Hi, I’m Jim Murphy, Principal Consulting Chemist at Waters, and I recently collaborated with Lucinda (Cohen) Hittle to present an exciting webinar on the use of microflow chromatography for macromolecules. Lucinda, a Director of Discovery Bioanalytics at Merck, presented an overview of the use of LC-MS for peptide analysis along with various case studies for in vitro and in vivo studies focused on the characterization of various DMPK properties in a discovery research environment. Lucinda provided great insight covering critical aspects of sample preparation strategies for proteins and presented case studies for GLP-1 and the ANP biomarker.
If you missed the webinar or you would like to revisit any subject matter you can watch the webinar on demand.
Lucinda addressed several questions at the conclusion of the webinar. Here are a few more questions that we received, but did not have time to answer:
Q1: Can you compare nano-LC to micro-LC?
Nanoflow LC, which operates at flow rates in the 100’s of nl/min, provides exceptional sensitivity. Sensitivity, however, comes at the cost of time and throughput. The gradient delay and sample loading volumes contribute to the much longer analysis times in nanoflow compared to microflow applications. LC-MS run times of an hour or longer are the average rather than the exception. Microflow can provide analysis times between 2.5 and 10 minutes per sample, which is comparable to the cycle times usually experienced in analytical flow analysis. Nanoflow systems have also been associated with poor robustness. Fluidic leaks in user-made fittings may have little to no impact on micro, or analytical flow analysis may have a large impact on nanoflow applications.
Microflow ultimately provides elevated sensitivity, high sample throughput, and system robustness.
Q2: Any thoughts on how larger microflow ID columns (like 300µm instead of 150µm) will enable microflow use for routine bioanalysis applications?
As the operating LC flow rate increases, increased robustness and throughput can be obtained with just a small decrease in sensitivity. When comparing a 300µm ID column with a 150µm ID, you may expect a decrease in sensitivity from 2-4x depending on the molecule. Operating at a 300µm ID and a flow rate of 8-12 µl/min will provide throughput which rivals that of analytical flow systems.
Q3: How does your experience with the ionKey system compare to conventional microflow systems?
ionKey provides ultimate sensitivity by integrating microflow UPLC directly into the source of the mass spectrometer. The plug-and-play design eliminates user-made connections and associated operator variability. This allows the user to obtain high quality data without requiring a high level of expertise.
Q4: What typical injection volumes are you using on ionKey?
The injection volumes associated with ionKey are very similar to analytical flow systems and can range from below 1 µl up to 50 µl. 5 µl injections may be placed directly into the iKey for many compounds and matrices, especially if the sample diluent is low in organic content. Sample trapping can be implemented to allow larger injection volume in routine analysis. Sample trapping columns usually have a larger inner diameter and operates at a higher flow rate. This allows for rapid sample loading and preconcentration for sample volumes above 5 µl.
Q5: Any preference on IP steps for moving the magnet beads (eg. Kingfisher) as opposed to moving the unbound components per your slide?
Lucinda says, “Regarding IP technique to move beads rather than unbound analyte/supernatant – we leverage our current robotic platform, Hamilton systems, and have been quite satisfied with them, so no plans to add Kingfisher capabilities. We have the luxury of a good amount of lab space so have not needed to consider smaller size systems. You can probably achieve comparable results with other approaches and robots, so I would encourage you to try.”
Q6: Arguably, the main challenge in plasma protein/peptide quantitation by LC/MS is not the analytical sensitivity of the platform but rather the abundant plasma proteins. Can lower flow rates improve on this particular challenge?
Lucinda says, “I would not recommend that you skip or put less effort into sample cleanup when switching from regular flow to microfluidic approaches like the Ion Key. If anything, I would invest more in cleaning the sample further to ensure you have rugged performance. There may be opportunities using the Ion Key trapping approach.”
Please be sure to watch the webinar on demand.