Automated and simplified two Dimensional LC and High-Resolution MS approach for the identification and characterization of unknown impurity in Mirabegron tablets

Importance of the Topic

Regulatory authorities impose strict requirements for detecting and identifying trace impurities in pharmaceuticals to ensure patient safety and product quality. Traditional one-dimensional liquid chromatography often lacks sufficient resolution and selectivity when analyzing complex formulations such as mirabegron tablets. Automated two-dimensional LC coupled with high-resolution mass spectrometry (2D-LC/HRMS) enhances separation power and delivers accurate mass data for reliable impurity characterization without altering established HPLC mobile phases.

Objectives and Study Overview

This work presents a simplified, fully automated 2D-LC/MS workflow utilizing multiple heart-cutting to isolate and structurally elucidate an unknown impurity in mirabegron tablets. The main goals are to improve peak capacity, achieve high mass accuracy (<2 ppm), and streamline data processing for routine impurity investigations.

Methodology and Instrumentation

• Sample extraction: Methanol-based preparation of tablet impurities.
• First-dimension LC: Agilent 1290 Infinity II pump system with 10 mM ammonium acetate buffer (pH 6.4) and acetonitrile:methanol (100:900, v/v) gradient over a 70-minute run at 1.5 mL/min.
• Multiple heart-cutting: Time-based cuts divert target peaks from 1D to 2D at predefined intervals.
• Second-dimension LC: Agilent pump delivering 10 mM ammonium acetate (pH 6.4) and acetonitrile gradient; 9 min per cut with a 10 min cycle time at 0.5 mL/min.
• Mass spectrometry: Agilent 6545 LC/Q-TOF in positive ESI mode (gas temp 250 °C, drying gas 14 L/min, capillary 3.5 kV, fragmentor 200 V).
• Data processing: Agilent MassHunter for molecular feature extraction and formula generation; Molecular Structure Correlator (MSC) with ChemSpider for in silico fragmentation matching.

Results and Discussion

The 2D-LC/MS approach successfully resolved the impurity from matrix components, generating high-resolution MS and MS/MS spectra with abundant fragment ions. Molecular formulae were assigned with mass errors below 2 ppm, and MSC software proposed candidate structures by correlating experimental and in silico fragments. The results demonstrate that automated 2D-LC/HRMS enables efficient structural elucidation of unknown impurities.

Benefits and Practical Applications

• Significantly enhanced peak capacity and selectivity over 1D LC.
• Automated workflow reduces method development time and operator expertise required.
• High-resolution mass data obtained without altering existing HPLC conditions.
• Suitable for routine QC, R&D, and regulatory laboratories handling complex pharmaceutical samples.

Future Trends and Opportunities

• Integration with orthogonal techniques (e.g., NMR, ion mobility) for comprehensive impurity confirmation.
• Advances in AI-driven software for fully automated structure elucidation and database searching.
• Extension to metabolite profiling and real-time process monitoring using 2D-LC/HRMS platforms.

Conclusion

The automated 2D-LC/MS workflow with multiple heart-cutting and high-resolution mass spectrometry offers a robust, user-friendly solution for impurity identification in pharmaceutical formulations. It provides superior separation, precise mass measurements, and streamlined data analysis to meet stringent regulatory demands.

References

1. Agilent Technologies. Heart-cut 2D-LC/MS approach for pharmaceutical impurity identification using an Agilent 6540 Q-TOF LC/MS System. Application Note 5991-1873 EN.
2. Divakaran S., et al. Two-Dimensional Liquid Chromatography (2D-LC) in Pharmaceutical Analysis: Applications Beyond Increasing Peak Capacity. Chromatographia, 2018, 81, 401–418.