Combined approach for high-throughput preparation and analysis of plasma samples from exposure studies

In drug discovery today, drug exposure is determined in preclinical efficacy and safety studies and drug effects are related to measured concentrations rather than to the administered dose. This leads to a strong increase in the number of bioanalytical samples, demanding the development of higher throughput methods to cope with the increased workload. Here, a combined approach is described for the high-throughput preparation and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of drug levels in plasma samples from the preclinical efficacy and safety studies, i.e. exposure studies. Appropriate pharmacokinetic (PK) compartmental models were fitted to data from PK screening studies in the rat, which were subsequently used to simulate the expected plasma concentrations of the respective exposure studies. Information on the estimated drug concentrations was used to dilute the samples to appropriate concentration levels. A Tecan Genesis RSP liquid handling system was utilized to perform automated plasma sample preparation including serial dilution of standard solutions, dilution of plasma samples, addition of internal standard solution and precipitation with acetonitrile. This robotic sample preparation process permitted two studies of 1-96 samples each to be run simultaneously. To ensure the performance of this method the accuracy and precision for diazepam were examined. Two novel drugs were used to illustrate the suggested approach. In conclusion, our method for sample preparation of exposure samples, based on the combined use of PK simulations, a liquid handling system and a fast LC/MS/MS method, increased the throughput more than three times and minimized the errors, while maintaining the required accuracy and precision.     

High-throughput purification of combinatorial libraries I: a high-throughput purification system using an accelerated retention window approach

We have developed a high-throughput purification system to purify combinatorial libraries at a 50-100-mg scale with a throughput of 250 samples/instrument/day. We applied an accelerated retention window method to shorten the purification time and targeted one fraction per injection to simplify data tracking, lower QC workload, and simplify the postpurification processing. First, we determined the accurate retention time and peak height for all compounds using an eight-channel parallel LC/UV/MS system, and calculated the specific preparative HPLC conditions for individual compounds. The preparative HPLC conditions include the compound-specific gradient segment for individual compounds with a fixed gradient slope and the compound-specific UV or ELSD threshold for triggering a fraction collection device. A unique solvent composition or solvent strength was programmed for each compound in the preparative HPLC in order to elute all compounds at the same target time. Considering the possible deviation of the predicted retention time, a 1-min window around the target time was set to collect peaks above a threshold based on UV or ELSD detection. Dual column preparative instruments were used to maximize throughput. We have purified more than 500 000 druglike compounds using this system in the past 3 years. We report various components of this high-throughput purification system and some of our purification results.     

Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter

The integration of complete analyses systems "on chip" is one of the great potentials of microfabricated devices. In this study we present a new pressure-driven microfabricated fluorescent-activated cell sorter chip with advanced functional integration. Using this sorter, fluorescent latex beads are sorted from chicken red blood cells, achieving substantial enrichments at a sample throughput of 12000 cells s(-1). As a part of the sorter chip, we have developed a monolithically integrated single step coaxial flow compound for hydrodynamic focusing of samples in flow cytometry and cell sorting. The structure is simple, and can easily be microfabricated and integrated with other microfluidic components. We have designed an integrated chamber on the chip for holding and culturing of the sorted cells. By integrating this chamber, the risk of losing cells during cell handling processes is eliminated. Furthermore, we have also developed integrated optics for cell detection. Our new design contributes to the ongoing efforts for building a fully integrated micro cell sorting and analysing system.     

A novel method for immediate post-purification purity determination of fractions collected during high-throughput purification

Following purification, the fractions of purified samples typically are analyzed to determine the relative purities of each fraction. We report a novel technique for performing post-purification analysis immediately after each preparative LC/MS run. The Single Pass Compound Purification and Analysis System (SPACPASS) samples and stores a representative aliquot from the fraction while it is being collected. Demonstrated for '1:1' fraction collections, this method of fraction purity assessment streamlined sample processing by reducing post-purification sample handling. For 97% of the collected fractions, this technique provided relative purities to within +/-5% when compared with more traditional post-purification analysis.     

