Combining mass spectra, retention time modelling, and charged aerosol detection for unambiguous peak annotation and uniform-response quantitation in polysorbate profiling

Importance of the Topic

Polysorbate-80 (PS80) is a widely used non-ionic surfactant in parenteral drug formulations. Its complex mixture of polyoxyethylene sorbitan and isosorbide esters lacks a chromophore, making accurate profiling challenging. Combining mass spectrometry (MS), retention time modelling and charged aerosol detection (CAD) addresses the need for unambiguous peak annotation and reliable quantitation of individual PS80 components, supporting stability testing, batch comparison and degradation studies.

Objectives and Overview of the Study

The study aimed to develop an ultra-high-performance liquid chromatography (UHPLC) method with simultaneous CAD and single-quadrupole MS detection. Key goals were to profile PS80 at the sub-class and single-component level, detect variations due to enzymatic degradation, and compare batch-to-batch differences with uniform response quantitation.

Methodology

A multi-step reversed-phase gradient was applied on a C18 column, combined with an inverse gradient make-up flow post-column to maintain constant solvent composition. The flow was split 1:1 between CAD and MS detectors via a capillary mixer to ensure stable detector responses. PS80 samples (2 mg/mL in water) were digested with recombinant human lipoprotein lipase at 37 °C for five days to induce degradation. Retention time modelling was performed using Chromeleon 7.3, correlating retention with mass or detected m/z values for peak assignment.

Used Instrumentation

• UHPLC: Thermo Scientific Vanquish Flex Duo for inverse gradient operation
• Detector 1: Vanquish Charge Aerosol Detector H (CAD)
• Detector 2: Thermo Scientific ISQ EM single-quadrupole mass spectrometer
• Column: Thermo Scientific Accucore C18, 2.1×100 mm, 2.6 µm, operated at 50 °C
• Mobile phases: (A) 5 mM ammonium formate pH 4.8; (B) 50/50 acetonitrile/isopropanol

Main Results and Discussion

The capillary mixer achieved homogeneous mixing and a stable split ratio, confirmed by constant solvent density on the MS branch. CAD chromatograms under inverse gradient revealed the distribution of PS80 sub-classes, with sorbitan-POEn-oleate dominating at retention times of 17–19 min. Retention time showed a linear dependency on esterification degree and compound class. MS data in positive and negative ion modes validated the identity of peaks, including detection of oleic acid released during lipase hydrolysis. Degradation studies demonstrated near-complete conversion of monoesters to polyols and the persistence of higher-order esters.

Contributions and Practical Applications

• Cumulative CAD with inverse gradient ensures accurate mass-balance of PS80 sub-classes.
• Combined retention time modelling and MS spectra enable single-component identity assignment without advanced MS expertise.
• The multidetector UHPLC system facilitates comprehensive PS80 analysis for stability testing, batch comparison and elucidation of degradation pathways.

Future Trends and Applications

Advancements may include coupling high-resolution MS for deeper structural elucidation, applying machine learning to retention time prediction, extending the approach to other complex surfactant or lipid systems, and integrating online process monitoring in biomanufacturing to ensure product consistency and quality.

Conclusion

The developed UHPLC method with simultaneous CAD and MS detection delivers unambiguous peak annotation and uniform-response quantitation of PS80 components. It supports detailed profiling, degradation monitoring and batch comparison while remaining accessible to laboratories without specialized MS training.

Reference

• Ispan DA, Carillo S, Bones J, Cook K, Steiner F, De Pra M. Combining mass spectra, retention time modelling, and charged aerosol detection for unambiguous peak annotation and uniform-response quantitation in polysorbate profiling. Thermo Fisher Scientific; 2022.