Innovative direct introduction-ion mobility-mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix: Application to 2'-fucosyllactose (2'-FL) in breast milk.

Identification and specific quantification of isomers in a complex biological matrix by mass spectrometry alone is not an easy task due to their identical chemical formula and therefore their same mass-to-charge ratio (m/z). Here, the potential of direct introduction combined with ion mobility-mass spectrometry (DI-IM-MS) for rapid quantification of isomers as human milk oligosaccharides (HMOs) was investigated. Differences in HMO profiles between various analyzed breast milk samples were highlighted using the single ion mobility monitoring (SIM2) acquisition for high ion mobility resolution detection. Furthermore, the Se+ (secretor) or Se- (non-secretor) phenotype could be assigned to breast milk samples studied based on their HMO contents, especially on the response of 2'-fucosyllactose (2'-FL) and lacto-N-fucopentaose I (LNFP I). The possibility of quantifying a specific isomer in breast milk by DI-IM-MS was also investigated. The standard addition method allowed the determination of the 2'-FL despite the presence of other oligosaccharides, including 3-fucosyllactose (3-FL) isomer in breast milk. This proof-of-concept study demonstrated the high potential of such an approach for the rapid and convenient quantification of isomers in complex mixtures.

2'-Fucosyllactose (2'-FL) changes infants gut microbiota composition and their metabolism in a host-free human colonic model.

BACKGROUND: Breast milk is critical for neonates, providing the necessary energy, nutrients, and bioactive compounds for growth and development. Research indicated that human milk oligosaccharides (HMOs) have been shown to shape a beneficial gut microbiota, as well as their metabolism (e.g. short-chain fatty acids). 2'-Fucosyllactose (2'-FL) is one major HMO that composed of 30% of total HMOs. OBJECTIVES: This study aimed to understand the impact of 2'-FL on the composition and metabolism of infant gut microbiota. METHODS: Our study utilized an in-vitro human colonic model (HCM) to investigate the host-free interactions between 2'-FL and infant gut microbiota. To simulate the infant gut microbiota, we inoculated the HCM system with eight representative bacterial species from infant gut microbiota. The effects of 2'-FL on the gut microbial composition and their metabolism were determined through real-time quantitative PCR and liquid-chromatography mass spectrometry (LC/MS). The obtained data were analyzed using Compound Discoverer 3.1 and MetaboAnalyst 4.0. RESULTS: Our study findings suggest that the intervention of 2'-FL in HCM resulted in a significant change in the abundance of representative bacterial species. PCR analysis showed a consistent increase in the abundance of Parabacteroides. distasonis in all three colon sections. Furthermore, analysis of free fatty acids revealed a significant increase in their levels in the ascending, transverse, and descending colons, except for caproic acid, which was significantly reduced to a non-detectable level. The identification of significant extracellular polar metabolites, such as glutathione and serotonin, enabled us to distinguish between the metabolomes before and after 2'-FL intervention. Moreover, correlation analysis revealed a significant association between the altered microbes and microbial metabolites. CONCLUSIONS: In summary, our study demonstrated the impact of 2'-FL intervention on the defined composition of infant gut microbiota and their metabolic pathways in an in vitro setting. Our findings provide valuable insights for future follow-up investigations into the role of 2'-FL in regulating the growth and development of infant gut microbiota in vivo.

Elimination of Residual Lacto-N-triose II for Lacto-N-tetraose Biosynthesis in Engineered Escherichia coli.

Lacto-N-tetraose (LNT) is an important neutral human milk oligosaccharide (HMO) and acts as a significant core structure for complex HMO biosynthesis. We previously achieved high-yield LNT biosynthesis (57.5 g/L) using fed-batch fermentation; however, residual lacto-N-triose II (LNTri II) was also found (21.58 g/L). Here, we re-engineered an efficient LNT-producing Escherichia coli with low LNTri II accumulation using genetically stable LNTri II-producing strains with a genomic insertion of lgtA (encoding ß1,3-N-acetylglucosaminyltransferase). Comparable and low titers of LNT (3.73-4.61 g/L) and LNTri II (0.33-0.63 g/L), respectively, were obtained by introducing ß1,3-galactosyltransferase. To reduce residual LNTri II, the E. coli transporter gene setA was disrupted, obviously reducing the accumulation of LNTri II and LNT. Next, the gene encoding ß-N-acetylhexosaminidase (BbhI) was introduced into LNT-producing strains or E. coli BL21(DE3) for single- or mixed-strain cultivation, respectively. Finally, LNT was obtained (30.13 g/L) in a cocultivation system of mixed engineered strains without undesired LNTri II.