Ternary-column system for high-throughput direct-injection bioanalysis by liquid chromatography/tandem mass spectrometry

As a continuation of our efforts to improve our high-flow on-line bioanalytical approach for high-throughput quantitation of drugs and metabolites in biological matrices by high-performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have developed a ternary-column on-line LC/MS/MS system with dual extraction columns used in parallel for purification and an analytical column for analysis. The advantage of the dual extraction column system is that sample analysis can take place in one of the extraction columns while the other column is being equilibrated. Thus, the equilibration time does not add to the run time, hence shortening the injection cycle time and increasing the sample throughput. Moreover, the use of two extraction columns in parallel increases the number of samples that can be injected before the system fails due to an overused extraction column. Such a system has successfully been used to develop and validate a positive ion electrospray LC/MS/MS bioanalytical method for the quantitative determination of a guanidine-containing drug candidate in rat plasma. The system used for this work utilized two Oasis HLB extraction columns (1 x 50 mm, 30 microm), one C18 analytical column (3.9 x 50 mm, 5 microm), a ten-port switching value and a tandem mass spectrometer. The on-line analysis was accomplished by the direct injection of 10 microL of the sample, obtained by mixing a rat plasma sample 1:1 with an aqueous internal standard solution. Selected reaction monitoring (SRM) was utilized for the detection of the analyte and internal standard. The standard curve range was 1.00-200 ng/mL. The intra- and inter-day precision and accuracy were within 6.6%. The on-line purification step lasted for only 0.3 min and total run time was only 1.6 min.     

Maximizing automation in LC/MS high-throughput analysis and purification

Here, we describe a system for LC/MS-based analysis and purification of compounds aiming at the minimization of manual interference in the overall process. Key elements of the concept are automated identification of the target compounds, automated assignment of optimized preparative gradients for purification of the target compounds, and automated purity assessment of fractions with subsequent pooling of validated product fractions. Additional support is provided by an automated solvent and waste management system. One person can easily process 100-200 compounds on a 150-mg scale per day on that system, while still the maximization of purity and yield after purification is guaranteed. Reduced demands with respect to purity or yield can lead to significantly higher throughput numbers.     

Rapid and high throughput separation technologies--steady state recycling and supercritical fluid chromatography for chiral resolution of pharmaceutical intermediates

The SSR and SFC techniques were used for the enantiomeric resolution of three pharmaceutical intermediates at various sample scales. The separation conditions, the sample purities and yields, the productivities and the solvent consumptions were discussed in three case studies in this paper. In case (I), the SSR process was used for a low selectivity resolution of 2.0 kg of pharmaceutical intermediate. By using this separation process, a productivity of 750 g racemate/kg stationary phase/day was achieved, while solvent usage was minimized ( approximately 200 l/kg racemate). Case (II) pertained to the effectiveness of the SSR process. Productivity using SSR techniques increased by a factor of 4.5, while solvent usage decreased by a factor of 4.1 when compared to the productivity and solvent usage of batch HPLC. Case (III) compared SFC purification to HPLC purification. The SFC process was more effective in terms of an increase in productivity and a reduction in solvent usage. Based on these results, it appears that SSR and SFC are very useful choices at the early stage of the drug development for a high throughput and a rapid turn around of samples.     

Acoustic deposition with NIMS as a high-throughput enzyme activity assay

Mass spectrometry (MS)-based enzyme assay has been shown to be a useful tool for screening enzymatic activities from environmental samples. Recently, reported approaches for high-specificity multiplexed characterization of enzymatic activities allow for providing detailed information on the range of enzymatic products and monitoring multiple enzymatic reactions. However, the throughput has been limited by the slow liquid-liquid handling and manual analysis. This rapid communication demonstrates the integration of acoustic sample deposition with nanostructure initiator mass spectrometry (NIMS) imaging to provide reproducible measurements of multiple enzymatic reactions at a throughput that is tenfold to 100-fold faster than conventional MS-based enzyme assay. It also provides a simple means for the visualization of multiple reactions and reaction pathways.     

