CPSA Digest 2001

Proceedings -Tuesday, October 9, 2001

TuOC4

Fast Gradient Elution LC/MS for Bioanalysis

Background:
Drug discovery and development in today's research and business environment requires the implementation of higher throughput assays for in vitro and in vivo studies. Examples of in vitro studies include metabolic stability, cytochrome P450 inhibition and drug transport; in vivo applications include pharmacokinetic studies. LC-MS technology has become the essential tool required for these advances to be successful. Several approaches for achieving high throughput analyses with LC-MS are available.

Premise:
Fast Gradients: The use of fast gradients allows for rapid elution of analytes via a chromatographic column (2.1 x 20 mm; 5 um particle size) and aids in maintaining high throughput LC-MS analyses. Gradients typically run from 95% water to 95% organic in from 1 to 2 min; mobile phase contains ammonium acetate or formic acid and flow rates range from 1 to 3 mL/min. Compounds generally afford peaks that are symmetrical with peak widths less than 0.05 min at baseline.

Method development time can be reduced using fast gradients on short columns compared with traditional isocratic conditions in high organic. In general, ion suppression is less with gradient elution than with isocratic elution because the signals of interest are better separated from the solvent front.

Monolithic Columns: the use of monolithic columns demonstrates an extremely small dependency of separation efficiency on flow rate. Typically, a 4.6 x 50 mm column operates at 4 to 6 mL/min. Performance is similar to that obtained using 3 um packed columns but with flow rates much greater than those normally employed. Thus, the advantage is that chromatographic run times can be reduced by a factor of 5 to 10 times without loss in performance (Dear et al.).

Alternative Strategies: Several other strategies can be used to increase throughput-- (1) On-line sample extraction using automated cartridge exchange, restricted access media columns or turbulent flow chromatography with high flow extraction columns, (2) staggered parallel analysis [see Figure below], and (3) MUX technology.

Value of the Technology
The use of fast gradient LC-MS for quantitative studies in drug metabolism allows for short run times, short method development times and good chromatographic resolution and efficiency. It is one of several strategies available to improve and maintain throughput required in today's research environment.

Links
Leslie Romanyshyn, Philip R. Tiller, Cornelis E. C. A. Hop, Bioanalytical applications of fast chromatography to high-throughput liquid chromatography/tandem mass spectrometric quantitation, Rapid Communications in Mass Spectrometry 14(18), 1662-1668 (2000).

Leslie Romanyshyn, Philip R. Tiller, Raul Alvaro, Antonio Pereira, Cornelis E. C. A. Hop, Ultra-fast gradient vs. fast isocratic chromatography in bioanalytical quantification by liquid chromatography/tandem mass spectrometry, Rapid Communications in Mass Spectrometry 15(5), 313-319 (2001).

C. Hop, American Pharmaceutical Review, in press.

Gordon Dear, Robert Plumb, Dave Mallett, Use of monolithic silica columns to increase analytical throughput for metabolite identification by liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 15(2), 152-158 (2001).

Jing-Tao Wu, The development of a staggered parallel separation liquid chromatography/tandem mass spectrometry system with on-line extraction for high-throughout screening of drug candidates in biological fluids. Rapid Communications in Mass Spectrometry 15(2), 73-81 (2001).