Polysorbate 80 profiling by HPLC with mass and charged aerosol detection

Importance of the Topic

Polysorbate 80 is a widely used non-ionic surfactant in pharmaceutical, biopharmaceutical, cosmetic, and food formulations. Its complex composition of hundreds of ethoxylated sorbitan and isosorbide esters with various fatty acids can strongly influence critical formulation properties such as micelle formation, solubilization capacity, and stability. Lot-to-lot variability of polysorbate 80 often leads to unexpected performance issues, making rapid and reliable raw-material fingerprinting essential for ensuring consistent drug product quality and reducing costly root-cause investigations.

Objectives and Study Overview

This study aimed to develop an HPLC-based fingerprinting method capable of:

• Resolving major component classes of polysorbate 80 according to their degree of esterification.
• Detecting batch-to-batch variability among different suppliers, grades, and production lots.
• Providing quantitative information without requiring individual reference standards.
• Allowing rapid identity confirmation of key ester species.

Methodology and Instrumentation

The method employs reversed-phase ultra-high-performance liquid chromatography (UHPLC) with an inverse gradient approach to ensure uniform detector response across all eluting components.

• Column: Accucore C18, 150 × 2.1 mm, 2.6 μm
• Mobile phases: A (5 mM ammonium formate, pH 4.8), B (50:50 isopropanol/acetonitrile)
• Gradient: 9–85% B over 26 min, ramp to 100% B, re-equilibration to 9% B (total run ∼56 min)
• Flow rate: 0.4 mL/min
• Sample concentration: 1 mg/mL (CAD), 0.5 mg/mL (MS)

Used Instrumentation

• Vanquish Flex UHPLC and Vanquish Flex Duo systems
• Vanquish Charged Aerosol Detector (CAD), 50 °C, 20 Hz data rate
• Vanquish Duo Inverse Gradient Kit
• ISQ EM single quadrupole mass spectrometer with HESI source (positive mode, m/z 350–2000)

Key Results and Discussion

CAD chromatograms consistently separated polysorbate 80 components into four clusters:

• Group 1: Non-esterified polyoxyethylene sorbitan and isosorbide
• Group 2: Sorbitan mono-oleate esters
• Group 3: Sorbitan di-oleate esters
• Group 4: Tri- and tetra-oleate esters

Inverse gradient mode yielded a uniform response across all groups, enabling direct area-based quantitation without calibration standards. Single quadrupole mass detection confirmed component identities via characteristic ammoniated adducts and ethylene oxide mass shifts. Quantitative linearity was excellent over 0.5–2.5 mg/mL (R² ≥ 0.998, CV ≤ 2.4%). Comparisons among sample lots revealed measurable differences in the relative abundance of polyols versus fatty acid esters, demonstrating the method’s sensitivity to supplier and batch variability.

Benefits and Practical Applications

The presented HPLC-CAD/MS workflow offers:

• Rapid fingerprinting of polysorbate 80 raw materials for QA/QC screening
• Early detection of lot-to-lot inconsistencies before formulation impact
• Reduced need for laborious quantification of all minor components
• Qualitative confirmation of major ester species by MS

Future Trends and Opportunities

Further developments may include:

• Extension to other polysorbate grades (20, 60) and surfactant classes
• Integration with high-resolution or tandem MS for deeper structural elucidation
• Automation of data processing and reporting within chromatography data systems
• Application in stability and degradation studies to monitor surfactant integrity over time
• Implementation as a process analytical technology (PAT) tool during manufacturing

Conclusion

The optimized UHPLC method with inverse gradient CAD and complementary single quadrupole MS detection provides a robust, sensitive, and user-friendly platform for routine polysorbate 80 profiling. It enables accurate classification and quantitation of ester classes, rapid identification of raw-material variability, and efficient confirmation of key species, supporting consistent formulation performance.

Reference

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