Structural characterization and in vitro analysis of the prebiotic activity of oligosaccharides from lotus (Nelumbo nucifera Gaertn.) seeds.

In the present study, lotus seed oligosaccharides (LOSs) were isolated from lotus (Nelumbo nucifera Gaertn.) seeds using preparative liquid chromatography. LOS structures were characterized using fourier transform infrared spectroscopy (FT-IR), acid hydrolysis, tandemmass spectrometry (MS/MS) and 1D/2D nuclear magnetic resonance (NMR) spectroscopy. Then, Lactobacillus acidophilus was used to evaluate the prebiotic activity of LOSs in vitro. The structural analysis revealed that the monosaccharide components of LOSs included glucose, mannose, fructose and galactose. The MS/MS results indicated that disaccharides, trisaccharides, trisaccharides and tetrasaccharides were the constituents of isolated oligosaccharide polymers LOS2, LOS3-1, LOS3-2, and LOS4, respectively. The FT-IR and 1D/2D NMR data confirmed that LOS3 and LOS4 had a linear structure consisting of (1 â†’ 6)-α-d-mannopyranosyl and glucopyranosyl residues. LOS3-1 and LOS4 effectively and selectively promoted the growth of an L. acidophilus strain, according to the results of the assays of optical density and the short-chain fatty acid (SCFA) content in the culture broth.

Microwave-assisted enzymatic hydrolysis to produce xylooligosaccharides from rice husk alkali-soluble arabinoxylan.

The prebiotic properties of xylooligosaccharides (XOS) and arabino-xylooligosaccharides (AXOS) produced from rice husk (RH) using microwave treatment combined with enzymatic hydrolysis were evaluated. The RH was subjected to microwave pretreatment at 140, 160 and 180 °C for 5, 10 and 15 min to obtain crude arabinoxylan (AX). Increasing microwave pretreatment time increased sugar content. Crude AX was extracted with 2% (w/v) sodium hydroxide at 25 °C for 24 h and used as a substrate for XOS production by commercial xylanases. Results showed that oligosaccharides produced by Pentopan Mono BG and Ultraflo Max provided xylobiose and xylotriose as the main products. AXOS was also present in the oligosaccharides that promoted growth of Lactobacillus spp. and resisted degradation by over 70% after exposure to simulated human digestion.

Recent advances on 2'-fucosyllactose: physiological properties, applications, and production approaches.

The trisaccharide, 2'-fucosyllactose (Fucα1-2Galß1-4Glc; 2'-FL), is the most abundant oligosaccharide in human milk. It has numerous significant biological properties including prebiotics, antibacterial, antiviral, and immunomodulating effects, and has been approved as "generally recognized as safe" (GRAS) by the Food and Drug Administration (FDA) and as a novel food (NF) by the European Food Safety Authority (EFSA). 2'-FL not only serves as a food ingredient added in infant formula, but also as a dietary supplement and medical food material in food bioprocesses. There is considerable commercial interest in 2'-FL for its irreplaceable nutritional applications. This review aims at systematically elaborating key functional properties of 2'-FL as well as its applications. In addition, several approaches for 2'-FL production are described in this review, including chemical, chemo-enzymatical, and cell factory approaches, and the pivotal research results also have been summarized. With the rapid development of metabolic engineering and synthetic biology strategies, using the engineered cell factory for 2'-FL large-scale production might be a promising approach. From an economic and safety point of view, microbial selection for cell factory engineering in 2'-FL bioprocess also should be taken into consideration.

Presence and Levels of Galactosyllactoses and Other Oligosaccharides in Human Milk and Their Variation during Lactation and According to Maternal Phenotype.

