# Exploring Animals and Human Milk Oligosaccharides: A Comprehensive Analysis using Hydrophilic Interaction Liquid Chromatography Coupled with Fluorescent and Mass Spectrometry Detectors

Sophie Le Gall, Loric Thoulouze, Yoann Richer, Eléonore Lepvrier, Isabelle Le Huërou-Luron

► To cite this version:

Sophie Le Gall, Loric Thoulouze, Yoann Richer, Eléonore Lepvrier, Isabelle Le Huërou-Luron. Exploring Animals and Human Milk Oligosaccharides: A Comprehensive Analysis using Hydrophilic Interaction Liquid Chromatography Coupled with Fluorescent and Mass Spectrometry Detectors. Journal of Food Composition and Analysis, 2025, pp.108074. 10.1016/j.jfca.2025.108074. hal-05190443

HAL Id: hal-05190443

https://hal.inrae.fr/hal-05190443v1

Submitted on 6 Aug 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Exploring animals and human milk oligosaccharides: A comprehensive analysis using hydrophilic interaction liquid chromatography coupled with fluorescent and mass spectrometry detectors

Sophie Le Gall<sup>a,b,*</sup> <sup>□</sup>, Loric Thoulouze<sup>a,b</sup>, Yoann Richer<sup>a,b</sup>, Eléonore Lepvrier<sup>c</sup>, Isabelle Le Huèrou-Luron<sup>d</sup>

## ARTICLE INFO

**Keywords:**

Milk oligosaccharides

2-AB labelling

Quantification

HILIC-FLD-MS

MOs database

**Chemical compounds studied in this article:**

3'-Sialyllactose, 3'-SL (PubChem CID: 123914)

6'-Sialyllactose, 6'-SL (PubChem CID: 122216319)

Xylohexaose, X6 (PubChem CID: 101656440)

Disialyllacto-*N*-tetraose, DSLNT (PubChem CID: 3085215)

Lacto-*N*-neotetraose, LNnT (PubChem CID: 121853)

Acetonitrile, ACN (PubChem CID: 6342)

Ammonium formate (PubChem CID: 2723923)

Acetic Acid (PubChem CID: 176)

Dimethyl sulfoxide, DMSO (PubChem CID: 679)

Sodium cyanoborohydride, BCN (PubChem CID: 5003444)

Formic Acid, FA (PubChem CID: 184)

Hydrochloric Acid, HCl (PubChem CID: 313)

6'-Galactosyllactose, N9 (PubChem CID: 25203970)

3'-Galactosyllactose, N7 (PubChem CID: 189088)

Gal(α1-3)Gal(β1-4)Glc, N6 (PubChem CID: 100962852)

## ABSTRACT

A methodology for the extraction, identification, and quantification of oligosaccharides present in both human and animal milks was developed. Our approach allows for the extraction of milk oligosaccharides from small volumes (50 µL), followed by their labelling with a fluorescent compound. Subsequently, these labeled oligosaccharides are subjected to analysis using Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry and Fluorescent Detectors, enabling both their identification and quantification. Our method successfully detected 99 distinct milk oligosaccharides across three matrices: human, porcine, and bovine milks, shedding light on their prevalence and significance. Notably, 80 distinct oligosaccharides in human milk were identified and quantified, compared to 21 in animal milks (20 in porcine and 10 in bovine milks, respectively). Furthermore, our findings revealed a common subset of six oligosaccharides shared between human and animal milks (porcine and bovine), including 3-FL, 3'-SL, and LNnT, underscoring their potential functional similarities.

**Abbreviations:** DSLN, disialyllactosamine; DSLNT, disialyllacto-*N*-tetraose; DFLNH, difucosyllacto-*N*-hexaose; DFpLNH, difucosyl-*para*-lacto-*N*-hexaose; Fuc, fucose; 2'-FL, 2'-fucosyllactose; 3-FL, 3-fucosyllactose; Gal, D-galactose; GalNAc, *N*-acetylgalactosamine; β3'-GL, β3'-galactosyllactose; β6'-GL, β6'-galactosyllactose; GNL, *N*-acetyl-galactosaminyl-lactose; Glc, D-glucose; GlcNAc, *N*-acetylglucosamine; LDFT, lactodifucotetraose; LNDFH, lacto-*N*-difucohexaose; LNFP, lacto-*N*-fucopentaose; LNH, lacto-*N*-hexaose; LNT, lacto-*N*-tetraose; LNnT, lacto-*N*-neotetraose; nonoLNP, lacto-*N*-novo-pentaose; LST, sialyllacto-*N*-tetraose; LNnH, lacto-*N*-neo-hexaose; MFLNH, monofucosyllacto-*N*-hexaose; Neu5Ac, *N*-Acetylneuraminic acid; Neu5Gc, *N*-Glycolylneuraminic acid; 3'-SL, 3'-sialyllactose; 6'-SL, 6'-sialyllactose; 6'-SLN, 6'-sialyl-*N*-acetyllactosamine; SLNnT, sialyllacto-*N*-neotetraose.

* Corresponding author at: INRAE, UR BIA, Nantes F-44316, France.

E-mail address: sophie.le-gall@inrae.fr (S. Le Gall).

<footnote><sup>a</sup> INRAE, UR BIA, Nantes F-44316, France</footnote>

<footnote><sup>b</sup> INRAE, BIBS Facility, CALIS Research Infrastructure, Nantes F-44316, France</footnote>

<footnote><sup>c</sup> SODIAAL INTERNATIONAL, Research and Innovation Center, 1-3 rue Jules Maillard de la Gournerie, Rennes 35000, France</footnote>

<footnote><sup>d</sup> Institut NuMeCan, INRAE, INSERM, University of Rennes, Saint Gilles 35590, France</footnote>
2'-Fucosyllactose, 2'-FL (PubChem CID: 170484)
3-fucosyllactose, 3-FL (PubChem CID: 16216990)
Lacto-N-difucohexaose I, LNDFH I (PubChem CID: 3082109)
Lacto-N-fucopentaose VI, LNFP VI (PubChem CID: 481147942)
Difucosyl-para-lacto-N-hexaose 2, DFpLNH II (PubChem CID: 76021528)
2-Aminobenzamide, 2-AB (PubChem CID: 6942)
Mannotriose, M3 (No PubChem CID)
6³,6⁴-α-D-Galactosyl-mannopentaose, GGM5 (No PubChem CID)

# 1. Introduction

Human and animal milks are complex biofluids essential for the growth of newborns. They contain macronutrients (lipids, proteins, and carbohydrates) and micronutrients (such as vitamins, minerals) that provide significant nutritional and health properties (for review: Boudry et al., 2021; Rousseaux et al., 2021; Hobbs et al., 2021). Among milk nutrients, milk oligosaccharides (MOs) are considered the fourth class of macronutrients, after lactose, lipids, and proteins, and are known to play a crucial role in shaping the infant gut microbiota (Sakanaka et al., 2019; Boudry et al., 2021; Rousseaux et al., 2021; Gallo et al., 2024). The concentration of MOs in milks varies during lactation. For example, human, porcine, and bovine colostrum's contain approximately 17.7 g/L, 16.5 g/L, and 1 g/L of MOs, respectively, while mature milks from these species contain approximately 11.3 g/L, 6.9 g/L, and 0.5 g/L, respectively (Nakamura et al., 2003; Fong et al., 2011; Difilippo et al., 2016; Sooyilmaz et al., 2021). Over 160 human milk oligosaccharides (HMOs) have been identified and classified into 20 structural series (Urashima et al., 2018). Despite this structural diversity, fewer than 20 HMOs account for over 90% of their total abundance (Zivkovic et al., 2011; Samuel et al., 2019; Tonon et al., 2019). In contrast, bovine and porcine milks, contain approximately 40–55 distinct oligosaccharides (Tao et al., 2008, 2009; Mariño et al., 2011; Fong et al., 2011; Albrecht et al., 2014; Difilippo et al., 2016; Wei et al., 2018). In bovine milk, sialylated oligosaccharides are predominant with *N*-acetylated (Neu5Ac) or *N*-glycolyl (Neu5Gc) modifications (Tao et al., 2008; Mariño et al., 2011). Among these, Neu5Ac-containing species represent over 98% of the total sialylated oligosaccharides (Albrecht et al., 2014), suggesting a minimal presence of Neu5Gc forms. Bovine milk also contains unique trisaccharides identified for the first time in the bovine colostrum (GNL and isoglobotriose) and lacto-*N*-novopentaose 1 [Galβ1–3(Galβ1–4GlcNAcβ1–6)Galβ1–4Glc] (Urashima et al., 1991), as well as minor neutral trisaccharide derived from the *N*-acetyl-lactosamine core (Gal-β(1,4)-GlcNAc) (Saito et al., 1987).

Understanding how maternal diet affects milk composition and the role of milk nutrients in preventing infant diseases and shaping the infant gut microbiota has become a major focus of research (van Leeuwen, 2019). Due to the strong impact of genetic and environmental variability influencing milk composition, such studies often involve large sample collections, making analysis both difficult and time-consuming. Researchers therefore need rapid and sensitive methods for extracting and quantifying MOs from large sample collections from human and animal milks (van Leeuwen, 2019). Several methods have been developed to identify and determine the relative abundance of oligosaccharides, while others allow for the quantitation of a limited number of MOs from various sources (Ninonuevo et al., 2006; Tao et al., 2010; Albrecht et al., 2014; Urashima et al., 2013; Davis et al., 2017; Yan et al., 2018; Gu et al., 2021; Hickey and Urashima, 2021). Early methods such as high performance liquid chromatography (HPLC) with amperometric detector allowed the quantitation of MOs (Thurl et al., 1996, 2010; Mudd

et al., 2016; Nijman et al., 2018), though sensitivity was limited. More advanced techniques include capillary electrophoresis (Bao et al., 2007; Difilippo et al., 2016; Vicaretti et al., 2018; Sarkozy et al., 2021), HPLC (Hydrophilic Interaction Chromatography (HILIC) or Porous graphitic carbon (PGC)) with mass spectrometry (Tao et al., 2010; Fong et al., 2011; Bao et al., 2013; Remoroza et al., 2018; Porfirio et al., 2020) and/or fluorescent detector (Mariño et al., 2011) have been developed. Recent studies showed the simultaneous and absolute quantification of 5–16 MOs using ultra performance liquid chromatography coupled with fluorescent detector (UPLC-FLD) and external MO standards (Huang et al., 2019; Yang et al., 2022), UPLC coupled to triple quadrupole-mass spectrometry in Multiple Reaction Monitoring mode (UPLC-QqQ-MS/MRM) (Xu et al., 2017; Zhang et al., 2019; Ma et al., 2018), graphitized carbon LC - electrospray ionization - MS (GCLC-EIMS) (Tonon et al., 2019), or UPLC-cyclic ion mobility-mass spectrometry (UPLC-cIM-MS) (Hou et al., 2024). However, the main limitation of these methods remains the limited availability of commercial MO standards. Labelling oligosaccharides with 2-Aminobenzamide (2-AB) offers several advantages, despite the additional step required in sample preparation. These advantages include increased hydrophobicity, enhanced fluorescent signals, and stoichiometric labelling of the reducing end of oligosaccharides (Ruhaak et al., 2010). Recent studies (Yang et al., 2022; Samuel et al., 2019; Huang et al., 2019; Austin & Bénet, 2018; Bénet et al., 2024) have demonstrated the efficiency of this approach for profiling and quantifying MOs, including an AOAC official method 2022.07 (Bénet et al., 2024) suitable for the determination of seven HMOs (2'-FL, 3-FL, DFL, LNT, LNnT, 3'-SL, and 6'-SL) in most infant formula. In the present study, a fast and straightforward approach was developed for extracting oligosaccharides from milk samples using a minimal volume (10–50 µL). A HILIC-FLD-MS method was used to simultaneously identify and quantify MOs labelled with 2-AB, using a single calibration curve with an appropriate internal standard (IS). Once validated, the method was applied to human, bovine, and porcine milk to explore similarities and species-specific features of MO composition.

# 2. Material and methods

## 2.1. Standards & chemicals

MO standards of 2'-FL (OF06739 [purity: min.95%]), 3-FL (OF05673 [purity: min.95%]), DSLNT (OD00173 [purity: min.85%]), LNnT (OL05765 [purity: min.95%]), LNFP VI (OL58467 [purity: min.90%]), LNDFH I (OL01664 [purity: 98%]), N9 (OG15070 [purity: min.95%]), N7 (OG15069 [purity: min.95%]) and N6 (OG44766 [purity: min.90%]) were purchased from Carbosynth and 3'-SL (35/01–0050 [purity: min.98%]), 6'-SL (35/02–0050 [purity: min.98%]), DFpLNH II (57/15–0010 [purity: min.95%]) were purchased from Isosep. Non-milk oligosaccharide standards of Mannotriose (M3, O-MTR [purity: min.95%]), Xylohexaose (X6, O-XHE [purity: min.95%]) and
6³,6⁴-α-D-Galactosyl-mannopentaose, (GGM5, O-GGM5 [purity: min.95%]) were purchased from Megazyme.

Acetonitrile (412392000 [purity: 99.9%]) was purchased from CARLO ERBA Reagents S.A.S. Acetic acid (33209-M [purity: min.99.8%]), Sodium cyanoborohydride (156159 [purity: 95%]), 2-Aminobenzamide (76884 [purity: min.99%]), Formic acid (F0507 [purity: 95%]) and Hydrochloric acid (320331 [purity: 37%]) were purchased from Sigma Aldrich Chimie SARL, France. Ammonium Formate ULC-MS (0001984153BS [purity: 98%]) and Dimethylsulfoxide (0004470501BS [purity: 99.9%]) were purchased from Biosolve Chimie, France.

## 2.2. Milk samples

Porcine milk samples were collected from Large White x Landrace sows (LW-LR) housed at INRAE (Saint Gilles, France). Colostrum was collected within 1–15 h post-parturition, and mature milk was collected on day 21 postpartum after an intramuscular injection of oxytocin (2 mL, Biocytocine®, Laboratoires Biové, Arques, France) to facilitate milking. A total of 24 colostrum and 24 mature milk samples were obtained and immediately frozen at –20 °C until analysis. Sixteen bovine raw milk samples representative of commercial milk were provided by the SODIAAL dairy cooperative (Rennes, France). Human milk samples were obtained from 21 lactating mothers (lactation range: 0.30 – 5.61 months post-partum, milk bank of Rennes University Hospital) as part of preclinical study (Charton et al., 2022). Since human colostrum is typically produced during the first week of lactation, the samples in this study were considered “mature”. This study was approved by the Institutional Review Board of South Mediterranean V, n°19.12.12.65653).

