Using the Systematic Screening Protocol and MaxPeak™ Premier Columns to Separate Seven Janus Kinase Inhibitors

Importance of the Topic

The development of robust analytical methods for structurally related compounds such as Janus kinase inhibitors is critical in pharmaceutical research and quality control. These molecules share similar chemical properties, making chromatographic separation challenging and time consuming. A systematic approach streamlines method development and ensures reliable, reproducible data for drug discovery and manufacturing.

Objectives and Study Overview

This application note details a structured method development process for simultaneous separation of seven Janus kinase inhibitors. By coupling a systematic screening protocol with MaxPeak Premier column technology, the study aimed to achieve baseline resolution rapidly under reversed-phase UHPLC conditions.

Methodology and Instrumentation

A two-step systematic screening protocol was applied:

• pH scouting using high and low pH mobile phases (200 mM ammonium hydroxide and 2 % formic acid) on a pH-stable bridged-ethyl hybrid C18 column to determine optimal retention conditions.
• Column and solvent screening across four MaxPeak Premier and Atlantis Premier columns, comparing acetonitrile versus methanol as organic modifiers to enhance selectivity.

Used Instrumentation

• UPLC system: ACQUITY UPLC H-Class Plus with Quaternary Solvent Manager, Sample Manager with Flow Through Needle, Column Manager, and QDa mass detector
• Columns: ACQUITY Premier BEH C18; CSH Phenyl-Hexyl; BEH Shield RP18; Atlantis Premier BEH C18 AX (2.1 × 50 mm, 1.7 µm)
• Detection: ESI positive mode MS full scan
• Software: Empower 3 Feature Release 4

Main Results and Discussion

High pH mobile phases provided increased retention of basic Janus kinase inhibitors. Methanol as the strong solvent delivered alternative selectivity compared to acetonitrile, enabling baseline resolution (USP resolution > 1.5) on the ACQUITY Premier BEH C18 column. The final method achieved complete separation in under 8 minutes, requiring only ten injections to identify optimal conditions. This process highlighted the benefits of combining pH scouting with solvent screening and hybrid surface columns.

Benefits and Practical Applications

• Rapid method development with minimal tuning, reducing total injections and development time.
• Robust separation of seven structurally similar kinase inhibitors with high resolution and reproducibility.
• Applicability to bioanalytical monitoring, pharmacokinetic studies, and quality control in pharmaceutical production.

Future Trends and Applications

The integration of automated screening protocols and advanced column hardware will continue to accelerate method development. High-performance surfaces and hybrid particles are expected to expand applications for challenging analytes, including other drug classes and biologics. Coupling these approaches with machine learning for data analysis may further streamline workflows and predict optimal conditions.

Conclusion

Using a systematic screening protocol combined with MaxPeak Premier column technology enabled fast and reliable separation of seven Janus kinase inhibitors. The optimized reversed-phase UHPLC method delivers baseline resolution and robust performance, demonstrating an efficient workflow for complex pharmaceutical analysis.

References

• Maziarz M, McCarthy S, Wrona M. Improving Effectiveness in Method Development by Using a Systematic Screening Protocol. Waters Application Note 720005026, 2014.
• Berthelette KD, Nguyen JM, Turner JE. Method Development of Ten Antibiotic Compounds Using a Systematic Screening Protocol. Waters Application Note 720007352, 2021.
• Berthelette KD, Turner JE, Walter TH, Haynes K. Using a Systematic Screening Protocol and MaxPeak HPS Technology to Develop a UHPLC Method for the Analysis of Deferoxamine. Waters Application Note 720007834, 2022.
• Hong P, McConville P. A Complete Solution to Perform a Systematic Screening Protocol for LC Method Development. Waters White Paper 720005268, 2022.
• Delano M, Walter TH, Lauber M, Gilar M, Jung MC, Nguyen JM, Boissel C, Patel A, Bates-Harrison A, Wyndham K. Using Hybrid Organic–Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC. Analytical Chemistry, 93, 5773–5781, 2021.