高通量蛋白质组学分析研究进展

基于质谱的蛋白质组学技术已经日趋成熟,可以对细胞和组织中的成千上万种蛋白质进行全面的定性和定量分析,逐步实现“深度覆盖”。随着生物医学日益增长的大队列蛋白质组学分析需求,如何在保持较为理想的覆盖深度下实现短时间、快速的“高通量”蛋白质组学分析已成为当前亟需解决的关键问题之一。常规的蛋白质组学分析流程通常包括样品前处理、色谱分离、质谱检测和数据分析。该文从以上4个方面展开介绍近10年以来高通量蛋白质组学分析技术取得的一系列研究进展,主要包括:(1)基于高通量、自动化移液工作站的蛋白质组样品前处理方法;(2)基于微升流速液相色谱与质谱联用的高通量蛋白质组检测方法;(3)利用灵敏度高、扫描速度快的质谱仪实现短色谱梯度分离下蛋白质组深度覆盖的分析方法;(4)基于人工智能、深度神经网络、机器学习等的蛋白质组学大数据分析方法。此外,对高通量蛋白质组学面临的挑战及其发展进行展望。总而言之,预期在不久的将来高通量蛋白质组学技术将会逐步“落地转化”,成为大队列蛋白质组学分析的利器。     

Cycling gradient capillary electrophoresis: a low-cost tool for high-throughput analysis of genetic variations

In the present work, we introduce a new type of DNA variation detection. This method represents a transfer of melting gel technique onto multicapillary electrophoresis DNA sequencing instrument with further improvements to achieve maximum sample throughput while maintaining a high performance. The main improvement comes from application of cycling (revolving) temporal temperature gradient in place of a single-sweep gradient, commonly used in similar gel-based techniques. This improvement enables utilization of multiple-injection technique, in which multiple samples are injected into the same capillary (or sets of capillaries) separated by predefined time intervals of partial electrophoresis. The periodic oscillation of the temperature results in identical separation conditions of all samples injected in such series. Using this novel approach, we demonstrate a dramatic increase in separation throughput by turning a standard commercial 96-capillary array instrument into a semicontinuous flow mutation detection system capable to screen over 15 000 samples in 24 h of operation on a single 96-capillary commercial instrument. This represents a 10-fold increase in sample throughput over the current comparable technology.     

Application of visual basic in high-throughput mass spectrometry-directed purification of combinatorial libraries

We present an approach to customize the sample submission process for high-throughput purification (HTP) of combinatorial parallel libraries using preparative liquid chromatography electrospray ionization mass spectrometry. In this study, Visual Basic and Visual Basic for Applications programs were developed using Microsoft Visual Basic 6 and Microsoft Excel 2000, respectively. These programs are subsequently applied for the seamless electronic submission and handling of data for HTP. Functions were incorporated into these programs where medicinal chemists can perform on-line verification of the purification status and on-line retrieval of postpurification data. The application of these user friendly and cost effective programs in our HTP technology has greatly increased our work efficiency by reducing paper work and manual manipulation of data.     

Pulse gradient, large-volume injection, high-throughput ultra-performance liquid chromatographic/tandem mass spectrometry bioanalysis for measurement of plasma amrubicin and its metabolite amrubicinol

In this communication, we report the development of a new ultra-performance liquid chromatographic/tandem mass spectrometry (UPLC-MS-MS) assay for measurement of amrubicin (an anthracycline anti-cancer agent) and its active metabolite, amrubicinol, in plasma. The enhanced electrospray ionization signal intensity of the analytes achieved by modifying the mobile phase with formic acid was associated with improvement in the lower limit of quantification. These favorable effects were electrolyte concentration-dependent. In order to maximize assay throughput, we used methanol protein precipitation to prepare the plasma samples, and simplified sample preparation by injecting 40 microL of the supernatant containing methanol at 87.5% (v/v) directly onto the UPLC column without any intermediary solvent evaporation step. The large-volume injection of highly organic supernatant sample increased matrix and elutropic effects, but these drawbacks were respectively overcome by using a 5mM formic acid-modified mobile phase and a new pulse gradient method. To our knowledge, this is the first report successfully using large-volume injection of strong organic samples with UPLC-MS-MS bioanalysis. The pulse gradient elution also resulted in band compression and enhanced the robustness of the chromatography. The promising new approach illustrated herein is extremely straightforward to optimize, and may be used for UPLC-MS-MS bioanalytical assay of other compounds.     

