inbuilt immunoassay controls showed excellent immunoassay and system stability over five days of unattended continuous operation. Titration curves for two biological warfare agents, Bacillus anthracis and Yersinia pestis, obtained using the automated SIA procedure were shown to be similar to those generated using a manual microtiter plate procedure.

Antibodies, Bacterial↗

Determination of 16 phthalate metabolites in urine using automated sample preparation and on-line preconcentration/high-performance liquid chromatography/tandem mass spectrometry.

We developed an on-line solid-phase extraction (SPE) method, coupled with isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) and with automated sample preparation, to simultaneously quantify 16 phthalate metabolites in human urine. The method requires a silica-based monolithic column for the initial preconcentration of the phthalate metabolites from the urine and a silica-based conventional analytical column for the chromatographic separation of the analytes of interest. It uses small amounts of urine (100 microL), is sensitive (limits of detection range from 0.11 to 0.90 ng/mL), accurate (spiked recoveries are approximately 100%), and precise (the inter- and intraday coefficients of variation are <10%). The method is not labor intensive, and, because pretreatment of the urine samples was performed automatically using an HPLC autosampler, involves minimal sample handling, thus minimizing exposure to hazardous chemicals. The method was validated on spiked, pooled urine samples and on urine samples from 43 adults with no known exposure to phthalates. The high sensitivity and high throughput (HPLC run time, including the preconcentration step, is 27 min) of this analytical method combined with the ease of use and effective automated sample preparation procedure make it suitable for large epidemiological studies to evaluate the prevalence of human exposure to phthalates.

Adult↗

Comparison of sample preparation methods for recovering Salmonella from raw fruits, vegetables, and herbs.

Methods for preparing raw fruits, vegetables, and herbs for enrichment or direct plating to determine the presence and populations of pathogenic bacteria vary greatly. A study was done to compare three sample processing methods (washing in 0.1% peptone, stomaching, and homogenizing) for their influence on recovery of Salmonella inoculated onto 26 types of raw produce. The mean numbers of Salmonella recovered from 10 fruits, 11 vegetables, and 5 herbs using all three processing methods were 7.17, 7.40, and 7.27 log10 CFU/sample, respectively. Considering all 26 types of produce and all processing methods, the number of Salmonella recovered ranged from 7.24 to 7.29 log10 CFU/sample, with no significant differences attributable to a particular sample processing method. Mean percent recoveries of Salmonella from washed, stomached, and homogenized produce were 39.4, 44.7, and 42.4%, respectively. Mean percent recoveries from fruits, vegetables, and herbs, regardless of sample preparation method, were 41.7, 50.1, and 25.9%, respectively. The number of Salmonella recovered from stomached and homogenized produce, but not washed produce, with pH < or = 4.53 was significantly less than the number recovered from produce with pH from 5.53 to 5.99, suggesting that the acidic environment in stomachates and homogenates was lethal to a portion of Salmonella. Reduced percent recoveries from herbs (pH 5.94 to 6.34) is attributed, in part, to antimicrobials released from plant cells during sample preparation. Overall, the type of processing method did not substantially affect the number of Salmonella recovered from the 26 types of raw produce representing a wide range of structural and morphological characteristics, composition, and pH. The influence of sample size, diluent composition, and processing time on efficiency of recovery of Salmonella and other pathogens needs to be evaluated before a method(s) for processing samples of raw produce can be recommended.

Colony Count, Microbial↗

Plan view TEM sample preparation for non-continuous and delaminating thin films.

The preparation of plan view samples for TEM examination is generally a straightforward process except where the film delaminates from the substrate or is non-continuous. A method is described where film is first stabilised using an epoxy, then transferred to an aperature grid and ion milled to produce an electron transparent sample. This technique provides a relatively simple and fast method to prepare these types of films, using the standard equipment and supplies found in most TEM sample preparation laboratories.

Microscopy, Electron↗

Application of a spotting sample preparation technique for the detection of pathogens in woody plants by RT-PCR and real-time PCR (TaqMan).

An extraction technique for reverse transcription-PCR (RT-PCR) detection of plant pathogens including viruses, bacteria and phytoplasma is described. The total nucleic acid of these plant pathogens was obtained by direct spotting of crude sap derived from infected leaf, petiole or cambial tissue onto two different types of membranes, positively charged Hybond N(+) Nylon and FTA membranes, and processed for use in PCR. Thirteen different plant viruses, Xylella fastidiosa (causal agent of Pierce's disease) and phytoplasmas were included in the experiment. A thermal treatment (95 degrees C for 10 min) of the Hybond N(+) Nylon discs in a buffered solution improved the detection, but for FTA membrane discs the thermal treatment was not required and the discs were directly placed in the PCR reaction cocktail. Specific amplification of genomic or ribosomal RNA fragments of these pathogens was obtained by one-step RT-PCR except for X. fastidiosa in which a fragment of the genomic DNA was used for amplification. The same sample preparation methods also worked well for real-time RT-PCR (TaqMan). The sample preparation techniques reported here could be used to store samples for future PCR test or for long distance shipment to a detection laboratory.

Edetic Acid↗