Application of Multiple Heart-Cutting 2D-LC in Method Development for Impurity Analysis

Importance of the Topic

Impurity profiling is a critical requirement in pharmaceutical and fine chemical development, mandated by regulatory guidelines to ensure product safety and efficacy. Coeluting or hidden impurities can compromise drug quality and patient safety, making advanced separation strategies necessary. Multiple heart-cutting two-dimensional liquid chromatography (2D-LC) offers a powerful approach to reveal and characterize such impurities, supporting robust method development and compliance with ICH Q3A (R2) impurity identification thresholds.

Objectives and Study Overview

This application note illustrates the use of the Agilent 1290 Infinity 2D-LC Solution with multiple heart-cutting to enhance impurity analysis. The goals were to:

• Detect every chromatographic peak in the first dimension and transfer it to a second dimension with different selectivity.
• Uncover potential coelutions between the main analyte (e.g., DEET) and trace impurities.
• Confirm impurity identities via spiking experiments.

Methodology and Instrumentation

The 2D-LC method combined a reversed-phase C18 column in the first dimension (ZORBAX RRHD Eclipse Plus C18, 2.1×150 mm, 1.8 µm) and a Poroshell 120 Bonus-RP column (4.6×50 mm, 2.7 µm) in the second dimension.

First-dimension conditions:

• Mobile phases: water/0.1% formic acid (A) and acetonitrile/0.1% formic acid (B)
• Gradient: 5% B to 95% B over 20 min, hold to 25 min; flow rate 0.2 mL/min

Second-dimension conditions:

• Mobile phases: water/0.1% formic acid (A) and methanol/0.1% formic acid (B)
• Gradient modulation: 10% B to 50% B over 22 s and 70% B to 95% B over 22 s; flow rate 2.0 mL/min; cycle time 2.0 min

Multiple heart-cutting setup:

• Time-based sampling of each first-dimension peak into 40 µL loops, with full-loop overfill for impurities (0.40 min) and dense sampling of the main peak (0.10 min) to capture coelutions.
• Two decks of heart-cut valves allow up to five simultaneous cuts per deck, alternating analysis and storage to maximize throughput.

Instrumentation:

• Agilent 1290 Infinity Binary Pumps (G4220A)
• Agilent 1290 Infinity Autosampler with Thermostat (G4226A, G1330B)
• Agilent 1290 Infinity Thermostatted Column Compartment (G1316C)
• Agilent 1290 Infinity Valve Drive with multiple heart-cutting valves (G4242-64000)
• Agilent 1290 Infinity Diode Array Detectors (G4212A)
• Agilent OpenLab CDS ChemStation and Infinity 2D-LC software

Main Results and Discussion

One-dimensional analysis on a C18 column showed partial coelution of two trace impurities with the main compound. Implementing multiple heart-cutting 2D-LC revealed:

• Complete separation of all detected impurities in the second dimension, including those coeluting in the first run.
• Identification of three impurities embedded within the main compound’s peak via dense heart-cut sampling.
• Confirmation of impurity identity by spiking the sample with a synthesized impurity standard and observing linear peak growth in the second dimension.

Benefits and Practical Applications

Multiple heart-cutting 2D-LC enhances impurity method development by:

• Ensuring no impurity remains hidden due to coelution.
• Accelerating optimization of chromatographic conditions without extensive column screening.
• Providing reliable quantification and identity confirmation through orthogonal separation.

Future Trends and Applications

Advancements poised to further leverage this technology include:

• Integration with high-resolution mass spectrometry for structural elucidation of unknown impurities.
• Automated, AI-driven optimization of heart-cut timing and selectivity.
• Expansion to complex formulations, biological matrices, and continuous manufacturing quality control.

Conclusion

The Agilent 1290 Infinity 2D-LC Solution with multiple heart-cutting proves to be a robust and versatile platform for pharmaceutical impurity analysis. By enabling orthogonal, sequential separations of every detected peak, it uncovers coelutions, confirms impurity identities, and streamlines method development to meet stringent regulatory standards.

References

1. ICH Q3A(R2): Impurities in New Drug Substances, October 2006.
2. Naegele, E., "Detection of Impurities by Heart-Cutting using the Agilent 1290 Infinity 2D-LC Solution," Application Note 5991-0834EN, Agilent Technologies, 2012.
3. Buckenmaier, S., "Multiple Heart-Cutting with the Agilent 1290 Infinity 2D-LC Solution," Technical Overview 5991-5615EN, Agilent Technologies, 2015.