Accepted: Fernandez Georgia Tech Andy Baker

Introduction

Small sugars represent an interesting class of molecules that are difficult to characterize using conventional ion mobility spectrometry (IMS). By adding a suitable covalent shift reagent, differences in collisional cross section (CCS) are amplified, allowing for better identification and characterization. 3-carboxy-5-nitrophenylboronic acid (3C5NBA) reacts with hydroxyl groups on the sugars to form bidentate derivatives depending on the available cis-diol reaction sites defined by the sugar composition, carbohydrate linkage, and conformation. High resolution IMS using a variable path length mobility separator and tandem-IMS strategies (further separation of mobility-selected sub-populations) can be used to probe the complex mixture of isomeric carbohydrate derivatives.

Methods

Monosaccharides (glucose, fructose, galactose, mannose) or disaccharides (sucrose, lactose, isomaltose, maltose, lactulose, cellobiose, melibiose, trehalose) were derivatized with 3C5NBA for 30 minutes. Water was added and the solutions analyzed by ESI- on a cyclic ion mobility enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer with a one meter cyclic mobility cell using nitrogen as the buffer gas. IMS path length and resulting IMS resolution were varied by increasing residence time in the mobility separator. Tandem IMS experiments were performed on mobility-resolved species by isolating selected ion populations in an external ion trap before reinjecting into the mobility separator. By changing the voltage offset between the ion trap and mobility separator during ion reinjection, fragmentation or structural changes were induced.

Preliminary Data

High resolution IMS experiments were used to characterize derivatized monosaccharides. The arrival time distribution (ATD) for derivatized mannose contained several peaks spread across a wide mobility range. After multiple passes, species with higher mobility overtake lower mobility species, limiting the attainable IMS resolution. By employing frontflush or backflush techniques, mobility-selected regions of the ATD can be selected for further analysis, enabling resolution of species with similar CCS. Using the frontflush technique and 10 passes after ejecting higher mobility species, the main peak in the ATD for mannose was resolved into two species. Similar experiments were used to increase resolution for disaccharides including isomaltose and cellobiose. A tandem-IMS technique where mobility-resolved species were injected from the cIM into an external ion trap and then reinjected to the cIM, followed by additional IM separation was used to increase resolution for components observed in maltose and sucrose.
Tandem-IMS experiments were performed to investigate the structure and interconversion of the resolved species observed with each of the derivatized carbohydrates. The ATDs from IMS-IMS experiments were compared to experiments where a voltage bias (65 V) was applied to the ion trap during reinjection. Changes in the ATD suggest changes in CCS or conversion to other species. The two resolved components from derivatized mannose did not interconvert, however the major component in the ATD of galactose was converted into a lower mobility component observed in the initial IM separation.

As expected, the added complications of disaccharide composition and linkage type or position resulted in more resolvable features in the ATD. The two components observed in trehalose were not interconvertible, however with isomaltose, a high mobility component was converted into the lower mobility component, as well as another minor component. Results obtained with maltose, lactose cellobiose, and sucrose showed many of the same interconversions.

Novel Aspect

Use of high resolution IMS and tandem-IMS experiments to probe structure and isomerization of multiple sugar-derivative isomers.

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Poster

Ion Mobility: Applications

Submitting Author:

Andrew Baker
Waters, Inc.
Pleasanton, CA
andy_baker@waters.com