Liquid chromatography-mass spectrometry approach for characterizing sucrose isomers in complex mono-floral honey.

Sucrose, which forms < 2% of the chemical content in honey samples, is known to have five structural isomers each with its own medicinal benefits. Unfortunately, studies characterizing the specific sucrose isomer(s) present in honey samples are limited. Herein, we introduce a contained electrospray ionization (cESI) method that can be coupled between liquid chromatography (LC) and tandem mass spectrometry (MS/MS). This LC-cESI-MS/MS platform leverages chloride adduction to enable sensitive differentiation and characterization of disaccharide isomers in complex honey samples. By integrating retention time and collision-induced dissociation (CID) MS/MS data, we achieved orthogonal analysis of six sucrose isomers. The MS/MS on the chloride adducts showed distinct fragment ions for each isomer. Additional optimization afforded nanomolar (nM) detection limits for all disaccharides analyzed via chloride adduction in negative-ion mode, a feature that showed superior sensitivity compared with conventional sodium adduction methods typically achieved in positive-ion mode. We identified four sucrose isomers (turanose, palatinose, maltulose, and trehalulose) in three mono-floral honey samples, of which turanose was the most abundant isomer. Sucrose itself could not be confirmed in any of the honey samples tested and leucrose was confirmed to be absent. Although the specific amounts of these isomers were not determined, principal component analysis showed that the abundances of the four identified structural isomers significantly differed in the three mono-floral honey samples. The current study forms the first report suggesting turanose to be the main sucrose isomer in the tested mono-floral honey. Such identification was made possible because of our ability to independently optimize LC and cESI spray solvents, and to enable online microdroplet chemistry via chloride adduction, which allowed the conventional CID-MS/MS to yield highly informative fragmentation.