## 2.3. Isolation and fluorescent labelling of MOs

Fifty µL of each milk sample were mixed with 100 µL of internal standard (IS) solution at 50 µg/mL, then diluted with milli-Q water to a final volume of 500 µL. For each sample, two IS standards were used: the 6³,6⁴-α-D-Galactosyl-mannopentaose (GGM5) (for recovery control established with the LNnT calibration curve mentioned below) and either mannotriose (M3) or xylohexaose (X6) for quantification, depending on the milk origin (human or animal). Samples were centrifuged at 20,000 × g for 20 min at 4 °C and the defatted supernatants were filtered using a 10 kDa molecular-weight cut-off ultrafiltration devices (10 kDa Amicon® Ultra 0.5 mL, UFC5010BK, Merck Millipore®). The resulting permeates were freeze-dried and reconstituted in 50 µL of milli-Q water. MOs were fluorescently derivatised by reductive amination with 2-AB by adding a 50 µL solution of 2-AB/NaBH₃CN (sodium cyanoborohydride) (50 and 63 mg/mL respectively) in 30 % (v/v) acetic acid-dimethylsulfoxide. The samples were incubated in the oven set at 60 °C for 4 h under orbital agitation at 150 × g (Multi Reax, Heidolph).

To assess the impact of sample preparation on MO recovery, clean up steps were either added or modified. To precipitate caseins, 10 µL of a 0.1 M hydrochloride solution was added to the defatted supernatant, and the resulting defatted/deprotonated supernatant was recovered through centrifugation (20,000 × g, 10 min, 4 °C, centrifuge 5804R, Eppendorf AG). In addition, ultrafiltration using 3 kDa cut-off filters (3 kDa Amicon® Ultra 0.5 mL, UFC5010BK, Merck Millipore®) was compared to the 10 kDa approach. Non-diluted samples were prepared by centrifuging and ultrafiltrating 200 µL of each milk sample, after which 50 µL from each resulting fraction was used for 2-AB labelling.

## 2.4. Labelled-oligosaccharides analyses by ultra-performance liquid chromatography – hydrophilic liquid chromatography with fluorescent and mass spectrometry detections (UPLC-FLD-MS)

Labelled MOs were analysed by ultra-performance liquid

chromatography–hydrophilic interaction liquid chromatography coupled with fluorescence and mass spectrometry detection (UPLC-FLD-MS), following a method adapted from Albrecht et al. (2014). The 2-AB-labelled MOs were separated using a Waters BEH glycan column (ACQUITY UPLC Glycan BEH Amide Column, 130 Å, 1.7 µm, 2.1 mm × 150 mm, 186004742, Waters) with fluorescence detection and mass spectrometer (ACQUITY UPLC H-class with FLR and SQD2 detectors, Waters). Solvent B was composed of a 50 mM ammonium formate buffer, pH 4.4, and solvent A was acetonitrile. The elution conditions were 15 % solvent B - 85 % solvent A (0–9 min), 15–42 % solvent B – 85–58 % solvent A (9–55.2 min) and 15 % solvent B - 85 % solvent A (55.3–65 min). The flow rate was set at 0.56 mL/min. The injection volume was 6 µL, and the samples were prepared in 80 % (v/v) acetonitrile (dilution by 12.5 for quantification analyses and 3 for identification by mass spectrometry). The separation temperature was 30 °C. The fluorescence detection wavelengths were λex = 330 nm and λem = 420 nm with a data collection rate of 20 Hz. MS analysis was carried out in positive and negative modes. The source capillary voltage was set to 3.2 kV or 2 kV in respectively positive or negative modes, the source temperature was set at 650 °C. The cone voltage was set in ramp mode from 65 to 130 V in positive mode and fixed at 50 V in negative mode. The mass range for MS scan was set to *m/z* 350–1550. Quantification was performed using an external standard calibration curve based on Lacto-N-neotetraose (LNnT, OL05765 Carbosynth) with a 12 concentration points ranging from 1 to 66 µmol·L⁻¹. All the data were processed using Empower3 chromatography workstation software (Waters Corporation).

## 2.5. Method validation

The method was validated based on ruggedness of 2-AB derivatisation, linearity, sensitivity (LoD and LoQ), and precision (repeatability and intermediate reproducibility).

Ruggedness was evaluated by varying incubation times and MO concentrations during derivatisation. Stability of labelled samples was evaluated after storage at 4 °C for several days.

Linearity of the UPLC-FLD method was assessed by plotting the ratio of peak areas (standard/IS) against the concentration ratios (standard/IS) across different concentrations of Lacto-N-neotetraose (LNnT), using fixed concentrations of neutral and sialylated IS.

The calculations of the limit of detection (LOD) and the limit of quantification (LOQ) for sensitivity were performed using neutral and sialylated standards. The LOD is expressed as the smallest amount of a single oligosaccharide showing a signal-to-noise (S/N) ratio above 3, whereas LOQ is the smallest amount of an individual oligosaccharide in the linearity.

Precision was assessed by calculating the repeatability and reproducibility of the peak area of representative oligosaccharides, which was calculated as the standard deviation (SD) from respectively 2 repetitions of extraction/labelling triplicates on different days and 4 independent analyses by two different operators. The concentrations were calculated using the regression equation and the recovery rate was calculated accordingly.

# 3. Results

## 3.1. Oligosaccharide extraction

Analysis of MOs by UPLC requires the removal of other milk macronutrients, particularly lipids and proteins, that can interfere with chromatographic separation and detection. In general, milk samples are first defatted by centrifugation, and proteins are removed by precipitation. Various protein precipitation strategies have been reported in the literature, including the use of organic solvents such as ethanol, acetonitrile, or chloroform/methanol mixtures. However, these methods present several drawbacks: some involve handling CMR (carcinogenic,
mutagenic, or toxic for reproduction) substances, while others are incompatible with hydrophilic interaction liquid chromatography (HILIC) mobile phases, often causing peak splitting or baseline distortion. Moreover, many of these approaches can result in substantial losses of oligosaccharides (Fong et al., 2011). Some studies go further by removing lactose by gel filtration and/or performing fractionation of neutral and acidic oligosaccharides with appropriate ion exchange phase in order to increase the detection of low concentration MOs (Albrecht et al., 2014; Mudd et al., 2016; Oursel et al., 2017; Remorozova et al., 2018). However, in the present study, we aimed to simplify the process by minimizing sample handling and avoiding purification steps that may compromise MO recovery, in line with recent recommendations (Huang et al., 2019).

Six different workflows (Wf) were designed and tested to evaluate the influence of individual preparation steps on MO recovery. The full description of each Wf is provided in Table 1.

Samples from human, bovine and porcine milks were used to compare the extraction steps. At first, the impact of lipid removal by dilution on IS recovery and/or oligosaccharide concentrations was studied and the relevance of an additional step of pellet washing was evaluated (Wf A to D).

The total amount of MOs recovered was higher after a ten-time water dilution prior to centrifugation (Wf-C vs Wf-A) for all type of milks considered (Table 2). Furthermore, the total amount of MOs and the IS recovery were higher with an additional step of pellet washing was included after lipid removal by centrifugation (Wf-B vs Wf-C and Wf-D vs Wf-A) (Table 2). In addition, except for the porcine colostrum, the concentrations of total human, bovine and porcine MOs were similar with (Wf-B) or without (Wf-D) the ten-time water dilution before lipid removal by centrifugation.

The final step of oligosaccharide extraction, which removes residual proteins and lipids, was mainly performed using ultrafiltration (UF). In some studies, caseins are removed by an additional acidification/centrifugation step using hydrochloric acid (HCl) prior UF. The molecular-weight cut-offs vary according to previous studies (3–10 kDa) (Spevacek et al., 2015; Albrecht et al., 2014; Tonon et al., 2019). In our study, the used of UF-3kDa and UF-10kDa after the lipid removal step (Wf E and C, respectively) and UF-3kDa with an additional protein removal step (Wf F) were compared (Table 2). Our results showed a significant decrease in both IS recovery and total HMOs using UF-3kDa (WfE) compared to 10 kDa (WfC) (Table 2). Moderate decreases in recovery were observed for animal milks, some of which were also impacted by the absence of washing pellet after the first step of delipidation. Our results showed that an additional step of protein removal with HCl as described in Albrecht et al. (2014) was not relevant for all milks tested (WfF) (Table 2).

## 3.2. 2-AB derivatisation and fluorescent labelling stability

2-AB labelling is a rapid and cost-effective method that has been widely used since the 1990s to label oligosaccharides (Bigge et al., 1995). It is recommended to add an excess of 2-AB to the oligosaccharide mixtures, and previous studies have reported that labelling with 2-AB is typically completed within 2–2.5 h of incubation at 65°C (Albrecht et al., 2014; Austin and Bénet, 2018; Huang et al., 2019). In our study,

complete labelling of oligosaccharides with a molecular weight exceeding 500 g/mol was achieved after 4 h of incubation at 60 °C (data not shown). Interestingly, we found that varying the excess of 2-AB labelling mixture (ranging from 6 to 40 times) had no discernible effect on the fluorescent signals (data not shown).

Furthermore, 2-AB labelling is known for its remarkable stability. Given that our method involved comparing a large collection of samples, we determined the maximum storage duration using porcine milk as a reference. As indicated in Table 3, the ratio between 2-AB labelled oligosaccharides, namely 3'-sialyllactose (3'-SL) and Gal(β1-3)Gal(β1-4) Glc (3'-GOS) (a major and a minor porcine MOs, respectively), and their internal standard, exhibited excellent stability after 40 days of storage at room temperature and 4°C and -20 °C, although slightly higher variability was observed at – 20 °C. Importantly, the absolute peak areas of both the MOs and the internal standard also remained consistent across all time points, confirming that neither the MO nor the standard underwent significant degradation. This indicates that the observed stability in their ratio reflects true chemical stability of both components, rather than compensatory degradation effects. Based on these results, we consider that the 2-AB labelling reaction is chemically stable regardless of the sample matrix, and we expect comparable stability when applied to other biological matrices such as bovine or human milk.

## 3.3. Identification of 2-AB labelled milk oligosaccharides and nomenclature

For quantification in milligrams per liter, this method involved determining the molar masses of oligosaccharides detected in porcine, bovine, and human milks. Molar mass determination was performed using simple quadrupole mass spectrometry (SQD2, Waters). Oligosaccharide identification was based on existing literature and a set of 12 commercially available oligosaccharides, namely 2'-FL, 3-FL, Gal(α1-3) Gal(β1-4)Glc (Isoglobotriose), 3'-GOS, 6'-GOS, 3'-SL, 6'-SL, LNnT, LNFP VI, LNDFH I, DSLNT, and DFpLNH II (Table 4, Supp. Fig. 1). Moreover, milk samples from mothers with three different serologic statuses —based on their genetic ability to express α(1–2)-fucosyltransferase FUT2 (Secretor gene, Se) and/or α(1–3/4)-fucosyltransferase FUT3 (Lewis gene, Le) in glandular epithelia—categorized as Se + Le + (or group 1), Se-Le + (or group 2), Se + Le- (or group 3). These genetic variations affect the structure of fucosylated HMOs (for review, see Boudry et al., 2021), allowing us to propose structures for some isomers.

First, the twelve commercial oligosaccharides enabled the unambiguous identification of the detected oligosaccharides nos. 1, 2, 5, 7, 10, 11, 13, 15, 20, 35, 46 and 55, as listed in Table 4. Among the twelve detected trisaccharides (nos. 1–5, 7–13), three isomers with molar masses of 623.2 Da were identified by mass spectrometry: isoglobotriose, 3'-GOS, and 6'-GOS. Since HILIC is a chromatographic technique highly sensitive to the fine structure of compounds, including the involved carbon and anomer of the glycosidic linkage, these results provide insight into the order of elution of these isomers. Isoglobotriose (no. 5, Table 4), characterized by an α(1–3)-linked galactose attached to the lactose core, eluted at 12.9 min. In contrast, 3'-GOS (no. 7, Table 4), which has a β(1–3)-linked galactose, eluted at 13.9 min. This suggests that for isomers involving the same carbons in the glycosidic linkage, the α anomer elutes before the β anomer. Additionally, among β-linked

<table><caption>**Table 1**
Minimal extraction step study. Summary of workflows (Wf) performed. IS: Internal Standard; UF: Ultrafiltration. Y: performed; N: not performed.</caption><thead><tr><th>Wf</th><th>Dilution &amp; IS</th><th>centrifugation</th><th>Wash pellet</th><th>protein removal</th><th>UF3kDa</th><th>UF10kDa</th></tr></thead><tbody><tr><td>A</td><td>N</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>Y</td></tr><tr><td>B</td><td>Y</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>Y</td></tr><tr><td>C</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>N</td><td>Y</td></tr><tr><td>D</td><td>N</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>Y</td></tr><tr><td>E</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td>Y</td><td>N</td></tr><tr><td>F</td><td>Y</td><td>Y</td><td>N</td><td>Y</td><td>Y</td><td>N</td></tr></tbody></table>
<caption>**Table 2**</caption>

Impact of the lipid removing steps (e.g. centrifugation ± after water dilution and ± washing pellet) and the ultrafiltration (UF) molecular-weight cut-offs on the internal standard (IS) recovery and total oligosaccharide concentrations (Conc) following workflow (Wf) B to D (lipid removing steps) and Wf C (UF-10 kDa), E (UF-3 kDa) and F (UF-3 kDa after protein removal with HCl). 6³,6⁴-α-D-Galactosyl-mannopentaose (GGM5) was used as IS for human and animals milks recovery measurement; detailed combination of extraction steps is given in Table 1.

