Automated sample treatment by flow techniques prior to liquid-phase separations

Sample preparation (SP) is an integral and important part of an analytical process. Lately, SP has been the topic of increased interest in research and development of novel advanced technologies. Major needs in this aspect are miniaturization, automation, and enrichment. Among other methodologies, flow techniques can be conveniently and effectively coupled to liquid-phase separation technologies for on-line sample preparation. This paper reviews the current trends in on-line automated sample preparation by flow-through techniques prior to liquid-phase separations. Strategies and interfaces developed to couple flow techniques with liquid chromatography, capillary electrophoresis, and mass spectrometry are described. Advantages and limitations of the coupling are discussed. The paper also highlights certain selected applications of these coupled systems.     

Determination of drugs in biological fluids by direct injection of samples for liquid-chromatographic analysis

The analysis of drugs in various biological fluids is an important criterion for the determination of the physiological performance of a drug. After sampling of the biological fluid, the next step in the analytical process is sample preparation. The complexity of biological fluids adds to the challenge of direct determination of the drug by chromatographic analysis, therefore demanding a sample preparation step that is often time-consuming, tedious, and frequently overlooked. However, direct on-line injection methods offer the advantage of reducing sample preparation steps and enabling effective pre-concentration and clean-up of biological fluids. These procedures can be automated and therefore reduce the requirements for handling potentially infectious biomaterial, improve reproducibility, and minimize sample manipulations and potential contamination. The objective of this review is to present an overview of the existing literature with emphasis on advances in automated sample preparation methods for liquid-chromatographic methods. More specifically, this review concentrates on the use of direct injection techniques, such as restricted-access materials, turbulent-flow chromatography and other automated on-line solid-phase extraction (SPE) procedures. It also includes short overviews of emerging automated extraction-phase technologies, such as molecularly imprinted polymers, in-tube solid-phase micro-extraction, and micro-extraction in a packed syringe for a more selective extraction of analytes from complex samples, providing further improvements in the analysis of biological materials. Lastly, the outlook for these methods and potential new applications for these technologies are briefly discussed.     

Determination of drugs in biological fluids by direct injection of samples for liquid-chromatographic analysis

The analysis of drugs in various biological fluids is an important criterion for the determination of the physiological performance of a drug. After sampling of the biological fluid, the next step in the analytical process is sample preparation. The complexity of biological fluids adds to the challenge of direct determination of the drug by chromatographic analysis, therefore demanding a sample preparation step that is often time-consuming, tedious, and frequently overlooked. However, direct on-line injection methods offer the advantage of reducing sample preparation steps and enabling effective pre-concentration and clean-up of biological fluids. These procedures can be automated and therefore reduce the requirements for handling potentially infectious biomaterial, improve reproducibility, and minimize sample manipulations and potential contamination.The objective of this review is to present an overview of the existing literature with emphasis on advances in automated sample preparation methods for liquid-chromatographic methods. More specifically, this review concentrates on the use of direct injection techniques, such as restricted-access materials, turbulent-flow chromatography and other automated on-line solid-phase extraction (SPE) procedures. It also includes short overviews of emerging automated extraction-phase technologies, such as molecularly imprinted polymers, in-tube solid-phase micro-extraction, and micro-extraction in a packed syringe for a more selective extraction of analytes from complex samples, providing further improvements in the analysis of biological materials. Lastly, the outlook for these methods and potential new applications for these technologies are briefly discussed.     

A Lateral Flow Assay for Quantitative Detection of Amplified HIV-1 RNA

Although the accessibility of HIV treatment in developing nations has increased dramatically over the past decade, viral load testing to monitor the response of patients receiving therapy is often unavailable. Existing viral load technologies are often too expensive or resource-intensive for poor settings, and there is no appropriate HIV viral load test currently available at the point-of-care in low resource settings. Here, we present a lateral flow assay that employs gold nanoparticle probes and gold enhancement solution to detect amplified HIV RNA quantitatively. Preliminary results show that, when coupled with nucleic acid sequence based amplification (NASBA), this assay can detect concentrations of HIV RNA that match the clinically relevant range of viral loads found in HIV patients. The lateral flow test is inexpensive, simple and rapid to perform, and requires few resources. Our results suggest that the lateral flow assay may be integrated with amplification and sample preparation technologies to serve as an HIV viral load test for low-resource settings.     

