Multiparallel microfluidic high-performance liquid chromatography for high-throughput normal-phase chiral analysis

The suitability of the Eksigent Express 800 microfluidic eight-channel HPLC instrument for multiparallel normal-phase chiral analysis in support of high-throughput pharmaceutical process research was investigated. Analysis of test mixtures containing the two enantiomers of benzoin and the closely related (R,S)-dihydrobenzoin, was carried out in a 96-well microplate, affording rapid (<2 h) and accurate assessment of enantiopurity. In a second example, use of the instrument to support high-throughput catalyst screening of the asymmetric hydrogenation of a prochiral unsaturated ester is presented, in which method development (gradient screening of four columns and two eluents, followed by optimization to afford a fast analytical method) and analysis of a 96-well microplate was carried out within a single working day. This represents a considerable improvement over conventional analysis techniques that usually take several days to complete.     

High-throughput manual parallel synthesis using SynPhase crowns and lanterns

The high-throughput manual solid-phase parallel synthesis of libraries comprising thousands of discrete samples using pellicular supports (i.e. SynPhase crowns and lanterns) and a suite of novel tools and techniques is described. Key aspects of this approach include the combination of a split-split-split synthesis strategy with spatial encoding to differentiate thousands of crowns, the rapid washing and filtration of up to 48 reaction vessels in parallel, the application of an inexpensive and environmentally friendly technique to remove trifluoroacetic acid from sixteen 96-well plates in parallel, and a high-throughput method for removing cleaved crowns from reusable pin racks. Tens of thousands of discrete samples have been produced in-house using this conceptually and operationally straightforward strategy.     

Determination of concentration and enantiomeric excess of amines and amino alcohols with a chiral nickel(II) complex

A chiral N,N'-dioxide-Ni(II) complex was found to exhibit highly enantioselective fluorescent recognition of chiral amines and amino alcohols. It can be used to determine their concentration and ee value. Using a 96-well plate, high-throughput screening for enantiomer discrimination was accomplished.     

A screening method for chiral selectors that does not require covalent attachment

A high-throughput screening protocol is proposed for chiral selector discovery. It is modeled after the protocol for biological screening of candidate drugs from chemical libraries. The procedure works based on target distribution between an aqueous phase and an organic phase. The target may be a racemate or separate enantiomers. Screening for noncovalent intermolecular association between target and candidate selectors is carried out by partitioning experiments in the presence and absence of the candidate chiral selectors in the organic phase (plasticized poly(vinyl chloride)). The partition ratio measurement uses 96-well plates for high throughput. The feasibility of this approach is validated by working with a known target/chiral selector pair, N-(3,5-dinitrobenzoyl)-alpha-phenylglycine and 2,2,2-trifluoro-1-(9-anthryl)ethanol. The validated protocol is applied to a small library of 12 cyclopropyl dipeptide isosteres. Eight bind the racemic target, econazole. Among them, one has measurable chiral selectivity. The advantage of the method is that it does not require the covalent attachment of either the analyte or the selector, and the required amount of the potential chiral selector is about 100 mug.     

Optimization of in-gel protein digestion system in combination with thin-gel separation and negative staining in 96-well plate format

Improvement of in-gel digestion efficiency is highly desirable for one- or two-dimensional gel electrophoretic separation and mass spectrometric (MS) analysis in proteomics, because the resultant increases in sequence coverage and MS signal intensity lead to higher confidence in protein identification. Here an optimized in-gel digestion system, in combination with thin-gel separation and negative staining in a high-throughput format using 96-well plates, is described. The combination of negative staining and protein separation on a 0.9 mm thick gel showed a clear improvement in in-gel digestion efficiency in comparison with the more typical protocols such as the combination of silver staining and a 1.0 mm gel. In addition, the use of 96-well plates to increase throughput did not decrease the efficiency of this strategy when the stirring of the gel pieces in processes such as destaining, washing, gel-shrinking and peptide extraction was performed by sonication instead of shaking the plates. This procedure was optimized and applied to identify proteins of the postsynaptic density fraction; 105 proteins were identified after SDS-PAGE separation.     

A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.     

