HPLC transfer and optimization of deoxycholic acid analysis using HPLC – Charged Aerosol Detector

Significance of the topic

The precise quantification of deoxycholic acid and related bile acids is critical in pharmaceutical and biopharmaceutical settings for quality control, impurity profiling and compliance with pharmacopeial standards.

Objectives and study overview

This work presents the transfer and optimization of a USP monograph HPLC method for deoxycholic acid analysis from the Vanquish Charged Aerosol Detector (CAD) to the enhanced Vanquish CAD HP. The aim was to compare sensitivity, detection limits, calibration performance and overall analytical efficiency between the two detector generations.

Methodology and instrumentation

• Instrumentation: Thermo Scientific Vanquish Flex UHPLC system fitted with Vanquish CAD and Vanquish CAD HP.
• Column: Acclaim 120 C18, 4.6×150 mm, 3 µm; temperature controlled at 30 °C or 60 °C depending on the mode.
• Mobile phases: 0.1 % formic acid in water (A) and acetonitrile (B); gradient elution over 38 min.
• Injection volume: 25 µL; autosampler maintained at 8 °C.
• Detector settings: CAD data rate 20 Hz, filter constant 2–10 s, evaporation tube at 50 °C; CAD HP data rate 50 Hz, filter 1.0 s, gas regulation analytical, PV adjusted between 1.5–2.4.
• Calibration: standard solutions from 0.5 to 10 µg/mL to evaluate linearity (R²) and residuals.

Main results and discussion

• Both detectors achieved excellent linearity (R² = 1.000) at optimized settings: PFV 1.2 for CAD and PV 1.8 for CAD HP.
• Vanquish CAD HP enabled simultaneous acquisition of four PV channels, reducing total analysis time.
• Sensitivity gains: signal-to-noise ratios increased from ~711 to ~928 for cholic acid and ~695 to ~916 for deoxycholic acid when using CAD HP.
• Limits of detection and quantification improved with CAD HP (LOD 0.3 µg/mL, LOQ 1.1 µg/mL for deoxycholic acid) versus CAD (LOD 0.4, LOQ 1.4 µg/mL).
• System suitability tests showed repeatability below 0.62 % RSD and met USP criteria for both detectors.

Benefits and practical applications of the method

• Enhanced sensitivity and lower detection limits support reliable trace-level quantification.
• Simultaneous multi-channel acquisition accelerates method development and optimization.
• Seamless transfer from legacy CAD instruments ensures continuity in regulated environments.

Future trends and potential uses

• Integration of advanced detector architectures for broader compound classes.
• Automation of parameter optimization using software-driven workflows.
• Expansion of charged aerosol detection to other semi-volatile and non-UV active analytes.

Conclusion

The transfer from Vanquish CAD to CAD HP successfully improved deoxycholic acid assay performance, delivering superior sensitivity, faster method adaptation and full compliance with USP requirements. The CAD HP system represents a robust solution for modern HPLC applications in pharmaceutical analysis.

Reference

1. United States Pharmacopeial Convention. USP-NF Deoxycholic Acid. Rockville, MD, 2024.
2. Bailey B, Gamache P, Acworth I, Lovejoy K. Guidelines for method transfer and optimization—from earlier model Corona detectors to Corona Veo and Vanquish charged aerosol detectors. Thermo Fisher Scientific; 2017.
3. Gamache P, Muellner T, Eggart B, Lovejoy K, Acworth I. Charged aerosol detection – use of the power function and robust calibration practices to achieve the best quantitative results. Thermo Fisher Scientific TN 73299; 2019.