<table><caption>**Table 3**</caption><thead><tr><th rowspan="2">Wf</th><th colspan="2">Conc (mg/mL)</th><th colspan="2">IS recovery (%)/Conc (mg/mL)</th><th colspan="2">IS recovery (%)/Conc (mg/mL)</th><th colspan="2">IS recovery (%)/Conc (mg/mL)</th></tr><tr><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>F</th><th>G</th><th>H</th></tr></thead><tbody><tr><td>Human milk</td><td>7.1</td><td>104/11.5</td><td>88/9.7</td><td>12.1</td><td>52/5.0</td><td>56/5.7</td><td></td><td></td></tr><tr><td>Bovine milk</td><td>0.20</td><td>98/0.31</td><td>92/0.31</td><td>0.24</td><td>70/0.22</td><td>60/0.21</td><td></td><td></td></tr><tr><td>Porcine<br>colostrum</td><td>1.4</td><td>86/3.4</td><td>58/2.0</td><td>1.6</td><td>51/1.7</td><td>59/1.8</td><td></td><td></td></tr><tr><td>Porcine milk</td><td>0.55</td><td>97/0.87</td><td>78/0.73</td><td>0.84</td><td>57/0.51</td><td>57/0.48</td><td></td><td></td></tr></tbody></table>

Ratio between porcine milk oligosaccharides (3'-GOS and 3'-SL) and their internal standard (IS: Xylohexaose (X6)) after storage at 3 different temperatures (Room Temperature (RT), 4 °C and -20 °C) during 40 days.

<table><thead><tr><th rowspan="2">storage T°C<br>during 40<br>days</th><th colspan="3">3'-GOS<br>(Galβ(1-3)Gal(β1-4)Glc)</th><th colspan="3">3'-SL<br>(Neu5Ac(α2-3)Gal(β1-4)Glc)</th></tr><tr><th>RT</th><th>4 °C</th><th>-20 °C</th><th>RT</th><th>4 °C</th><th>-20 °C</th></tr></thead><tbody><tr><td>Mean (ratio;<br>n = 6)</td><td>2.1</td><td>2.2</td><td>2.6</td><td>7.1</td><td>7.3</td><td>8.1</td></tr><tr><td>Standard<br>deviation</td><td>0.1</td><td>0.2</td><td>0.5</td><td>0.6</td><td>0.9</td><td>1.5</td></tr><tr><td>Coeff. of<br>variation</td><td>5.66%</td><td>11.03%</td><td>18.91%</td><td>9.10%</td><td>12.06%</td><td>18.43%</td></tr></tbody></table>

isomers to the lactose core, the linkage in position (1-3) (3'-GOS) eluted before the linkage in position (1-6) (6'-GOS), suggesting that the elution order depends on the position of the carbon involved in the linkage. Based on these observations, we propose the structure for the fourth isomer with a *m/z* of 623.2 Da and eluted at 14.4 min (no. 8, Table 4) as Gal(β1-4)Gal(β1-4)Glc. This is based on the fact that Gal(α1-4) or Gal (α1-6) linkages have not been reported in the literature in any MOs, except for Gal(α1-4)Gal(β1-4)Glc (globotriose) which has been only reported in bottlenose dolphin milk and thus very rare in other mammal milks (Uemura et al., 2005).

Two other isomers were also identified unambiguously using commercial standards: 3'-SL (no. 11, Table 4) and 6'-SL (no. 13, Table 4). These findings confirm that, for the same sugar and anomer, the linkage in position (2-3) elutes before the linkage in position (2-6). Based on this observation, the two additionally isomers detected at *m/z* 793.3 Da (no. 9 and no. 12, Table 4) were unambiguously identified as 3'-SLN and 6'-SLN previously described in Albrecht et al. (2014) in that elution order, confirming that the linkage in position (2-3) elutes before the linkage in position (2-6). The two remaining trisaccharides (nos. 3 and 4, Table 4) were detected with an *m/z* of 664.3 Da and therefore correspond to the addition of an *N*-acetyl-hexosamine to the lactose body. The structure was identified using literature information, specifically the HILIC elution order established by Albrecht et al. (2014).

The structures of the six detected tetrasaccharides (nos. 14-17, 19 and 23, Table 4), were primarily based on Albrecht et al. (2014), with reference to tetrasaccharides nos.16, 17, and 19, and the commercially available tetrasaccharide LNnT (no. 15). This result confirms that the elution order depends on the carbon position in the glycosidic bond. Specifically, for the isomers with an *m/z* of 826.3 Da (nos. 15 and 17), which differ only by the β(1-3) and β(1-6) linkages of *N*-acetyllactosamine to the lactose core. A third isomer (no. 14, Table 4) with an *m/z* of 826.3 Da was detected and eluted before LNnT. The intensity of the fluorescence-detected peak and the fact that a known isomer of LNnT has already been described in the literature suggest that this oligosaccharide is LNT, which differs from LNnT by having a lacto-*N*-biose I instead of *N*-acetyllactosamine. This is consistent with the proposed

elution order, as the (1-3) linkage is eluted before the (1-4) linkage. However, the sixth tetrasaccharide (no. 23, Table 4), detected at 25.4 min in porcine milk and colostrum with an *m/z* of 785.3 Da, corresponds to four hexoses linked together. A similar tetrasaccharide with the same *m/z*, but an unknown structure, was previously reported in porcine milk by Albrecht et al. (referred to as N13). Given its late elution time, the oligosaccharide detected in our study is likely a distinct structure.

Next, eleven pentasaccharides were detected (nos. 18, 20-22, 24-27 and 29-30 and 34, Table 4). Among these, a commercial standard unambiguously identified no. 20 as lacto-*N*-fucopentaose VI (LNFP VI), eluted at 24.1 min. Five other LNFP VI isomers were also detected (no. 18, 21, 22, 24 and 25). The HILIC elution order for this family of LNFP isomers has previously been described (Huang et al., 2019, using three commercial oligosaccharides: LNFP V, I and II; Remoroza et al., 2018, LNFP I, V, III and II). Combining this information, the proposed order is: LNFP V, LNFP I, LNFP III and LNFP II. Additionally, LNFP VI (no. 20, Table 4) shares structural similarity with LNFP V, differing only in the β(1-4) and β(1.3) linkages between the last galactose residue and the *N*-acetylglucosamine residue. Therefore, the oligosaccharide detected at 23.8 min (no. 18, Table 4) is proposed to be LNFP V. Since LNFP I is the most abundant oligosaccharide in women serologic groups 1 and 3 and absent in group 2, the detected oligosaccharide number 21 likely corresponds to LNFP I. Finally, LNFP III and II were attributed to isomers no. 24 and 25 (Table 4), consistent with the absence of LNFP II in woman serologic group 3.

For the remaining pentasaccharides, *m/z* values and retention times led to the identification of additional isomers, including N4010a and N4010b (nos. 26 and 29), and the well-known LSTA, LSTb, and LSTc (or 6'-SLnNT) (nos. 27, 30, and 34). These assignments are consistent with previous reports (Remoroza et al., 2018; Albrecht et al., 2014).

Fourteen hexasaccharides were detected (nos. 28, 31-33, 35-42, 45-46). These included five isomers with an *m/z* of 1118.4 Da, corresponding to lacto-*N*-diflucohexaose (LNDFH) isomers, and five isomers with an *m/z* of 1191.4 Da, corresponding to lacto-*N*-hexaose (LNH) isomers. Among LNDFH isomers, only three were described and identified in HILIC. LNDFH I was unambiguously attributed to no. 35 using a commercial oligosaccharide. Notably, LNDFH I is the most abundant isomer of this family, and its synthesis requires FUT2 and FUT3. In our study, LNDFH I was detected only in the serologic woman group 1 Se + Le + and was absent of groups 2 and 3, confirming the attribution (see no. 35, Table 4). Consistent with the HILIC elution order described in Remoroza et al. (2018) and considering that LNDFH II is not synthesized in the serologic group 3 while LNDFH III is synthesized in all three serologic groups, these two isomers were attributed to nos. 33 and 32 respectively (Table 4). Among the isomers with an *m/z* of 1191.4, LNH and LNnH were previously identified (Remoroza et al., 2018; Tonon et al., 2019) with close retention times, with LNH being less retained than LNnH. Since LNH is the most abundant isomer in this family, it was assigned to the oligosaccharide detected at 31.6 min (no. 40), and LNnH was proposed for no. 41. The structures of two other isomers from this family have also been identified (van Leeuwen, 2019): para-LNH and
<caption>Table 4</caption>

Structures and their concentrations in porcine, bovine and human milks. *confirmed by commercial standard. || co-eluted. ** doubly charged ion. Galactose; N-acetyl-galactosamine; N-acetyl-glucosamine; Fucose; N-acetyl-neuraminic acid/Sialic acid; lactose core; Lacto-N-biose I; N-acetyllactosamine; Lewis b antigen in Se + Le +; Lewis a antigen in Se-Le + (Ashline et al., 2014; Remoroza et al., 2020; van Leeuwen et al., 2018).

<table><thead><tr><th rowspan="3">no.</th><th rowspan="3">code</th><th rowspan="3">Structure</th><th rowspan="3">Name</th><th rowspan="3">Scheme</th><th rowspan="3">RT (min)</th><th rowspan="3">2AB-MO MW</th><th rowspan="3">m/z obs (** doubly charged ion)</th><th colspan="4">Human milk</th><th colspan="4">Not detected (Ø), detected only (T) and detected & quantified (Concentration in g/L ± SD (Number of samples; Coefficient of Variation)) milk oligosaccharides (MOs)</th></tr><tr><th colspan="2">Porcine milk / Colostrum</th><th colspan="2">Cow milk</th><th colspan="2">Se+Le+ (Group 1)</th><th colspan="2">Se-Le+ (Group 2)</th><th colspan="2">Se+Le- (Group 3)</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>1</td><td>N1</td><td>Fuc(α1-2)Gal(β1-4)Glc</td><td>2'-fucosyl-lactose (2'-FL)</td><td></td><td>7.2*</td><td>608.24</td><td>607.2</td><td>0.108 / 0.030 ± α0.003 (24; 11.3)</td><td>Ø</td><td>2.404 ± 0.349 (16; 14.5)</td><td>Ø</td><td>3.346</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2</td><td>N2</td><td>Gal(β1-4)(Fuc(α1-3))Glc</td><td>3-fucosyl-lactose (3-FL)</td><td></td><td>8.3*</td><td>608.24</td><td>607.2</td><td>0.016 / 0.051 ± α0.008 (24; 15.6)</td><td>0.020 ± 0.003 (16; 15.5)</td><td>0.417 ± 0.099 (16; 23.8)</td><td>1.213 ± 0.213 (4; 17.5)</td><td>0.130</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>3</td><td>N4</td><td>GalNAc(α1-3)Gal(β1-4)Glc</td><td>N-acetyl-galactosa minyl-lactose (GNL) or α3'-GalNAcL</td><td></td><td>10.6</td><td>665.26</td><td>664.3</td><td>0.065 ± 0.007 (16; 12.2)</td><td>Ø</td><td>Ø</td><td>Ø</td><td>Ø</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>4</td><td>N5</td><td>GlcNAc(β1-3)Gal(β1-4)Glc</td><td>LNTri II (Urashima et al., 2018)</td><td></td><td>12.1</td><td>665.26</td><td>664.3</td><td>0.020 / Ø</td><td>Ø</td><td>Ø</td><td>Ø</td><td>Ø</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>5</td><td>N6</td><td>Gal(α1-3)Gal(β1-4)Glc</td><td>Isoglobo-triose (or β3'-GL Urashima et al., 2018)</td><td></td><td>12.9*</td><td>624.23</td><td>623.2</td><td>0.018 / 0.096 ± α0.008 (24; 8.3)</td><td>0.025 ± 0.004 (16; 14.6)</td><td>Ø</td><td>Ø</td><td>Ø</td><td>Ø</td><td></td><td></td><td></td><td></td></tr><tr><td>6</td><td>-</td><td>Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]Glc</td><td>Lacto-difucotetraose (LDFT or Difucosyllactose (DF-L) (van Leeuwen et al., 2019))</td><td></td><td>13.8</td><td>754.58</td><td>753.6</td><td>Ø</td><td>Ø</td><td>0.232 ± 0.093 (16; 40.0)</td><td>Ø</td><td>0.097</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>7</td><td>N7</td><td>Gal(β1-3)Gal(β1-4)Glc</td><td>3'-GOS (Huang et al., 2019) or β3'-GL (Difillippo et al., 2016) or 3'-GalL (Remoroza et al., 2019)</td><td></td><td>13.9*</td><td>624.23</td><td>623.2</td><td>0.035 / 0.289 ± α0.022 (24; 7.6)</td><td>0.020 ± 0.004 (16; 18.3)</td><td>T</td><td>0.003 ± 0.002 (4; 72.4)</td><td>T</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>8</td><td>-</td><td>Gal(β1-4)Gal(β1-4)Glc (Hypothesis structure)</td><td>B4'-GL (Urashima et al., 2018)</td><td></td><td>14.4</td><td>624.23</td><td>623.2</td><td>Not detected / α0.082 ± 0.007 (24; 8.4)</td><td>Ø</td><td>Ø</td><td>Ø</td><td>Ø</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 4 (continued)**</caption><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>20</td><td>-</td><td>Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]Glc</td><td>Lacto-N-fucopentaose VI (LNFP VI)</td><td>24.1*</td><td>973.36</td><td>972.4</td><td>Ø</td><td>Ø</td><td>0.035 ± 0.014 (16; 38.6)</td><td>0.230 ± 0.038 (4; 16.6)</td><td>0.014</td></tr><tr><td>21</td><td>-</td><td>Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc</td><td>Lacto-N-fucopentaose I (LNFP I)</td><td>24.6</td><td>973.36</td><td>972.4</td><td>Ø</td><td>Ø</td><td>1.069 ± 0.336 (16; 31.5)</td><td>Ø</td><td>1.975</td></tr><tr><td>22</td><td>-</td><td>-</td><td>Lacto-N-fucopentaose (LNFP) isomer</td><td>-</td><td>25.2</td><td>973.36</td><td>972.4</td><td>Ø</td><td>Ø</td><td>T</td><td>0.002 ± 0.001 (4; 44.3)</td><td>0.003</td></tr><tr><td>23</td><td>N13</td><td>Gal-Gal-Gal(β1-4)Glc</td><td>Tetrasaccharide (unknown structure)</td><td>-</td><td>25.4</td><td>786.3</td><td>785.3</td><td>Not detected / 0.041 ± 0.008 (24; 18.5)</td><td>Ø</td><td>Ø</td><td>Ø</td><td>Ø</td></tr><tr><td>24</td><td>N17</td><td>Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc</td><td>Lacto-N-fucopentaose III (LNFP III)</td><td>25.9</td><td>973.36</td><td>972.4</td><td>0.027 / 0.042 ± 0.007 (24; 16.4)</td><td>Ø</td><td>0.217 ± 0.063 (16; 29.1)</td><td>0.232 ± 0.045 (4; 19.6)</td><td>0.301</td></tr><tr><td>25</td><td>-</td><td>Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc</td><td>Lacto-N-fucopentaose II (LNFP II)</td><td>26.1</td><td>973.36</td><td>972.4</td><td>Ø</td><td>Ø</td><td>0.334 ± 0.092 (16; 27.6)</td><td>1.677 ± 0.445 (4; 26.6)</td><td>Ø</td></tr><tr><td>26</td><td>-</td><td>Gal(β1-4)[Gal(β1-3)]GlcNAc(β1-3)Gal(β1-4)Glc</td><td>N4010a (Remorozae et al. 2018)</td><td>26.8</td><td>989.36</td><td>988.4</td><td>Ø</td><td>Ø</td><td>0.003 ± 0.003 (15; 74.2)</td><td>0.010 ± 0.002 (4; 17.8)</td><td>0.004</td></tr><tr><td>27</td><td>-</td><td>Neu5Ac(α2-3)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc</td><td>Sialylacto-N-tetraose a (LSTa)</td><td>27.8</td><td>1118.4</td><td>1117.4</td><td>Ø</td><td>Ø</td><td>0.023 ± 0.011 (16; 49.0)</td><td>0.029 ± 0.007 (4; 24.0)</td><td>0.033</td></tr><tr><td>28</td><td>-</td><td>-</td><td>lacto-N-difucohexaose (LNDFH) isomer unknown structure</td><td>-</td><td>28.0</td><td>1119.42</td><td>1118.4</td><td>Ø</td><td>Ø</td><td>0.026 ± 0.025 (16; 97.4)</td><td>Ø</td><td>0.011</td></tr><tr><td>29</td><td>N21</td><td>Gal(β1-4)GlcNAc(β1-6)[Gal(β1-3)]Gal(β1-4)Glc</td><td>Lacto-N-novopentaose I (novoLNPI) (or N4010b, Remorozae et al. 2018)</td><td>28.3</td><td>989.36</td><td>988.4</td><td>0.031 / 0.042 ± 0.005 (24; 11.7)</td><td>Ø</td><td>T</td><td>T</td><td>T</td><td>T</td></tr><tr><td>30</td><td>-</td><td>Gal(β1-3)[Neu5Ac(α2-6)]GlcNAc(β1-3)Gal(β1-4)Glc</td><td>Sialylacto-N-tetraose b (LSTb)</td><td>28.5</td><td>1118.4</td><td>1117.4</td><td>Ø</td><td>Ø</td><td>0.050 ± 0.015 (16; 31.0)</td><td>0.060 ± 0.009 (4; 14.9)</td><td>0.037</td></tr></tbody></table>

