Anomeric discrimination and rapid analysis of underivatized lactose, maltose, and sucrose in vegetable matrices by U‐HPLC–ESI‐MS/MS using porous graphitic carbon

Abstract

Lactose intolerance is a common condition caused by intestinal lactase deficiency, and a lactose-free diet represents the simplest way to avoid gastrointestinal symptoms. The emerging use of dietary supplements requires analytical tools that are capable of assessing these analytes, particularly for those based on dry herbal extracts that contain lactose together with maltose and sucrose, because of cross-contamination and/or deliberate addition as excipient. Electrospray ionization mass spectrometry (ESI-MS) and MS/MS are valuable detection methods for underivatized disaccharides; however, the absence of distinctive ions and collision-induced dissociation (CID) fragmentation patterns does not allow discrimination of stereoisomers without good chromatographic resolution. We developed an ultrahigh performance liquid chromatography-ESI (U-HPLC-ESI) approach, based on porous graphitic carbon (PGC) columns, working at 5 °C to separate and detect the disaccharides in their anomeric forms as formate adducts obtained directly in-column by eluting with formate buffer/acetonitrile gradient mixtures. Using a Paul trap, we monitored the adducts [M + HCOO](-) at m/z 387 in ESI negative mode (MS(1)) as well as the CID fragment ion [M - H](-) at m/z 341 (MS(2)) and used MS(3) fragment ions at m/z 178 and 161 to confirm disaccharides identity in complex vegetable matrices. Complete resolution of lactose α- and β-anomers, maltose α- and β-anomers, and sucrose was obtained with R ≥ 2.0 for all peaks and selectivity α = 1.2 between α- and β-anomers of lactose. The limits of detection were in the range of 3-7 µg/l (ppb) for the target disaccharides. Because of the rapidity and good anomeric discrimination, the described method represents an alternative tool to investigate the mutarotation phenomenon for reducing disaccharides.