Semi-preparative high-resolution recycling liquid chromatography

Area of Science:

• Analytical Chemistry
• Chromatography
• Pharmaceutical Analysis

Background:

• Drug and impurity purification faces challenges like poor resolution, solvent mismatches, and low impurity abundance.
• Achieving high purity (>90%) and yield (>99%) for trace impurities is critical for structure elucidation.

Purpose of the Study:

• To develop and test a semi-preparative high-resolution system using twin column recycling liquid chromatography for purifying trace impurities.
• To optimize the system for maximizing speed-resolution and production rate while addressing purification constraints.

Main Methods:

• A semi-preparative high-resolution system with twin columns, binary pump, sample manager, column oven, and fraction manager was constructed.
• Optimization involved selecting particle size for maximum speed-resolution and determining optimal cycles and injection volume for production rate.
• The process was automated for sample cleaning, impurity transfer, and collection, tested with a trace impurity in estradiol.

Main Results:

• The system achieved significant enrichment (factor ~5000) of a trace impurity from a concentrated estradiol solution.
• Optimization of column particle size and recycling cycles maximized speed-resolution and production rate.
• Despite automation and enrichment, the collected impurity amount (~120 μg) was insufficient for NMR, with only 50% purity.

Conclusions:

• The developed recycling liquid chromatography system demonstrates potential for trace impurity purification from APIs.
• LC-MS and UV spectra suggested the impurity was the enol tautomer of estrone.
• Further optimization is needed to achieve sufficient quantity and purity for definitive structure elucidation via NMR.