Analysis of Tween 80 by highperformance liquid chromatography with diode array detection

Significance of the Subject

Tween 80 (polysorbate 80) is a nonionic surfactant widely employed as an emulsifier, stabilizer, solubilizer, and anticalcification agent in pharmaceuticals, vaccines, and tissue treatments. Accurate quantitation of this heterogeneous, low-chromophore excipient is essential for quality control and product stability.

Objectives and Study Overview

This study presents the development and validation of a rapid, simple isocratic HPLC method with max plot diode array detection (DAD) for the quantification of Tween 80 in 0.02 M phosphate buffer. Key performance parameters—specificity, linearity, precision, accuracy, and detection limits—were evaluated to demonstrate suitability for routine analysis.

Methodology and Instrumentation

The assay employs reversed-phase HPLC under isocratic conditions using an Agilent InfinityLab Poroshell 120 EC-C18 column. Detection is performed by recording the maximum absorbance across 195–400 nm at each time point (max plot DAD). Validation steps included:

• Preparation of standard and sample solutions in 0.02 M phosphate buffer
• Injection volume: 20 µL; flow rate: 0.4 mL/min; column temperature: 30 °C
• Mobile phase: 20:80 water (0.1% H₃PO₄) : acetonitrile (0.1% H₃PO₄)
• Assessment of specificity, linearity (1–20 mg/mL), precision, accuracy, LOD/LOQ, and solution stability

Results and Discussion

Max plot chromatograms revealed multiple peaks between 1.8 and 14.2 min corresponding to Tween 80 components. Summation of all peaks in this window provided consistent quantitation, overcoming variability in individual peak ratios. Linearity was excellent across 1–20 mg/mL (R² > 0.99995). Intraday precision (n=6) yielded RSD < 0.3%. Accuracy at mid and high levels ranged from 98 to 100%; low-level recovery variability indicated an LOQ near 1 mg/mL. Matrix interference was negligible (<5% of standard signal). Five-day stability tests showed shifts in peak distribution but maintained overall quantifiable response.

Benefits and Practical Applications of the Method

• Fast, isocratic HPLC workflow without gradient programming
• Uses commonly available DAD instead of specialized detectors
• Robust quantitation of heterogeneous polysorbate mixtures
• Applicable to quality control and stability monitoring in pharmaceutical excipients

Future Trends and Potential Applications

The method may be extended to more complex matrices (e.g., protein formulations) with suitable sample preparation. Future enhancements include coupling with mass spectrometry or charged aerosol detection for greater sensitivity, automated peak integration algorithms, and advances in column technologies to improve resolution of individual polysorbate species.

Conclusion

A straightforward HPLC–DAD approach using max plot detection has been validated for accurate, precise, and sensitive quantification of Tween 80 in phosphate buffer. Its simplicity and reliance on standard instrumentation facilitate rapid implementation in routine pharmaceutical testing.

Instrumentation

• Agilent InfinityLab HPLC system
• Poroshell 120 EC-C18 column (3.0 × 100 mm, 2.7 µm)
• Diode array detector (max plot, 195–400 nm)
• Isocratic pump and auto-sampler

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