Determination of Fatty Acids Composition in Polysorbates 80 and 20 Pharmaceutical Raw Materials by HPLC with Mass Detection

Significance of the Topic

Polysorbates 80 and 20 are essential non-ionic surfactants used in pharmaceuticals, food and cosmetics to stabilize emulsions and enhance formulation stability and texture.

Objectives and Study Overview

The aim of this work was to establish HPLC methods coupled with mass spectrometry for direct determination of free fatty acid composition in hydrolyzed polysorbate samples, streamlining the workflow compared with traditional GC-FID procedures.

Methodology and Instrumentation

Sample Preparation

• Hydrolysis of polysorbates using 1 M KOH at 40 °C for 6 h
• Neutralization with formic acid and dilution in 50:50 water/ethanol
• Filtration through GHP syringe filters

Chromatographic and Detection Conditions

• Arc HPLC System with ACQUITY QDa Detector and isocratic solvent manager
• Mobile phase: ammonium acetate buffer, acetonitrile, isopropyl alcohol wash
• XBridge BEH C18 column at 60 °C, 2.0 mL/min flow rate
• Negative electrospray ionization (ESI-) with single ion recording (SIR) for quantitation

Used Instrumentation

• Arc HPLC System with column heater/cooler and passive pre-heater
• ACQUITY QDa Detector
• Xevo G2-XS QTof Mass Spectrometer
• ACQUITY UPLC I-Class System
• Empower Software for data analysis

Main Results and Discussion

The proposed HPLC-MS methods achieved baseline separation of all USP specified fatty acids in polysorbates 80 and 20. Mass spectral data enabled rapid identification, while SIR channels provided accurate quantitation within USP criteria. Unknown peaks corresponding to positional isomers of linoleic (conjugated linoleic acids) and oleic acids (cis-vaccenic and elaidic) were detected and confirmed using QTof mass accuracy. Base hydrolysis conditions proved effective for complete fatty acid release.

Benefits and Practical Applications

• Elimination of derivatization and direct injection of hydrolyzed samples
• Faster quality control compared with GC-FID methods
• Enhanced detection of additional fatty acid isomers
• Compliance-ready workflow integrated with Empower Software

Future Trends and Opportunities

Extending this approach to other excipients and degradation product profiling using high-resolution MS could further improve product characterization. Automation of sample preparation and data processing will support high-throughput QC environments. Emerging MS technologies may enable real-time monitoring of excipient stability.

Conclusion

The developed HPLC-MS workflows offer a rapid, robust and accurate solution for fatty acid composition analysis in polysorbates, fulfilling USP requirements while simplifying sample preparation and expanding the range of detectable analytes.

Reference

1. Martos A, Koch W, Jiskoot W, Wuchner K, Winter G, Friess W, Hawe A. Trends on Analytical Characterization of Polysorbates Their Degradation Products in Biopharmaceutical Formulations. Journal of Pharmaceutical Sciences 106(7):1722–1735, 2017.
2. Wang Z, Wang Y, Tie C, Zhang J. A Fast Strategy for Profiling and Identifying Pharmaceutical Excipient Polysorbates by Ultra-High Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry. Journal of Chromatography A 1609:460450, 2021.
3. United States Pharmacopeia. Monograph for Polysorbate 80. USP–NF 2021 Issue 1. US Pharmacopeia Convention, 2020.
4. United States Pharmacopeia. Monograph for Polysorbate 20. USP–NF 2021 Issue 1. US Pharmacopeia Convention, 2020.
5. Hu M, Niculescu M, Zhang XM, Hui A. High Performance Liquid Chromatographic Determination of Polysorbate 80 in Pharmaceutical Suspensions. Journal of Chromatography A 984:233–236, 2003.