Two-Dimensional Liquid Chromatography for Small Molecule Pharmaceutical Analysis – More Knowledge in Less Time

Significance of the Topic

The pharmaceutical industry faces increasing pressure to accelerate the delivery of safe therapeutics while managing complex molecular structures and diverse impurity profiles. Traditional one-dimensional liquid chromatography often struggles to fully resolve co-eluting impurities, particularly during early-stage process development when impurity knowledge is limited. Two-dimensional liquid chromatography (2D-LC) offers enhanced separation power and selectivity, enabling more comprehensive analysis of known and unknown impurities in less time.

Objectives and Study Overview

This study presents Bristol Myers Squibb’s implementation of 2D-LC methods for small molecule pharmaceutical analysis, focusing on three key applications:

• Peak purity assessment using orthogonal reversed-phase dimensions combined with photodiode array (PDA) and mass spectrometric detection
• Quantitative impurity profiling of challenging atropisomeric mixtures
• Trace-level impurity enrichment via trapping-mode 2D-LC for low-abundance mutagenic species

Methodology and Instrumentation

2D-LC workflows were developed to expand separation space and improve sensitivity:

• Peak purity checks employed heart-cutting from an existing 1D method into orthogonal stationary phases selected via an online column database
• Quantitative analysis of atropisomers used high-resolution fraction sampling with multiple cuts and summation of peak areas
• Trapping mode experiments incorporated repeated enrichment cycles on a dedicated trap column for mutagenic impurity quantification

Instrumentation

• H-Class UPLC system with OpenLab CDS (Empower) control
• Reversed-phase columns: Ascentis Express C18, Zorbax Bonus-RP, Pinnacle DB BiPh, BEH Shield RP18
• Photodiode array detector operating at 220–320 nm
• Triple quadrupole mass spectrometer (full scan MS)
• Automated switching valves for heart-cutting and trapping

Main Results and Discussion

• Orthogonal 2D-LC–MS achieved separation of co-eluting isomeric impurities not resolved in 1D, with no loss of chromatographic efficiency and detection sensitivity at sub-percent levels.
• Atropisomeric analysis of BMS-986142 demonstrated consistent quantification (%RSD < 1.0 for total peak areas) and recovery (> 96%) across multiple injections and fractionation schemes.
• Trapping-mode 2D-LC enabled enrichment factors exceeding 20-fold, with linear calibration (R² > 0.9997) and recoveries near 100% for a low-level mutagenic impurity at ppm concentrations.

Benefits and Practical Applications

• Enhanced confidence in impurity profiling by resolving unknown unknowns and confirming peak purity with coupled PDA and MS
• Accurate quantitative analysis of complex mixtures without extensive method development time
• Enrichment of trace-level analytes supports regulatory requirements for mutagenic impurity control
• Turnkey commercial solutions and standardized workflows facilitate routine implementation in quality control and process development labs

Future Trends and Potential Applications

Advancements in hardware, software, and column chemistries will further streamline 2D-LC adoption:

• Faster cycle times through improved solvent compatibility and valve technology
• Automated method scouting and data processing leveraging machine learning
• Integration with orthogonal detection modes such as ion mobility and high-resolution MS
• Expansion to multi-dimensional separations beyond two dimensions.

Conclusion

Two-dimensional liquid chromatography has emerged as a versatile and powerful tool for pharmaceutical impurity analysis, delivering deeper structural insights and robust quantification in less time. Its adoption is poised to grow as solvent-mismatch challenges are overcome and commercial solutions become more accessible, making 2D-LC a potential default approach for complex small molecule analyses.

Reference

1. Baghdady YZ, Stoll DR. System suitability testing for two-dimensional liquid chromatography. LCGC. 2022;40:292.
2. Wang Q, He B, Shackman J. Quantitative analysis of atropisomeric impurities in pharmaceutical candidates via two-dimensional LC. J Pharm Biomed Anal. 2021;193:113730.
3. Lin Z, Wang Q, Zhou Y, Shackman J. Trapping-mode two-dimensional LC for trace-level mutagenic impurity quantification. J Chromatogr A. 2023;1700:464043.