(T1330-09-53) Achieving the Ultimate Laboratory Flexibility with a Four Channel Multi-Method Enabled Multiplexing Liquid Chromatography Mass Spectrometry Platform

Purpose: As laboratory demands increase and assays move towards LCMS analysis high throughput capabilities become more important while maintaining ease of operation and robustness. This study demonstrates high throughput capabilities of a multi-method high throughput multiplexing LCMS system. The ability and performance consideration of having the ability to run different assays simultaneously on one system will be explored.

Methods: Data was collected using the Shimadzu Nexera QX Multi-Method enabled Multiplexing LCMS instrument, comprised of quad-injection equipped autosampler, column oven, 6 x binary pumps, and low-pressure gradient pump (for needle inner rinse) coupled to a Shimadzu LCMS-8060NX mass spectrometer. Data was generated on urine samples that were extracted following an established solid-phase extraction procedure. Assays contained within the scope of the project had analytes of interest included benzodiazepine and synthetic cathinone drug class panels with calibration ranges ranging between 0.2 ng/mL to 2000 ng/mL. Dedicated methods were developed for both needle and stream rinsing utilizing Analytical Intelligence (AI) features built into instrument control software (QX Solution). The intelligent sample submission algorithms allows for up to four sequences schemes to be achieved with the goal of optimizing sample run time over multiple assays across the four LC streams.

Results: The two methods use different mobile phase and column. Therefore, after the first assay finished the system required columns to be changed as well as mobile phases in single method multiplex analysis. In multimethod approach, the system used 2-channels for benzodiazepines and 2-channels for bath salts, effectively performing overlapping 2-plex analysis of both assays simultaneously within the 4-plex configuration. Using the multimethod approach when factoring in the manual process that would be required to switch system when running sequentially, multimethod saved approximately 30 minutes of run time per 2 plates (1 plate for each assay) in this method combination (8.3 hr vs. 8.8 hr). The Nexera QX system provided capability to overlap multiple methods on one system without the user having to pause within batches of samples to change mobile phases and columns. The unattended operation of the system while multiplexing different methods together achieved an overall faster batch run time. Depending on the combination of methods, further time reduction would be possible by multiplexing based on the balance of LC run time and MS acquisition window. The systems flexibility also allows for more then 2 methods to run within one system, the system can run 4 independent methods concurrently. It is important during the analysis that no effect on the quality of data be observed by using multiple assays multiplexed together, as highlighted in the results excellent linearity for the calibration curves was maintained ( >0.993) with accuracies within 20%. Quality control samples were also run and all QC samples passed acceptance criteria of the laboratory. Robustness was evaluated by repeated analysis of samples plate for both assays, over 1000 injections were performed for each assays and retention time stability as well as internal standard response was evaluated. Retention times over the 1000 injections were less then 5% variability and internal standard response across all the analytes was less then 10%.

Conclusion: The innovation in the hardware design along with AI software features provide an extremely robust and simple user interface allowing maximum productivity when running multiple assays simultaneously. The flexibility of the system allows for numerous assays to be configured on a single platform reducing analyst intervention to change mobile phases and columns between different assays.

Nexera QX Four Channel Multiplexing LCMS Instrumentation Schematic, Multi-Method Capable Operation



Summary of calibration data for both assays tested on the QX system along with accuracies for standards and control samples.



Time comparison between running methods sequentially on QX vs. running them in multi-method configuration with both assays running concurrently.