Best practices for liposome analysis with the charged aerosol detector

Importance of the Topic

Liposome formulations containing cholesterol, DSPE-PEG2000, and hydrogenated soy phosphatidylcholine (HSPC) are widely used as drug carriers in oncology and vaccine development. Accurate quantitation of these lipids in final products is essential for ensuring consistent drug potency and safety. Traditional UV detectors struggle with nonchromophoric compounds, whereas charged aerosol detection (CAD) provides uniform, sensitive response for nonvolatile analytes, making it an ideal choice for liposome quality control.

Objectives and Study Overview

The primary goal was to streamline column selection and system setup for liposome lipid analysis using CAD, establish reliable quantitation strategies, and reduce analyst workload. A comparison test was conducted across four laboratories using multiple Thermo Scientific Vanquish Flex UHPLC and Core HPLC systems, three different C18 columns, and evaluation of method performance against ASTM E3297-21 criteria.

Methodology

• Sample preparation and calibration followed ASTM E3297-21 guidelines, with standards at 0.5–300 µg/g in methanol.
• A step gradient HPLC method (40–90 % methanol) at 35 °C separated cholesterol, DSPE-PEG2000, HSPC 1 (C16:0) and HSPC 2 (C18:0).
• Three fully porous C18 columns (3–3.5 µm, 150 × 3 mm) were tested.
• CAD parameters, including evaporation tube temperature and power function value (PFV 1.0 or 1.2), were optimized for linear response over broad concentration ranges.

Used Instrumentation

• Thermo Scientific Vanquish Flex Quaternary UHPLC and Vanquish Core Quaternary/Binary HPLC systems with CAD detectors.
• Thermo Scientific Hypersil GOLD C18, ethylene-bridged hybrid C18, and fully porous silica C18 columns.
• Standard laboratory sample handling equipment: heated sonicator, vortex mixer, centrifuge, amber glass vials.

Main Results and Discussion

• System suitability was met in all 12 column-system-site combinations: resolution > 1.5, peak area RSD < 5 %, and calibration R² ≥ 0.995.
• Critical resolution between cholesterol and DSPE-PEG2000 improved by increasing gradient delay volume; HSPC 1/HSPC 2 selectivity was enhanced by reducing the high-organic step from 90 % to 80 % B on column 3.
• CAD linearization via log-log and quadratic fitting after data collection both yielded acceptable calibration across 0.2–300 µg/g. PFV adjustments (1.0 and 1.2) enabled direct linear calibration for different concentration windows (0.5–50 and 10–300 µg/g respectively).
• Limits of quantification ranged from 0.2 to 8 µg/g depending on lipid, PFV, and column choice.

Benefits and Practical Applications

• Universal detection of nonchromophoric lipids and impurities without the need for chromophores.
• Robust performance across multiple UHPLC/HPLC platforms and column chemistries.
• Flexible calibration strategies reduce the number of standards and analyst time.
• Simple method adjustments (mixer volume, gradient composition, PFV) tailor resolution and sensitivity.

Future Trends and Applications

• Implementation of inverse gradients for single-curve quantitation of all lipids and related impurities.
• Automation and AI-driven optimization of CAD parameters and gradient settings.
• High-throughput liposome profiling and real-time monitoring in biomanufacturing.
• Integration with mass spectrometry and alternative detectors for comprehensive liposomal characterization.

Conclusion

This study confirms that HPLC-CAD analysis of liposomal lipids is robust, reproducible, and adaptable across multiple columns and systems. Following ASTM E3297-21, laboratories can achieve precise quantitation with minimal method adjustments, supporting quality control in pharmaceutical development.

Reference

• ASTM International. E3297-21: Standard Test Method for Lipid Quantitation in Liposomal Formulations by HPLC-CAD (2022).
• Thermo Fisher Scientific. Technical Guide 73914: Factors Influencing CAD Performance.
• Thermo Fisher Scientific. White Paper 72711: UHPLC Method Transfer Guide.
• Thermo Fisher Scientific. Technical Note 73449: CAD with Inverse Gradient.
• Eckardt M., Kubicova M., Simat T.J. J. Chromatogr. A 1572 (2018) 187–202.
• Thermo Fisher Scientific. Application Note 001630: UHPLC/UV/CAD/MS for Extractables and Leachables.
• Gamache P.H., Charged Aerosol Detection for Liquid Chromatography, Wiley (2017).
• Thermo Fisher Scientific. Technical Note 73299: CAD Calibration Practices.
• Thermo Fisher Scientific. Technical Note 732998: CAD Power Function Optimization.