Among the human milk oligosaccharides (HMOS), the galactosyllactoses (GLs) are only limitedly studied. This study aims to describe the presence and relative levels of HMOS, including GLs, in human milk (HM) according to maternal Secretor and Lewis (SeLe) phenotype and lactation stage. Relative levels of 19 HMOS were measured in 715 HM samples collected in the first 4 months postpartum from 371 donors participating in the PreventCD study. From a subset of 24 Dutch women (171 HM samples), samples were collected monthly up to 12 months postpartum and were additionally analyzed for relative and absolute levels of ß6'-GL, ß3'-GL and α3'-GL. Maternal SeLe phenotype or HM group was assigned based on the presence of specific fucosylated HMOS. Most HMOS, including ß6'- and ß3'-GL, were present in the vast majority (≥75%) of HM samples, whereas others (e.g., LNDFH II, 2'-F-LNH and α3'-GL) only occurred in a low number (<25%) of samples. Clear differences were observed between the presence and relative levels of the HMOS according to the maternal phenotype and lactation stage. Absolute concentrations of ß6'-GL and ß3'-GL were higher in HM group IV samples compared to samples of the other three HM groups. ß3'-GL was also higher in HM group II samples compared to HM group I samples. ß3'-GL and ß6'-GL were stable over lactation stages. In conclusion, presence and levels of HMOS vary according to HM group and lactation stage. Not all HMOS behave similarly: some HMOS depend strongly on maternal phenotype and/or lactation stage, whereas others do not. ß3'-GL and ß6'-GL were present in low concentrations in over 75% of the analyzed HM samples and showed differences between HM groups, but not between the lactation stages.

Longitudinal Changes in Human Milk Oligosaccharides (HMOs) Over the Course of 24 Months of Lactation.

BACKGROUND: Human milk oligosaccharides (HMOs) are complex glycans that are highly abundant in human milk. While over 150 HMOs have been identified, it is unknown how individual HMOs change in concentration over 24 months of lactation. OBJECTIVES: To understand how HMO concentrations change over 24 months of lactation. METHODS: Breast milk samples were collected from participants in a longitudinal cohort study of Hispanic mother-infant pairs at 1, 6, 12, 18, and 24 months postpartum. Concentrations of 19 of the most abundant HMOs were measured using HPLC. Because the parent study is ongoing and not all participants have finished all time points yet, the sample sizes ranged per time point (n = 207 at 1 month; n = 109 at 6 months; n = 83 at 12 months; n = 59 at 18 months; and n = 28 at 24 months). Approximately 88% of participants were classified as HMO secretors-a genetic factor that affects concentrations of HMOs such as 2'fucosyllactose (2'FL) and lacto-N-fucopentaose I-while the remaining 12% were classified as nonsecretors. Mixed models were used to examine changes in HMO concentrations and relative abundances over the course of lactation. RESULTS: The majority of HMOs significantly decreased in concentration over the course of lactation. The exceptions were 2'FL, sialyl-lacto-N-tetraose b, and disialyl-lacto-N-tetraose, which did not change with time, and 3-fucosyllactose (3FL) and 3'-sialyllactose (3'SL), which significantly increased. The concentration of 3FL increased 10-fold, from 195 (IQR 138-415) µg/mL at 1 month to 1930 (1100-2630) µg/mL at 24 months, while 3'SL increased 2-fold, from 277 (198-377) µg/mL to 568 (448-708) µg/mL over the same time period. CONCLUSIONS: These results indicate that HMOs do not decrease in concentration uniformly across lactation. In particular, 3FL and 3'SL increased over the course of lactation in this cohort. Future studies are required to fully understand the functions of these HMOs.

Discrimination of isomeric trisaccharides and their relative quantification in honeys using trapped ion mobility spectrometry.

Carbohydrates play a myriad of critical roles as key intermediaries for energy storage, cell wall constituents, or also fuel for organisms. The deciphering of multiple structural isomers based on the monosaccharides composition (stereoisomers), the type of glycosidic linkages (connectivity) and the anomeric configuration (α and ß), remains a major analytical challenging task. The possibility to discriminate 13 underivatized isomeric trisaccharides were reported using electrospray ionization coupled to trapped ion mobility spectrometry (ESI-TIMS). After optimization of scan ratio enhancing both the mobility resolving power (R) and resolution (r), fingerprints from 5 different honeys were obtained. Seven trisaccharides with relative content varying from 1.5 to 58.3%, were identified. It was demonstrated that their relative content and/or their ratio could be used to ascertain origin of the honeys. Moreover, such direct approach constitutes an alternative tool to current longer chromatographic runs, paving the way to a transfer as suitable routine analysis.

