Investigation of 3-Carboxy-5-Nitrophenyl-Boronic Acid Derivatized Mono- and Disaccharides using a Q-ToF fitted with a Cyclic IMS Mobility Separator

Introduction

Small sugars represent an interesting class of molecules that are difficult to characterize using ion mobility spectrometry (IMS).  By adding a suitable covalent derivatizing agent, differences in collisional cross section 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.   Extending resolution in IMS is most frequently performed by increasing path length or residence time in the ion mobility separator.  A novel geometry cyclic IMS cell has been developed and incorporated into a Q-ToF.  By extending the delay time between ion injection and ejection, effective path length and resulting IMS resolution are increased dramatically.

Methods

Monosaccharides (glucose, fructose, galactose, mannose) or disaccharides (sucrose, lactose, isomaltose, maltose, lactulose, cellobiose, melibiose, trehalose) were derivatized by incubating 5 micrograms of sugar with 10 micrograms of 3C5NBA in 15 µL water for 2 hours.  Water (400 µL) was added and analyzed using ESI- on a prototype IMS Q-ToF fitted with a one meter cyclic IMS cell operated with nitrogen as the mobility gas. IMS path length and resulting IMS resolution were increased by increasing residence time in the cyclic IMS separator.  MS/MS spectra of mobility resolved analytes were generated by elevating the voltage applied to a travelling wave ion guide placed before the ToF entrance.  Similar experiments were performed on a commercial SYNAPT G2- Si (Waters Corporation) for comparison.

Preliminary Data

Monosaccharides derivatized with 3C5NBA had two or three isomeric forms that could be well resolved using one to three cycles (effective path length of one, two, or three meters).  Tandem MS/MS spectra of these mobility resolved isomers showed a number of diagnostic fragments that could be used to unambiguously determine the carbohydrate identity. For the disaccharides, the combination of carbohydrate composition, linkage, and anomers resulted in much more complex arrival time distributions, with cellobiose having five resolvable features, and sucrose and maltose having three.  Tandem MS/MS spectra for these species were also collected and used for characterization of the different isomers.  Using the commercially available IMS instrument (SYNAPT G2 Si), lactose (Gal-Glc) and trehalose (Glc-Glc) had single peaks in their arrival time distribution (ATD).  Using up to 8 cycles, corresponding to an effective path length of 8 meters, and a100 msec residence time resulted in the resolution of these single features into two well resolved components that were again characterized by post mobility MS/MS. Even at long analysis times (100 msec) in the comparatively high pressure of the mobility separator, ion losses were small, no more than a factor of two for the carbohydrate derivatives studied here.

Novel Aspect

Increasing mobility separation path length resolves multiple sugar-derivative isomers based on sugar constitution-configuration and derivative formation.