High-Resolution Sampling 2D-LC for Pharmaceutical Impurity Analysis

Significance of the topic

In pharmaceutical quality control, detecting impurities present at trace levels beneath a high-concentration active pharmaceutical ingredient (API) is critical for patient safety and regulatory compliance. Conventional one-dimensional (1D) liquid chromatography may fail to resolve coeluting impurities that differ by structural similarity and large concentration ratios. High-resolution sampling two-dimensional liquid chromatography (HiRes 2D-LC) addresses this challenge by fractionating the entire API peak from the first dimension into multiple narrow cuts, which are then separated in a second dimension to reveal hidden low-level impurities.

Objectives and overview of the study

This application note demonstrates the use of HiRes 2D-LC to detect impurities at or below 0.05% relative to the main API peak. Two model systems were investigated: mixtures of three chlorodifluorobenzoic acid isomers at relative concentrations ranging from 0.05% to 100%, and deamidated bovine insulin as a real-world example of a structurally related impurity. Key performance metrics—limit of quantification, accuracy, and precision—were evaluated under different detector configurations.

Methodology and instrumentation

The HiRes 2D-LC workflow comprises:

• First dimension: Agilent 1290 Infinity II high-resolution reversed-phase separation of the sample peak.
• Time-based sampling: Up to ten discrete 4–9 s cuts spanning the full width of the first-dimension peak are collected into 40 µL loops.
• Second dimension: Fast gradient elution on a complementary column, with sequential analysis of each cut.
• Detection: UV diode array detectors (DAD) with 10 mm cell or HDR-DAD solution for extended dynamic range; and coupling to an Agilent 6545 Q-TOF mass spectrometer using a diverter valve to exclude salt-rich fractions.

The main instrumentation included two high-speed pumps, a multisampler with cooling, multicolumn thermostats, DAD and HDR-DAD modules, a duo valve with multiple heart-cut loops, and, for MS experiments, an Agilent Jet Stream electrospray source. Data processing utilized Agilent OpenLAB CDS ChemStation with 2D-LC software and the 2D Chromatogram Creator in MassHunter.

Main results and discussion

Chlorodifluorobenzoic acid isomers:
The method resolved a low-level isomer (5-chloro-2,4-difluorobenzoic acid) hidden under a dominant isomer (3-chloro-2,4-difluorobenzoic acid). Using conventional DAD, signal-to-noise ratios for the trace isomer at 0.05% relative concentration were near the limit of quantification, yielding accuracy of 71.6% and RSD up to 7.95%. Employing the HDR-DAD solution to increase injection volume improved quantitation: S/N ratios rose above LOQ, RSDs fell below 1.6%, and accuracy reached 96.4–108.5%.

Deamidated insulin:
Time-based HiRes sampling isolated the tailing region of the intact insulin peak into ten cuts. Sequential 2D-LC/MS analysis generated total ion chromatograms for each cut, revealing a deamidation product coeluting in the 1D separation. Mass spectra extracted from the relevant cuts confirmed the presence of a +0.98 Da mass shift consistent with one deamidation event, demonstrating the ability to detect and identify low-level biopharmaceutical variants.

Benefits and practical applications

• Enhanced selectivity: HiRes 2D-LC uncovers coeluting impurities hidden under dominant peaks.
• Regulatory compliance: Achieves detection at ICH reporting thresholds (0.05% relative to API).
• Quantitative reliability: HDR-DAD extends dynamic range to quantify high- and low-level analytes in a single run.
• MS compatibility: Salt exclusion via diverter valve protects the ion source and enables structural confirmation of trace species.

Future trends and possibilities

The integration of advanced detectors (e.g., high-resolution MS, ion mobility) with HiRes sampling workflows will further improve impurity profiling. Automation of cut optimization, real-time data evaluation using AI-driven algorithms, and miniaturized 2D-LC systems could accelerate method development and routine monitoring in pharmaceutical and biopharmaceutical environments.

Conclusion

High-resolution sampling 2D-LC is a powerful approach for detecting and quantifying trace impurities that coelute with APIs in complex matrices. By segmenting the main peak into narrow fractions and applying fast second-dimension separations with high-dynamic-range detection or MS confirmation, this technique meets stringent quality control requirements and enhances confidence in impurity characterization.

References

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2. International Conference on Harmonization Q3A(R2): Impurities in New Drug Substances, 2006.
3. Petersson P.; Haselmann K.; Buckenmaier S. Multiple Heart-Cutting Two-Dimensional Liquid Chromatography Mass Spectrometry: Towards Real-Time Determination of Related Impurities of Bio-Pharmaceuticals in Salt Based Separation Methods. J. Chromatogr. A 2016, 1468, 95–101.
4. Stephan S. High-Resolution Sampling 2D-LC with the Agilent 1290 Infinity II 2D-LC Solution. Agilent Technologies Technical Overview, 2016.
5. Schneider S. 2D-LC/MS Characterization of Charge Variants Using Ion Exchange and Reversed-Phase Chromatography Multiple Heart-Cutting 2D-LC Analysis of Innovator versus Biosimilar Monoclonal Antibodies. Agilent Technologies Application Note, 2016.