Automated DBS Extraction Prior to Hilic/RP LC–MS/MS Target Screening of Drugs

This article describes a rapid LC–MS/MS target screening method based on an automated extraction of 5 μL dried blood spots (DBS), two 5 min chromatographic runs on orthogonal phase columns (RP and Hilic) and a data dependent acquisition (DDA) of product ions spectra for the reliable identification of the detected compounds. The extraction step was performed in 2 min by using the LC autosampler itself in 96-well plates. This procedure was evaluated using 22 model compounds frequently encountered in forensic investigations, i.e., cocaine, benzodiazepines, amphetamines, opioids, antidepressants and antipsychotics. These investigations showed that even if the extraction step was reduced to a minimum, the extraction recoveries were satisfactory (median value of 40 %) and allowed for the detection of the model compounds in their therapeutic ranges, with the exception of morphine. Moreover, the use of two different chromatographic columns broadened the number of screening targets to those that behaved poorly under RP conditions, such as amphetamines or glucuronides, while keeping chromatographic gradients very short. This procedure was applied to 34 authentic post-mortem cases. It allowed the detection of 89 % of the compounds that were quantified in the routine procedures and the formal identification of 77 % of the compounds using their product ions spectra. These results were considered more than satisfactory compared to routine screening alone (GC–MS and LC-DAD, 55 % compound identification). The method described in this article is therefore a powerful approach for a fast, reliable and efficient target screening of drugs in forensic and clinical investigations.     

Chiral liquid chromatography-mass spectrometry for high-throughput screening of enzymatic racemase activity

In finding suitable biocatalysts for processes in chemical industry, expression libraries are constructed containing typically >10,000 clones. Search for a desired activity is done by examination of all the clones in one or more libraries using a high-throughput screening assay. Here we describe a method for the screening of the enzymatic racemase activity of clones from an expression library on alpha-amino-epsilon-caprolactam (ACL) using a fast chiral LC separation and ionspray-MS as the detection technique. After substrate incubation with S-ACL, the 96-well microplates were centrifuged to remove cell material. The conversion of S-ACL to R-ACL was monitored by quantitation of the R-ACL enantiomer. Separation of the two ACL enantiomers was performed on a Crownpak CR+ column within 1 min. A Gilson 215 autosampler with a 889 multiple injection probe was used for injecting the samples into the LC system. The total analysis time for a 96-well microplate was 56 min. The MS was operated in the positive-ion mode using selected ion monitoring at m/z 129 [M+H]+ of ACL. Using this method over 12,000 samples were analyzed without loss in performance of the system. The LC column remained stable without loss of resolution and the MS system did not show loss in sensitivity throughout the screening. Inter-day reproducibility was within 15%.     

Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.     

Automated carbohydrate profiling by capillary electrophoresis: a bioindustrial approach

Automated, high-resolution, quantitative, high-throughput analysis of mono- and oligosaccharides, produced by enzymatic digestion of cellohexaose (model substrate) and lignocellulosic biomass, is demonstrated using high-performance capillary electrophoresis in conjunction with a single-step fluorophore labeling strategy for sensitive laser-induced fluorescence detection. Unattended batch sample processing from 96-well plates enabled reliable industrial-scale carbohydrate analysis. Excellent resolution of mono- and oligosaccharides was achieved under suppressed electroosmotic flow conditions, using either covalently or dynamically coated fused-silica capillary columns. The proposed approach readily supports the demands of bioindustrial operation environments with respect to high-throughput carbohydrate profiling.     

A simple and affordable method for high-throughput DNA extraction from animal tissues for polymerase chain reaction

We have developed a very simple and inexpensive method for high-throughput DNA extraction from animal tissues. The procedure contains three steps (digestion, heating, and centrifugation) and it is compatible with the 96-well plate format commonly used in polymerase chain reaction (PCR) amplifications. The duration for processing a plate is about 1.5 h; therefore, one researcher can isolate DNA from up to 1000 samples during a single workday. A small piece of tissue (ca. 10-20 mg) yields enough template for at least 50-70 PCR amplifications, as demonstrated by using the processed samples as templates successfully for long distance PCR, multiplex PCR, and randomly amplified polymorphic DNA (RAPD) assay. The application of our method is expected to facilitate studies that require high-throughput DNA isolation for PCR amplification, such as genotyping by microsatellites for mapping and genetic diversity studies, as well as mutant screening in zebrafish.     

"Paralogs", sorbent families for protein separations.

Novel peptide-based (paralog) sorbents are evaluated with respect to performance, reproducibility and reusability in a 96-well test plate screening format, and to utility in protein separations. The results demonstrate that this approach to constructing sorbents provides a new and generally applicable set of tools for separating proteins.     

