Isomer-specific quantification of human milk oligosaccharides based on Girard reagent P-d0/d5 derivatization and trapped ion mobility spectrometry-mass spectrometry

Human milk oligosaccharides (HMOs), the third most abundant components in breastmilk, show high structural diversity with many isomers. Here, we proposed a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) strategy for the differentiating and isomer-specific quantification of HMOs combined with Girard reagent P-d 0 /d 5 (GP-d 0 /d 5 ) derivatization . Three functional HMO isomer pairs including LNT/LNnT, 3'-SL/6'-SL, and 2'-FL/3'-FL were investigated by the proposed method. The GP-derivatized HMO isomers showed enhanced ionization efficiency (8-fold) and achieved mobility separation with ΔCCS % ≥ 3.7% in TIMS-MS. With the GP-d 5 derivatized HMOs as an internal standard, HMOs in real samples could be quantified by derivatization with GP-d 0 , and each isomeric species could be further determined based on their corresponding mobility peaks. The recoveries of spiked HMOs are obtained at 72.4 − 109%. Thus, an isomer-specific quantification strategy for HMO isomers was developed based on the GP-d 0 /d 5 derivatization and TIMS-MS.