(continued on next page)
<table><caption>Table 4 (continued)</caption><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>39</td><td>-</td><td>Neu5Ac(α2-3)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc & Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]GlcGal(β1-4)GlcNAc(β1-6)[Gal(β1-3)GlcNAc(β1-3)]Gal(β1-4)GlcGal(β1-4)GlcNAc(β1-6)[Gal(β1-3)GlcNAc(β1-4)]Gal(β1-4)Glc</td><td>Sialyl-lacto-N-fucopentaose II (SLNFP II) or F-LSTa Urashima et al., 2018 / Fucosylated sialyllacto-N-tetraose (F-LSTc)</td><td>31 ||</td><td>1264.46</td><td>631.2**</td><td>Ø</td><td>Ø</td><td>T</td><td>T</td><td>Ø</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>40</td><td>-</td><td>Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-4)GlcGal(β1-4)GlcNAc(β1-6)[Gal(β1-3)GlcNAc(β1-4)]Gal(β1-4)Glc</td><td>Lacto-N-hexaose (LNH)</td><td>31.6</td><td>1192.44</td><td>1191.4</td><td>Ø</td><td>Ø</td><td>0.099 ± 0.035 (16; 35.8)</td><td>0.063 ± 0.023 (4; 36.2)</td><td>0.138</td><td></td><td></td><td></td><td></td></tr><tr><td>41</td><td>N24</td><td>Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-4)GlcGal(β1-4)GlcNAc(β1-6)[Gal(β1-3)GlcNAc(β1-4)]Gal(β1-4)Glc</td><td>Lacto-N-neohexaose (LNH)</td><td>32,0</td><td>1192.44</td><td>1191.4</td><td>0.035 / 0.058 ± 0.006 (24; 10.8)</td><td>Ø</td><td>T</td><td>Ø</td><td>Ø</td><td></td><td></td><td></td><td></td></tr><tr><td>42</td><td>-</td><td>Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-4)GlcGal(β1-4)GlcNAc(β1-6)[Gal(β1-3)GlcNAc(β1-4)]Gal(β1-4)Glc</td><td>A3111a or LSTd Urashima et al., 2018</td><td>32.3 ||</td><td>1264.46</td><td>631.2**</td><td>Ø</td><td>Ø</td><td>0.016 ± 0.006 (16; 38.2)</td><td>0.050 ± 0.010 (4; 19.0)</td><td>0.003</td><td></td><td></td><td></td><td></td></tr><tr><td>43</td><td>-</td><td>Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)GlcNAc(β1-4)Glc</td><td>Iso-fucosyllacto-N-hexaose I (IFLNH I) (Remorozza et al., 2018) Or Monofucoparalacto-N-hexaose (MFpLNH I)</td><td>32.7</td><td>1338.52</td><td>1337.5</td><td>Ø</td><td>Ø</td><td>T</td><td>Ø</td><td>T</td><td></td><td></td><td></td><td></td></tr><tr><td>44</td><td>-</td><td>Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)[Gal(β1-4)]Glc</td><td>Monofucosyllacto-N-hexaose (MFLNH I) or Fucosylated d-lacto-N-hexaose I (F-LNH I) (van Leeuwen et al., 2019)</td><td>33.2</td><td>1338.52</td><td>1337.5</td><td>Ø</td><td>Ø</td><td>0.010 ± 0.007 (15; 72.5)</td><td>Ø</td><td>0.020</td><td></td><td></td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>Table 4 (continued)</caption><tbody><tr><td>52</td><td>-</td><td>Fuc(α1-2 )Gal(β1-3)GlcNAc (β1-3)[Gal(β1-4)][Fuc(α 1-3)]GlcNAc(β1-6)][Gal(β1-4)Glc</td><td>Difucosylla cto-N-hexaose a (DFLNH a) or DFLNH I (van Leeuwen et al., 2019)</td><td>37.2</td><td>1484.57</td><td>741.3**</td><td>Ø</td><td>Ø</td><td>0.228 ± 0.039 (16; 17.3)</td><td>Ø</td><td>0.659</td></tr><tr><td>53</td><td>A37</td><td>Neu5Ac( α2-6)Gal(β1-4)GlcNAc (β1-3)[Gal(β1-4)GlcNAc (β1-6)][Gal(β1-4)Glc</td><td>6-sialyl-lacto-N-neohexaose (6-SLnH) (animal milks) or S-LNnH II (human milk)</td><td>37.6</td><td>1483.6</td><td>740.8**</td><td>0.037 / 0.058 ± 0.007 (24; 12.9)</td><td>Ø</td><td>0.066 ± 0.021 (16; 31.9)</td><td>0.052 ± 0.017 (4; 31.9)</td><td>0.103</td></tr><tr><td>54</td><td>-</td><td>Gal(β1-4)[Fuc(α 1-3)]GlcNAc(β1-3)Gal(β1-4)[Fuc(α 1-3)]GlcNAc(β1-3)Gal(β1-4)Glc</td><td>Difucosyl-para-lacto-N-neohexaose (DFpLNnH)</td><td>38,0</td><td>1484.57</td><td>741.3**</td><td>Ø</td><td>Ø</td><td>0.066 ± 0.020 (16; 29.8)</td><td>0.095 ± 0.024 (4; 25.0)</td><td>0.137</td></tr><tr><td>55</td><td>-</td><td>-</td><td>Difucosylla cto-N-hexaose isomers (DFLNHb/ DFLNH II/DFpLNH II) or DF-para-LNH Urashima et al., 2018)</td><td>38.2*</td><td>1484.57</td><td>741.3**</td><td>Ø</td><td>Ø</td><td>0.113 ± 0.037 (16; 33.1)</td><td>0.604 ± 0.126 (4; 20.9)</td><td>Ø</td></tr><tr><td>56</td><td>-</td><td>Fuc(α1-2 )Gal(β1-3)[Fuc(α 1-4)]GlcNAc(β1-3)[Gal(β1-4)GlcNAc (β1-6)][Gal(β1-4)Glc</td><td>Difucosylla cto-N-hexaose c (DFLNH c) or DFLNH III (van Leeuwen et al., 2019)</td><td>38.5</td><td>1484.57</td><td>741.3**</td><td>Ø</td><td>Ø</td><td>0.072 ± 0.031 (16; 43.6)</td><td>Ø</td><td>Ø</td></tr><tr><td>57</td><td>-</td><td>Fuc(α1-2 )Gal(β1-4)[Fuc(α 1-3)]GlcNAc(β1-3)[Gal(β1-4)][Fuc(α 1-3)]GlcNAc(β1-6)][Gal(β1-4)Glc</td><td>Trifucosyll acto-N-neohexaose e (TFLNnH)</td><td>38.8</td><td>1630.63</td><td>814.3**</td><td>Ø</td><td>Ø</td><td>0.012 ± 0.008 (16; 68.1)</td><td>Ø</td><td>0.036</td></tr></tbody></table>

(continued on next page)
<table><caption>Table 4 (continued)</caption><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>58</td><td>-</td><td>Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)GlcGal(β1-3)[Neu5Ac(α2-6)]GlcNAc(β1-3)[Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)]Gal(β1-4)GlcGal(β1-3)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)]Gal(β1-4)Glc</td><td>5130c (or F-LNO III Urashima et al., 2018)</td><td>39.3</td><td>1703.65</td><td>850.9**</td><td>Ø</td><td>Ø</td><td>0.004 ± 0.003 (15; 79.3)</td><td>Ø</td><td>0.016</td></tr><tr><td>59</td><td>-</td><td>Fucosylated sialylated Lacto-N-Hexaose I (FS-LNH I, Urashima et al., 2018)</td><td></td><td>39.8</td><td>1629.68</td><td>813.8**</td><td>Ø</td><td>Ø</td><td>0.191 ± 0.052 (16; 27.2)</td><td>0.139 ± 0.040 (4; 29.1)</td><td>0.367</td></tr><tr><td>60</td><td>-</td><td>Fucosyllacto-N-octaose I (FLNO I) (Urashima et al., 2018)</td><td></td><td>40.3</td><td>1703.65</td><td>850.9**</td><td>Ø</td><td>Ø</td><td>0.021 ± 0.009 (16; 44.7)</td><td>0.024 ± 0.005 (4; 20.5)</td><td>0.029</td></tr><tr><td>61</td><td>-</td><td>Neu5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc</td><td>A4121c</td><td>40.6</td><td>1629.68</td><td>813.8**</td><td>Ø</td><td>Ø</td><td>0.012 ± 0.016 (15; 133.1)</td><td>0.044 ± 0.009 (4; 19.9)</td><td>0.084</td></tr><tr><td>62</td><td>-</td><td>Trifucopara-lacto-N-hexaose (TFpLNH) or TFpLNH I (Urashima et al., 2018)</td><td></td><td>40.9</td><td>1630.63</td><td>814.3**</td><td>Ø</td><td>Ø</td><td>0.275 ± 0.054 (16; 19.6)</td><td>Ø</td><td>Ø</td></tr><tr><td>63</td><td>-</td><td>Fucosyllacto-N-octaose (FLNO) isomer</td><td></td><td>40.9</td><td>1703.65</td><td>850.9**</td><td>Ø</td><td>Ø</td><td>0.099 ± 0.024 (4; 24.5)</td><td>T</td><td></td></tr></tbody></table>

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

(continued on next page)
<table><caption>Table 4 (continued)</caption><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>64</td><td>-</td><td>Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)]GlcNAc(β1-6)]Gal(β1-4)Glc</td><td>Iso-fucosyllacto-N-neooctaose (F-isolFNnO I (Urashima et al., 2018))</td><td>41.2</td><td>1703.65</td><td>850.9**</td><td>Ø</td><td>Ø</td><td>0.007 ± 0.008 (9; 110.8)</td><td>0.007 ± 0.004 (4; 53.8)</td><td>0.021</td></tr><tr><td>65</td><td>-</td><td>Fucosyllacto-N-octaose (FLNO) isomer</td><td>-</td><td>41.4</td><td>1703.65</td><td>850.9**</td><td>Ø</td><td>Ø</td><td>0.012 ± 0.013 (10; 100.3)</td><td>Ø</td><td>0.033</td></tr><tr><td>66</td><td>-</td><td>Disialylated Fucosyllacto-N-hexaose II) DS-FLNH II Or Fucosyldisialylacto-N-hexaose II (FDS-LNH II Remorozae et al. 2019) or FDS-LNH I (Urashima et al., 2018)</td><td>41.6 ||</td><td></td><td>1920.7</td><td>959.4**</td><td>Ø</td><td>Ø</td><td>T</td><td>Ø</td><td>T</td></tr><tr><td>67</td><td>-</td><td>N5230a or DF-isolNO II (Urashima et al., 2018)</td><td></td><td></td><td>1849.7</td><td>923.9**</td><td>Ø</td><td>Ø</td><td>T</td><td>Ø</td><td>T</td></tr><tr><td>68</td><td>-</td><td>Neu5Ac(α2-3)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc</td><td>A4122a or FDS-LNH III (Urashima et al., 2018)</td><td>41.9</td><td>1920.7</td><td>959.4**</td><td>Ø</td><td>Ø</td><td>0.002 ± 0.003 (14; 123.0)</td><td>0.010 ± 0.004 (4; 42.3)</td><td>Ø</td></tr><tr><td></td><td></td><td></td><td>A4221a or DFS-LNH III (Urashima et al., 2018) or b</td><td>41.9</td><td>1775.7</td><td>886.8**</td><td>Ø</td><td>Ø</td><td>Ø</td><td>T</td><td>Ø</td></tr></tbody></table>

