Quantitative aspects in on-line comprehensive two-dimensional liquid chromatography for pharmaceutical applications

Area of Science:

• Analytical Chemistry
• Chromatography
• Pharmaceutical Analysis

Background:

• Complex pharmaceutical samples necessitate advanced analytical techniques for impurity profiling.
• On-line comprehensive two-dimensional liquid chromatography (LCxLC) offers high peak capacity, suitable for complex mixtures.
• Regulatory requirements demand sensitive and precise methods for pharmaceutical impurity control.

Purpose of the Study:

• To optimize on-line RPLCxRPLC (reversed-phase liquid chromatography x reversed-phase liquid chromatography) for quantitative analysis of pharmaceutical impurities.
• To achieve conditions meeting regulatory standards for impurity detection at low concentrations.
• To evaluate the method's sensitivity, linearity, repeatability, and precision.

Main Methods:

• Optimization of RPLCxRPLC conditions using a Pareto-optimality approach, focusing on sensitivity and low dilution factors.
• Quantitative analysis based on relating impurity concentration to the sum of second dimension peak areas.
• Application of the developed method to analyze a drug substance batch.

Main Results:

• Achieved a detection limit below 0.05% for impurities relative to the active pharmaceutical ingredient (API).
• Demonstrated excellent linearity (R² > 0.999), repeatability (RSD < 1% for 2D retention times), and precision (RSD < 1.5% for sum of 2D peak areas).
• Successfully detected impurities as low as 0.04% in a drug substance sample.

Conclusions:

• On-line RPLCxRPLC is a powerful technique for the quantitative analysis of pharmaceutical samples.
• The optimized method meets stringent regulatory requirements for pharmaceutical impurity control.
• This approach enables sensitive and reliable detection of impurities in drug development stages.