Bioconversion of alpha-Damascone by Botrytis cinerea

Bioconversion of alpha-damascone (compound 1) was studied with four strains of Botrytis cinerea in grape must (pH 3.2). As biotransformation products of compound 1, 3-oxo-alpha-damascone, cis- and trans-3-hydroxy-alpha-damascone, gamma-damascenone, 3-oxo-8, 9-dihydro-alpha-damascone, and cis- and trans-3-hydroxy-8,9-dihydro-alpha-damascone were identified. In addition, acid-catalyzed chemical transformation of compound 1 to the diastereomers of 9-hydroxy-8,9-dihydro-alpha-damascone was observed. Identifications were performed by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e., on-line HRGC-mass spectrometry and HRGC-Fourier transform infrared spectroscopy, after extractive sample preparation.     

Critical appraisal of the side population assay in stem cell and cancer stem cell research

The "Side Population" (SP) discrimination assay is a flow cytometry method used to detect stem cells based on the dye efflux properties of ABC transporters. We discuss the SP assay and its applications in stem cell?biology, with an emphasis on the technical challenges related to sample preparation, data acquisition, analysis, and interpretation. We highlight the value of multicolor phenotyping, the impact of DNA ploidy, and the importance of distinguishing graft versus host cells for an appropriate SP discrimination. To improve the consistency and reliability of data between laboratories, we propose a set of recommendations for SP assay data reporting.     

A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis

A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96‐well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on‐bead digested by using Single‐Pot solid‐phase sample preparation (SP3). The whole IP‐SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter‐aided sample preparation) or a longer incubation protocol. Taken together, the IP‐SP3 protocol is a fast and economical approach easily applicable for large‐scale protein interactome analysis.     

New trends in sample preparation: on-line microextraction in packed syringe (MEPS) for LC and GC applications Part III: Determination and validation of local anaesthetics in human plasma samples using a cation-exchange sorbent, and MEPS-LC-MS-MS

The need for on-line sample preparation for high-throughput applications in bioanalysis has increased during the past decade. In this paper a robust and on-line sample preparation technique, micro extraction in packed syringe (MEPS) has been developed and validated. The method is a miniaturized, fully automated, solid-phase extraction (SPE) technique that can be connected on-line to GC or LC without any modification of the chromatographs. The performance of MEPS as sample preparation method is illustrated by the determination of local anaesthetics in human plasma samples on-line with high performance liquid chromatography (HPLC) and tandem mass spectrometry. The sampling sorbent was 1mg silica based benzenesulphonic acid cation exchanger that was inserted in a 250 microl syringe. Ropicavine and two of its metabolites (PPX and 3-OH-ropivacine), lidocaine and bupivacine were used as model substances. The accuracy values of quality control samples (QC) were between 95% and 109%, and precision (relative standard deviation, R.S.D.) had a maximum deviation of 9% for the analytes.     

Microfluidic components for protein characterization

The use of microfluidic components to create an analytical toolbox for the very rapidly growing field of proteomics is described. This toolbox provides novel generic analytical solutions that are highly adaptable for analysis of various biomolecules, ranging from high to low abundant. The components are fabricated using silicon micromachining and consist of a microchip immobilised enzyme reactor (microIMER), a piezoelectric microdispenser and high-density nanovial target plates. This microtechnology based platform interfaces matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF-MS) to a wide range of upstream sample handling and/or analytical techniques. Examples of applications such as rapid on-line digestion (12 s) and sample preparation of proteins, interfacing to capillary liquid chromatography (100 attomol sensitivity), and in-vial chemistry on femtomol amounts of sample are presented.     

On-line monitoring of monoclonal antibodies in animal cell culture using a grating coupler

A grating coupler was used for the on-line determination of monoclonal antibodies produced in perfused animal cell bioreactor. The device was connected with the culture vessel via a flow-injection analysis (FIA) system, which was controlled automatically. Specific antimouse lgG antibodies were immobilized on the surface of the sensor-chip. After injection of the sample, the binding of mouse lgG was observed in real time. The regeneration of the binding sites of the immobilized antibodies using an acidic solution allowed the on-line detection of produced monoclonal antibodies in the range of 10 to 150 mug/mL. In contrast to other techniques coupled to bioprocesses, the developed method represents a regenerable direct immunosensor. Results were compared with standard ELISA techniques (off-line) and a competitive immunochemical assay using the grating coupler (off-line). (c) 1993 John Wiley & Sons, Inc.     

On-line cleanup for 2-aminobenzamide-labeled oligosaccharides

Analysis of 2-aminobenzamide-labeled oligosaccharides requires removal of excess labeling reagents before chromatography. Manual cleanup is time-consuming and not optimal for routine analysis, so an on-line solid-phase extraction was developed. Labeled oligosaccharides are trapped on an amide phase in a small guard column, the excess reagents are washed away, and then the sample is transferred to the analytical column for analysis. The on-line protocol shortened the sample preparation time and has been applied for the analysis of oligosaccharides and N-glycans released from glycoproteins.     

