MSACL 2025 Abstract

Characterizing the Structural Heterogeneity of Polysorbates Using High Resolution Ion Mobility-Mass Spectrometry (HRIM-MS) Strategies

Kyle E. Lira, Jody C. May, John A. McLean
Vanderbilt University, Nashville, TN

Kyle Lira (Presenter) Vanderbilt University

Presenter Bio: Kyle was born and raised in Long Beach, CA and received a B.S. in both Chemistry and Humanities from Azusa Pacific University (Azusa, CA). Since joining the McLean research group at Vanderbilt University in 2021, Kyle’s research broadly involves the application of IM-MS techniques to understand and characterize analytically challenging molecules. Recently, this has included characterizing the molecular composition of polysorbate, a synthetic surfactant utilized commercially in cosmetics, food, and pharmaceutical formulations to help increase solubility and bioavailability. His work seeks to provide valuable resources to support ongoing pharmaceutical and life sciences research.

Relevant Financial Disclosures (within past 24 months, reported on Mar 18, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Polysorbates (PS) are synthetic surfactants commonly utilized commercially to increase the solubility and bioavailability of molecules of interest in cosmetics, foods, and pharmaceuticals. Commercial PS products (e.g., Tween) consist of heterogeneous mixtures of numerous species with similar structures; consequently, PS are analytically challenging to characterize. Typically, MS techniques are utilized in PS analysis, yet these cannot resolve isomeric species which are prevalent in PS mixtures. Using ion mobility (IM)—a gas phase separation technique that discriminates ions by size, shape and charge—structurally-similar compounds, including isomers, can be resolved. From IM experiments, a reproducible chemical descriptor known as a collision cross section (CCS) can be determined for individual species. Here, we evaluate the capability of various high resolution ion mobility (HRIM) techniques for the characterization of two common polysorbates (PS-20 and PS-80). Additionally, IM profiles and CCSs are compared to measurements obtained on a drift tube ion mobility (DTIMS) platform which is capable of measuring CCS values from first principles.

OBJECTIVES:
The goal of this work is to integrate analytical data from various HRIM techniques towards a unified ion mobility resource that will support pharmaceutical and life sciences research. To this end, each IM technique is leveraged for specific analytical strengths.

METHODS:
Data for PS-20 and PS-80 (USP) were acquired in positive mode via direct infusion (10 µg/mL) on a DTIMS (6560 IM-QTOF, Agilent Technologies), a traveling wave structures for lossless ion manipulation IM (TWSLIM, MOBIE, MOBILion Systems) coupled to MS (6546 QTOF, Agilent), a trapped IM-MS (timsTOF Pro 2, Bruker Corporation), and a cyclic IM-MS (cIM, SELECT SERIES, Waters Corporation). DTIMS measurements were obtained in 4-bit multiplexing mode to enable high resolution demultiplexing (HRdm, Agilent). Arrival times were determined using vendor software (HRIM Data Processor, MOBILion; IM-MS Browser, Agilent; Compass DataAnalysis, Bruker; and MassLynx, Waters). CCS values were determined using previously-documented calibration procedures incorporating a MS tuning mixture (ESI-L, Agilent), with CCS values from HRIM techniques validated against drift tube measurements (single-field calibration).

RESULTS:
A target list obtained from DTIMS-MS analysis was used to support PS identifications from HRIM analyses. First, the MS distributions for each PS structural series were extracted from the IM-MS datasets, and both the weight- and number-averaged mass distributions (Mw, Mn) were determined for each oligomer series. It was found that Mw and Mn were comparable, providing a basis for measurement alignment and standardization. Next, IM-MS conformational space correlations—which provide information on structural series, number of fatty acids, type of adduct, and charge state—were found to be generally consistent between DTIMS-MS and HRIM-MS analysis once aligned in CCS space. While most IM profiles observed using DTIMS (Rp ~60) do not exhibit multiple peak features, HRIM analysis revealed that some PS species were candidates for isomeric separation. For further validation, TIMS and cIM were utilized in a targeted mode to optimize IM resolution and confirm the presence of isomers. Mass accuracy values for PS identifications typically are better than ±5.0 ppm and are strongly correlated to ion abundance.

CONCLUSION OR DISCUSSION:
Preliminary results suggest that the previous DTIMS-MS measurements are reproducible across multiple IM platforms, with some features containing multiple isomers under a single IM profile. For most PS species, IM profiles are consistently comprised of single peaks even under high IM resolution, indicating that CCS values obtained from drift tube IM are also applicable for HRIM, which allows direct comparison of various IM techniques via CCS alignment.