Abstract: In this study, a comprehensive comparison of the technical performance and practical applicability of liquid chromatography (LC) and ion chromatography (IC) was conducted in the determination of seven human milk oligosaccharides (HMOs) in infant formula. We introduced a dual-internal standard quantification strategy into LC. In IC, we optimized the gradient elution program and established a specific enzymatic hydrolysis system. Both methods demonstrated good specificity, and all seven HMOs showed excellent linearity (R2 > 0.999) over a wide concentration range. The limit of quantification (LOQ) of LC ranged from 1.8 to 3.6 mg/100 g for solid samples and from 0.2 to 0.4 mg/100 g for liquid samples. The spiked recoveries at the LOQ level were between 81.1% and 118.8%, and those within the determination range were 91.3%−108.0%, with relative standard deviations (RSDs) of 1.8%−2.8%. The LOQs of IC ranged from 18 to 36 mg/100 g for solid samples and from 2 to 4 mg/100 g for liquid samples. The spiked recoveries at the LOQ level were 80.7%−118.5%, and those within the determination range were 90.9%−109.4%, with RSDs of 1.7%−3.7%. The validation results confirmed that both methods were suitable for the determination of the seven HMOs in infant formula. This study further clarified the applicable scenario of each method. Due to its lower LOQ, LC was more suitable for detecting endogenous HMOs in milk powder, whereas IC, with simpler pretreatment, was more applicable for the determination of goat milk-based formula. In terms of practicality, IC offered advantages in ease of operation and analytical timeliness. This research fills a gap in the systematic comparison between LC and IC for HMO detection, provides experimental support for method selection in different application contexts, and offers a valuable reference for the establishment of relevant food safety standards.

Key words: human milk oligosaccharides; liquid chromatography; ion chromatography; infant formula; comparative study