A UPLC Method for Analysis of Metformin and Related Substances by Hydrophilic Interaction Chromatography (HILIC)

Importance of the Topic

Metformin hydrochloride is a widely prescribed oral antidiabetic agent for type 2 diabetes mellitus with over 48 million prescriptions filled annually in the US alone. However, its highly polar nature and weak UV absorbance at 218 nm pose analytical challenges in reversed-phase chromatography. Hydrophilic interaction chromatography (HILIC) offers a powerful alternative for polar basic compounds, enabling improved retention and selectivity.

Study Objectives and Overview

The study aimed to develop and validate a rapid UPLC–HILIC method for simultaneous separation of metformin and six related impurities. Using the ACQUITY UPLC H-Class system equipped with a BEH Amide column, the work focused on optimizing chromatographic parameters to reduce run time, solvent consumption, and overall analysis cost while maintaining robustness for routine quality control use.

Methodology and Instrumentation

Method development began by adapting an existing HPLC procedure (isocratic, phosphate buffer/ACN, 30 min) to UPLC conditions using the Waters Column Calculator. Two HILIC stationary phases (BEH HILIC and BEH Amide) were screened. Key parameters adjusted included organic mobile phase ratio, buffer cation (Na+ vs. K+), ionic strength, flow rate, and temperature. Data processing and trending were performed with Empower 2 CDS.

Used Instrumentation

• ACQUITY UPLC H-Class system with quaternary solvent manager, sample manager, and PDA detector
• ACQUITY UPLC BEH Amide column (2.1 × 150 mm, 1.7 µm) with pre-column filter
• Empower 2 Chromatography Data Software

Main Results and Discussion

Initial transfer to BEH HILIC showed inadequate retention and co-elution. The BEH Amide phase provided baseline separation except for slight overlap of impurities B and D. Increasing organic content improved retention of B and D but co-eluted with E. Elevating temperature enhanced resolution but risked co-elution of E and B at higher temperatures. Substituting sodium phosphate with potassium phosphate (20 mM, pH 2.2) at 40 °C and 0.5 mL/min achieved optimal resolution of all critical pairs within a 5-min isocratic run. A routine use study (1800 injections) revealed pressure drift due to sample particulates; implementing a 0.2 µm filtration step stabilized pressure profiles.

Benefits and Practical Applications

• Six-fold reduction in run time (30 min to 5 min)
• 80% decrease in solvent consumption (65 L to 11 L per 1500 injections)
• Estimated cost savings of $8800 in acetonitrile per 1500 injections
• Improved throughput and resource utilization in QC laboratories
• Enhanced method robustness through informatics-driven troubleshooting

Future Trends and Applications

• Integration of HILIC with high-resolution mass spectrometry for impurity profiling
• Automated method development and AI-driven optimization workflows
• Use of eco-friendly solvents and microfluidic systems to reduce waste
• Real-time monitoring and inline filtration for continuous quality control

Conclusion

The optimized UPLC–HILIC method offers rapid, cost-efficient, and robust separation of metformin and related impurities. Strategic manipulation of mobile phase composition, buffer cation, ionic strength, and temperature, combined with informatics tools, ensures reliable routine performance and significant operational savings.

Reference

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