20
September 2009
Turbulent Flow Chromatography: an Evolving Solution for Bioanalysis
by Christophe Chassaing1 , Sarah Robinson2
1 Pfizer Global Research and Development, Sandwich, Kent, CT13 9NJ, UK 2 Thermo Fisher Scientific, Hemel Hempstead, HP2 7GE, UK
Keywords: Biological fluids, Trace analysis, On-line sample extraction, Turbulent Flow Chromatography, High Throughput Liquid Chromatography, Tandem Mass Spectrometry
Bioanalysis has evolved considerably over the last ten years with the rapid implementation of three key technology levers: robotics for automated sample extraction, fast liquid chromatography coupled to mass spectrometry for high throughput trace analysis and finally Laboratory Information Management Systems (LIMS) for seamless data processing and data interpretation. This article describes how Turbulent Flow Chromatography (TFC), by allowing on-line extraction of crude biological matrices, has played an important role in addressing both old and new challenges of modern bioanalysis. After a brief reminder of TFC principles, the authors will focus on describing how the TFC platform has evolved to become the tool of choice for optimal analysis cost, speed and data quality. Practical considerations such as assay sensitivity and column robustness will be discussed in detail. In addition to some examples of current applications, future technology advances will also be presented as TFC adapts to an evolving Drug Metabolism and Pharmacokinetic (DMPK) environment.
Introduction The determination of small drugmolecules in biological fluids,mainly plasma and urine, has remained for a long time a very challenging task. This was due to both the complexity of the biologicalmatrices requiring time consuming sample preparation and the need for long analytical runs, typically using reversed-phase liquid chromatography (RP-LC), to achieve the appropriate separations of remaining endogenous peaks. The recent implementation of automated off-line 96/384-well plate extraction (including protein precipitation, liquid-liquid or solid- phase extraction), or on-line extraction (including TurboFlow chromatography (TFC)) has allowed fast sample clean-up and partly removed the bottleneck associated with sample preparation. The routine usage of tandemmass spectrometers (MS/MS) for quantitation has provided a highly selective means ofmonitoring the peak of interest without the need to develop a lengthy RP-LC method.With short analytical run times (e.g. less than 2minutes) andminimal sample preparation, the original bottlenecks associated with biological samples have gradually been addressed. Bioanalysis as we know it has become amature analytical field.
Nowadays it is not unusual for pharmaceutical companies to use a short suite of genericmethods suitable to support most in-vitro and in-vivo DMPK samples. TFC/MS/MS is part of Pfizer’s generic tool box for routine bioanalysis. A description of how the technique has evolved in our labs is provided. The advantages and limitations of the TFC platformare also discussed in detail.
In this context of well established and fit-for- purpose practices, the need for further investments and wide-spread implementation of new technologies could be perceived as challenging. However, this is without taking into consideration the constantly evolving opportunities arising in both the drug discovery and development arenas. The need to bring new drugs to patients even faster is raising the bar for all analytical applications in termof reduced cost, increased speed and data quality. Specific examples where TFC can greatly impact the drug development process are also discussed in this article.
TFC Principles TurboFlow methods are based on the direct injection of biological samples without previous extraction or treatment onto a column packed with large particles.
These large particles have an additional level of selectivity via the stationary phase chemistry added to them. After the sample is injected onto a TurboFlow column the high flow rate (cf. 1.5 – 5.0 mL/min) generates turbulent flow conditions inside the column. Since 100% aqueous mobile buffers are used, the small analyte molecules are retained via diffusion into the particle pores, while the proteinaceous material is washed to waste (Figure 1a). Once the compounds of interest are extracted from the biological matrix, they are eluted from the TurboFlow column onto the analytical column with a volume of solvent, which has been stored in a holding loop. The holding loop should have a volume at least ten times that of the TurboFlow column and is typically filled with organic mobile phase (for reversed stationary phase) or pH buffered solutions (for ion exchange phases). As the analytes are released from the TurboFlow column they are transferred with the pumping solvent (at a considerably lower flow rate than that used during loading) through the tee rotor-seal in the second valve and mixed with the pumping solvent from the analytical system. The analyte molecules
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