High-throughput Sample Preparation Research Articles

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-Throughput N-Glycan Analysis with Rapid Magnetic Bead-Based Sample Preparation.

N-glycan profiling of therapeutic glycoproteins is essential to ensure the activity and efficacy of these promising new-generation drugs. The N-linked glycan moieties of these entities highly affect circulation half-life, immunogenicity and receptor-binding activity as well as physicochemical and thermal stability properties. In addition, more than half of the biopharmaceuticals are glycoproteins representing multibillion dollar worldwide business, further emphasizing the importance of their analysis. In the biomedical field, on the other hand, revealing disease-related glycan structure alterations holds the promise of the discovery of new biomarkers for early diagnostics. Therefore, there is a great demand for widely applicable, high-throughput sample preparation and analysis methods for N-glycan profiling of glycoproteins. One of the newest exciting developments of the field is the magnetic bead based glycoprotein sample preparation technique. A detailed protocol of this method is given in this chapter in conjunction with rapid capillary electrophoresis analysis of the prepared samples by laser induced fluorescence detection (CE-LIF). N-glycans are digested by the endoglycosidase PNGase F and the released carbohydrates are labeled with the charged fluorophore dye of aminopyrenetrisulfonate (APTS). Effective glycan capture by magnetic microparticles enabled fast, easily automated sample preparation both in individual (single vial) and 96-well plate formats, including excess dye removal. Rapid separation of APTS labeled IgG glycans is also shown utilizing an optimized CE-LIF protocol.

High-throughput and sensitive analysis of urinary heterocyclic aromatic amines using isotope-dilution liquid chromatography-tandem mass spectrometry and robotic sample preparation system.

Heterocyclic aromatic amines (HCAA) are listed by the US Food and Drug Administration (FDA) as harmful or potentially harmful constituents of tobacco smoke. However, quantifying HCAA exposure is challenging. In this study, we developed a sensitive, precise, and accurate isotope dilution, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify urinary HCAAs in smokers and nonsmokers. The high-throughput robotic sample preparation system could handle a throughput of over 300 samples per day, while maintaining intra-day and inter-day imprecision and bias ≤10%. The limits of detection of carcinogenic HCAAs ranged from 0.31 to 0.83pg/mL. The validated method was applied to measure HCAAs in urine collected from smokers and non-smokers. This sensitive and efficient analytical method is ideal to support large-scale biomonitoring studies of HCAA exposure. Graphical Abstract LC/MS/MS and robotic sample preparation system for urinary HCAA analysis.

A high-throughput nanofibers mat-based micro-solid phase extraction for the determination of cationic dyes in wastewater

This study used nanofibers mat (NFsM)-based micro-SPE (μ-SPE) in 96-well plate format as a novel high-throughput sample preparation technique prior to the determination of cationic dyes in wastewater using HPLC-DAD. P-Toluene sulfonate (PTS−) doped polypyrrole (PPy) functionalized NFsM (PTS-PPy NFsM) was optimized as SPE sorbent in aspects of PPy form (particles and NFsM) and its doped anions (Cl− and PTS−), which showed good extraction efficiency and adsorption capacity for cationic dyes (Auramine-O, Chrysoidine and Rhodamine-B). Under the optimal conditions, the limits of detection (LODs) were between 0.3 and 0.5μg/L, and the linearity was achieved in the concentration ranging from 1.0 to 150.0μg/L with correlation coefficients (R) between 0.992 and 0.999. Compared with the published SPE methods, this approach demonstrated its advantages such as shorter extraction time (0.3min per sample), lower requirement sorbent (2.0mg), lower volume of organic solvent (0.7mL), higher recovery (90.1–99.1%) and precision (RSDs<6.9%). With this developed method, we have successfully analyzed the dyeing industry wastewater, which meets the Discharge Standards of Water Pollutants for Dyeing and Finishing of Textile Industry in China. The concentrations of three analyzed cationic dyes were from 2.9 to 13.9μg/L. The NFsM-based μ-SPE technique is practically a high-throughput sample preparation procedure that can accurately assess the pollutants in the wastewater from dyeing industry.

