Best practices for liposome analysis with the Charged Aerosol Detector

Importance of the topic

Liposomal drug carriers play a critical role in targeted cancer therapy. Accurate quantitation of key lipids such as cholesterol, DSPE-PEG2000 and HSPC ensures consistent drug potency and stability. The Charged Aerosol Detector (CAD) offers a universal, sensitive response for nonvolatile lipid species, overcoming the limitations of UV detection and enabling robust quantitative analysis in complex formulations.

Objectives and study overview

This work evaluates best practices for lipid quantitation using the new ASTM E3297-21 standard with HPLC-CAD. The study compares column chemistries and system configurations across four laboratories in three countries. Key goals include verifying system suitability, assessing method reproducibility, and defining optimal calibration and gradient conditions for reliable lipid ratio determination.

Methodology and instrumentation

Sample preparation involved liposomal formulations spiked with cholesterol, DSPE-PEG2000 and HSPC. Calibration curves covered 0.2 to 300 μg/g. Three C18 column types (fully porous Hypersil GOLD, ethylene-bridged hybrid, silica) of 150×3 mm were tested on Thermo Scientific Vanquish Flex UHPLC and Vanquish Core HPLC systems equipped with quaternary or binary pumps. Chromatographic conditions included 0.7 mL/min flow, step gradient from 40% to 90% organic, and constant column temperature at 35 °C. The CAD was operated at 35 °C evaporation temperature with power function values of 1.0 and 1.2. Data were acquired and processed in Chromeleon CDS using peak grouping and power-function (log-log) calibration fitting.

Key results and discussion

• Resolution between lipid pairs exceeded Rs>1.5 on all columns, ensuring clear peak separation.
• Peak area reproducibility was excellent, with %RSD below 3% for a mid-range standard across all sites and columns.
• Log-log calibration curves achieved R2>0.995 across the full concentration range; quadratic and weighted linear fits also met ASTM criteria.
• Limit of quantification was improved at PFV 1.2, reaching 0.2‒0.5 μg/g for cholesterol and DSPE-PEG2000, and 0.5 μg/g for HSPC.
• DSPE-PEG2000 and HSPC coelute in the same gradient step, enabling quantification with a single calibration curve. Nonvolatile impurities eluting alongside can also be quantified similarly.
• The Hypersil GOLD 150×3 mm, 3 µm column consistently passed system suitability tests across all configurations, making it the preferred choice.

Benefits and practical applications

• Single-curve quantitation of multiple lipid components accelerates analysis and reduces calibration efforts.
• High inter-laboratory reproducibility supports method transfer and regulatory compliance in pharmaceutical QC.
• CAD’s uniform response for nonvolatile species allows surrogate calibration for unknown lipids, simplifying method development.
• Wide dynamic range accommodates both trace-level and high-concentration measurements in formulation development.

Future trends and opportunities

The method can be further refined by exploring inverse gradient strategies to extend linear range and by evaluating novel stationary phases for enhanced selectivity. Integration of CAD data with automated data-handling workflows and machine learning could streamline quality control. Expansion to other lipid classes and coupling with orthogonal detectors may broaden applications in lipidomics and biopharmaceutical analysis.

Conclusion

The ASTM E3297-21 HPLC-CAD protocol delivers a robust, reproducible approach for quantifying liposomal lipids across multiple laboratories. Its reliable resolution, low quantification limits and simplified calibration make it highly suitable for routine QC and research in pharmaceutical and lipid analysis settings.

Instrumental setup

• HPLC systems: Thermo Scientific Vanquish Flex UHPLC (quaternary pump) and Vanquish Core HPLC (binary pump)
• Columns: Hypersil GOLD C18, EBH C18, and silica C18 (150×3 mm, 3–3.5 µm)
• Detector: Thermo Scientific Corona Veo Charged Aerosol Detector
• Autosampler and column compartment temperature control at 10 °C and 35 °C, respectively

References

1. ASTM International Standard E3297-21. Standard test method for lipid quantitation in liposomal formulations using HPLC-CAD. April 2022. DOI: 10.1520/E3297-21.
2. Thermo Fisher Scientific Technical Guide 73914. Getting the most out of your charged aerosol detector.
3. Thermo Fisher Scientific White Paper 72711. Instrument parameter guide for (U)HPLC method transfer.
4. Thermo Fisher Scientific Technical Note 73449. Why use charged aerosol detection with inverse gradient?
5. Gamache PH, ed. Charged aerosol detection for liquid chromatography and related separation techniques. John Wiley & Sons; 2017. DOI: 10.1002/9781119390725.
6. Thermo Fisher Scientific Technical Note 73299. CAD power function and robust calibration practices.