HPLC throughput improvements

A. Metal Oxide Phases IMPROVEMENTS IN DETECTORS IMPROVEMENTS IN HPLC THROUGHPUT... 

This chapter is intended to serve as a general overview of new and emerging HPLC technologies and is divided into four sections simplifying sample preparation, new column technologies, improvements in detectors, and improvements in HPLC throughput. 

Simplifying Sample Preparation New Column Technologies Improvements in Detectors Improvements in HPLC Throughput... 

FIGURE 5.11 Metabolite profiling throughput improvements associated with transitioning from HPLC-TopCount to UPLC-ViewLux. (Reprinted from Dear, G.J. et al., J. Chromatogr. B Anal. Technol. Biomed. Life Sci., 868, 49, 2008. With permission.)... 

Preparative HPLC has been widely used for purification of peptides and small molecules for over a decade. Its inherent serial nature, lack of automation, and severe throughput limits, would seem to make it unsuitable for purification of large and diverse compound libraries. Recent advances in automation, detection, and method development, however, now make it possible to purify hundreds of compounds per day using a single instrument. Further throughput improvements may come from parallel HPLC systems which have been recently reported. 

Ultra-performance HPLC (UPLC) utilizes sub-2-pm porous particles inside packed microbore columns up to 150 mm long. Significant improvements in terms of resolution, analysis time, and detection sensitivity have been reported. A side-by-side comparison of HPLC and UPLC was made to determine concentrations of alprazolam in rat plasma.10 UPLC provided a four-fold reduction in terms of LC/MS/MS cycle time that translated into higher sample throughput. Another important... 

Other techniques to improve throughput are instrumentation based and may involve multiple HPLC systems. The simplest method involves the automated use of solid phase extraction cartridges for sample cleanup followed by direct injection into the mass spectrometer [114], Coupling of multiple HPLC systems to one mass spectrometer allows one column to equilibrate and separate while another column to flow into the mass spectrometer. Multiple HPLC systems may be configured such that the mass spectrometer is only exposed to each serial HPLC eluent as the analyte of interest is eluted [115,116]. Although multiple H P LC-based methods may increase throughput, they also typically decrease sensitivity and may confound data workup and interpretation. 

From Analytical Chemists, a partial answer to this problem was the development and validation of new methods that permit an improvement in terms of productivity ( high-throughput ), sensitivity and selectivity, especially using very recent hyphenated analytical assays, such as HPLC-MS/MS, GC-MS/MS or further complex couplings, that can provide more complete information in a single analysis. 

Over the years, in addition to developments with ELISA reagents such as labels, there have been improvements in automation. This has enabled ELISA to be utilized as a high-throughput tool. Typically, ELISAs can be performed in several hours to days. The most common practice is to precoat the microtiter plate for an overnight incubation period, with the remainder of the steps performed the following day. While ELISAs are fast when compared to other assays such as bioassays, which can take days to weeks, they might be considered slow when compared to methods like HPLC, in which the time from sample injection to chromatogram is a matter of minutes. 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

This chapter discusses HPLC methodologies used to analyze dissolution samples, the automation of methods, and improved throughput and productivity. 

The earlier chapters in this book focus on the current state-of-the-art in HPLC and provide useful practical advice for analyzing pharmaceuticals by HPLC. In this chapter, we plan to offer a glimpse into the new developments in HPLC and discuss how these advances can help improve the performance of HPLC. The progress in HPLC technology has been driven by the need to improve sample throughput because project time lines are short, and the pressure to bring the next blockbuster drug to... 

From a DMPK perspective, a common goal is to be able to compare multiple compounds based on their absorption, distribution, metabolism and excretion (ADME) properties as well their preclinical PK properties [8, 12-22]. Therefore, lead optimization typically is performed as an iterative process that uses the DMPK data to select structural modifications that are then tested to see whether the DMPK properties of the series have been improved. This iterative process is shown schematically in Fig. 13.2. Clearly an important element for the successful lead optimization of a series of NCEs is the ability to perform the DMPK assays in a higher throughput manner. The focus of this chapter will be to discuss ways that mass spectrometry (MS), particularly HPLC-MS/MS can be used to support the early PK studies for NCEs in a higher throughput manner. 

In addition to increasing throughput, researchers are finding ways to utilize the increased sensitivity of the new HPLC-MS/MS systems. For example, Xu et al. [113] recently described the development of a low sample volume assay for preclinical studies. In this assay, only a lO-pL plasma sample volume is required for the analysis. The small volume is prepared by protein precipitation (1 6 = plasmaiacetonitrile) using a special low volume 96-well plate. Only 5 pL of the precipitated sample is injected onto the HPLC-MS/MS system. In spite of these low volumes, the example assay is reported to have a limit of quantitation (LOQ) of 0.1 ng mL h It can be predicted that there will be more reports of improved LOQs and reduced sample volumes as new LC-MS/MS instrumentation is introduced to more laboratories. 

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