A liquid chromatography – tandem mass spectrometry method to measure a selected panel of uremic retention solutes derived from endogenous and colonic microbial metabolism

Chronic kidney disease (CKD) is associated with an increased risk of mortality and cardiovascular disease, which is, at least partly, mediated by the accumulation of so-called uremic retention solutes. Although there has been an increasing interest in the behavior of these solutes, derived from both the endogenous and colonic microbial metabolism, methods to simultaneously and accurately measure a broad panel of relevant uremic retention solutes remain scarce.We developed a highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. A high throughput sample preparation was used with extraction of analytes from 50 μl serum using Ostro plate technology. For most solutes, stable isotopes labelled metabolites were used as internal standards. Chromatography was achieved using an Acquity UPLC CSH Fluoro Phenyl column. The total run time was 8 min, the mobile phase was a gradient of 0.1% formic acid in Milli-Q water and pure methanol at a flow rate of 0.5 ml min−1. Detection was performed using a tandem mass spectrometer with alternated positive and negative electrospray ionization.Calibration curves were linear for all solutes. Precision was assessed according to the NCCLS EP5-T guideline, being below 15% for all metabolites. Mean recoveries were between 83 and 104% for all metabolites. The validated method was successfully applied in a cohort of 488 patients with CKD.We developed and validated a sensitive and robust UPLC-MS/MS method for quantification of 15 uremic retention solutes derived from endogenous and colonic microbial metabolism. This method allows for studying the behavior and relevance of these solutes in patients with CKD.

Multifunctional Device for Nucleic Acid Extraction Based on Magnetic Separation and Its Co-Working with Liquid Handling System for High Throughput Sample Preparation

Multifunctional Device for Nucleic Acid Extraction Based on Magnetic Separation and Its Co-Working with Liquid Handling System for High Throughput Sample Preparation

High-Throughput Quantitative Lipidomics Analysis of Nonesterified Fatty Acids in Human Plasma.

We present a high-throughput, nontargeted lipidomics approach using liquid chromatography coupled to high-resolution mass spectrometry for quantitative analysis of nonesterified fatty acids. We applied this method to screen a wide range of fatty acids from medium-chain to very long-chain (8 to 24 carbon atoms) in human plasma samples. The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important ω-3 and ω-6 polyunsaturated fatty acids. We used 51 fatty acid species to demonstrate the quantitative capability of this method with quantification limits in the nanomolar range; however, this method is not limited only to these fatty acid species. High-throughput sample preparation was developed and carried out on a robotic platform that allows extraction of 96 samples simultaneously within 3 h. This high-throughput platform was used to assess the influence of different types of human plasma collection and preparation on the nonesterified fatty acid profile of healthy donors. Use of the anticoagulants EDTA and heparin has been compared with simple clotting, and only limited changes have been detected in most nonesterified fatty acid concentrations.

An Automated High-Throughput Sample Preparation Protocol for LC-MS/MS Analysis of Glycopeptides

An Automated High-Throughput Sample Preparation Protocol for LC-MS/MS Analysis of Glycopeptides

State‐of‐the‐art technologies for rapid and high‐throughput sample preparation and analysis of N‐glycans from antibodies

Glycosylation is a PTM that occurs during production of many protein-based biologic drugs and can have a profound impact on their biological, clinical, and pharmacological properties. Quality by design, process optimization, and advance in manufacturing technology create a demand for robust, sensitive, and accurate profiling and quantification of antibody glycosylation. Potential drawbacks in antibody glycosylation profiling include the high hands-on time required for sample preparation and several hours for data acquisition and analysis. Rapid and high-throughput (HTP) N-glycan profiling and characterization along with automation for sample preparation and analysis are essential for extensive antibody glycosylation analysis due to the substantial improvement of turnaround time. The first part of this review article will focus on the recent progress in rapid and HTP sample preparation and analysis of antibody glycosylation. Subsequently, the article will cover a brief overview of various separation and mass spectrometric methods for the rapid and HTP analysis of N-glycans in antibodies. Finally, we will discuss the recent developments in process analytical technologies for the screening and quantification of N-glycans in antibodies.

A novel strategy for the determination of polycyclic aromatic hydrocarbon monohydroxylated metabolites in urine using ultra-high-performance liquid chromatography with tandem mass spectrometry.

In this study, a novel analytical approach for the determination of 11 monohydroxylated polycyclic aromatic hydrocarbon metabolites (OH-PAHs) in urine was developed and validated. The rapid, simple and high-throughput sample preparation procedure based on ethyl acetate extraction and subsequent purification by dispersive solid-phase extraction (d-SPE) employing a Z-Sep sorbent is used for the first time. For the identification/quantification of target compounds, ultra-high-performance liquid chromatography (U-HPLC) interfaced with tandem mass spectrometry (MS/MS) was applied. The results of validation experiments performed on the Standard Reference Material (SRM) 3673 (organic contaminants in non-smokers' urine) were in accordance with the certified values. The method recoveries ranged from 77 to 114 % with the relative standard deviation lower than 20 % and the quantification limits in the range of 0.010-0.025 ng mL(-1) (except for benzo[a]pyren-3-ol with 0.9 ng mL(-1)). Within the pilot study, the new method was used for the analysis of OH-PAHs in 50 urine samples. The concentrations of ΣOH-PAHs were in the range of 0.87-63 ng mL(-1) (1600-33,000 ng g(-1) creatinine), with naphthalen-2-ol (2-OH-NAP) and phenanthren-1-ol (1-OH-PHEN) being the most abundant exposure biomarkers detected in all samples.

Modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis.

A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This resulted in an optimised protocol that was able to recover all viruses present in the mock-virome and strongly alters the ratio of viral versus bacterial and 16S rRNA genetic material in favour of viruses (from 43.2% to 96.7% viral reads and from 47.6% to 0.19% bacterial reads). Furthermore, our study indicated that most of the currently used virome protocols, using small filter pores and/or stringent centrifugation conditions may have largely overlooked large viruses present in viromes. We propose NetoVIR (Novel enrichment technique of VIRomes), which allows for a fast, reproducible and high throughput sample preparation for viral metagenomics studies, introducing minimal bias. This procedure is optimised mainly for faecal samples, but with appropriate concentration steps can also be used for other sample types with lower initial viral loads.

Fast and sensitive HPLC method for the determination of neopterin, kynurenine and tryptophan in amniotic fluid, malignant effusions and wound exudates.

A new HPLC method for the determination of neopterin, kynurenine and tryptophan using a second-generation monolith stationary phase and high-throughput sample preparation procedure based on microplates was developed and fully validated. As the stationary phase a monolithic C18 Chromolith high-resolution column with dimensions of 4.6 × 100 mm connected to a monolithic 4.6 × 10-mm security guard was used. Separation was achieved using 15 mM phosphate buffer (KH2PO4 +K2HPO4·3H2O at pH 3) and acetonitrile in gradient mode. Target analytes were determined in 5.5 minutes in amniotic fluid, effusions and wound exudates with a limit of quantification (LOQ) of 1.25 nM for neopterin, 2.5 µM for tryptophan and 0.25 µM for kynurenine. The method was applied to real clinical sample measurements, and it will be used to monitor neopterin, kynurenine and tryptophan levels in biological fluids to assess the patient response to therapy and clinical status.

High-throughput glycomics: optimization of sample preparation.

Glycosylation affects structure, folding, and function of numerous proteins. Aberrant glycosylation has been shown to be associated with different diseases. A wide range of analytical methods is available for glycan analysis of antibodies (mainly IgG), but analysis of plasma glycans is less established due to additional challenges encountered with higher complexity of the sample. Here we describe development and optimization of a high-throughput sample preparation method for hydrophilic interaction liquid chromatography and ultra-performance liquid chromatography analysis of plasma N-glycans. Clean-up of labeled glycans was found to be the largest source of variation, and we tested cellulose, silica gel, Bio-Gel, and hydrophilic GHP filter as stationary phases for solid-phase extraction. All stationary phases were shown to be suitable for purification of labeled glycans, but GHP filter plate in combination with cold 96% acetonitrile had the highest reproducibility and was easiest to work with. The method was further optimized with Plackett-Burman screening design and validated in terms of analysis of major step variation and between-day and between-person variation. The developed method is fast, cost-effective, and easy to perform, and it has very good reproducibility during long period of time, enabling the detection of biological variability of the plasma N-glycome.

Screening of Chlamydomonas reinhardtii Populations with Single-Cell Resolution by Using a High-Throughput Microscale Sample Preparation for Matrix-Assisted Laser Desorption Ionization Mass Spectrometry.

The consequences of cellular heterogeneity, such as biocide persistence, can only be tackled by studying each individual in a cell population. Fluorescent tags provide tools for the high-throughput analysis of genomes, RNA transcripts, or proteins on the single-cell level. However, the analysis of lower-molecular-weight compounds that elude tagging is still a great challenge. Here, we describe a novel high-throughput microscale sample preparation technique for single cells that allows a mass spectrum to be obtained for each individual cell within a microbial population. The approach presented includes spotting Chlamydomonas reinhardtii cells, using a noncontact microarrayer, onto a specialized slide and controlled lysis of cells separated on the slide. Throughout the sample preparation, analytes were traced and individual steps optimized using autofluorescence detection of chlorophyll. The lysates of isolated cells are subjected to a direct, label-free analysis using matrix-assisted laser desorption ionization mass spectrometry. Thus, we were able to differentiate individual cells of two Chlamydomonas reinhardtii strains based on single-cell mass spectra. Furthermore, we showed that only population profiles with real single-cell resolution render a nondistorted picture of the phenotypes contained in a population.

The potential of electromembrane extraction for bioanalytical applications.