(continued on next page)
<table><caption>Table 4 (continued)</caption><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>69</td><td>-</td><td>Neu5Ac(α2-3)Gal(β1-3)[Neu5Ac(α2-6)]GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc</td><td>Disialyllacto-N-hexaose (DSLNH) or DS-LNH II (Urashima et al., 2018)</td><td>42.2</td><td>1774.72</td><td>886.4**</td><td>Ø</td><td>Ø</td><td>T</td><td>0.004 ± 0.003 (4; 83.7)</td><td>0.021</td></tr><tr><td>70</td><td>-</td><td>-</td><td>Difucosylla cto-N-neooctaos e II (DFLNnO II) or DFIGiso-LNnO Urashima et al., 2018)</td><td>42.4</td><td>1849.71</td><td>923.9**</td><td>Ø</td><td>Ø</td><td>0.021 ± 0.010 (16; 46.8)</td><td>0.001 ± 0.001 (2; 119.8)</td><td>0.018</td></tr><tr><td>71</td><td>-</td><td>-</td><td>Difucosylla cto-N-octaose II (DFLNO II)</td><td>42.6</td><td>1849.71</td><td>923.9**</td><td>Ø</td><td>Ø</td><td>0.007 ± 0.008 (13; 104.1)</td><td>0.025 ± 0.002 (4; 9.6)</td><td>Ø</td></tr><tr><td>72</td><td>-</td><td>Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)[Neu5Ac(α2-6)]Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc</td><td>Monosialyl difucosylla cto-N-Hexaose (MSDFLNH) or DFS-LNH I (Urashima et al., 2018)</td><td>42.9</td><td>1775.67</td><td>886.8**</td><td>Ø</td><td>Ø</td><td>0.053 ± 0.038 (14; 71.5)</td><td>Ø</td><td>Ø</td></tr><tr><td>73</td><td>-</td><td>-</td><td>Difucosylla cto-N-octaose (DFLNO) isomer</td><td>-</td><td>42.9</td><td>1849.71</td><td>923.9**</td><td>Ø</td><td>Ø</td><td>T</td><td>Ø</td><td>0.070</td></tr><tr><td>74</td><td>-</td><td>Gal(β1-3)GlcNAc(β1-3)[Gal(β1-3)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc</td><td>Lacto-N-decaose (LND) or Lacto-N-neo-decaose (LNnD) Ashline et al., 2014)</td><td>43.4</td><td>1922.7</td><td>960.4**</td><td>Ø</td><td>Ø</td><td>T</td><td>T</td><td>0.059</td></tr><tr><td>-</td><td>-</td><td>-</td><td>Difucosylla cto-N-octaose (DFLNO) isomer</td><td>-</td><td>1849.71</td><td>923.9**</td><td>Ø</td><td>Ø</td><td>0.023 ± 0.007 (16; 32.0)</td><td>0.026 ± 0.011 (4; 43.5)</td><td>T</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 4 (continued)**</caption><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>75</td><td>-</td><td>-</td><td>Difucosylla<br>cto-N-<br>octaose I<br>(DFLNO I)</td><td>43.7</td><td>1849.71</td><td>923.9**</td><td>Ø</td><td>Ø</td><td>0.012 ±<br>0.007 (16;<br>62.9)</td><td>0.031 ±<br>0.036 (2;<br>115.5)</td><td>0.007</td></tr><tr><td>76</td><td>-</td><td>-</td><td>Difucosylla<br>cto-N-<br>neooctaos<br>e I<br>(DFLNnO I)</td><td>44.0</td><td>1849.71</td><td>923.9**</td><td>Ø</td><td>Ø</td><td>0.030 ±<br>0.012 (16;<br>38.9)</td><td>0.179 ±<br>0.025 (4;<br>14.1)</td><td>Ø</td></tr><tr><td>77</td><td>-</td><td>-</td><td>Trifucosyll<br>acto-N-<br>octaose<br>(TFLNO)<br>isomer</td><td>-</td><td>44.6</td><td>1996.77</td><td>997.4**</td><td>Ø</td><td>Ø</td><td>T</td><td>0.006 ±<br>0.002 (4;<br>25.8)</td><td>0.002</td></tr><tr><td>78</td><td>-</td><td>-</td><td>Trifucosyll<br>acto-N-<br>octaose<br>(TFLNO)<br>isomer</td><td>-</td><td>44.9</td><td>1996.77</td><td>997.4**</td><td>Ø</td><td>Ø</td><td>0.027 ±<br>0.008 (16;<br>31.1)</td><td>Ø</td><td>0.108</td></tr><tr><td>79</td><td>-</td><td>unknown<br>structure</td><td>Trifucosyll<br>acto-N-<br>octaose<br>(TFLNO)<br>isomer</td><td>-</td><td>45.2</td><td>1996.77</td><td>997.4**</td><td>Ø</td><td>Ø</td><td>0.016 ±<br>0.006 (16;<br>39.7)</td><td>Ø</td><td>0.058</td></tr><tr><td>80</td><td>-</td><td>unknown<br>structure</td><td>Trifucosyll<br>acto-N-<br>octaose<br>(TFLNO)<br>isomer</td><td>-</td><td>45.5</td><td>1996.77</td><td>997.4**</td><td>Ø</td><td>Ø</td><td>0.010 ±<br>0.005 (16;<br>47)</td><td>0.027 ±<br>0.014 (4;<br>50.1)</td><td>0.034</td></tr><tr><td>81</td><td>-</td><td>unknown<br>structure</td><td>Tetrafucos<br>yllacto-N-<br>octaose<br>(TeFLNO)<br>isomer</td><td>-</td><td>45.5</td><td>2142.83</td><td>1070.4*<br>*</td><td>Ø</td><td>Ø</td><td>Ø</td><td>T</td><td>T</td></tr><tr><td>82</td><td>-</td><td>unknown<br>structure</td><td>Trifucosyll<br>acto-N-<br>octaose<br>(TFLNO)<br>isomer</td><td>-</td><td>45.7</td><td>1996.77</td><td>997.4**</td><td>Ø</td><td>Ø</td><td>0.015 ±<br>0.008 (16;<br>57.5)</td><td>0.008 ±<br>0.002 (4;<br>24.0)</td><td>0.007</td></tr><tr><td>83</td><td>-</td><td>unknown<br>structure</td><td>Trifucosyll<br>acto-N-<br>octaose<br>(TFLNO)<br>isomer</td><td>-</td><td>45.9</td><td>1996.77</td><td>997.4**</td><td>Ø</td><td>Ø</td><td>0.030 ±<br>0.008 (16;<br>27.2)</td><td>Ø</td><td>0.011</td></tr><tr><td>84</td><td>-</td><td>unknown<br>structure</td><td>Trifucosyll<br>acto-N-<br>octaose<br>(TFLNO)<br>isomer</td><td>-</td><td>46.2</td><td>1996.77</td><td>997.4**</td><td>Ø</td><td>Ø</td><td>Ø</td><td>0.066 ±<br>0.009 (4;<br>14.0)</td><td>Ø</td></tr><tr><td>85</td><td>-</td><td>unknown<br>structure</td><td>Trifucosyll<br>acto-N-<br>octaose<br>(TFLNO)<br>isomer</td><td>-</td><td>46.3</td><td>1996.77</td><td>997.4**</td><td>Ø</td><td>Ø</td><td>0.035 ±<br>0.006 (16;<br>17.2)</td><td>Ø</td><td>0.016</td></tr></tbody></table>

(continued on next page)
<table><caption>Table 4 (continued)</caption><tr><td>86</td><td>-</td><td>unknown structure</td><td>Trifucosyll acto-N-octaose (TFLNO) isomer</td><td>-</td><td>46.7</td><td>1996.77</td><td>997.4**</td><td>Ø</td><td>Ø</td><td>0.005 ± 0.004 (13; 69.5)</td><td>0.079 ± 0.064 (3; 80.8)</td><td>Ø</td></tr><tr><td>87</td><td>-</td><td>unknown structure</td><td>Tetrafucos yllacto-N-octaose (TeFLNO) isomer</td><td>-</td><td>47.0</td><td>2142.83</td><td>1070.4* *</td><td>Ø</td><td>Ø</td><td>T</td><td>T</td><td>0.035</td></tr><tr><td>88</td><td>-</td><td>unknown structure</td><td>Tetrafucos yllacto-N-octaose (TeFLNO) isomer</td><td>-</td><td>47.2</td><td>2142.83</td><td>1070.4* *</td><td>Ø</td><td>Ø</td><td>0.012 ± 0.007 (13; 60.9)</td><td>0.006 ± 0.004 (4; 64.9)</td><td>0.048</td></tr><tr><td>89</td><td>-</td><td>unknown structure</td><td>Tetrafucos yllacto-N-octaose (TeFLNO) isomer</td><td>-</td><td>47.6</td><td>2142.83</td><td>1070.4* *</td><td>Ø</td><td>Ø</td><td>Ø</td><td>0.007 ± 0.008 (4; 109.8)</td><td>0.010</td></tr><tr><td>90</td><td>-</td><td>unknown structure</td><td>Tetrafucos yllacto-N-octaose (TeFLNO) isomer</td><td>-</td><td>47.8</td><td>2142.83</td><td>1070.4* *</td><td>Ø</td><td>Ø</td><td>0.020 ± 0.009 (15; 47.4)</td><td>0.006 ± 0.007 (2; 117.7)</td><td>Ø</td></tr><tr><td>91</td><td>-</td><td>unknown structure</td><td>Tetrafucos yllacto-N-octaose (TeFLNO) isomer</td><td>-</td><td>48.1</td><td>2142.83</td><td>1070.4* *</td><td>Ø</td><td>Ø</td><td>0.040 ± 0.008 (16; 20.5)</td><td>0.059 ± 0.005 (4; 8.9)</td><td>0.009</td></tr><tr><td>92</td><td>-</td><td>unknown structure</td><td>Tetrafucos yllacto-N-octaose (TeFLNO) isomer</td><td>-</td><td>48.5</td><td>2142.83</td><td>1070.4* *</td><td>Ø</td><td>Ø</td><td>0.009 ± 0.006 (16; 68.3)</td><td>0.011 ± 0.003 (4; 30.0)</td><td>0.005</td></tr><tr><td>93</td><td>-</td><td>unknown structure</td><td>Tetrafucos yllacto-N-octaose (TeFLNO) isomer</td><td>-</td><td>48.7</td><td>2142.83</td><td>1070.4* *</td><td>Ø</td><td>Ø</td><td>0.013 ± 0.005 (16; 38.1)</td><td>Ø</td><td>Ø</td></tr><tr><td>94</td><td>-</td><td>unknown structure</td><td>Pentafucos yllacto-N-octaose (PFLNO) isomer</td><td>-</td><td>48.7</td><td>2289.0</td><td>1143.5* *</td><td>Ø</td><td>Ø</td><td>Ø</td><td>T</td><td>0.026</td></tr><tr><td>95</td><td>-</td><td>unknown structure</td><td>Pentafucos yllacto-N-octaose (PFLNO) isomer</td><td>-</td><td>49.3</td><td>2289.0</td><td>1143.5* *</td><td>Ø</td><td>Ø</td><td>0.008 ± 0.004 (16; 50.9)</td><td>Ø</td><td>0.005</td></tr><tr><td>96</td><td>-</td><td>unknown structure</td><td>Pentafucos yllacto-N-octaose (PFLNO) isomer</td><td>-</td><td>49.7</td><td>2289.0</td><td>1143.5* *</td><td>Ø</td><td>Ø</td><td>0.012 ± 0.006 (16; 45.2)</td><td>0.008 ± 0.001 (4; 16.8)</td><td>0.008</td></tr><tr><td>97</td><td>-</td><td>unknown structure</td><td>Pentafucos yllacto-N-octaose (PFLNO) isomer</td><td>-</td><td>50.0</td><td>2289.0</td><td>1143.5* *</td><td>Ø</td><td>Ø</td><td>0.005 ± 0.003 (16; 51.9)</td><td>Ø</td><td>Ø</td></tr></table>

(continued on next page)
<table><caption>Table 4 (continued)</caption><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>98</td><td>-</td><td>unknown structure</td><td>Pentafucos yllacto-N-octaose (PFLNO) isomer</td><td>-</td><td>50.2</td><td>2289,0</td><td>1143.5*<br/>*</td><td>Ø</td><td>Ø</td><td>Ø</td><td>0.023 ±<br/>0.004 (4;<br/>19.3)</td><td>Ø</td></tr><tr><td>99</td><td>-</td><td>unknown structure</td><td>Pentafucos yllacto-N-octaose (PFLNO) isomer</td><td>-</td><td>50.7</td><td>2289,0</td><td>1143.5*<br/>*</td><td>Ø</td><td>Ø</td><td>0.014 ±<br/>0.006 (16;<br/>43.6)</td><td>0.005 ±<br/>0.002 (4;<br/>39.7)</td><td>Ø</td></tr><tr><td></td><td></td><td></td><td>Total MOs</td><td></td><td></td><td></td><td></td><td>0.848 / 2.687<br/>± 0.327</td><td>0.336 ±<br/>0.042</td><td>10.242 ±<br/>0.893 (16;<br/>8.7)</td><td>9.066 ±<br/>0.664 (4;<br/>7.3)</td><td>11.302</td></tr></tbody></table>

para-LNnH. Consequently, the oligosaccharides eluting at 30 and 30.3 min were proposed to be para-LNH and para-LNnH, respectively (nos. 36 and 37, Table 4).