Scale-up of free-flow electrophoresis: II. Purification of alcohol dehydrogenase from a crude yeast extract by field step electrophoresis and combined field step-zone electrophoresis

Results of the purification of alcohol dehydrogenase (ADH) by field step electrophoresis and combined field step-zone electrophoresis are presented. In field step electrophoresis, optimization of voltage, residence time and pH of the sample solution led to a maximal purification factor of 2.8 and a yield of 89% ADH. The limit of loading capacity was reached at a protein concentration of the sample solution of approximately 4 g/L, allowing a maximal throughput of 1.14 g/h with a yield of 86% and a 2.8-fold purification in the Elphor VaP 22 apparatus. With a production scale apparatus a throughput of 2.07 g/h without any loss of separation quality could be achieved. By introducing the sample solution into the separation chamber through 3 inlets, simultaneously, the throughput was increased to 3.2 g/h with a purification factor of 2.7 and a yield of 82% ADH. For the combined field step-zone electrophoresis method a maximum purification factor of 3.6 and a yield of 80% ADH were achieved. The loading capacity was limited to a 4.13 g/L protein concentration of the sample solution, resulting in a throughput of 440 mg/h. Injecting the sample solution simultaneously into 3 inlets resulted in a maximum throughput of 1.92 g/h with 3.1-fold purification and a yield of 80% ADH. Zone electrophoresis, field step electrophoresis and a combination of both are compared with respect to resolution, throughput and the application potential in a protein purification scheme. A scale-up to 3 g/h is possible in zone electrophoresis and field step electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Realities of high-throughput liquid chromatography/mass spectrometry purification of large combinatorial libraries: a report on overall sample throughput using parallel purification

We report on the development of a validated, streamlined high-throughput process for the purification of parallel-synthesis-derived combinatorial libraries. The steps involved in this library purification process include dissolution of dry films of crude synthetic material, dual-column LC/MS purification, dual-column postpurification analysis, quantitation, reformatting, and submission of pure compounds for registration. Although the purification and postpurification analysis times decreased essentially linearly as a function of the number of HPLC columns employed, it was not possible to decrease the total purification process time linearly as a function of the number of columns employed in the system. This was due primarily to the fact that numerous steps in the total purification process are independent of sample analysis and purification (e.g., evaporation, reconstitution, and reformatting, etc.). Additionally, experiments were also performed to assess whether separate gradient pumps were necessary for each channel of this two-channel LC/MS or if acceptable results could be reliably obtained by splitting the flow from one set of gradient pumps between two HPLC columns. On the basis of the parallel, two-column LC/MS system employed in this work, throughput estimates were extrapolated to more massively parallel systems (e.g., four-channel LC/MS).     

Ultrahigh-pressure dual online solid phase extraction/capillary reverse-phase liquid chromatography/tandem mass spectrometry (DO-SPE/cRPLC/MS/MS): a versatile separation platform for high-throughput and highly sensitive proteomic analyses

Capillary RPLC/ESI-MS (cRPLC/ESI-MS) is one of the most powerful analytical tools for current proteomic research. The development of cRPLC techniques coupled online to a mass spectrometer has focused on increasing the separation efficiency, detection sensitivity, and throughput. Recently, the use of high-pressure (over 10,000 psi) LC systems that utilize long, small inner diameter capillary columns has gained much attention for proteomic analyses. In this study, we developed an ultrahigh-pressure dual online SPE/capillary RPLC (DO-SPE/cRPLC) system. This LC system employs two online SPE columns and two capillary columns (75 microm inner diameter x 1 m length) in a single separation system, and has a maximum operating pressure of 10,000 psi. This DO-SPE/cRPLC system is capable of providing high-resolution separation in addition to several other advantageous features, such as high reproducibility in terms of the LC retention time, rapid sample injection, online desalting, online sample enrichment of dilute samples, and increased throughput as a result of essentially removing the column equilibration time between successive experiments. We coupled the DO-SPE/cRPLC system online to a tandem mass spectrometer to allow high-throughput proteomic analyses. In this paper, we demonstrate the efficiency of this DO-SPE/cRPLC/MS/MS system by its use in the analyses of proteomic samples exhibiting different levels of complexity.     