Modern requirements in the field of bioanalysis often involve miniaturized, high-throughput sample preparation techniques that consume low amounts of both sample and potentially hazardous organic solvents. Electromembrane extraction is one technique that meets several of these requirements. In this principle analytes are selectively extracted from a biological matrix, through a supported liquid membrane and into an aqueous acceptor solution. The whole extraction process is facilitated by an electric field across the supported liquid membrane, which greatly reduces the extraction time. This review will give a thorough overview of recent advances in bioanalytical applications involving electromembrane extraction, and discuss both possibilities and challenges of the technique in a bioanalytical setting.

Determination of selected veterinary antimicrobials in poultry excreta by UHPLC-MS/MS, for application in Salmonella control programs.

The most important source of Salmonella spp. infection in humans is by the consumption of contaminated poultry products. Due to the risk of resistance development and its transfer from animals to humans, the Belgian Royal Decree concerning the eradication of Salmonella (C-2007/22784) prohibits treatment of poultry with antimicrobials against zoonotic Salmonella spp. To uncover illicit use, an analytical method using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the determination of antimicrobial residues in poultry excreta was developed and validated for classes having an active spectrum against Salmonella spp. in poultry: β-lactams (amoxicillin and penicillin V), fluoroquinolones (enrofloxacin, difloxacin, and flumequine), polymyxins (colistin), sulfonamides in combination with trimethoprim (sulfachloropyridazine, sulfadiazine, and sulfaclozine), and tetracyclines (chlortetracycline and doxycycline). A generic and high-throughput sample preparation was developed. Extraction of samples was performed by ultrasonication using a combination of acetonitrile and McIlvaine buffer, followed by centrifugation and filtration prior to analysis. The method was validated according to Commission Decision 2002/657/EC for linearity, apparent recovery/trueness, repeatability, reproducibility, limit of quantification, limit of detection, specificity, matrix effect, and storage stability in matrix. To demonstrate the applicability of the method, an in vivo experiment was conducted. For each antimicrobial class, one registered drug was selected and administered in the drinking water to two laying hens. Excreta samples were collected every 12h during and until 2days after treatment and analyzed using the developed method.

Extraction and sample preparation.

As everyone knows, the role played by extraction and sample preparation in the analytical sciences cannot be overemphasized. Despite tremendous advances in chromatography, detection, and other aspects of analysis, extraction and sample preparation remain a preanalysis ritual of critical importance. It has been estimated that around 50% to 70% possibly even more of the time and effort that goes into an analytical process comprises extraction and sample preparation. Sample preparation procedure can vary in the degree of selectivity, speed, and convenience, depending on the approach and conditions used, as well as on the geometric configurations of the extraction phase and conditions. Proper design of the extraction devices and procedures facilitates rapid and convenient on-site implementation, coupled with separation/quantification and/or automation. This special issue addresses the research studies on the sample preparation, analytical extraction, and sample clean-up techniques. For example, M. Jablonska-Czapla reported a review on chemical speciation and provided numerous examples of the hyphenated technique usage (e.g., the LC-ICP-MS application in the speciation analysis of chromium, antimony, arsenic, or thallium in water and bottom sediment samples). T. Perez-Palacios et al. investigated the use of a mixer mill as the homogenization tool for the extraction of free amino acids in meat samples, with the main goal of analyzing a large number of samples in the shortest time and minimizing sample amount and solvent volume. It takes less time and requires lower amount of sample and solvent than conventional techniques. N. Sher et al. studied colorimetric visible spectrophotometric quantification methods for amino acids, namely, tranexamic acid and pregabalin. Both drugs contain the amino group, and when reacted with 2,4-dinitrophenol and 2,4,6-trinitrophenol they give rise to yellow colored complexes showing absorption maximum at 418 nm and 425 nm, respectively, based on the Lewis acid base reaction. J. A. Rodriguez et al. reported magnetic solid phase extraction of tartrazine from nonalcoholic beverages. The method involves the extraction and clean-up by activated carbon covered with magnetite dispersed in the sample, followed by the magnetic isolation and desorption of the analyte by basified methanol. The proposed methodology saves time and is less expensive than the reference method. M. Khorshid et al. reported QuEChERS (quick, easy, cheap, effective, rugged, and safe) method for extraction followed by solid phase extraction for sample purification and gas chromatography mass spectrometer, GCMS, for determination of 16 PAHs in fish at low LOQ level. E. A. Pfannkoch et al. reported combination QuEChERS and SBSE methods for extraction and concentration PAHs from fish and shellfish. I. Amin et al. reported a semiautomated extraction protocol of HCV-RNA using Favorgen RNA extraction kit. The kit provided protocol was modified by replacing manual spin steps with vacuum filtration. The assay performance was evaluated by real time qPCR based on Taqman technology. W. Wang et al. reported a high throughput sample preparation method utilizing mixed-mode solid phase extraction (SPE) in 96-well plate format for the determination of free arachidonic acid in plasma by LC-MS/MS. J. Knutsson et al. reported an uncertainty budget for the determination of fully labile Cu in water using a DGT passive sampler. H.-R. Lee et al. reported two different ionization techniques including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the analysis of cholesteryl esters (CEs). The ESI technique proved to be effective in ionizing more kinds of CEs than the APCI technique. J. Wang et al. evaluated various existing protein extraction buffers with zebrafish liver tumor samples and found that sodium deoxycholate (DOC) based extraction buffer with heat denaturation was the most effective approach for highly efficient extraction of proteins from complex tissues such as the zebrafish liver tumor. B. B. Burckhardt and S. Laeer developed sample preparation exemplified by solid phase extraction for the bioanalytical method development of low-volume assays for pediatric studies according to international agency guidelines. A. Rehman et al. suggested that Oxalis corniculata has good antibacterial, antifungal, and insecticidal properties and can be used for the treatment of infections and control of insects. The plant extracts could be a new source for antibiotics and pesticides with minimum noxious effects on the environment. Further studies may also lead to isolating and characterizing the active compounds of the plant extracts and elucidating their biological mechanisms of action.