Four hexasaccharide isomers with an *m/z* of 631.2 Da co-eluted at respectively 31 min and 32.3 min (nos. 39 & 42). Four structures have been described in the literature: S-LNFP II, F-LSTc, A3111a and A3111b (Remoroza et al., 2018). Since SLNFP II is absent from serologic group 3 (Le-), it can only correspond to no. 39 (Table 4). In agreement with the assignments and similar co-elutions determined by Remoroza et al. (2018), the structure of the remaining three isomers have been proposed (see Table 4, nos. 39 and 42). The two last hexasaccharides detected (nos. 45 & 46) with an *m/z* of 1279.5 and 703.8 Da correspond, based on their mass, to 6-SLNnP I and DSLNT, previously identified in bovine milk (Albrecht et al., 2014; Marìno et al., 2011) and human milk (Huang et al., 2019; Zhang et al., 2019; Samuel et al., 2019; Remoroza et al., 2018; van Leeuwen, 2019). DSNLT was also unambiguously identified using a commercial oligosaccharide and its detected *m/z*.

Eight heptasaccharides from which six isomeric heptasaccharides at *m/z* 1337.5 (nos. 43–44, 47–50 and 53, Table 4) were also detected. Their molar mass suggests that they belong to the monofucosyllacto-N-hexaose (MFLNH) family. According to the literature, their known abundance and serologic groups (Remoroza et al., 2018; van Leeuwen, 2019; Samuel et al., 2019), five of these six isomers have been identified and separated by HILIC, allowing us to attribute nos. 43, 44, 47, 48 and 49. The sixth unknown isomer with an *m/z* of 1337.5 co-eluted with another heptasaccharide with an *m/z* of 740.8 Da (no. 50). A last heptasaccharide eluted at 37.6 min and detected with an *m/z* of 740.8 Da (no. 53). Two possible structures were described in literature (Albrecht et al., 2014; Remoroza et al., 2018; Urashima et al., 2018): S-LNH and 6-SLNnH/S-LNnH II. Of the two, only 6-SLNnH was identified in both porcine milk (6-SLNnH) and human milk (under the designation S-LNnH II). This oligosaccharide has therefore been assigned to no. 53 (Table 4), with S-LNH assigned to no. 50 (Table 4).

Eight octasaccharides (nos. 51–52, 54–56, 59, 61 and 69, Table 4) were detected and correspond to difucosylated neutral oligosaccharides (nos. 51–52, 54–56, Table 4), fucosylated sialylated oligosaccharides (59 and 61) and disialylated oligosaccharides (no. 69). Among the five difucosylated neutral oligosaccharides, no. 55 was unambiguously assigned using a commercial oligosaccharide DFpLNH II. As expected, this oligosaccharide was not detected in the serologic group 3. Two other isomers were described in literature (Remoroza et al., 2018; Tonon et al., 2019; van Leeuwen, 2019). Since DFLNH III is not synthesized in serologic groups 2 (Se-) and 3 (Le-), it was assigned to no. 56. Since DFLNH I is not synthesized in serologic group 2 (Se-), it was assigned to no. 52. DFLNnH and DFpLNnH were assigned to be respectively nos. 51

and 54 (Table 4). Two octasaccharide isomers, no. 59 and 61 (Table 4), were attributed according to Remoroza et al., 2018. The latter octasaccharide no. 69 was unambiguously attributed to DSLNH according to its molar mass and literature (Remoroza et al., 2018).

Eleven nonasaccharides were detected (nos. 57–58, 60, 62–68 and 72, Table 4), one of which (no. 66) is co-eluted with a decasaccharide. Nine structures were described in Remoroza et al., 2018. Using possible structures based on serologic groups in women and the elution order described in Remoroza et al. (2018), structures nos. 57 and 62 (TFLNnH and TFpLNH, respectively), nos. 58, 60, and 64 (5130c, FLNO, and FLNnO, respectively), nos. 66 and 67 (DS FLNH II and A4122a respectively), and nos. 68 and 72 (A4221a/b and MSDFLNH, respectively) were attributed.

Eight decasaccharides were detected (nos. 66, 70–71, 73–76, Table 4), including one that coeluted (no. 66) with a nonasaccharide and two distinct decasaccharides that co-eluted (no. 74). According to Remoroza et al. (2018), six decasaccharides were structurally described. Five isomers with an *m/z* of 923.9 Da were assigned according to literature (Remoroza et al., 2018) and/or serologic groups (nos. 66, 70, 71, 75 and 76). The decasaccharide at *m/z* 960.4 (no. 74) was assigned to LND or its isomer LNnD (Table 4).

Finally, 23 oligosaccharides with a degree of polymerization ranging from 11 to 14 were detected, including tri, tetra and penta-fucosyllacto-N-octaose isomers, of which tri- and tetra-fucosyllacto-N-octaose structures were described in Urashima et al. (2018). However, it was not possible to propose a structure without further structural characterization.

Despite the identification of more than 150 different HMO structures in the literature (Urashima et al., 2018), only 18 HMOs represent the major oligosaccharides over the first 4 months of lactation (for review, Boudry et al., 2021). Our method allowed the detection and/or identification of 90 HMOs of which 82 in the Lewis-positive Secretor group 1 (Se + Le+), 66 in the Lewis-positive non-Secretor group 2 (Se-Le+) and 72 in the Lewis-negative Secretor group 3 (Se + Le-). Most of these HMOs were previously described and some have been quantified (Bao et al., 2013; Nijman et al., 2018; Huang et al., 2019; Remoroza et al., 2018 van Leeuwen et al., 2018; Tonon et al., 2019; Samuel et al., 2019; Zhang et al., 2019; Gu et al., 2021; Tan et al., 2022; Li et al., 2022). Additional unknown isomers of HMOs were detected and quantified in our study (Table 4). However, as mentioned above, further studies were needed in order to identify their fine structure.

In animal milks, 20 MOs were identified from porcine (both mature milk and colostrum), and 10 from bovine milk, as previously reported (Albrecht et al., 2014; Difilippo et al., 2016; Mudd et al., 2016; Marìno et al., 2011; Fong et al., 2011; Liu et al., 2014; Yang et al., 2022). Only
six MOs were shared by human, bovine and porcine milks: one neutral fucosylated MO, 3-FL, three neutral MOs, LNNt, 3'-GOS and 6'-GOS, and two sialylated MOs, 3'-SL and 6'-SL. Interestingly, eight additional MOs were shared between porcine and human milks including two neutral fucosylated MOs, 2'-FL and LNFP III, three neutral MOs, LNT, novoLNP I and LNNH, and three sialylated MO, 6'-SLN, 6'-SLNt (also named LST c) and 6'-SLNNH. No other oligosaccharides were shared between bovine and human milks. On the other hand, only one oligosaccharide (no. 4, Table 4) was unique to bovine milk, while six were unique to porcine milk/colostrum and seventy-nine were exclusive to human milk.

Thus, by integrating MS analyses, retention times, standard oligo-
saccharides, and relevant literature, our approach allowed us to suc-
cessfully detect and/or identify 99 distinct oligosaccharides from
human, bovine and porcine milk samples analyzed with certainty
(Table 4). Additionally, our study provides an opportunity to compare
the nomenclature used in studies involving human and animal milks for
naming oligosaccharides. Notably, it is not uncommon for the same
oligosaccharide to be assigned different names in separate studies or
between investigations focused on human milk or animal milks. For
example, the oligosaccharide Gal(β1-3)Gal(β1-4)Glc was codified N7
(animal milks, Albrecht et al., 2014) and named 3'-GOS (human milk,
Huang et al., 2019), β3'-GL (porcine milk, Difillippo et al., 2016) and
3'GalL (human milk, Remoroza et al., 2018). Similarly, the isomers of
HMOs with a molar mass of 1338.5 Da (2-AB coupled oligosaccharides)
are referred to as MFLNH, FLNH, or IFLNH (Remoroza et al., 2018,
Samuel et al., 2019, van Leeuwen et al., 2018). To clarity oligosaccha-
ride nomenclature, we have consolidated all the information men-
tioned above into a comprehensive table (Table 4).

3.4. Method validation

Quantification methods typically use standards for each individual
oligosaccharide. However, due to the strong structural variability of
MOs, standards for each individual MO are not readily available, and
those available are expensive. It is therefore necessary to develop a
method where the signal is not affected by the composition and/or
chemical structure of each oligosaccharide. Reductive amination with
fluorescent molecules offers a solution to this problem by allowing
molar quantification using an external calibration curve (Ruhaak et al.,
2010; Austin and Benet, 2018).

In our method, Lacto-*N*-neotetraose (LNnT), a commercially avail-
able HMO was used as an external standard, while xylohexaose (X6) and
mannotriose (M3) were selected as IS for animal and human milks,
respectively.

The linearity of the calibration curve using purchased LNNt was
validated for four purchased MOs (3'-GOS, 6'-GOS, 3'-SL, 6'-SL) and
three other purchased non-MOs (M3, X6 and GGM5) from 240 pg to
1.2 µg (Table 5).

The method also demonstrated high reproducibility and repeat-
ability (Table 6). For example, the precision range (CV) in reproduc-
ibility tests varied from 5.9 % to 17.3 % at low concentration levels
below 0.1 g/L, and from 6.0 % to 15.7 % at high concentration level
above 0.1 g/L. The standard deviation ranged from 4 mg/L to 8 mg/L at
concentration below 0.1 g/L and from 21 mg/L to 149 mg/L at con-
centration above 0.1 g/L (Table 6).

3.5. *Oligosaccharides quantification from human and animals milks*

Our method allowed the quantification of 20 (17 in porcine mature
milk and 18 in porcine colostrum), 10 (in bovine mature milk) and 80
MOs (68 in human milk group 1 (Se + Le+), 56 in human milk group 2
(Se-Le+) and 60 in human milk group 3 (Se + Le-)) in porcine, bovine
and human milks, respectively (Table 4). The total concentration of MOs
in animal milks was, as expected, lower (ranging from 0.336 g/L in
bovine milk to 0.848 g/L in porcine milk) compared to human milk (Se
+ Le+: 10.35 g/L; Se-Le+: 9.21 g/L; Se+Le-: 11.27 g/L).

Our findings revealed that in human milks, 21 MOs were detected at concentrations exceeding 100 mg/L across all serologic groups. Specifically, in Group 1 (Se+Le+), four HMOs (2'-FL, LNT, LNFP I, and LNDFH I) surpassed the 1 g/L threshold, while in Group 2 (LNT, 3-FL, and LNFP II) and in Group 3 (2'-FL, LNFP I, and LNT) three oligosaccharides in each group exceeded this threshold. Furthermore, within Group 1, fifteen HMOs were quantified at concentration ranging from 100 to 600 mg/L, including 3-FL, MFLNH III, LNFP II, 6'-SL, and DFLNH a. In Group 2, ten HMOs were quantified at concentration ranging from 100 to 600 mg/L, such as DFpLNH, MFLNH III, LNDFH II, LNFP III, and 6'-SL. In Group 3, the ten quantified HMOs ranging from 100 to 600 mg/L include MFLNH III, 6'-SL, LNFP III, FS-LNH I, and LNNt. All remaining HMOs exhibited concentration ranging from 1 to 94 mg/L.

Our findings reveal that in porcine colostrum and porcine milk at 21
days of lactation, sialylated oligosaccharides dominated, constituting
66 % and 58 % of total porcine MOs (PMOs), respectively. Among these
matrices, 3'-SL alone accounted for a significant portion, ranging from
54 % to 34 %. In contrast, our analysis of the bovine milk panel con-
tradicted existing literature, as sialylated oligosaccharides comprised
only 41 % of total bovine MOs (BMOs). Within bovine milk, 3'-SL alone
contributed to 22 % of the total, and the GNL oligosaccharide repre-
sented 19 % of BMOs in our study.

**4. Discussion and conclusions**

As previously emphasized by Huang et al. (2019), the establishment
of a simple and high-throughput sample preparation method is essential
to ensure accurate and reliable quantification. In the present study, a
rapid and straightforward approach to extract, identify, and quantify
MOs in diverse milk samples was successfully adapted and implemented.

One of our primary objectives was to validate a quantification
method using small sample volumes, specifically in the context of pre-
clinical or clinical trials where milk volumes from mice or human is
limited. Previous studies have used sample volumes ranging from 50 µL
to 2 mL. In our method, the use of 50 µL of milk was validated for all
milks tested. Furthermore, as demonstrated by Huang et al. (2019) in
their quantification of 19 HMOs, our method was tested with porcine
milk and successfully applied with sample volumes as low as 10 µL (data
not shown). While further studies are needed to confirm its applicability
to other types of milk, this method shows promise when sample volumes
are limited.

Various methods for MOs extraction have been employed in the
literature, including solvent-based approaches utilizing chloroform/
methanol or acetonitrile, as well as solvent-free methods. Notably, Liu
et al. (2014) demonstrated that ultrafiltration was more effective in
extracting MOs compared to solvent-based methods. Considering the
potential hazards to human health and the environment associated with
solvent use, the exploration of MO extraction without solvents was
favored. However, the optimal extraction protocol for each milk sample
to achieve maximum MO recovery remains a question. In our study, the
experiments were conducted to determine the minimal extraction steps
required for optimal MO recovery in each milk sample. A ten-fold
dilution with water, followed by centrifugation to collect the superna-
tant, an additional pellet-washing step to ensure complete MO recovery,
and ultimately an ultrafiltration process employing a 10-kDa
molecular-weight cut-off membrane, yielded satisfactory results. This
optimized protocol allowed us to extract MOs effectively while mini-
mizing the use of hazardous solvents, promoting both human and
environmental safety.

Furthermore, the method sensitivity using fluorescent labelling with
2-AB, a cost-effective and stable chromophore, was confirmed with the
detection and/or identification of up to 82 distinct MOs in a single run
directly from the total oligosaccharide extract, without the need for a
preliminary fractionation step previously used to separate neutral and
sialylated MOs (Mudd et al., 2016; Difilippo et al., 2016; Wei et al.,
2018; Remoroza et al., 2018).
Hydrophilic interaction chromatography (HILIC), well-known for its ability to provide excellent reproducibility and efficient separation of isomers, is frequently used for the analysis of large sample collections after oligosaccharide extraction (For review, van Leeuwen, 2019). However, the gradient used is the key to perform the correct separation of isomers. By comparison with Li et al. (2022), which offered the advantage of fast analysis time for human milk, but resulted in poor separation of isomers belonging to the LNFP, LNDFH, and MFLNH families, the longer analysis time of our method allowed for the detection and quantification of a greater number of isomers. Thus, while our HILIC method produced minimal co-elutions of minor oligosaccharides (e.g., SLNFP II and F-LSTc in human milk), it offered enhanced capabilities for characterizing a broader range of MOs and isomers, which can be valuable for comprehensive analyses.