Microfluidic devices for sample preparation and rapid detection of foodborne pathogens

Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line.     

Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery

The scientific community has shown great interest in the field of mass spectrometry-based proteomics and peptidomics for its applications in biology.Proteomics technologies have evolved to produce larg...     

Advances in on-line monitoring and control of mammalian cell cultures: Supporting the PAT initiative

In recent years, much attention has been directed towards the development of global methods for on-line process monitoring, especially since the Food and Drug Administration (FDA) launched the Process Analytical Technology (PAT) guidance, stimulating biopharmaceutical companies to update their monitoring tools to ensure a pre-defined final product quality. The ideal technologies for biopharmaceutical processes should operate in situ, be non-invasive and generate on-line information about multiple key bioprocess and/or metabolic variables. A wide range of spectroscopic techniques based on in situ probes have already been tested in mammalian cell cultures, such as near infrared (NIR), mid infrared (MIR), 2D fluorescence and dielectric capacitance spectroscopy; similarly, the electronic nose technique based on chemical array sensors has been tested for in situ off-gas analysis of mammalian cell cultures. All these methods provide series of spectra, from which meaningful information must be extracted. In this sense, data mining techniques such as principal components regression (PCR), partial least squares (PLS) or artificial neural networks (ANN) have been applied to handle the dense flow of data generated from the real-time process analyzers. Furthermore, the implementation of feedback control methods would help to improve process performance and ultimately ensure reproducibility. This review discusses the suitability of several spectroscopic techniques coupled with chemometric methods for improved monitoring and control of mammalian cell processes.     

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation

A recently introduced silicon microextraction chip (SMEC), used for on-line proteomic sample preparation, has proved to facilitate the process of protein identification by sample clean up and enrichment of peptides. It is demonstrated that a novel grid-SMEC design improves the operating characteristics for solid-phase microextraction, by reducing dispersion effects and thereby improving the sample preparation conditions. The structures investigated in this paper are treated both numerically and experimentally. The numerical approach is based on finite element analysis of the microfluidic flow in the microchip. The analysis is accomplished by use of the computational fluid dynamics-module FLOTRAN in the ANSYS software package. The modeling and analysis of the previously reported weir-SMEC design indicates some severe drawbacks, that can be reduced by changing the microextraction chip geometry to the grid-SMEC design. The overall analytical performance was thereby improved and also verified by experimental work. Matrix-assisted laser desorption/ionization mass spectra of model peptides extracted from both the weir-SMEC and the new grid-SMEC support the numerical analysis results. Further use of numerical modeling and analysis of the SMEC structures is also discussed and suggested in this work.     

An on-line approach to monitor ethanol fermentation using FTIR spectroscopy

Fermentation process control is currently limited by its inability to measure parameters such as substrate, product, and biomass concentrations rapidly for consistent on-line feedback. Physical and chemical parameters, such as temperature and pH, currently can be obtained on-line using appropriate sensors. However, to obtain information on the concentration of the substrate, product, and biomass, samples must be taken off-line for measurement. With the use of spectroscopic techniques, real-time monitoring of process constituents such as product and substrate is possible. Spectroscopic techniques are rapid and nondestructive, require minimal or no sample preparation, and can be used to simultaneously assess several constituents in complex matrices. The production of ethanol is the largest fermentation process in terms of production volume and economic value as a result of its prominence in the food, agricultural, and fuel industries. This study attempts to develop an on-line ethanol fermentation monitoring technique using Fourier transform infrared (FTIR) spectroscopy with a flow-through ATR capability. Models developed using multivariate statistics, employed to obtain on-line FTIR measurements, were successfully validated by off-line HPLC analysis and spectrophotometry data. Standard errors of prediction (SEP) values of 0.985 g/L (R2 = 0.996), 1.386 g/L (R2 = 0.998), and 0.546 (R2 = 0.972) were obtained for ethanol, glucose, and OD, respectively. This work demonstrates that FTIR spectroscopy could be used for rapid on-line monitoring of fermentation.     

Solid-phase extraction or liquid chromatography coupled on-line with gas chromatography in the analysis of biological samples

This review provides an overview of the on-line coupling of solid-phase extraction or liquid chromatography with gas chromatography for the analysis of biological samples. Principles relevant to techniques are briefly presented and selected applications are described. Benefits of the coupled systems are discussed.