Determination and quantification of 2’-O-fucosyllactose and 3-O-fucosyllactose in human milk by GC–MS as O-trimethylsilyl-oxime derivatives

[Display omitted] •Isolation of 2’-O-fucosyllactose and 3-O-fucosyllactose from human milk.•Separation of these isomeric trisaccharides by a gas chromatographic method for the very first time.•Identification and quantification of the trimethylsilylated trisaccharides by validated GC–MS method.•Fragmentation pattern analysis of the derivatized trisaccharides. Human milk oligosaccharides possess various biological functions by protecting the infant from several bacterial and viral infections, modulating the immune system, serving as prebiotics and also contributing to the brain development. Hence, huge effort is underway by manufacturers to produce infant formulas enriched with human milk oligosaccharides which could mimic its diverse biological role the most. For this purpose, quantification of the natural oligosaccharide composition of the human milk is a key task. This study reports a fit for purpose GC–MS method for the quantification of the TMS ether oxime derivatives of 2’-O-fucosyllactose and 3-O-fucosyllactose, the two most abundant trisaccharides in human milk. The EI fragmentation pattern of the linkage isomers is discussed in details, focusing also on specific fragment ions. The GC–MS method with external standard calibration was applied for the monitoring of concentration changes of the trisaccharides throughout the first week of lactation in human milks samples collected from two volunteers. The results showed high concentration of both 2’-FL (4525–6266μg/mL in donor A and 2694–3551μg/mL in donor B) and 3-FL (271–441μg/mL in donor A and 99–208μg/mL in donor B), while no significant change has been observed throughout the one-week lactation period. The presented GC–MS method can serve as a quality control technique for the infant formulas and also offers an alternative to existing chromatographic methods to investigate HMOs in milk samples.