Abstract: A method was developed for the simultaneous determination of six human milk oligosaccharides (HMOs) in infant formula using high performance liquid chromatography-fluorescence detector (HPLC-FLD). The samples were first enzymatically digested and precipitated with organic solvents to remove interfering substances, and then separated using a hydrophilic chromatographic column. Under the optimal conditions, the six HMOs showed good linearity in their respective concentration ranges, with determination coefficients (R2) above 0.999. The recoveries of the spiked samples ranged from 95.91% to 105.03%, with relative standard deviations (RSD) of less than 2%. The limits of detection (LOD) and quantification (LOQ) were in the range of 0.003 6–0.006 4 and 0.012 0–0.021 4 g/100 g, respectively. The developed method is characterized by high sensitivity and good stability, and can meet the requirements of the determination of the six HMOs in infant formula.

Key words: human milk oligosaccharides, infant formula, high performance liquid chromatography, determination, standard