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Advancing High-Throughput HPLC
Expanding technology offers improved speed and enhanced resolution.

By Vicki Glaser

HPLC has myriad applications in drug discovery, from analytical to preparative and from early stage research through product formulation and manufacturing.  The range of uses for these instruments continues to expand with the introduction of chromatographic systems offering higher pressures, longer columns, and small-particle media that enable improved speed and enhanced resolution. The field is regularly sprouting new acronyms to describe higher pressure, higher throughput LC including UPLC, UHPLC, and RRLC. The options multiply when LC is integrated with mass spectroscopy (MS) in its numerous guises.

A recent paper in the Journal of Proteome Research (2007;6(2):552-558), describes the use of reverse-phase UPLC-oaTOF-MS (orthogonal acceleration time-of-flight MS) as a lipidomics tool for analyzing targeted lipids in complex biological mixtures. UltraPerformance LC, or UPLC, is the trademark of Waters’ Acquity systems. The authors describe the ability to produce chromatograms with typical peak base widths of 3s and peak capacities in excess of 200 in 10 minutes.

A study of UPLC-MS for quantitative analysis of serum metabolites yielded several conclusions: UPLC-based separation resulted in 20% more detected components than HPLC; length of separation was a critical parameter; and UPLC offered advantages over HPLC in terms of retention time reproducibility and signal-to-noise ratios (Anal Chem 2006;78(10):3289-3295). Another report (Anal Chem 2006;78(5):1697-1706) found that the use of high pressures (>1500 bar, using smaller diameter reverse-phase supports) improved protein recovery compared to conventional pressures (160 bar). The authors concluded that increased pressure reduced (and in some cases eliminated) run-to-run protein carryover and enhanced protein recovery.

Higher Pressures, Smaller Particles

Anton Jerkovich, a principal scientist at Novartis, touts the speed of analysis as the main advantage of higher-pressure HPLC technology for his work in drug product analytics and formulations development. Jerkovich describes, on average, a 5-fold increase in the speed of separations. This can accelerate methods development and generate cost savings, especially as a drug nears production stage and lower flow rates enabled by higher pressures result in substantially lower solvent usage.

Rapid adoption of the new technology could be slowed by the need to revalidate existing methods. “All of our stakeholders would have to be onboard in terms of methods transfer to other sites,” says Jerkovich. “We have to reach that tipping point before we can fully adopt the technology as a common application.”

Thermo Fisher Scientific’s Accela high speed chromatography system (UHPLC) can accommodate conventional HPLC and ultrahigh pressure/small particle LC. Use of Thermo’s TurboFlow technology enables injection of biological samples directly into the Accela. Agilent’s 12 Rapid Resolution Liquid Chromatography (RRLC) system offers column dimensions from 1 to 4.6 ID and 10 to 300 mm length, and particle sizes from 1.5 to 10 µm. The system enables up to a 20-fold increase in speed and 60% higher resolution than conventional HPLC, says Agilent. Recent additions to Waters’ Acquity family include the TQD tandem quadruple UPLC/MS/MS system, a new fluorescence detector, and HSS T3 columns with 1.8µ high-strength silica particles.

Accelerating Method Development

Peter Sajonz, research fellow, Christopher Welch, associate director, and colleagues at Merck are using the Eksigent ExpressLC-800 eight-channel HPLC system to perform multiparallel, high-throughput chiral method development and analysis. The group was able to demonstrate the ability of the multiparallel microfluidic system for use in rapid development of fast analytical HPLC methods for racemic test mixtures. The development of chiral assays with runtimes of 1-2 minutes typically required about 1.5 hours (Chiralty 2006; 18(10):803). 

A related application supported the use of the ExpressLC-800 for catalyst screening of an asymmetric hydrogenation reaction. Multiparallel normal-phase HPLC screening facilitated the rapid identification of a method to resolve the two enantiomers of the cis product from starting material (J Chromatogr 2007;doi:10.1016/j.chrom.2007.01.067).

“We have demonstrated that it is possible to do method development and to run a full 96-well plate for catalyst screening in one day,” says Sajonz, “ a process that could typically take several days to complete.”

Copyright 2007, Cambridge Healthtech Institute. All Rights Reserved.



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