EAS: Improving the Quantitation of Unknown Impurity Analysis Using Dual-Gradient HPLC with Charged Aerosol Detection

Significance of the Topic

The accurate quantification of unknown impurities and trace compounds is critical in pharmaceutical development, cleaning validation, and quality control. Traditional HPLC detectors such as UV and evaporative light scattering often suffer from variable sensitivity and nonlinear response under gradient conditions, leading to substantial errors when reference standards are unavailable.

Study Objectives and Overview

This work evaluates a dual‐gradient HPLC/UHPLC platform coupled with charged aerosol detection (CAD) and a postcolumn inverse gradient. The goals are to:

• Stabilize nebulization efficiency under gradient elution
• Assess response consistency across diverse nonvolatile analytes
• Demonstrate quantification of unknown compounds using a single calibrant

Methodology

Two experiments were designed. The first compared CAD response variability for five APIs with and without an inverse gradient. The second tested low‐level quantification of nine compounds spanning a range of chemical classes and retention behaviors. Mixtures were prepared at known mass concentrations and serially diluted. Peak areas were recorded to evaluate reproducibility, linearity, and recovery.

Used Instrumentation

• UltiMate 3000 RSLC system with dual gradient pumps
• Acclaim RSLC 120 C18 3 µm column (3.0 × 33 mm)
• DAD UV detector (210 nm, 254 nm)
• Corona ultra CAD (nitrogen at 35 psi)
• Mobile phases: 10 mM ammonium acetate pH 4.5 and acetonitrile

Main Results and Discussion

Implementation of the inverse gradient yielded dramatic improvements:

• Five‐compound set: CAD response deviation reduced from 19% to 4.4% RSD.
• Nine‐compound set: Linear calibration curves (r^2 ≥ 0.999), limits of detection 1–5 ng on column, limits of quantitation 6–11 ng.
• Recovery across all nine analytes using a single calibrant showed 66% within ±25% and 87% within ±50% of expected values.
• In comparison, UV at 210 nm and 254 nm showed >100% RSD for the same mixture.

Practical Benefits and Applications

The combined dual‐gradient/CAD approach enables:

• Uniform mass‐sensitive detection of nonvolatile impurities without reference standards
• Improved accuracy for mass balance and impurity reporting
• Streamlined workflows in impurity profiling, degradant analysis, cleaning validation, and library compound screening
• Time and cost savings by reducing the need for multiple methods and standards

Future Trends and Opportunities

Potential developments include:

• Expansion to semivolatile and polar compound classes
• Integration with high‐resolution mass spectrometry for structural elucidation
• Automation of inverse gradient optimization
• Broader application in environmental, food, and petrochemical analysis

Conclusion

The use of a postcolumn inverse gradient with charged aerosol detection on a dual‐gradient HPLC platform markedly improves response uniformity and quantitation accuracy for unknown impurities. This strategy allows reliable single‐calibrant quantification and enhances sensitivity under gradient elution, representing a robust alternative to UV and ELSD methods.

References

1. Dependence of Response on Chemical Structure, ESA—A Dionex Company, Application Brief 70-8913 Rev B.
2. Matthews D. et al., Improving Quantitative Measurements for the ELSD, Chromatographia, 2004, 60, 625–633.
3. Lane T. et al., Toward Single Calibrant Quantification in HPLC, Anal. Chem., 2005, 77, 4354–4365.
4. Górecki T., Lynen F., Scucs R., Sandra P., Universal Response in Liquid Chromatography Using Charged Aerosol Detection, Anal. Chem., 2006, 78, 3186–3192.