High-throughput sample preparation and analysis using 96-well membrane solid-phase extraction and liquid chromatography-tandem mass spectrometry for the determination of steroids in human urine

A 96-well solid-phase extraction (SPE) system is used to rapidly prepare human urine samples for high-throughput quantitative analysis of two steroids, equilenin and progesterone, by liquid chromatography-tandem mass spectrometry using deuterated estrone as the internal standard. We define high-throughput here as analysis of 384 samples in a 24 h period. A total of 384 samples and standards were extracted by an individual in one day and subsequently analyzed within a 24 h period. The inter- and intratray accuracy and precision obtained over the course of these injections was within 8% coefficient of variation when analyzed by atmospheric pressure chemical ionization mass spectrometry using positive ion detection. A semiautomated sample processing workstation was used to add internal standard and then process 96 samples at a time. The recovery of the analytes from the SPE was approximately 85%. The accuracy and precision obtained was comparable to that ordinarily obtained using manual sample preparation techniques.     

Miniaturization of a panel of high-throughput yeast-cell-based nuclear receptor assays in 384- and 1536-well microplates

A panel of luminescent Saccharomyces cerevisiae cell-based nuclear receptor assays, consisting of human estrogen receptors α and β, androgen receptor, and aryl hydrocarbon receptor, was miniaturized from the standard 96-well microplate format to high-throughput 384- and 1536-well microplate formats. In these assays, firefly luciferase lacking the peroxisome targeting sequence was used as a reporter and D-luciferin substrate was pre-mixed with the yeast cells before the incubation step, eliminating cell lysis and substrate addition steps, and allowing multiple readings at any desired time point. All of the assays were highly functional in the 384-well format, and most functioned well in the 1536-well format. The detection limit of the estrogen receptor α assay was even lower in the miniaturized microplate formats than in the original 96-well format. The panel of yeast-cell-based nuclear receptor assays can be used for high-throughput chemical testing and environmental monitoring of potential endocrine-disrupting activity of compounds and samples.     

A high-throughput microchip-based glycan screening assay for antibody cell culture samples

A high-throughput screening assay was developed to quantify major glycan species in the crude mammalian cell culture samples for monoclonal antibodies (mAbs). This method utilizes high-speed microchip electrophoresis separation following a fast sample preparation procedure. Using a 96-well ultra-filtration membrane, interfering species in the cell culture media were efficiently removed as the samples were concentrated. A commercial microchip electrophoresis instrument was used for high-speed separation, allowing each sample to be analyzed in less than 1 min. This method is well suited for the purpose of high-throughput antibody glycan profiling during cell culture expression, including clone selection and cell culture process optimization. The relative levels of high mannose (HM), fucosylated and galactosylated glycan species in the Fc domain can be determined for hundreds of crude cell culture samples in a few hours.     

High-throughput quantitative bioanalysis by LC/MS/MS

This review article discusses the most recent significant advances in the sample preparation and mass spectrometry aspects of high-throughput bioanalysis by LC/MS/MS for the quantitation of drugs, metabolites and endogenous biomolecules in biological matrices. The introduction and implementation of automated 96-well extraction has brought about high-throughput approaches to the biological sample preparation techniques of solid-phase extraction, liquid-liquid extraction and protein precipitation. The fast-flow on-line extraction technique is a different high-throughput approach that has also significantly speeded up analysis by LC/MS/MS. The use of pierceable caps for biological tubes further enhances the analysis speed and improves the safety in handling biological samples. The need for adequate chromatographic separation in order to eliminate interferences due to metabolites and/or matrix effects in LC/MS/MS is discussed. To highlight our limited understanding of atmospheric pressure ionization mass spectrometry, results from recent investigations that appear to be counter-intuitive are presented. Looking ahead to the future, multiplexed LC/MS/MS systems and capillary LC are presented as areas that can bring about further improvements in analysis speed and sensitivity to quantitative bioanalysis by LC/MS/MS.     

