Orthogonal Separation of Pharmaceutical Impurities by the Agilent 1260 Infinity II SFC/UHPLC Hybrid System

Significance of the Topic

The identification and quantification of trace impurities in active pharmaceutical ingredients (APIs) are essential for ensuring drug safety, efficacy, and compliance with International Council for Harmonisation (ICH) guidelines. Orthogonal chromatographic techniques such as supercritical fluid chromatography (SFC) and ultra-high-performance liquid chromatography (UHPLC) offer complementary selectivities that increase the likelihood of detecting structurally diverse impurities.

Objectives and Study Overview

This application note examines the use of the Agilent 1260 Infinity II SFC/UHPLC Hybrid System equipped with a variable wavelength detector (VWD) for low-level impurity profiling in the model API metoclopramide. By comparing separations in both SFC and UHPLC modes, the study aims to demonstrate enhanced impurity detection through orthogonal selectivity and to evaluate method performance at a 0.03 % impurity level.

Methodology and Instrumentation

The hybrid system integrates SFC control, binary pump, multisampler, and a VWD compatible with high pressure. Key elements include:

• SFC mode: CO₂ mobile phase with methanol containing 10 mM ammonium formate, ZORBAX Rx-SIL column, 150 bar backpressure, 55 °C column temperature.
• UHPLC mode: Water with 0.1 % formic acid (A) and acetonitrile with 0.1 % formic acid (B), ZORBAX SB-C18 column, 30 °C, 1.0 mL/min flow.
• Detection: VWD at 270 nm with 20 Hz data rate.
• Samples: Metoclopramide and five known impurities spiked at 200 µg/mL for development and at 0.03 % in a 10 mg/mL API solution for trace detection.

Main Results and Discussion

UHPLC separations resolved degradation impurities B, C, and D but failed to distinguish impurity A from the API, while impurity G co-eluted with peak tailing. Under SFC conditions, degradation products eluted early (1.5–2.7 min) and structurally similar impurities A and G eluted later (4.0–6.2 min) with better baseline separation. Trace-level analysis in SFC mode yielded signal-to-noise ratios above 12 for all impurities, retention time RSDs below 0.4 % and peak area RSDs under 2.1 %, demonstrating reliable detection at the 0.03 % level.

Benefits and Practical Applications

Seamless switching between UHPLC and SFC on one platform enables orthogonal screening without additional instrumentation. The approach improves impurity coverage, accelerates method development, and supports compliance with strict regulatory limits for impurity reporting and identification.

Future Trends and Opportunities

• Coupling hybrid systems with mass spectrometry for enhanced identification confidence.
• Automated method scouting workflows that leverage orthogonal selectivity.
• Application of advanced data processing and chemometrics to further improve impurity detection.
• Extension of hybrid separation strategies to other complex pharmaceutical matrices.

Conclusion

The Agilent 1260 Infinity II SFC/UHPLC Hybrid System with VWD provides robust, orthogonal separations that significantly enhance the detection and quantification of low-level pharmaceutical impurities. By combining UHPLC and SFC modes in a single platform, this method meets stringent regulatory requirements and offers high productivity and confidence for impurity profiling.

References

1. ICH Harmonized Tripartite Guideline Q3B(R2): Impurities in New Drug Products.
2. Agilent Application Note 5994-1351EN: Improved Sensitivity for Low-Level Impurity Detection with the Agilent 1260 Infinity II SFC System Featuring an Agilent 1260 Infinity II Variable Wavelength Detector.
3. Agilent Technical Overview 5991-8276EN: Orthogonal Chromatographic Separations using the Agilent 1260 Infinity II SFC/UHPLC Hybrid System.