Regarding animal milks, our results provide new insights into the oligosaccharide composition of porcine colostrum, porcine milk and bovine milk. In porcine colostrum and milk, sialylated oligosaccharides were found to be the predominant type, corresponding to 66% and 58% of total PMOs, respectively. Notably, the oligosaccharide 3'-SL stood out as the most abundant component, ranging from 54% to 34% of the total PMO content.

Furthermore, our method allowed to identify and quantify 10 BMOs, a notable improvement over previous study, which quantified only five to six BMOs (Fong et al., 2011; Yang et al., 2022). Our quantification results were fairly close to what Yang et al. (2022) found. However, while Fong et al. (2011) determined the concentration of GNL in bovine colostrum and mature milk, the concentration was much smaller than that in this study. In addition, Yang et al. (2022) did not find GNL, which is, with 3'-SL, the main BMO in our study, while their chromatogram displayed a large unidentified peak that could potentially correspond to GNL, suggesting the presence of this oligosaccharide in significant quantities. This observation aligns with our results, providing additional confirmation for the presence of GNL in bovine milk. Thus, our analysis of bovine milk contradicted the prevailing belief that sialylated oligosaccharides were the primary constituents, as they accounted for only 41% of the total BMOs. Among this group, 3'-SL accounted for 22% of the total, which was notably lower compared to the porcine milk samples. This disparity suggests variations in the oligosaccharide profiles between porcine and bovine milks, indicating species-specific differences in their composition. Of particular interest is the GNL oligosaccharide, which represented 19% of BMOs in our study.

Overall, the analysis of MOs remains a complex and challenging task, but advances in analytical techniques have enabled researchers to gain a better understanding of the composition, structure, and biological activities of these important milk compounds.

The application of the method to other animal milks is of considerable interest for advancing our understanding of the evolution of MOs (Urashima et al., 2022) and their implications for human health and nutrition. This approach offers the possibility to explore and understand the specific composition of oligosaccharides in milks from other animal species, to study the impact of the environment and diet on the composition of these milks as well as their effects on human nutrition (Boudry et al., 2021; Le Bourgot et al., 2024). One limitation for future studies is the potential for variability in sample collection and handling

<caption>**Table 5**</caption>

Linearity of the calibration curve of four milk oligosaccharides and 3 standard oligosaccharides using LNnT.

<table><caption>**Table 6**</caption><thead><tr><th>OS</th><th>MW-2AB (Da)</th><th>Linear range (ng)</th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td>Gal(β1-3)Gal(β1-4)Glc (3'-GOS)</td><td>624.24</td><td>0.24-1200</td><td>0.9983</td></tr><tr><td>6'-GOS or β6'-GL (N9)</td><td>624.24</td><td>0.24-1200</td><td>0.9991</td></tr><tr><td>M3</td><td>624.24</td><td>0.24-1200</td><td>0.9992</td></tr><tr><td>3'-SL (3'-sialyllactose)</td><td>753.28</td><td>0.24-1200</td><td>0.9988</td></tr><tr><td>6'-SL (6'-sialyllactose)</td><td>753.28</td><td>0.24-1200</td><td>0.9987</td></tr><tr><td>X6</td><td>930.33</td><td>0.24-1200</td><td>0.9987</td></tr><tr><td>GGM5</td><td>1272.45</td><td>0.24-1200</td><td>0.9969</td></tr></tbody></table>

Method reproducibility and repeatability assessed by the concentration of 4 different oligosaccharides of 3 matrices, their SD and CV.

<table><thead><tr><th rowspan="2">Matrix</th><th rowspan="2">Compound Name</th><th colspan="2">Reproducibility (n = 10)</th><th colspan="2">Repeatability (n = 6-15)</th></tr><tr><th>Mean ± SD (g/L)</th><th>CV</th><th>Mean ± SD (g/L)</th><th>CV</th></tr></thead><tbody><tr><td rowspan="4">Human milk</td><td>2'-FL</td><td>2.487 ± 0.149</td><td>6.0</td><td>2.358 ± 0.035</td><td>1.5</td></tr><tr><td>LDFT</td><td>0.331 ± 0.029</td><td>8.9</td><td>0.308 ± 0.003</td><td>1.1</td></tr><tr><td>LNnT</td><td>0.174 ± 0.027</td><td>15.7</td><td>0.149 ± 0.002</td><td>1.6</td></tr><tr><td>DFLNH a</td><td>0.037 ± 0.004</td><td>10.3</td><td>0.034 ± 0.002</td><td>5.0</td></tr><tr><td rowspan="4">Bovine milk</td><td>GNL</td><td>0.066 ± 0.008</td><td>12.4</td><td>0.067 ± 0.004</td><td>5.7</td></tr><tr><td>3'-SL</td><td>0.076 ± 0.006</td><td>8.3</td><td>0.074 ± 0.004</td><td>5.3</td></tr><tr><td>6'-SL</td><td>0.029 ± 0.004</td><td>14.0</td><td>0.031 ± 0.002</td><td>5.2</td></tr><tr><td>LNnT</td><td>0.025 ± 0.004</td><td>17.3</td><td>0.028 ± 0.002</td><td>5.5</td></tr><tr><td rowspan="4">Porcine colostrum</td><td>3'-GOS</td><td>0.303 ± 0.021</td><td>6.9</td><td>0.284 ± 0.021</td><td>7.3</td></tr><tr><td>6'-SL</td><td>0.097 ± 0.008</td><td>8.6</td><td>0.092 ± 0.006</td><td>6.9</td></tr><tr><td>6'-SLNT</td><td>0.044 ± 0.005</td><td>11.2</td><td>0.045 ± 0.006</td><td>13.6</td></tr><tr><td>LNnH</td><td>0.061 ± 0.004</td><td>5.9</td><td>0.057 ± 0.008</td><td>13.3</td></tr></tbody></table>

prior to analysis, which may influence MO composition. Such variability remains an important consideration for future large-scale or cross-study comparisons. To conclude, we acknowledge the importance of analytical standardization, particularly to ensure reproducibility and comparability across laboratories and studies. Given the growing interest in milk oligosaccharide research, it would be highly relevant to conduct a more comprehensive validation study aimed at standardizing the method developed here, especially in the context of biological matrices.

A similar effort was recently undertaken by Bénet et al. (2024), who proposed a standardized approach for the quantification of 7 HMOs in infant formula products, demonstrating that harmonized methods can significantly improve data robustness and inter-study consistency. Following that example, a future objective would be to expand the validation of our method — which already allows for sensitive, high-throughput analysis of a wide range of MOs from small milk volumes — across multiple laboratories and sample types. Such standardization efforts would support broader applications of this method, particularly in clinical and nutritional research involving both human and animal milks.

### CRediT authorship contribution statement

Sophie Le Gall: Writing – review & editing, Writing – original draft, Supervision, Resources, Methodology, Investigation, Funding acquisition, Conceptualization. Yoann Richer: Formal analysis. Loric ThouLouze: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Isabelle Le Huérou-Luron: Writing – review & editing, Resources. Eléonore Lepvrier: Resources.

### Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
## Acknowledgements

This work was supported by INRAE (Human Nutrition Department) and SODIAAL INTERNATIONAL. We gratefully acknowledge the milk bank of the Rennes University Hospital Centre, the Rennes pig experimental facilities (UE3P, INRAE, F-35590 Saint-Gilles, France), and the dairy cooperative SODIAAL INTERNATIONAL (F-35000 Rennes, France) for respectively providing human, porcine and bovine milks.

All analyses were performed on the BIBS instrumental platform (http://www.bibs.inrae.fr/bibs_eng/, UR1268 BIA, IBiSA, Phenome-Emphasis-FR ANR-11-INBS-0012, PROBE infrastructure, CALIS infrastructure, Biogenouest).

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jfca.2025.108074.

## Data availability

Data will be made available on request.

## References

Albrecht, S., Lane, J.A., Marino, K., Al Busadah, K.A., Carrington, S.D., Hickey, R.M., Rudd, P.M., 2014. A comparative study of free oligosaccharides in the milk of domestic animals. Br. J. Nutr. 111 (7), 1313-1328. https://doi.org/10.1017/S0007114513003772.

Ashline, D.J., Yu, Y., Lasanajak, Y., Song, X., Hu, L., Ramani, S., Prasad, V., Estes, M.K., Cummings, R.D., Smith, D.F., Reinhold, V.N., 2014. Structural characterization by multistage mass spectrometry (MSn) of human milk glycans recognized by human rotaviruses. Mol. Cell. Proteom. 13 (11), 2961-2974. https://doi.org/10.1074/mcp/M114.039925.

Austin, S., Bénet, T., 2018. Quantitative determination of non-lactose milk oligosaccharides. Anal. Chim. Acta 1010, 86-96. https://doi.org/10.1016/j.aca.2017.12.036.

Bao, Y., Chen, C., Newburg, D.S., 2013. Quantification of neutral human milk oligosaccharides by graphitic carbon high-performance liquid chromatography with tandem mass spectrometry. Anal. Biochem. 433 (1), 28-35. https://doi.org/10.1016/j.ab.2012.10.003.

Bao, Y., Zhu, L., Newburg, D.S., 2007. Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis. Anal. Biochem. 370 (2), 206-214. https://doi.org/10.1016/j.ab.2007.07.004.

Bénet, T., Frei, N., Spichtig, V., Cuany, D., Austin, S., 2024. Determination of seven human milk oligosaccharides (HMOs) in infant formula and adult nutritionals: first action 2022.07. J. AOAC Int. 107 (2), 286-302. https://doi.org/10.1093/jaoacint/qsa001.

Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., Parekh, R.B., 1995. Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal. Biochem. 230 (2), 229-238. https://doi.org/10.1006/abio.1995.1468.

Boudry, G., Charton, E., Le Huérou-Luron, I., Ferret-Bernard, S., Le Gall, S., Even, S., Blat, S., 2021. The relationship between breast milk components and the infant gut microbiota. Front. Nutr. 8, 629740. https://doi.org/10.3389/fnut.2021.629740.

Charton, G., Bourgeois, A., Bellanger, A., Le-Gouar, Y., Dahirel, P., Romé, V., Randuineau, G., Cahu, A., Moughan, P.J., Montoya, C.A., Blat, S., Dupont, D., Deglaire, A., Le Huérou-Luron, I., 2022. Infant nutrition affects the microbiota-gut-brain axis: comparison of human milk vs. infant formula feeding in the piglet model. Front. Nutr. 9, 976042. https://doi.org/10.3389/fnut.2022.976042.

Davis, J.C., Lewis, Z.T., Krishnan, S., Bernstein, R.M., Moore, S.E., Prentice, A.M., Mills, D.A., Lebrilla, C.B., Zivkovic, A.M., 2017. Growth and morbidity of Gambian infants are influenced by maternal milk oligosaccharides and infant gut microbiota. Sci. Rep. 7, 40466. https://doi.org/10.1038/srep40466.

Difilippo, E., Pan, F., Logtenberg, M., Willem, R.H., Braber, S., Fink-Gremmels, J., Schols, H.A., Gruppen, H., 2016. Milk oligosaccharide variation in sow milk and milk oligosaccharide fermentation in piglet intestine. J. Agric. Food Chem. 64 (10), 2087-2093. https://doi.org/10.1021/acs.jafc.6b00497.

Fong, B., Ma, K., McJarrow, P., 2011. Quantification of bovine milk oligosaccharides using liquid chromatography-selected reaction monitoring-mass spectrometry. J. Agric. Food Chem. 59 (18), 9788-9795. https://doi.org/10.1021/jf202035m.

Gallo, V., Arienzo, A., Tomassetti, F., Antonini, G., 2024. Milk bioactive compounds and gut microbiota modulation: the role of y-gou proteins and milk oligosaccharides. Foods 13 (6), 907. https://doi.org/10.3390/foods13060907.

Gu, F., Kate, G.A.T., Arts, I.C.W., Penders, J., Thijs, C., Lindner, C., Nauta, A., van Leusen, E., van Leeuwen, S.S., Schols, H.A., 2021. Combining HPAEC-PAD, PGC-LC-MS, and 1D. J. Agric. Food Chem. 69 (23), 6495-6509. https://doi.org/10.1021/acs.jafc.0c07446.

R. Hickey T. Urashima (2021) Indigenous oligosaccharides in bovine milk. Encyclopedia of Dairy Science the third edition 706-722 doi: 10.1016/B978-0-12-374407-4.00 279-X.

Hobbs, M., Jahan, M., Ghorashi, S.A., Wang, B., 2021. Current perspective of sialylated milk oligosaccharides in mammalian milk: implications for brain and gut health of newborns. Foods 10 (2), 473. https://doi.org/10.3390/foods10020473.

Hou, H., Wang, M., Yang, S., Yang, X., Sun, W., Sun, X., Guo, Q., Debrah, A.A., Zhenxia, D., 2024. Evaluation of prebiotic glycan composition in human milk and infant formula: profile of galacto-oligosaccharides and absolute quantification of major milk oligosaccharides by UPLC-cyclic IM-MS and UPLC-MS/MS. J. Agric. Food Chem. 72 (14), 7980-7990.

Huang, X., Zhu, B., Jiang, T., Yang, C., Qiao, W., Hou, J., Han, Y., Xiao, H., Chen, L., 2019. Improved simple sample pretreatment method for quantitation of major human milk oligosaccharides using ultrahigh pressure liquid chromatography with fluorescence detection. J. Agric. Food Chem. 67 (44), 12237-12244. https://doi.org/10.1021/acs.jafc.9b03445.

Le Bourgot, C., Lollier, V., Richer, Y., Thoulouze, L., Svilar, L., Le Gall, S., Blat, S., Le Huérou-Luron, L., 2024. Maternal short chain fructo-oligosaccharides supplementation during late gestation and lactation influences milk components and offspring gut metabolome: a pilot study. Sci. Rep. 14 (1), 4236. https://doi.org/10.1038/s41598-024-54813-3.

