Method for the Analysis and Quantitation of Pharmaceutical Counterions Utilizing Hydrophilic Interaction Liquid Chromatography

Significance of the Topic

The selection and quantification of pharmaceutical counterions play a critical role in ensuring drug quality and therapeutic efficacy. Many active pharmaceutical ingredients (APIs) require soluble salt forms to improve bioavailability and stability. Accurate analysis of both anionic and cationic counterions, alongside the free base API, is essential for regulatory compliance and quality control.

Objectives and Study Overview

This work aims to develop a single hydrophilic interaction liquid chromatography (HILIC) method coupled with evaporative light scattering detection (ELSD) to separate, detect, and quantify multiple pharmaceutical counterions and their corresponding free base APIs. The study evaluates method reproducibility, separation efficiency, and quantitative linearity.

Methodology and Instrumentation

HILIC separation is achieved using a zwitterionic sulfobetaine-based stationary phase, leveraging a high-organic mobile phase to form an aqueous layer for analyte partitioning based on polarity. ELSD provides universal detection for non-chromophoric ions by measuring scattered light from dried solute particles.

• LC System: ACQUITY Arc Premier LC System
• Detector: Waters 2424 Evaporative Light Scattering Detector
• Column: Atlantis Premier BEH Z-HILIC, 4.6 x 100 mm, 2.5 μm
• Software: Waters Empower 3
• Mobile Phases: Acetonitrile, water, 200 mM ammonium formate, 2% formic acid
• Flow Rate: 1.4 mL/min; Column Temp: 40 °C; Injection Volume: 10 μL

Main Results and Discussion

The optimized HILIC–ELSD method achieved baseline separation of multiple counterions with retention time relative standard deviations (RSD) below 5% and area RSD below 6%. Representative drug salt analyses demonstrated clear resolution between free base APIs and their counterions, enabling quantitation of sodium, chloride, and potassium ions.
Calibration curves constructed over defined ranges (Na+: 4–60 mM; Cl−: 2–20 mM; K+: 8–40 mM) exhibited correlation coefficients (R2) >0.997, confirming quantitative reliability. System maintenance, including periodic ELSD cleaning and mobile phase preparation, ensured baseline stability over more than 100 injections.

Benefits and Practical Applications

This unified HILIC–ELSD approach streamlines pharmaceutical counterion analysis by:

• Combining anion and cation quantitation in a single run
• Eliminating the need for multiple chromatographic modes
• Enabling detection of non-chromophoric species
• Supporting quality control and regulatory workflows

Future Trends and Potential Applications

Advancements may include extending the method to a wider array of counterions, integrating mass spectrometric detection for enhanced specificity, and automating sample preparation and instrument cleaning workflows. Further functionalization of HILIC phases could offer improved selectivity for complex drug formulations.

Conclusion

The presented HILIC–ELSD method delivers a robust, reproducible, and quantitative platform for simultaneous analysis of pharmaceutical counterions and API free bases. Its high throughput and versatility address key challenges in drug substance quality control.

References

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• Walter T, Berthelette K, et al. Introducing Atlantis BEH Z-HILIC: A zwitterionic stationary phase based on hybrid particles. Waters Application Note 720007311; 2021.
• Waters Corporation. 2424 Evaporative Light Scattering Detector Operator’s Guide. Rev B; 2006.