Composition and potential as a prebiotic functional food of a Giant Willow Aphid (Tuberolachnus salignus) honeydew honey produced in New Zealand.

The Giant Willow Aphid (Tuberolachnus salignus, GWA) is an invasive pest insect in New Zealand, which excretes honeydew. European honeybees collect this honeydew and make it into a type of honey that crystallizes in the comb, representing a significant loss to apiarists. This crystallization has been ascribed to high concentrations of oligosaccharides, particularly melezitose. In this research, the first carbohydrate profile of GWA honeydew honey, a sample of GWA honeydew honey was found to contain 37.8% total oligosaccharides of which 27.4% was melezitose, and 2.5% gluconic acid (higher than typical honeydew honeys); 41.2% monosaccharides (lower than typical honeydew honeys); and 0.054% salicylic acid (higher than previous estimates). Melezitose extracted from GWA honeydew honey was not significantly hydrolyzed in crude human-stomach and human-small-intestine simulations and may therefore meet the prebiotic criterion of human indigestibility.

Development of fluorescent Escherichia coli for a whole-cell sensor of 2'-fucosyllactose.

2'-Fucosyllactose (2'-FL), a major component of fucosylated human milk oligosaccharides, is beneficial to human health in various ways like prebiotic effect, protection from pathogens, anti-inflammatory activity and reduction of the risk of neurodegeneration. Here, a whole-cell fluorescence biosensor for 2'-FL was developed. Escherichia coli (E. coli) was engineered to catalyse the cleavage of 2'-FL into L-fucose and lactose by constitutively expressing α-L-fucosidase. Escherichia coli ∆L YA, in which lacZ is deleted and lacY is retained, was employed to disable lactose consumption. E. coli ∆L YA constitutively co-expressing α-L-fucosidase and a red fluorescence protein (RFP) exhibited increased fluorescence intensity in media containing 2'-FL. However, the presence of 50 g/L lactose reduced the RFP intensity due to lactose-induced cytotoxicity. Preadaptation of bacterial strains to fucose alleviated growth hindrance by lactose and partially recovered the fluorescence intensity. The fluorescence intensity of the cell was linearly proportional to 1-5 g/L 2'-FL. The whole-cell sensor will be versatile in developing a 2'-FL detection system.

Real-world study in infants fed with an infant formula with two human milk oligosaccharides.

Introduction: Introduction: human milk oligosaccharides (HMOs) are an important component of human milk supporting the development of a balanced intestinal microbiota and immune protection in breastfed infants. Randomized controlled trials (RCTs) have demonstrated that infant formulas supplemented with the HMOs 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) are safe, well-tolerated, and support normal growth. This Real-World Evidence (RWE) study aimed to evaluate growth and tolerance in infants consuming a formula supplemented with 1 g/L of 2'FL and 0.5 g/L of LNnT, and included a mixed-feeding group never studied before in RCTs. Participants and methods: this open-label, prospective study was conducted at six centers in Spain, and included healthy, exclusively breastfed infants (BF group), an exclusively formula-fed group (FF) who received a milk-based formula with 2' FL and LNnT, and a group mixed fed with both formula and human milk (MF), for 8 weeks. Co-primary outcomes were growth (anthropometry) and gastrointestinal tolerance (Infant Gastrointestinal Symptom Questionnaire, IGSQ). Secondary outcomes included formula satisfaction and adverse events (AEs). Results: 159 infants completed the study (66 FF, 48 MF, and 45 BF). Mean z-scores for growth were similar between all groups and within ± 0.5 of WHO medians at week 8. Composite IGSQ scores demonstrated low GI distress in all groups, with no significant group differences at baseline, week 4, or week 8. Incidence of AEs was low overall, and comparable across groups. Conclusions: in this RWE study examining a HMO-supplemented infant formula, growth and tolerance outcomes were similar to RCT findings, supporting the effectiveness of this early feeding option.

