Separation and Analysis of Polysorbate 80 in the Presence of Human Serum Immunoglobulin G Using Agilent Bond Elut Lipid Extraction SPE Cartridges

Importance of the Topic

Biotherapeutic proteins are susceptible to aggregation and surface adsorption under interfacial stress conditions. Polysorbate 80 is a nonionic surfactant widely used to stabilize formulations by reducing protein aggregation and adsorption. Accurate quantification of Polysorbate 80 in complex matrices such as human serum immunoglobulin G is essential for maintaining product quality and ensuring regulatory compliance.

Study Objectives and Overview

This application note describes a selective extraction and analysis method for Polysorbate 80 in diluted aqueous solutions and in human serum IgG formulations. The method employs Agilent Bond Elut Lipid Extraction SPE cartridges and compares two detection techniques, liquid chromatography with diode array detection and evaporative light scattering detection. Key performance parameters including sensitivity, linearity, accuracy, precision, and specificity were evaluated.

Methodology and Instrumentation

Sample preparation uses Bond Elut Lipid Extraction cartridges with EMR—Lipid sorbent to selectively retain polysorbates. The workflow includes cartridge conditioning with acetonitrile and water, sample loading under positive pressure, washing to remove salts and proteins, and elution of Polysorbate 80 with 80 percent acetonitrile in water. Final extracts are analyzed by LC/DAD and LC/ELSD.

Instrumentation Used

• Agilent 1260 Infinity II Bio-inert LC system
• Agilent diode array detector and evaporative light scattering detector
• Agilent InfinityLab Poroshell 120 EC-C18 columns (3.0×100 mm and 3.0×50 mm)
• OpenLab and ChemStation software
• HPLC grade solvents, human serum IgG, formic acid, phosphoric acid, phosphate buffer

Main Results and Discussion

• LC/DAD detection achieved a limit of detection of 0.1 mg/mL and limit of quantitation of 0.2 mg/mL. LC/ELSD provided a lower limit of detection of 0.03 mg/mL and limit of quantitation of 0.04 mg/mL.
• Calibration ranges were 0.2 to 0.6 mg/mL for LC/DAD and 0.04 to 0.6 mg/mL for LC/ELSD, with coefficients of determination above 0.99. Polynomial regression was used for the broader ELSD range and linear fits for narrower segments.
• Quantitation accuracy ranged from 86 to 106 percent and repeatability (RSD) was within 2 to 6 percent. No interfering peaks were observed in blank IgG controls.

Benefits and Practical Applications

The SPE-LC methods effectively remove protein matrix interferences, enabling reliable Polysorbate 80 quantitation in both development and quality control environments. Orthogonal detection strategies offer flexibility to meet varying sensitivity and calibration range requirements.

Future Trends and Potential Applications

The EMR—Lipid extraction approach can be extended to other nonionic surfactants such as Polysorbate 20, Solutol HS 15, Triton X-100, and Poloxamers. Integration with mass spectrometric detection could provide detailed compositional insights and high throughput screening capabilities in biopharmaceutical workflows.

Conclusion

Agilent Bond Elut Lipid Extraction cartridges combined with LC/DAD and LC/ELSD detectors provide a robust and efficient workflow for the separation, analysis, and quantitation of Polysorbate 80 in the presence of human serum IgG. The methods deliver excellent sensitivity, accuracy, precision, and specificity for biopharmaceutical development and quality control.

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