Liquid chromatography/tandem mass spectrometry with fluorous derivatization method for selective analysis of sialyl oligosaccharides

RATIONALE A separation‐oriented derivatization method using a specific fluorous affinity between perfluoroalkyl‐containing compounds was applied to selective liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis of sialyl oligosaccharides. The perfluoroalkyl‐labeled sialyl oligosaccharides could be selectively retained on an LC column with the perfluoroalkyl‐modified stationary phase and effectively distinguished from non‐derivatized species. METHODS Sialyl oligosaccharides (3′‐sialyllactose, 6′‐sialyllactose, sialyllacto‐ N ‐tetraose a, sialyllacto‐ N ‐tetraose b, sialyllacto‐ N ‐tetraose c, and disialyllacto‐ N ‐tetraose) were derivatized with 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11‐heptadecafluoroundecylamine via amidation in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride (condensation reagent). The obtained derivatives were directly injected onto the fluorous LC column without any pretreatments and then detected by positive electrospray ionization MS/MS. RESULTS The method enabled accurate determination of the sialyl oligosaccharides in biological samples such as human urine and human milk, because there was no interference with matrix‐induced effects during LC/MS/MS analysis. The limits of detection of the examined sialyl oligosaccharides, defined as signal‐to‐noise ( S / N ) = 3, were in the range 0.033–0.13 nM. Accuracy in the range 95.6–108% was achieved, and the precision (relative standard deviation) was within 9.4%. CONCLUSIONS This method enabled highly selective and sensitive analysis of sialyl oligosaccharides, enabling accurate measurement of even their trace amounts in biological matrices. The proposed method may prove to be a powerful tool for the analysis of various sialyl oligosaccharides. Copyright © 2014 John Wiley & Sons, Ltd.