Introducción: Introducción: los oligosacáridos de la leche materna (HMO) contribuyen a desarrollar la inmunoprotección y la microbiota intestinal. Los ensayos aleatorizados (RCT) han demostrado que las fórmulas enriquecidas con 2'fucosilactosa (2'FL) y lacto-N-neotetraosa (LNnT) son seguras, bien toleradas y favorecen el crecimiento. El objetivo de este estudio ha sido valorar el crecimiento, la seguridad y la tolerancia digestiva en lactantes alimentados con una fórmula enriquecida con 1 g/L de 2'FL y 0,5 g/L de LNnT, con datos de la vida real (RWE), incluyendo un grupo de alimentación mixta no estudiado antes en los RCT. Participantes y métodos: estudio prospectivo abierto en seis hospitales españoles que incluyó lactantes sanos alimentados con leche materna (BF), con fórmula enriquecida en 2'FL y LNnT (FF) o con mezcla de ambas (MF), durante ocho semanas. Se valoraron el crecimiento (antropometría), la tolerancia gastrointestinal (cuestionario IGSQ) y los acontecimientos adversos. Resultados: 159 lactantes completaron el estudio (66, 48 y 45, en los grupos FF, MF y BF, respectivamente). Las puntuaciones Z antropométricas a la semana 8 fueron similares entre los grupos y se hallaron dentro del rango de ± 0,5 de la normalidad. Las puntuaciones IGSQ compuestas mostraron un bajo malestar digestivo, sin diferencias significativas entre los grupos, al inicio y en las semanas 4 y 8. La incidencia de eventos adversos fue baja y comparable entre los grupos. Conclusiones: en este estudio RWE que evaluó una fórmula para lactantes enriquecida en HMO, los resultados sobre el crecimiento, la tolerancia y la seguridad fueron similares a los obtenidos en los RCT, respaldando su eficacia como alimentación temprana opcional.

Glycosynthase reaction meets the flow: Continuous synthesis of lacto-N-triose II by engineered ß-hexosaminidase immobilized on solid support.

The D746E variant of Bifidobacterium bifidum ß-N-acetyl-hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis of lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides. Here, we develop a flow process for the glycosynthase reaction, which is the regioselective ß-1,3-glycosylation of lactose from a d-glucosamine 1,2-oxazoline donor. Using the glycosynthase immobilized on agarose beads (∼30 mg/g) packed into a fixed bed (1 ml), we show stable continuous production of LNT II (145-200 mM) at quantitative yield from the donor substrate. The wild-type ß-N-acetyl-hexosaminidase used under exactly comparable conditions gives primarily (∼85%) the hydrolysis product d-glucosamine. By enabling short residence times (2 min) that are challenging for mixed-vessel types of reactor to establish, the glycosynthase flow reactor succeeds in an effective uncoupling of the LNT II formation (∼80-100 mM/min) from the slower side reactions (decomposition of donor substrate, enzymatic hydrolysis of LNT II) to obtain optimum synthetic efficiency. Our study thus provides a strong case for the application of flow chemistry principles to glycosynthase reactions and by that, it reveals the important synergy between enzyme and reaction engineering for biocatalytic synthesis of oligosaccharides.

Quantifying the human milk oligosaccharides 2'-fucosyllactose and 3-fucosyllactose in different food applications by high-performance liquid chromatography with refractive index detection.

In recent years, production of biosynthesized human milk oligosaccharides (HMOs) has become scalable to industrial standards. As a result, infant formula fortified with 2'-fucosyllactose (2'-FL), the most abundant HMO in human breast milk, is now commercially available. 2'-FL and 3-fucosyllactose (3-FL), another abundant HMO, are thought to be beneficial for infant health and development. Products containing HMOs are projected to expand in the future, showing the need for robust, easily applicable analytical methods for the quantitative assessment of HMOs in different food applications. We present here a validated high-performance liquid chromatography method for the quantification of 2'-FL and 3-FL in whole milk, infant formula, and cereal bars. The sample preparation was simple dispersion and extraction of the sample. The samples were analyzed by hydrophilic interaction liquid chromatography with refractive index detection and a runtime of 19 min. The method had a high degree of linearity (R2 > 0.9995) in the range 0.2 to 12 mg/mL. The recovery for 2'-FL was 88% to 105% and for 3-FL 94% to 112%. The limit of detection (LOD) for whole milk was 0.1 mg/mL for 2'-FL and 0.2 mg/mL for 3-FL. In infant formula and cereal bars, the LOD was 0.6 mg/g for both 2'-FL and 3-FL. To show the practical application of this method, it was successfully utilized in stability studies of 2'-FL and 3-FL in whole milk, UHT milk, and yoghurt. The method provides a means of simultaneous and robust quantification of 2'-FL and 3-FL in various food matrices with high accuracy and high reproducibility. PRACTICAL APPLICATION: 2'-Fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) are two of the most abundant human milk oligosaccharides (HMOs) present in human breast milk. We present a fast HPLC method for the robust quantification of these two compounds in infant formula, whole milk, UHT milk, cereal bars, and yoghurt. This method can easily be set up by food producers and researchers to analyze the dosage of 2'-FL and 3-FL in their product or perform shelf life studies in different food applications.