Low-cost multi-core inertial microfluidic centrifuge for high-throughput cell concentration

We developed a low-cost multi-core inertial microfluidic centrifuge (IM-centrifuge) to achieve a continuous-flow cell/particle concentration at a throughput of up to 20 mL/min. To lower the cost of our IM-centrifuge, we clamped a disposable multilayer film-based inertial microfluidic (MFIM) chip with two reusable plastic housings. The key MFIM chip was fabricated in low-cost materials by stacking different polymer-film channel layers and double-sided tape. To increase processing throughput, multiplexing spiral inertial microfluidic channels were integrated within an all-in-one MFIM chip, and a novel sample distribution strategy was employed to equally distribute the sample into each channel layer. Then, we characterized the focusing performance in the MFIM chip over a wide flow-rate range. The experimental results showed that our IM-centrifuge was able to focus various-sized particles/cells to achieve volume reduction. The sample distribution strategy also effectively ensured identical focusing and concentration performances in different cores. Finally, our IM-centrifuge was successfully applied to concentrate microalgae cells with irregular shapes and highly polydisperse sizes. Thus, our IM-centrifuge holds the potential to be employed as a low-cost, high-throughput centrifuge for disposable use in low-resource settings.     

Fast and efficient extraction methods for the analysis of polychlorinated biphenyls and polybrominated diphenyl ethers in biological matrices

This paper describes fast and simple extraction methods for the determination of polychlorinated biphenyls and polybrominated diphenyl ethers in biological matrices. Four extraction protocols were tested. The first protocol used microwave-assisted extraction combined with two purification steps. The second one was similar, except that microwave-assisted extraction was replaced by accelerated solvent extraction. The third one combined extraction/purification by accelerated solvent extraction with final purification on a silica gel column. The last one combined microwave-assisted extraction with purification on an acidic silica gel column. The protocols were tested on various matrices: a spiked matrix, two certified matrices (SRM 2977, WMF 01), and natural matrices (mysids and fish). All of the protocols produced good performance in terms of recovery and reproducibility. The two last protocols showed promising results in terms of applicability to natural matrices, as they required a minimum of sample handling and minimal amounts of solvent and time. These methods allowed at least 24 samples to be handled per day, and could easily be used for routine analysis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Parallel ultra-high flow rate liquid chromatography with mass spectrometric detection using a multiplex electrospray source for direct, sensitive determination of pharmaceuticals in plasma at extremely high throughput

Recent years have seen increasing usage of large particle size stationary phases and ultra-high flow rate liquid chromatography/mass spectrometry (LC/MS) for rapid determination of pharmaceuticals in plasma without prior sample preparation. This lack of sample preparation prior to analysis, together with the extremely high throughput of the chromatography, makes the technique extremely attractive to the bioanalyst. Further, the introduction of multiple sprayer interfaces to mass spectrometers provides the potential for even higher throughput. In this paper, we present parallel ultra-high flow rate liquid chromatography using four columns in parallel and a four-way multiple sprayer interface to the mass spectrometer. We have applied this on both the narrow-bore and capillary scale. This technique enables the quantification of drugs from four plasma samples simultaneously, at nanogram per millilitre concentrations, from small aliquots of plasma without sample preparation and with throughputs of up to 120 samples per hour.     

Free flow electrophoresis as a method for the purification of enzymes from E. coli cell extract

The application of the four techniques of free flow electrophoresis (zone electrophoresis, isotachophoresis, isoelectric focusing and field step electrophoresis) for the purification of proteins from a complex protein mixture was investigated. For this purpose alpha-amylase (EC 3.2.1.1) from Aspergillus oryzae was added and reisolated from E. coli cell extract. The chosen enzyme and the biological extract are models for many industrial separation problems. In optimized experiments purity, purification factor, yield, throughput and efficiency were calculated. The best results were obtained with field step electrophoresis in combination with zone electrophoresis. High purity (0.82 mg enzyme/mg total protein) and high throughput (111 mL sample/h) were achieved using this technique. Field step electrophoresis gave the best throughput (330 mL sample/h), but low purity (0.63 mg enzyme/mg total protein). This technique can also be used for a simple concentration of the sample. With zone electrophoresis a purity of more than 0.95 mg enzyme/mg total protein was obtained, which was the best of all techniques. However, the enzyme concentration was decreased due to dilution with buffer solution after the separation. Isotachophoresis was the most difficult technique, combined with a relatively low recovery of 31% of the enzyme activity. In a purification scheme, free flow electrophoresis is able to substitute one or even several chromatography steps with a negligible loss of biological activity.     

Toward a high-throughput approach to quantitative proteomic analysis: Expression-dependent protein identification by mass spectrometry

The isotope-coded affinity tag (ICAT) [1] technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.