Quantification of paclitaxel, its degradants, and related substances using UHPLC with charged aerosol detection

Significance of the topic

Paclitaxel is a vital chemotherapeutic agent extracted or semi-synthesized from Taxus species. Accurate quantification of paclitaxel, its related substances, and degradation products is mandatory to ensure drug safety and compliance with regulatory thresholds. Conventional UV/Vis detection struggles with analytes lacking chromophores or with widely varying extinction coefficients. Charged aerosol detection (CAD) offers near-uniform response for nonvolatile analytes but requires compensation for mobile phase gradients.

Objectives and Study Overview

This study aimed to demonstrate: 1) quantification of multiple impurities using a single calibrant by leveraging CAD’s inherent uniform response; 2) the use of Thermo Scientific™ Vanquish™ Flex Duo UHPLC with inverse gradient compensation to maintain consistent solvent composition at the CAD nebulizer during gradient elution.

Methodology and Instrumentation

The separation utilized an Accucore™ Pentafluorophenyl (PFP) column (2.1×150 mm, 2.6 μm) with water (A) and acetonitrile (B). The analytical gradient (23–60 % B over 25 min at 0.3 mL/min) was counterbalanced by a mirrored compensation gradient (23–60 % A) directed post-column to the CAD. Detection combined a Variable Wavelength Detector at 227 nm and a Charged Aerosol Detector (evaporation temperature 50 °C, 5 Hz). Forced thermal degradation of paclitaxel (65 °C, 2 h) generated degradants for comprehensive profiling. Chromeleon™ CDS v7.2.8 enabled data acquisition and global calibration.

Instrumentation

• Thermo Scientific™ Vanquish™ Flex Duo UHPLC system with Dual Pump F, Split Sampler FT, Column Compartment H
• Charged Aerosol Detector F and Variable Wavelength Detector F
• Accucore™ PFP column, 2.1×150 mm, 2.6 μm
• GenPure™ xCAD Plus ultrapure water system; LC-MS grade acetonitrile
• Digital Heating Shaking Drybath for forced degradation

Main Results and Discussion

CAD calibration curves for paclitaxel, Impurity C, and cephalomannine exhibited slopes within 2 % deviation over 0.5–10 μg/mL, confirming uniform response. In contrast, UV response varied by up to 63 %. Without inverse gradient compensation, CAD peak areas of early eluting impurities were underestimated and late eluters overestimated, yielding >10 % quantitation errors. Implementing inverse gradient provided stable solvent composition at the detector, minimizing response bias. The combined UV/CAD approach allowed detection of UV-active but CAD-invisible volatile degradants, ensuring comprehensive impurity profiling.

Benefits and Practical Applications

• Single-calibrant quantification of multiple related species reduces the need for individual impurity standards.
• Inverse gradient compensation ensures unbiased CAD response throughout gradient elution.
• Combination of CAD and UV detection offers thorough profiling of both nonvolatile and UV-active compounds, ideal for stability and forced-degradation studies.

Future Trends and Potential Applications

• Extension of inverse-gradient CAD workflows to additional pharmaceutical APIs and complex matrices.
• Integration with high-resolution mass spectrometry for structural elucidation of unknown degradants.
• Automation and AI-driven data evaluation to accelerate impurity classification and regulatory compliance.
• Implementation in routine QA/QC laboratories for robust method transfer and standardization.

Conclusion

The Vanquish™ Flex Duo UHPLC system with inverse gradient compensation enables reliable, uniform CAD quantification of paclitaxel and related substances using a single calibrant. Coupling CAD with UV detection delivers a powerful multi-detector platform for accurate impurity and degradation analysis, enhancing drug development and quality control workflows.

References

1. World Health Organization. Cancer Fact Sheet No. 297. Accessed November 20, 2017.
2. International Council for Harmonisation (ICH). ICH Guidelines. Accessed November 20, 2017.
3. Gamache P. Charged Aerosol Detection for Liquid Chromatography and Related Separation Techniques. Wiley-VCH; 2017.
4. Thermo Fisher Scientific. Charged Aerosol Detection brochure. Accessed November 20, 2017.
5. Thermo Fisher Scientific. Technical Note: Charged Aerosol Detection (CAD) – Factors Affecting Uniform Analyte Response. In press.