Development and validation of a LC-MS/MS method based on a new 96-well Hybrid-SPE<Superscript>™</Superscript>-precipitation technique for quantification of CYP450 substrates/metabolites in rat plasma

A rapid and selective high-throughput HESI-LC-MS/MS method for determining eight cytochrome P450 probe drugs in one-step extraction and single run was developed and validated. The four specific probe substrates midazolam, dextromethorphan, tolbutamide, theophylline and their metabolites 1-hydroxymidazolam, dextrorphan, hydroxyl(methyl)tolbutamide, 1,3-dimethyluric acid, together with the deuterated internal standards, were extracted from rat plasma using a novel 96-well Hybrid-SPE™-precipitation technique. The bioanalytical assay was based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using selected reaction monitoring for drug (-metabolite) quantification. All analytes were separated simultaneously in a single run that lasted less than 11 min. The intra- and inter-day precisions for all eight substrates/metabolites were 1.62–12.81% and 2.09–13.02%, respectively, and the relative errors (accuracy) for the eight compounds ranged from −9.62% to 7.48% and −13.84% to 8.82%. Hence, the present method provides a robust, fast and reproducible analytical tool for the evaluation of four major drug metabolising cytochrome P450 (3A4, 2C9, 1A2 and 2D6) activities with a cocktail approach in rats to clarify herb–drug interactions. The method can be used as a basic common validated high-throughput analytical assay for in vivo interaction studies.     

A fluorescent high-throughput assay for double-stranded DNA: the RediPlate PicoGreen assay

The fluorescent PicoGreen reagent for detection and quantitation of double-stranded DNA has been adapted for high-throughput screening: the RediPlate PicoGreen double-stranded DNA assay format. In the RediPlate PicoGreen assay format, the PicoGreen reagent is predistributed and co-dried into either 96- or 384-well microplates with the excipient trehalose. The user resuspends the dried reagents upon adding DNA, and measures the resulting fluorescence after a five minute incubation. Replicate fluorescence measurements on nominally identical wells have less than a 5% coefficient of variation. The assay is linear from 5 to 500 ng/ml DNA in a 200 micro l volume. The RediPlate PicoGreen assay format retains the advantages of the original PicoGreen reagent - sensitivity, speed, and specificity - but in a high-throughput format.     

A 96-well Plate Based Dot-ELISA Array for Simultaneous Detection of Multi-Drugs

Abstract

Drug abuse is rising day by day and becoming a big problem in countries around the globe. The existing drug testing methods could not effectively cope with the problems in the sensitivity and specificity of the diversified drugs. Dot-ELISA, as a complement of ELISA, has been widely used in many fields. In this study, we optimized and applied the Dot-ELISA technique as a maneuverable assay for rapid detection of multiple drug abuses via a kind of 96-well plates-based Dot-ELISA array. In total 92 positive urine samples were tested with high-throughput and all results were confirmed by commercial drug testing kits.     

Peptide protein binding assay using ImageFlashPlates or Imaging Beads

Imaging devices used for the measurement of radioligand-receptor binding assays are typically based on charge-coupled device (CCD) cameras, which are more sensitive for red-shifted scintillation. In the past, red-shifted scintillants had only been integrated into microspheres, referred to as scintillation proximity assay (SPA) Imaging Beads. More recently, ImageFlashPlates have been developed that emit light at 615 nm when exposed to beta-radiation. In this article, we report the establishment of peptide-protein binding assays using either streptavidin-coated ImageFlashPlates or Imaging Beads in a low volume 384-well format. In these assays, we employed a biotinylated peptide X and a [33P]-phosphorylated protein Y as the binding partner. The FlashPlates required a washing step, the bead-filled microtiter plates (MTPs) needed a centrifugation step for optimal performance in the scintillation measurements. Both the peptide X-loaded FlashPlates and the beads displayed saturable binding of [33P]-phosphorylated protein Y with a similar scintillation efficiency. A KD value of about 30 nmol/l was measured using the bead-based assay. Due to the washing step in the FlashPlate experiment, approximately two-thirds of the [33P]-phosphorylated protein Y were withdrawn from equilibrium binding. This resulted in correspondingly lower scintillation signals for the FlashPlate experiment. For this reason, the FlashPlate produced a Z' value of 0.64 that was lower than the Z' value of 0.87 for the beads. Using a reference inhibitor in a competition assay produced similar IC50 values for the bead-based assay as for the FlashPlate. Depending on the local automation environment either the centrifugation step for the beads or the washing step for the FlashPlates may be considered more or less of a challenge. Low volume 384-well high-throughput screening (HTS) applicable assay formats are achievable using either the ImageFlashPlates or the Imaging Beads.