Quality assessment of Polysorbates 80 and 20 Pharmaceutical Raw Materials by Measuring Fatty Acids Composition using HPLC with Mass Detection

Significance of the Topic

Polysorbates 80 and 20 are essential non-ionic surfactants employed as excipients in numerous pharmaceutical formulations. Ensuring their compositional purity and compliance with pharmacopeial standards is critical to final product safety and efficacy. Traditional GC-FID assays involve hydrolysis and derivatization steps that can be time-consuming and resource intensive.

Aims and Study Overview

This work aimed to develop and validate a streamlined HPLC-MS approach for direct measurement of free fatty acid profiles in hydrolyzed polysorbate samples. The method sought to meet United States Pharmacopeia criteria for polysorbate quality, while reducing sample preparation complexity and enhancing detection of both specified and unexpected fatty acid species.

Methodology and Instrumentation

Sample preparation involved alkaline hydrolysis of polysorbate 80 and 20 with 1 M KOH at 40 °C for 6 hours, neutralization with formic acid, and dilution in water/ethanol (50:50). Chromatographic separation utilized a C18 reversed-phase column (2.1×150 mm, 1.7 μm) on an ACQUITY UPLC system, employing a gradient of 10 mM ammonium acetate in water and acetonitrile at 60 °C. Mass detection was performed in negative electrospray ionization mode with single ion recording over 75–350 m/z. Key instrumentation included:

• Arc HPLC and ACQUITY UPLC systems
• XBridge BEH C18 and UPLC BEH C18 columns
• Xevo G2-XS QTof Mass Spectrometer for accurate mass verification
• ACQUITY QDa Detector for routine quantitation

Main Results and Discussion

The HPLC-MS method successfully resolved all USP-prescribed fatty acids in both polysorbates. Additional isomeric peaks not defined by the GC-FID monograph were detected and identified. Unknown peaks at m/z 279 were determined to be conjugated linoleic acid isomers (Δ9,11 and Δ10,12), specifically cis-vaccenic and elaidic acids. Peaks at m/z 281 corresponded to positional oleic acid isomers eluting adjacent to the oleic standard. Quantitative results for specified fatty acids met USP acceptance criteria.

Benefits and Practical Applications

The presented HPLC-MS workflow offers multiple advantages:

• Eliminates derivatization and simplifies sample preparation
• Accelerates analysis turnaround suitable for high-throughput QC
• Enhances specificity and confidence through accurate mass data
• Integrates with compliant-ready Empower software for streamlined reporting

Future Trends and Potential Applications

Emerging directions include adapting this platform to monitor surfactant degradation pathways, expanding to other biopharmaceutical excipients, and integrating automated high-throughput screening. Advances in high-resolution MS may further improve detection of trace impurities and degradation products.

Conclusion

The developed HPLC-MS method provides a rapid, sensitive, and reliable alternative to GC-FID for assessing fatty acid composition in polysorbates 80 and 20. It achieves USP compliance while offering enhanced identification capabilities and streamlined operation, making it well suited for routine pharmaceutical quality control.

References

1. Martos A, Koch W, Jiskoot W, Wuchner K, Winter G, Friess W, Hawe A. Trends on Analytical Characterization of Polysorbates and Their Degradation Products in Biopharmaceutical Formulations. Journal of Pharmaceutical Sciences. 2017;106:1722–1735.
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. 2021;1609:460450.
3. United States Pharmacopeia. Polysorbate 80 Monograph. USP–NF 2021 Issue 1. The United States Pharmacopeia Convention; 2020.
4. United States Pharmacopeia. Polysorbate 20 Monograph. USP–NF 2021 Issue 1. The United States Pharmacopeia Convention; 2020.