An Optimized High Throughput Clean-Up Method Using Mixed-Mode SPE Plate for the Analysis of Free Arachidonic Acid in Plasma by LC-MS/MS.

A high throughput sample preparation method was developed utilizing mixed-mode solid phase extraction (SPE) in 96-well plate format for the determination of free arachidonic acid in plasma by LC-MS/MS. Plasma was mixed with 3% aqueous ammonia and loaded into each well of 96-well plate. After washing with water and methanol sequentially, 3% of formic acid in acetonitrile was used to elute arachidonic acid. The collected fraction was injected onto a reversed phase column at 30°C with mobile phase of acetonitrile/water (70 : 30, v/v) and detected by LC-MS/MS coupled with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The calibration curve ranged from 10 to 2500 ng/mL with sufficient linearity (r 2 = 0.9999). The recoveries were in the range of 99.38% to 103.21% with RSD less than 6%. The limit of detection is 3 ng/mL.

Solid-phase extraction strategies to surmount body fluid sample complexity in high-throughput mass spectrometry-based proteomics.

For large-scale and standardized applications in mass spectrometry- (MS-) based proteomics automation of each step is essential. Here we present high-throughput sample preparation solutions for balancing the speed of current MS-acquisitions and the time needed for analytical workup of body fluids. The discussed workflows reduce body fluid sample complexity and apply for both bottom-up proteomics experiments and top-down protein characterization approaches. Various sample preparation methods that involve solid-phase extraction (SPE) including affinity enrichment strategies have been automated. Obtained peptide and protein fractions can be mass analyzed by direct infusion into an electrospray ionization (ESI) source or by means of matrix-assisted laser desorption ionization (MALDI) without further need of time-consuming liquid chromatography (LC) separations.

High throughput quantification of prohibited substances in plasma using thin film solid phase microextraction

Simple, fast and efficient sample preparation approaches that allow high-throughput isolation of various compounds from complex matrices are highly desired in bioanalysis. Particularly sought are methods that can, without sacrificing time, easily remove matrix interferences capable of inducing ionization suppression/enhancement, or causing detrimental effects in instrumental performance. In this work, an automated high-throughput sample preparation method using thin film solid phase microextraction (SPME) for the analysis of multiple prohibited substances in plasma is proposed. A biocompatible SPME extraction phase made of hydrophilic–lipophilic balance particles immobilized with polyacrylonitrile (PAN) demonstrated satisfactory extraction capabilities for 25 compounds of a wide range of polarities (logP from −2 to 6.8). Due to the well-known biocompatible characteristics of PAN-based SPME coatings, minimum sample handling was required. Experimental conditions for pre-conditioning, extraction, wash and desorption were carefully optimized for the proposed method. By taking full advantage of the 96 thin film handling capability of the automated system, a preparation time of approximately 1.5min per sample can be achieved. Satisfactory results in terms of absolute matrix effects were found for the majority of the studied analytes, given that 24 out of 25 compounds exhibited values in the range of 100 and 120%. The method was validated in terms of linearity (R2>0.99), inter and intra-day accuracy (85–130%) and precision (<20%) and limits of quantitation (0.25–10ngmL−1 for most compounds).