(T1430-05-30) The Use of Short 10 mm Columns for Ultra-fast LC-MS Analyses

Purpose: The increasing number of samples that are seen in high throughput analytical laboratories such as such as bioanalytical and drug discovery labs means that fast LC-MS analysis, using triple quadrupole and high resolution (HRMS) instrumentation, is becoming essential. Over the last two decades, many labs have dramatically increased sample throughput by converting to UHPLC, using shorter columns packed with sub-2 micron particles. However, the performance of modern mass spectrometers has continued to evolve, with ultra-fast data acquisition capabilities allowing further reductions in analytical run times when coupled to specially designed, high throughput columns. This poster assesses the design of 10 mm columns from a kinetic performance perspective and demonstrates their use to achieve high throughput separations for various pharmaceutical samples.

Methods: Kinetic plots were used to design the optimal column format for high throughput analysis. Kinetic and van Deemter plots were generated from ketoprofen efficiency data (k = 10) on a range of particle sizes, which were used to define the column specifications (2 µm fully porous particles in 10 x 2.1 mm hardware). The impact of extra-column dispersion on this column format was assessed theoretically and experimentally by systematically varying post-column tubing volume on a Sciex QTRAP® 6500+ LC-MS/MS system and assessing the chromatographic performance of a set of injected NSAIDs. Other considerations, including MS dwell time and LC dwell volume were also assessed. Finally, a series of rapid LC-MS applications to demonstrate the utility of these short columns were developed.

Results: The use of the modelling approach identified an optimum particle size and column format. Packing methods were developed to provide reproducible and robust cartridge-based columns, stable up to 1000 bar. The dispersion experiments demonstrated that for optimal performance, minimisation of post column volumes is important, with the maximum performance obtained by direct connection of the column to a grounded integral MS valve inlet. Critically, however, the LC-MS system used could be configured to also permit column temperature control within the column oven with minimal loss of performance (8.9 - 12.8% decrease in peak width and 2.0 – 4.7 % loss in peak height). Using this approach, a method for the analysis of a set of immunosuppressant drugs requiring a column temperature of 80 °C, was developed with the analysis achieved in under 1 minute. The potential for accelerating existing methods was then demonstrated by taking an existing method for the determination of drugs of abuse in plasma (16 analytes in 22 minutes) and redeveloping on the 10 x 2.1 mm format column. Using this approach, the separation of 16 components could be achieved using a 0.7 minute gradient and 1 minute cycle time. Excellent quantitative performance was demonstrated.

Conclusion: The work presented in this poster demonstrates the design and application of a 10 mm novel format LC column to achieve rapid LC-MS analysis. Through careful consideration of the instrumental setup and method parameters utilised, excellent performance was achieved and sub-one minute separations obtained.

Van Deemter Plots of typical 2, 3 and 5 micron and predicted back pressures for representative plots

Typical Kinetic Plots for 1.7 micron particle packed columns

HTP-MS column and image of column in implementation connected to valve of grounded MS source