Analysis of Metformin and its Impurities Using Hydrophilic Interaction Liquid Chromatography (HILIC) Coupled With Ultraviolet (UV) and Mass Spectrometry (MS) Detection

Significance of the Topic

Type II diabetes affects millions worldwide, making Metformin the frontline therapy in glycemic control. Ensuring the purity and potency of Metformin drug substances is critical for patient safety and regulatory compliance. Hydrophilic interaction liquid chromatography (HILIC) combined with ultraviolet (UV) and mass spectrometric (MS) detection offers a targeted approach to retain, separate and identify highly polar analytes and related impurities.

Objectives and Study Overview

This application note aims to develop and validate a rapid, robust HILIC-UV-MS method for:

• Quantitative determination of Metformin in pharmaceutical samples.
• Simultaneous separation and identification of six known impurities.
• Assessment of method reproducibility across multiple column lots.

Methodology and Instrumentation

Sample standards of Metformin and impurities (A–F) were prepared in 80:20 acetonitrile:water. Calibration standards ranged from 50 to 150 µg/mL Metformin. Tablet formulations were pulverized, diluted to 100 µg/mL, and filtered through a 0.2 µm nylon syringe filter.

Instrumentation:

• LC System: Arc™ Premier.
• Column: Atlantis™ Premier BEH™ Z-HILIC, 4.6×100 mm, 2.5 µm, 40 °C.
• Detectors: Waters 2998 PDA at 240 nm; ACQUITY QDa™ MS (ESI+; 40–300 amu; capillary 0.8 kV; cone 15 V).
• Mobile phases: (A) 90:10 acetonitrile:water with 10 mM ammonium formate/0.1% formic acid; (B) water with same additives; flow 1.4 mL/min; gradient elution.
• Software: Empower™ 3 for data acquisition and processing.

Main Results and Discussion

• Precision:

• Area %RSD ≤6%; retention time %RSD ≤1% over six injections.

• Specificity: No interference observed in blank injections.
• MS detection enabled identification of all impurities, including non-chromophoric impurity F. Extracted ion chromatograms confirmed m/z values matching PubChem data.
• Linearity: Calibration curve for Metformin (50–150 µg/mL) yielded R² ≥ 0.999.
• Filter recovery: Filtered vs unfiltered standards both quantitated at 97.9 µg/mL, demonstrating negligible analyte loss.
• Sample analysis: Tablet sample measured at 86.4 µg/mL Metformin with clear peak resolution. Spiked impurities (10 µg/mL) were baseline-separated from the drug peak.
• Column reproducibility: Three different Atlantis Premier BEH Z-HILIC lots produced equivalent retention profiles and area counts, confirming lot-to-lot consistency.

Benefits and Practical Applications

• Single HILIC-UV-MS method achieves both quantitation and impurity profiling.
• Compatibility with MS detection provides insight into non-UV-active substances.
• Robust linearity and precision support routine quality control in pharmaceutical manufacturing.

Future Trends and Potential Applications

The integration of high-resolution MS technologies may enhance impurity elucidation. Further miniaturization to UPLC formats and automation could accelerate throughput. Expanding this approach to other polar drug classes will strengthen quality assurance across pharmaceutical pipelines.

Conclusion

This study demonstrates that HILIC with a BEH Z-HILIC column, combined with UV and MS detection, provides a reliable, reproducible and sensitive platform for Metformin analysis and impurity characterization. The method’s precision, linearity and column lot consistency make it suitable for regulatory-compliant quality control workflows.

References

• Centers for Disease Control and Prevention. What is Diabetes? 2021.
• Johns Hopkins Medicine. Metformin: Patient Guide to Diabetes. 2016.
• Walter T. et al. Introducing Atlantis BEH Z-HILIC. Waters Application Note 720007311, 2021.
• Clements B., Maziarz M., Rainville P. HILIC Quantitation of Pharmaceutical Counterions. Waters App Note 720007564, 2022.
• PubChem. Cyanoguanidine CID 10005.
• PubChem. (4,6-Diamino-1,3,5-triazin-2-yl)guanidine CID 24196656.
• PubChem. N,N-Dimethyl-1,3,5-triazine-2,4,6-triamine CID 11469186.
• PubChem. Melamine CID 7955.
• PubChem. 1-Methylbiguanide CID 15486.
• PubChem. Dimethylamine hydrochloride CID 10473.