Li, J., Bi, Y., Zheng, Y., Cao, C., Yu, L., Yang, Z., Chai, W., Yan, J., Lai, J., Liang, X., 2022. Development of high-throughput UPLC-MS/MS using multiple reaction monitoring for quantitation of complex human milk oligosaccharides and application to large population survey of secretor status and Lewis blood group. Food Chem. 397, 133750. https://doi.org/10.1016/j.foodchem.2022.133750.

Liu, Z., Moate, P., Pocks, B., Rochfort, S., 2014. Simple liquid chromatography-mass spectrometry method for quantification of major free oligosaccharides in bovine milk. J. Agric. Food Chem. 62 (47), 11568-11574. https://doi.org/10.1021/jf5037849.

Ma, L., McJarrow, P., Mohamed, H.J.B.J., Liu, X., Welman, A., Fong, B.Y., 2018. Lactational changes in the human milk oligosaccharide concentration in Chinese and Malaysian mothers' milk. Int. Dairy J. 87, 1-10. https://doi.org/10.1016/j.idairyj.2018.07.015.

Mariño, K., Lane, J.A., Abrahams, J.L., Struwe, W.B., Harvey, D.J., Marotta, M., Hickey, R.M., Rudd, P.M., 2011. Method for milk oligosaccharide profiling by 2-aminobenzamide labeling and hydrophilic interaction chromatography. Glycobiology 21 (10), 1317-1330. https://doi.org/10.1093/glycob/cwr067.

Mudd, A.T., Salcedo, J., Alexander, L.S., Johnson, S.K., Getty, C.M., Chichlowski, M., Berg, B.M., Barile, D., Dilger, R.N., 2016. Porcine milk oligosaccharides and sialic acid concentrations vary throughout lactation. Front. Nutr. 3, 39. https://doi.org/10.3389/fnut.2016.00039.

Nakamura, T., Kawase, H., Kimura, K., Watanabe, Y., Ohtani, M., Arai, I., Urashima, T., 2003. Concentrations of sialyloligosaccharides in bovine colostrum and milk during the prepartum and early lactation. J. Dairy Sci. 86 (4), 1315-1320. https://doi.org/10.3168/jds.S0022-0302(03)73715-1.

Nijman, R.M., Liu, Y., Bunyatratchata, A., Smilowitz, J.T., Stahl, B., Barile, D., 2018. Characterization and quantification of oligosaccharides in human milk and infant formula. J. Agric. Food Chem. 66 (26), 6851-6859. https://doi.org/10.1021/acs.jafc.8b01515.

Ninonuevo, M.R., Park, Y., Yin, H., Zhang, J., Ward, R.E., Clowers, B.H., German, J.B., Freeman, S.L., Killeen, K., Grimm, R., Lebrilla, C.B., 2006. A strategy for annotating the human milk glycome. J. Agric. Food Chem. 54 (20), 7471-7480. https://doi.org/10.1021/jf0615810.

Oursel, S., Cholet, S., Junot, C., Fenaille, F., 2017. Comparative analysis of native and permethylated human milk oligosaccharides by liquid chromatography coupled to high resolution mass spectrometry. J. Chromatogr. B 1071, 49-57. https://doi.org/10.1016/j.jchromb.2017.03.028.

Porfirio, S., Archer-Hartmann, S., Moreau, G.B., Ramakrishnan, G., Haque, R., Kirkpatrick, B.D., Azadi, P., 2020. New strategies for profiling and characterization of human milk oligosaccharides. Glycobiology 30 (10), 774-786. https://doi.org/10.1093/glycob/cwaa028.

Remorozoa, C.A., Mak, T.D., De Leoz, M.L.A., Mirokhin, Y.A., Stein, S.E., 2018. Creating a mass spectral reference library for oligosaccharides in human milk. Anal. Chem. 90 (15), 8977-8988. https://doi.org/10.1021/acs.analchem.8b01176.

Remorozoa, C.A., Liang, Y., Mak, T.D., Mirokhin, Y., Sheetlin, S.L., Yang, X., San Andres, J.V., Power, M.L., Stein, S.E., 2020. Increasing the coverage of a mass spectral library of milk oligosaccharides using a hybrid-search-based bootstrapping method and milks from a wide variety of mammals. Anal. Chem. 92 (15), 10316-10326. https://doi.org/10.1021/acs.analchem.0c00342.

Rousseau, A., Brosseau, C., Le Gall, S., Piloquet, H., Barbarot, S., Bodinier, M., 2021. Human milk oligosaccharides: their effects on the host and their potential as therapeutic agents. Front. Immunol. 12, 680911. https://doi.org/10.3389/fimmu.2021.680911.

Ruhaak, L.R., Zauner, G., Huhn, C., Bruggink, C., Deelder, A.M., Wuhrer, M., 2010. Glycan labeling strategies and their use in identification and quantification. Anal. Bioanal. Chem. 397 (8), 3457-3481. https://doi.org/10.1007/s00216-010-3532-z.

Saito, T., Itoh, T., Adachi, S., 1987. Chemical structure of three neutral trisaccharides isolated in free form from bovine colostrum. Carbohydr. Res. 165 (1), 43-51. https://doi.org/10.1016/0008-6215(87)80076-9.

Samuel, T.M., Binia, A., de Castro, C.A., Thakkar, S.K., Billeaud, C., Agosti, M., Al-Jashi, I., Costeira, M.J., Marchini, G., Martinez-Costa, C., Picaud, J.C., Stiris, T., Stoicescu, S.M., Vanpéé, M., Domellöf, M., Austin, S., Sprenger, N., 2019. Impact of maternal characteristics on human milk oligosaccharide composition over the first 4 months of lactation in a cohort of healthy European mothers. Sci. Rep. 9 (1), 11767. https://doi.org/10.1038/s41598-019-48337-4.
Sakanaka, M., Gotoh, A., Yoshida, K., Odamaki, T., Koguchi, H., Xiao, J.Z., Kitaoka, M., Katayama, T., 2019. Varied pathways of infant gut-associated Bifidobacterium to assimilate human milk oligosaccharides: prevalence of the gene set and its correlation with Bifidobacteria-rich microbiota formation. *Nutrients* 12 (1), 71. https://doi.org/10.3390/nu12010071.

Sarkozy, D., Borza, B., Domokos, A., Varadi, E., Szigeti, M., Meszaros-Matwiejuk, A., Molnar-Gabor, D., Guttman, A., 2021. Ultrafast high-resolution analysis of human milk oligosaccharides by multicapillary gel electrophoresis. *Food Chem.* 341 (Pt 2), 128200. https://doi.org/10.1016/j.foodchem.2020.128200.

Soyyilmaz, B., Mikš, M.H., Röhrig, C.H., Matwiejuk, M., Meszaros-Matwiejuk, A., Vigsnaes, L.K., 2021. The mean of milk: a review of human milk oligosaccharide concentrations throughout lactation. *Nutrients* 13 (8), 2737. https://doi.org/10.3390/nu13082737.

Spevacek, A.R., Smilowitz, J.T., Chin, E.L., Underwood, M.A., German, J.B., Slupsky, C. M., 2015. Infant maturity at birth reveals minor differences in the maternal milk metabolome in the first month of lactation. *J. Nutr.* 145 (8), 1698–1708. https://doi.org/10.3945/jn.115.210252.

Tao, N., DePeters, E.J., Freeman, S., German, J.B., Grimm, R., Lebrilla, C.B., 2008. Bovine milk glycome. *J. Dairy Sci.* 91 (10), 3768–3778. https://doi.org/10.3168/jds.2008-1305.

Tao, N., DePeters, E.J., German, J.B., Grimm, R., Lebrilla, C.B., 2009. Variations in bovine milk oligosaccharides during early and middle lactation stages analyzed by high-performance liquid chromatography-chip/mass spectrometry. *J. Dairy Sci.* 92 (7), 2991–3001. https://doi.org/10.3168/jds.2008-1642.

Tao, N., Ochonicky, K.L., German, J.B., Donovan, S.M., Lebrilla, C.B., 2010. Structural determination and daily variations of porcine milk oligosaccharides. *J. Agric. Food Chem.* 58 (8), 4653–4659. https://doi.org/10.1021/jf100398u.

Tan, J., Lee, H.H., Wong, L., Fong, C., Ong, A., Lin, Q., Xiao, Y., 2022. Simultaneous determination of neutral and acidic human milk oligosaccharides (HMOs) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). *Appl. Food Res.* 2 (2), 100153. https://doi.org/10.1016/j.afres.2022.100153.

Thurl, S., Muller-Werner, B., Sawatzki, G., 1996. Quantification of individual oligosaccharide compounds from human milk using high-pH anion-exchange chromatography. *Anal. Biochem.* 235 (2), 202–206. https://doi.org/10.1006/abio.1996.0113.

Thurl, S., Munzert, M., Henker, J., Boehm, G., Müller-Werner, B., Jelinek, J., Stahl, B., 2010. Variation of human milk oligosaccharides in relation to milk groups and lactational periods. *Br. J. Nutr.* 104 (9), 1261–1271. https://doi.org/10.1017/S0007114510002072.

Tonon, K.M., Miranda, A., Abrão, A.C.F.V., de Morais, M.B., Morais, T.B., 2019. Validation and application of a method for the simultaneous absolute quantification of 16 neutral and acidic human milk oligosaccharides by graphitized carbon liquid chromatography - electrospray ionization - mass spectrometry. *Food Chem.* 274, 691–697. https://doi.org/10.1016/j.foodchem.2018.09.036.

Uemura, Y., Asakuma, S., Nakamura, T., Arai, I., Taki, M., Urashima, T., 2005. Occurrence of a unique sialyl tetrasaccharide in colostrum of a bottlenose dolphin (Tursiops truncatus). *Biochim. et Biophys. Acta (BBA)-Gen.* Subj. 1725 (3), 290–297. https://doi.org/10.1016/j.bbagen.2005.05.01.

Urashima, T., Saito, T., Ohmisya, K., Shimazaki, K., 1991. Structural determination of three neutral oligosaccharides in bovine (Holstein-Friesian) colostrum, including the novel trisaccharide; GalNAc alpha 1-3 Gal beta 1-4Glc. *Biochim. Biophys. Acta* 1073 (1), 225-229. https://doi.org/10.1016/0304-4165(91)90207-w.

Urashima, T., Taufik, E., Fukuda, K., Asakuma, S., 2013. Recent advances in studies on milk oligosaccharides of cows and other domestic farm animals. *Biosci. Biotechnol. Biochem.* 77 (3), 455–466. https://doi.org/10.1271/bbb.120810.

Urashima, T., Hirabayashi, J., Sato, S., Kobata, A., 2018. Human milk oligosaccharides as essential tools for basic and application studies on galectins. *Trends Glycoseci. Glycotechnol.* 30 (172), SE51–SE65. https://doi.org/10.4052/tigg.1734.1SE.

Urashima, T., Katayama, T., Sakanaka, M., Fukuda, K., Messer, M., 2022. Evolution of milk oligosaccharides: origin and selectivity of the ratio of milk oligosaccharides to lactose among mammals. *Biochim. et Biophys. Acta (BBA)-Gen.* Subj. 1866 (1), 130012. https://doi.org/10.1016/j.bbagen.2021.130012.

van Leeuwen, S.S., Stoutjesdijk, E., Ten Kate, G.A., Schaafsma, A., Dijck-Brouwer, J., Muskiet, F.A., Dijkhuizen, L., 2018. Regional variations in human milk oligosaccharides in Vietnam suggest FucTx activity besides FucT2 and FucT3. *Sci. Rep.* 8 (1), 16790. https://doi.org/10.1038/s41598-018-34882-x.

van Leeuwen, S.S., 2019 Nov 6. Challenges and pitfalls in human milk oligosaccharide analysis. *Nutrients* 11 (11), 2684. https://doi.org/10.3390/nu11112684.

Vicaretti, S.D., Mohtarudin, N.A., Garner, A.M., Zandberg, W.F., 2018. Capillary electrophoresis analysis of bovine milk oligosaccharides permits an assessment of the influence of diet and the discovery of nine abundant sulfated analogues. *J. Agric. Food Chem.* 66 (32), 8574–8583. https://doi.org/10.1021/acs.jafc.8b01041.

Wei, J., Wang, Z.A., Wang, B., Jahan, M., Wang, Z., Wynn, P.C., Du, Y., 2018. Characterization of porcine milk oligosaccharides over lactation between primiparous and multiparous female pigs. *Sci. Rep.* 8 (1), 4688. https://doi.org/10.1038/s41598-018-23025-x.

Xu, G., Davis, J.C., Goonatilleke, E., Smilowitz, J.T., German, J.B., Lebrilla, C.B., 2017. Absolute quantitation of human milk oligosaccharides reveals phenotypic variations during lactation. *J. Nutr.* 147 (1), 117–124. https://doi.org/10.3945/jn.116.238279.

Yan, J., Ding, J., Jin, G., Yu, D., Yu, L., Long, Z., Guo, Z., Chai, W., Liang, X., 2018. Profiling of sialylated oligosaccharides in mammalian milk using online solid phase extraction-hydrophilic interaction chromatography coupled with negative-ion electrospray mass spectrometry. *Anal. Chem.* 90 (5), 3174–3182. https://doi.org/10.1021/acs.analchem.7b04468.

Yang, B., Zhang, M., Zhao, J., Qiao, W., Chen, J., Hou, J., Chen, L., 2022. Development and validation of UPLC method for quantitative determination of major bovine milk oligosaccharides and their heat stability. *Int. Dairy J.* 126, 105005. https://doi.org/10.1016/j.idairyj.2021.105005.

Zhang, W., Wang, T., Chen, X., Pang, X., Zhang, S., Obaroakpo, J.U., Shilong, J., Lu, J., Lv, J., 2019. Absolute quantification of twelve oligosaccharides in human milk using a targeted mass spectrometry-based approach. *Carbohydr. Polym.* 219, 328–333. https://doi.org/10.1016/j.carbpol.2019.04.092.

Zivkovic, A.M., German, J.B., Lebrilla, C.B., Mills, D.A., 2011. Human milk glycobiome and its impact on the infant gastrointestinal microbiota. *Proc. Natl. Acad. Sci. USA* 108 Suppl. 1, 4653–4658. https://doi.org/10.1073/pnas.1000083107.