Development of a quantitative assay for 2´-fucosyllactose via one-pot reaction with α1,2-fucosidase and l-fucose dehydrogenase.

2'-Fucosyllactose (2'-FL) is the most abundant milk oligosaccharide in human breast milk and it has several benefits for infant health. The quantification of 2'-FL in breast milk or in samples from other sources generally requires lengthy analyses. These methods cannot be used to simultaneously detect 2'-FL in numerous samples, which would be more time-efficient. In this study, two genes, namely α1,2-fucosidase from Xanthomonas manihotis and l-fucose dehydrogenase from Pseudomonas sp. no. 1143, were identified, cloned and overexpressed in E. coli. The recombinant enzymes were produced as 6 × His-tagged proteins and were purified to homogeneity using Ni2+ affinity chromatography. The purified α1,2-fucosidase and l-fucose dehydrogenase are monomers with molecular masses of 63 kDa and 36 kDa, respectively. Both enzymes have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37 °C, making it possible to develop a coupled enzyme reaction in a single buffer system for the quantitative determination of 2'-FL in a large number of samples simultaneously. This method can be used to quantify 2'-FL in infant formulas and in samples collected from different phases of the biotechnological production of this oligosaccharide. Furthermore, the method is applicable for the rapid screening of active variants during the development of microbial strains producing 2'-FL.

Infrared-assisted extraction followed by high performance liquid chromatography to determine angoroside C, cinnamic acid, and harpagoside content in Scrophularia ningpoensis.

BACKGROUND: Angoroside C, cinnamic acid, and harpagoside are bioactive constituents in Scrophularia ningpoensis. Currently, an infrared-assisted extraction (IRAE) method coupled with high-performance liquid chromatography with ultraviolet detection (HPLC-UV) for the analysis of bioactive constituents in this plant is lacking. METHODS: A method based on HPLC following IRAE has been developed for quantifying angoroside C, cinnamic acid, and harpagoside in Scrophularia ningpoensis. Four main factors, namely, extraction solvent, solid/liquid ratio, illumination time, and distance between the infrared lamp and the round-bottom flask, were optimized for extraction. Furthermore, conventional ultrasonic extraction (USE) and microwave-assisted extraction (MAE) were also investigated to validate the developed method. RESULTS: The optimal extraction conditions were as follows: ethanol concentration, 37.5%; solid/liquid ratio, 1:25; illumination time, 10 min; and distance between infrared lamp and round-bottom flask, 3 cm. The results of method validation demonstrated that the developed method meets the requirement of analysis. CONCLUSION: The results show that the IRAE-HPLC is a simple, accurate, and green analytical preparatory method for the potential extraction and quantification of angoroside C, cinnamic acid, and harpagoside in Scrophularia ningpoensis.

Characterization of Electrospray Ionization (ESI) Parameters on In-ESI Hydrogen/Deuterium Exchange of Carbohydrate-Metal Ion Adducts.

