Impurity Profiling Using UPC2/MS

Importance of the Topic

The accurate profiling of impurities in pharmaceutical products is critical for ensuring drug safety, efficacy, and regulatory compliance. UltraPerformance Convergence Chromatography coupled with mass spectrometry (UPC 2/MS) offers unique orthogonal selectivity to conventional liquid chromatography methods, enabling more comprehensive detection and characterization of known and unknown impurities.

Study Objectives and Overview

This work describes the application of the ACQUITY UPC 2 System coupled to an SQD mass spectrometer to investigate impurity profiles of metoclopramide standards and an expired formulation. Key aims included:

• Identifying chromatographic anomalies in impurity standards
• Determining degradation pathways and stability of impurity F solutions
• Profiling impurities in an aged metoclopramide sample

Methodology and Instrumentation

Samples of USP impurity standards C and F were prepared in methanol; expired metoclopramide was extracted in methanol at 2 mg/mL. Separation employed a 3.0×100 mm BEH 2-EP column (1.7 µm) at 50 °C with CO₂ (A) and 1 g/L ammonium formate in 50:50 methanol/acetonitrile plus 3% formic acid (B). A gradient from 5% to 30% B over 5 minutes and a 2.0 mL/min flow rate were used. Detection combined a PDA (200–410 nm) and single-quadrupole MS (150–1200 Da, ES+, ES–). Empower 3 managed data acquisition and review.

Results and Discussion

Impurity C exhibited a doublet on a Fluoro-Phenyl UPC 2 column; MS spectra confirmed the peaks as monomer/dimer forms rather than contaminants. Impurity F standard solutions developed additional peaks and a brown tint upon storage. Mass shifts (+58, +44, +10 Da, etc.) indicated methylation, methoxylation, hydrolysis, and dechlorination reactions. These findings led to reducing working standard shelf life to three days and prompted further diluent studies. Analysis of the expired drug revealed 12 impurities (including seven unknowns) with retention times and m/z values catalogued. Four of the degradation products matched those seen in impurity F studies, confirming similar transformation pathways.

Benefits and Practical Applications

• Orthogonal separations increase impurity coverage beyond reversed-phase LC.
• Rapid MS interrogation enables prompt differentiation between related species (e.g., monomer vs. dimer).
• Stability profiling informs standard preparation guidelines and method refinement.
• Automated data review in Empower 3 streamlines impurity confirmation and unknown identification.

Future Trends and Opportunities

Advancements may include integrating high-resolution MS/MS for structural elucidation of unknowns, exploring alternative stationary phases to further enhance selectivity, and adopting real-time monitoring for in-process impurity control. Expanded UPC 2 applications could address complex biologics and polar compound profiling.

Conclusion

The coupling of UPC 2 with single-quadrupole MS provides a versatile platform for comprehensive impurity profiling in pharmaceuticals. It accelerates method development decisions, uncovers degradation pathways, and improves quality control practices for active pharmaceutical ingredients.

Instrumentation

• ACQUITY UPC 2 System with BEH 2-EP 3.0×100 mm, 1.7 µm column
• PDA detector (200–410 nm)
• ACQUITY SQD Mass Spectrometer (150–1200 Da, ES+, ES–)
• Empower 3 Software for data acquisition and review

References

1. Ahuja S, Alsante K. Handbook of Isolation and Characterization of Impurities in Pharmaceutical Compounds. Elsevier, 2003.
2. Jones MD et al. Analysis of an Unknown Degradant Found in Quetiapine Fumarate. Waters Application Note 720003079EN, 2009.
3. Jones MD et al. Identification and Structural Elucidation of Impurities of Quetiapine Hemifumarate. Waters Application Note 720003081EN, 2009.
4. Jones MD, Potts W. UPC 2 Method Development for Achiral Impurity Analysis. Waters Application Note 720004577, 2013.