A Sensitive Detection Technique for Analysis of Lipids in Liposomal Formulations Using an Agilent 1290 Infinity II ELSD

Significance of the Topic

The accurate quantification of lipids in liposomal drug delivery systems is critical for ensuring formulation stability, controlled release properties and therapeutic efficacy. Conventional detectors relying on UV absorption cannot detect lipids lacking chromophores, making evaporative light scattering detection (ELSD) an ideal choice for comprehensive lipid analysis.

Objectives and Study Overview

This study evaluates the performance of the Agilent 1290 Infinity II ELSD in combination with an Agilent 1260 Infinity II LC to achieve sensitive, reproducible detection of four lipids commonly used in liposomal formulations—tricaprylin, dipalmitoylphosphatidylglycerol (DPPG), cholesterol and dierucoylphosphatidylcholine (DEPC). Key aims include determining detection limits, calibration linearity and method repeatability for a single-run separation.

Used Instrumentation

• Agilent 1260 Infinity II LC system with binary pump (G7112B) and vialsampler with column compartment (G7129A)
• Agilent 1290 Infinity II evaporative light scattering detector (G4261B)
• Agilent ZORBAX Eclipse Plus C8 column (4.6 × 250 mm, 5 µm)
• Agilent OpenLab CDS software v2.3.0

Methodology

Sample Preparation:

• Stock Solutions: 1 000 µg/mL in methanol for each lipid, stored at 2–8 °C
• Calibration Standards: 20–100 µg/mL in methanol (levels 1–5)
• LOD Solutions: 1 µg/mL for tricaprylin, cholesterol, DPPG; 1.5 µg/mL for DEPC

HPLC Conditions:

• Column Temperature: 30 °C
• Mobile Phase: ammonium acetate in water (Solution A) and in methanol (Solution B)
• Gradient: 90% B at 0 min to 100% B at 15 min, hold until 23 min, return to 90% B at 23.1 min, re-equilibrate until 30 min
• Flow Rate: 1.0 mL/min
• Injection Volume: 10 µL (100 µL for LOD measurements)

ELSD Settings:

• Evaporator Temperature: 35 °C
• Nebulizer Temperature: 35 °C
• Nitrogen Gas Flow: 1.6 SLM
• PMT Gain: 1.0
• Data Rate: 10 Hz; Smoothing: 3 s

Results and Discussion

The method achieved limits of detection down to 1 µg/mL for tricaprylin, cholesterol and DPPG and 1.5 µg/mL for DEPC, with signal-to-noise ratios between 4.7 and 8.6. Quadratic calibration curves provided the best fit across 20–100 µg/mL, yielding correlation coefficients (R²) of 0.991–0.999. Repeatability tests (n = 6 at 100 µg/mL) demonstrated retention time RSD ≤0.05% and peak area RSD ≤3.1%, confirming excellent method precision. All four analytes were resolved in a single 30-minute run.

Benefits and Practical Applications

This ELSD-based approach enables sensitive, reproducible quantitation of both volatile and nonvolatile lipids without derivatization or reliance on chromophores. Its compatibility with gradient elution permits simultaneous separation of multiple lipid classes, making it well suited for pharmaceutical quality control, formulation development and routine lipid profiling in biopharmaceutical labs.

Future Trends and Applications

Advances in ELSD optics and subambient operation offer further sensitivity gains for volatile lipids. Coupling ELSD with mass spectrometry and expanding its use to novel lipid-based nanocarriers will enhance lipidomics workflows. Automated data processing and integration with QC software will streamline high-throughput lipid analysis.

Conclusion

The Agilent 1290 Infinity II ELSD combined with an Agilent 1260 Infinity II LC provides a robust, high-sensitivity platform for lipid analysis in liposomal formulations. The method achieves low microgram-per-milliliter detection limits, excellent linearity and precision while resolving multiple lipids in a single run, addressing key needs in pharmaceutical analytics.

References

• Improved Sensitivity with the Agilent 1290 Infinity Evaporative Light Scattering Detector. Agilent Technologies Technical Overview, publication number 5991-2394EN.
• Jeschek D, et al. A Versatile, Quantitative Analytical Method for Pharmaceutical Relevant Lipids in Drug Delivery Systems. J. Pharmaceut. Biomed. 2016;119:37–44.
• Van Hoogevest P, Wendel A. The Use of Natural and Synthetic Phospholipids as Pharmaceutical Excipients. Eur. J. Lipid Sci. Technol. 2014;116(9):1088–1107.