Quantitative analysis of 3’- and 6’-sialyllactose in human milk samples by HPLC-MS/MS: A validated method for the comparison of two consecutive lactation periods in the same woman

•The isomeric 3′- and 6′-sialyllactose were determined by LC–MS/MS in human milk.•Separation of alditol forms on porous graphitic carbon column was performed.•A validated quantitative method was developed.•Sialyllactose concentrations of consecutive lactations monitored for the first time. Human milk oligosaccharides (HMOs) are complex carbohydrates that exist naturally in mother’s milk. Due to their myriad beneficial biological effects, HMOs are in the focus of current researches. Some of these complex carbohydrates are already commercially available utilizing their health-promoting benefits not just in infant formulas but in dietary supplements. Among the acidic oligosaccharides 3′- and 6′-sialyllactoses (SLs) are the most abundant in human milk. Extending the health benefits of these trisaccharides, 3′-SL and 6′-SL are expected to be active ingredients in infant- and follow-on formula and novel foods, therefore reliable and easy-to-perform analytical methods are urgently needed for their characterization. Herein we report a graphitized carbon liquid chromatography-mass spectrometry-based method for the separation and quantitation of sialyllactoses in human milk. Compounds were separated as their alditol forms and were quantified using maltotriose as internal standard. The developed bioanalytical method was validated in terms of selectivity, accuracy, precision, recovery, carry-over, matrix effects and stability. Two consecutive lactation periods of the same individual were monitored for the first time and a slight decrease in SL concentrations were found during the first month of the second lactation.