The conformations of glycans are crucial for their biological functions. In-electrospray ionization (ESI) hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is a promising technique for studying carbohydrate conformations since rapidly exchanging functional groups, e.g., hydroxyls, can be labeled on the timeframe of ESI. However, regular application of in-ESI HDX to characterize carbohydrates requires further analysis of the in-ESI HDX methodology. For instance, in this method, HDX occurs concurrently to the analyte transitioning from solution to gas-phase ions. Therefore, there is a possibility of sampling both gas-phase and solution-phase conformations of the analyte. Herein, we differentiate in-ESI HDX of metal-adducted carbohydrates from gas-phase HDX and illustrate that this method analyzes solvated species. We also systematically examine the effects of ESI parameters, including spray solvent composition, auxiliary gas flow rate, sheath gas flow rate, sample infusion rate, sample concentration, and spray voltage, and discuss their effects on in-ESI HDX. Further, we model the structural changes of a trisaccharide, melezitose, and its intramolecular and intermolecular hydrogen bonding in solvents with different compositions of methanol and water. These molecular dynamic simulations support our experimental results and illustrate how an individual ESI parameter can alter the conformations we sample by in-ESI HDX. In total, this work illustrates how the fundamental processes of ESI alter the magnitude of HDX for carbohydrates and suggest parameters that should be considered and/or optimized prior to performing experiments with this in-ESI HDX technique. Graphical Abstract ᅟ.

Determination of 2'-Fucosyllactose and Lacto-N-neotetraose in Infant Formula.

Human milk oligosaccharides (HMO) are the third most abundant solid component of human milk. It is likely that they are responsible for at least some of the benefits experienced by breast-fed infants. Until recently HMO were absent from infant formula, but 2'-fucosyllactose (2'-FL) and lacto-N-neoteraose (LNnT) have recently become available as ingredients. The development of formula containing these HMO and the quality control of such formula require suitable methods for the accurate determination of the HMO. We developed two different approaches for analysis of 2'-FL and LNnT in formula; high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD). In lab trials using blank formula spiked with the two oligosaccharides, both approaches worked well with recoveries of 94⁻111% (HPAEC-PAD) and 94⁻104% (HILIC-FLD) and RSD (iR) of 2.1⁻7.9% (HPAEC-PAD) and 2.0⁻7.4% (HILIC-FLD). However, when applied to products produced in a pilot plant, the HPAEC-PAD approach sometimes delivered results below those expected from the addition rate of the ingredients. We hypothesize that the oligosaccharides interact with the formula matrix during the production process and, during sample preparation for HPAEC-PAD those interactions have not been broken. The conditions required for labeling the HMO for detection by the FLD apparently disrupt those interactions, and result in improved recoveries. It is likely that both analytical approaches are appropriate if a suitable extraction process is used to recover the HMO.

Human Milk Oligosaccharides in Colostrum and Mature Milk of Chinese Mothers: Lewis Positive Secretor Subgroups.

To study the variability in human milk oligosaccharide (HMO) composition of Chinese human milk over a 20-wk lactation period, HMO profiles of 30 mothers were analyzed using CE-LIF. This study showed that total HMO concentrations in Chinese human milk decreased significantly over a 20-wk lactation period, independent of the mother's SeLe status, although with individual variations. In addition, total acidic and neutral HMO concentrations in Chinese human milk decreased over lactation, and levels are driven by their mother's SeLe status. Analysis showed that total neutral fucosylated HMO concentrations in Chinese human milk were higher in the two secretor groups as compared to the nonsecretor group. On the basis of the total neutral fucosylated HMO concentrations in Chinese human milk, HMO profiles within the Se+Le+ group can be divided into two subgroups. HMOs that differed in level between Se+Le+ subgroups were 2'FL, DF-L, LNFP I, and F-LNO. HMO profiles in Dutch human milk also showed Se+Le+ subgroup division, with 2'FL, LNT, and F-LNO as the driving force.

Quantification of Soluble Sugars and Sugar Alcohols by LC-MS/MS.

Sugars are simple carbohydrates composed primarily of carbon, hydrogen, and oxygen. They play a central role in metabolism as sources of energy and as building blocks for synthesis of structural and nonstructural polymers. Many different techniques have been used to measure sugars, including refractometry, colorimetric and enzymatic assays, gas chromatography, high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy. In this chapter we describe a method that combines an initial separation of sugars by high-performance anion-exchange chromatography (HPAEC) with detection and quantification by tandem mass spectrometry (MS/MS). This combination of techniques provides exquisite specificity, allowing measurement of a diverse range of high- and low-abundance sugars in biological samples. This method can also be used for isotopomer analysis in stable-isotope labeling experiments to measure metabolic fluxes.