Analysis of Fatty Acids in Polysorbate 80 Using High-Performance Liquid Chromatography (HPLC) with Charged Aerosol Detection (CAD)

Significance of the topic

Polysorbate 80 is a widely used non-ionic surfactant in pharmaceutical, food and cosmetic products. Its complex, heterogeneous lipid composition and the absence of strong chromophores in constituent fatty acids complicate routine quality control. Conventional USP workflows rely on lengthy reflux/sample preparation and GC-FID analysis. Development of a fast, robust HPLC method with charged aerosol detection (CAD) addresses the need for higher throughput, sensitive quantitation and streamlined QC for raw material release and stability testing.

Objectives and overview of the study

The study aimed to develop and optimize a high-throughput reversed-phase HPLC method coupled to CAD for the quantitative profiling of fatty acids released from polysorbate 80. Key goals were to (1) optimize CAD and chromatographic parameters to maximize sensitivity and linearity, (2) establish limits of quantitation (LOQ) and system suitability, and (3) demonstrate accurate fatty acid composition determination including identification of an additional peak (petroselinic acid) observed in polysorbate samples.

Methodology

• Sample preparation: Polysorbate 80 samples were saponified in 90:10 1 M KOH/methanol at 1.5 mg/mL (incubation 6 h at 40 °C). After saponification, samples were acidified with formic acid, extracted into methyl tert‐butyl ether (MTBE), dried under nitrogen and reconstituted in 75:25 acetonitrile/water (v/v).
• Chromatography: Reversed-phase separation on XBridge BEH C18, 4.6 × 100 mm, 3.5 µm, column temperature 60 °C; mobile phases: A = 0.05% formic acid in water, B = 0.05% formic acid in acetonitrile; flow rate 1.5 mL/min; injection volume 35 µL; sample tray at 10 °C; gradient elution to resolve common fatty acids.
• Detection and data processing: Charged aerosol detection with optimization of evaporator temperature, filter time constant and digital power function value (PFV). Data acquisition and reporting used Empower 3.6.0.

Instrumentation used

• Arc HPLC system with column heater/cooler and active pre-heater (Waters).
• XBridge BEH C18 column, 4.6 × 100 mm, 3.5 µm.
• Waters Charged Aerosol Detector (CAD) configured with: PFV = 1.00, sampling rate = 10 points/s, filter time constant = 1.4 s, ion trap voltage = 20 V. Evaporator temperature optimized at 25 °C.

Main results and discussion

• Detector optimization: Evaporator temperature had a strong effect on signal; the highest CAD response was observed at 25 °C. A filter time constant of 1.4 s maximized signal-to-noise (S/N) for fatty acids. The PFV digital correction of the CAD signal improved linearity; PFV = 1.00 produced R2 ≥ 0.99 for most analytes across 0.05–25 µg/mL, although myristic acid showed less ideal linearity.
• Analytical performance: Limits of quantitation (LOQ), defined as S/N ≥ 10, ranged from approximately 0.9 to 3.5 ng on column depending on the fatty acid. System suitability over 10 injections of a 10 µg/mL standard mixture showed excellent repeatability: peak area %RSD ≤ 0.48% and retention time %RSD ≤ 0.02%.
• Fatty acid profiling and identification: Chromatograms of saponified polysorbate 80 revealed the expected fatty acids (myristic, palmitoleic, palmitic, linoleic, oleic, stearic) plus an additional peak after oleic acid. Spiking with a petroselinic acid standard confirmed this peak identity. Relative composition was calculated per the USP polysorbate 80 monograph by comparing individual fatty acid peak areas to the total fatty acid-related peak area, including petroselinic acid; results met USP criteria for the tested materials.
• Method advantages and limitations: CAD provided robust detection for non-chromophoric fatty acids and eliminated the need for derivatization or GC-FID. However, CAD response is inherently non-linear for some analytes and requires PFV/digital processing for optimal calibration. Short-chain or low-mass species (e.g., myristic acid) may show deviating linearity and should be monitored during validation.

Benefits and practical applications of the method

• High-throughput QC: The HPLC-CAD workflow reduces sample preparation time versus USP reflux and GC-FID approaches and is amenable to routine QC throughput in raw material release testing.
• Broad applicability: The method quantifies a comprehensive panel of fatty acids released from polysorbate 80 and can incorporate additional identified species (petroselinic acid) in composition calculations.
• Regulatory readiness: Integration with Empower software and robust system suitability metrics supports traceable, compliance-ready reporting in regulated environments.

Future trends and possibilities for use

• Broader validation and inter-laboratory studies to support method transfer and potential adoption as an alternative or complement to USP GC-based procedures.
• Automation of sample preparation and extraction to further increase throughput and reduce manual variability.
• Hybrid detection strategies: coupling CAD profiling with orthogonal identification by LC–MS/MS for confirmation of minor or unexpected fatty acids across different polysorbate lots and suppliers.
• Extension to other polysorbates and complex excipient matrices, and development of standardized CAD data processing (PFV and filter settings) to improve cross-platform reproducibility.

Conclusion

A high-throughput HPLC method with CAD detection was developed and optimized for quantitative fatty acid profiling of polysorbate 80. Optimization of CAD parameters (evaporator temperature, filter time constant and PFV) and chromatographic conditions produced low LOQs (0.9–3.5 ng on column), excellent repeatability and linearity across a practical calibration range for most analytes. The method accurately determined fatty acid composition, including confirmation of petroselinic acid, and offers a faster, compliance-ready alternative to traditional GC-FID-based USP workflows for polysorbate 80 quality control.

References

1. Ilko D, Braun A, Germershaus O, Meinel L, Holzgrabe U. Fatty Acid Composition Analysis in Polysorbate 80 with High Performance Liquid Chromatography Coupled to Charged Aerosol Detection. European Journal of Pharmaceutics and Biopharmaceutics. 2015;94:569–574.
2. Maziarz M, Harden S, Rainville R. Determination of Fatty Acid Composition in Polysorbate 80 using HPLC with Charged Aerosol Detection. Waters Application Note 720009340. 2026.
3. United States Pharmacopeia. USP Monograph for Polysorbate 80, USP–NF 2021 Issue 1. Official 01-May-2020.