High-throughput quantification of multiple human milk oligosaccharides by ion mobility-mass spectrometry: From standard addition to single-point calibration

Quantifying human milk oligosaccharides (HMOs) in breast milk is challenging due to structural isomerism, matrix effects, and the lack of a true blank milk. Here, a multiplexed standard-addition strategy was proposed to quantify simultaneously nine HMOs in breast milk using ion mobility-mass spectrometry with single ion mobility monitoring (SIM²)-stitching acquisition on a TIMS-Tof instrument. Although, both ordinary (OLS) and weighted (WLS) least squares regressions yielded comparable results in terms of precision and accuracy, the WLS method accounted for heteroscedastic response variance and reduced dependence on the calibration range for endogenous concentration estimates. This method enabled the HMO determination in breast milk samples ranging from 0.06 to 0.92 g L⁻¹. As a simplified alternative to multi-point calibration, the performance of a single-point standard addition strategy was evaluated. This provided HMO levels very similar to those determined by multipoint standard addition, demonstrating the potential of this simple quantification strategy for high-throughput applications.