Posted by Ion mobility–mass spectrometry (IM-MS) has gained significant popularity across a wide range of research fields due to its ability to separate and identify isomers, conformers, or isobaric compounds with very similar exact masses. Ion mobility separates gas-phase ions based on their size, charge, and shape as they are guided through an inert buffer gas by an electric field. The cyclic ion mobility–mass spectrometer (cIM-MS) is an advanced version of traditional linear IM-MS instruments. It features a traveling wave ion mobility cell with a unique cyclic geometry. The main advantage of cIM-MS over linear IM-MS is that the path length can be adjusted and optimized for specific separation challenges. When coupled with chromatography, cIM-MS creates a multidimensional separation system, with high resolving power and peak capacity.
During the production of Sugammadex several process-related byproducts form, one of the major impurities is the hexakis(6-deoxy-6-(2-carboxyethyl)thio)-γ-cyclodextrin (Di-OH-SGM), which lacks two substituents compared to Sugammadex. Di-OH-SGM has four possible regioisomers, typically labelled as isomer AB, AC, AD, and AE, representing the glucose rings that are not fully modified.
The main objective of this study is to apply the cIM-MS technique to cyclodextrin analysis by separating, identifying, and characterizing the four regioisomers of Di-OH-SGM using cIM-MS and HPLC-cIM-MS. Two alternative cIM methods were developed specifically for the separation of these isomers.
Method 1 is a multipass cIM separation, when the components travelled different number of passes in the cIM cell, three of the components overtook the slowest isomer several times. This “wrap-around” occurred because the collision cross section (CCS) value of the slowest isomer was much larger than the other three isomers’, and 19 passes were necessary to separate the faster three isomers. Method 2 is a so-called “slicing” experiment which consists of two main experimental phases. In phase 1 only one isomer was separated from the other three unresolved peaks followed by its identification by MS/MS. In phase 2 the three unresolved isomers were ejected into the prestore cell and stored in the cell to eject the separated peak from the cIM cell. Subsequently, the unresolved isomers were reinjected from the prestore into the cIM cell followed by the separation and MS/MS fragmentation of these peaks.
Separation of the isomeric fragment ions was also carried out. Due to the layout of the instrument, the isomeric mixture of the precursor ions was infused directly into the ESI source and activated in the source. Isomeric fragment ions, produced by in-source fragmentation, were selected by the quadrupole mass filter, and then the ions were separated in the cIM cell and detected by the TOF.
In addition to direct cIM analysis of the isomer mixture, a 2D separation method was developed in which the four isomers were first separated by HPLC and then further resolved by cIM. In the heat-map of this 2D separation the arrival time distribution is plotted against the retention time. Comparing the speed of the cIM and HPLC methods, the cIM method, with a 1 min acquisition time, is much faster than the 50 min HPLC method. The separation of isomers AE and AD proved to be the most challenging for both cIM and HPLC. Baseline separation of these isomers was achieved with the more time-consuming HPLC method (Rs = 1.51). In contrast, the cIM provided a partial separation (Rs = 0.82), but this slight overlap did not affect the MS/MS assignments. Although several HPLC conditions were tested in this study, baseline separation of the first two eluting peaks was only achieved with the time-intensive 50 min HPLC method, which cannot be considered as a routine HPLC separation.
Péter Soma Szakály, Dávid Papp, Arnold Steckel, Erzsébet Varga, Gitta Schlosser;Journal of the American Society for Mass Spectrometry, 36, 2, 258–264 (2025)
The paper was selected by the Hungarian Academy of Sciences for the title of “outstanding paper of the month” (Feb. 2025)