Pulsed Amperometric Detection Research Articles

Electroactive sugars, organic acids and sugar alcohol analysis in wine using anion-exchange chromatography with electrochemical detection

The present work describes an easy, rapid and sensitive alternative methodology for simultaneous detection of sugars (glucose, fructose and arabinose), organic acids (gluconic, glucuronic and glucaric acids) and sugar alcohol (arabitol) in wine samples. The method is based on high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) at gold electrode under detection potential of 0.26 V vs Pd|PdO. The optimized chromatographic anion-exchange separation indicates good separation and detection using a binary gradient with 0.28 mol L−1 CH3COONa containing 0.10 mol L−1 NaOH and ultrapure water as mobile phase with a flow rate at 1.0 mL min−1. Analytical curves presented linear relationship from 1.0 × 10−7 and 1.0 × 10−3 mol L−1 with detection limits (S/N = 3) from 3.0 × 10−8 (glucose) to 9.0 × 10−7 mol L−1 (gluconic acid). The present method was successfully applied in the determination of sugars, organic acids and sugar alcohols in wine, without time consuming pre-treatment of the sample, no need for organic solvent, fast relative execution time, satisfactory separation, high sensitivity and accuracy.

Physicochemical and Biological Characterization of RTXM83, a New Rituximab Biosimilar.

RTXM83 is a rituximab biosimilar with proven clinical safety and efficacy. It is the first rituximab biosimilar developed and approved in South America and is currently marketed in several Latin American, Middle Eastern and African countries. The aim of this study was to present the physicochemical and biological characterization studies utilized to demonstrate the similarity between RTXM83 and its reference product. Primary and higher order protein structures were analysed using peptide mapping with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), fluorescence spectroscopy and circular dichroism, and micro-differential scanning calorimetry, among other techniques. Charge variants were determined by cation-exchange chromatography (CEX)and capillary isoelectric focusing(cIEF). Glycosylation and glycoforms distribution were analysed using MS, normal phase high-performance liquid chromatography (NP-HPLC) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Size variants were evaluated by size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (SV-AUC), dynamic light scattering (DLS), and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Biological characterization included binding assays for complement C1q, CD20, and several Fc receptors (FcRs), as well as potency determination for in vitro apoptosis induction, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). RTXM83 and the reference product showed identical primary sequences and disulfide bridge patterns, and similarity at higher order protein structures, post-translational modification profiles (amino acid modifications, charge variants, and glycosylation) and levels of purity and process-related impurities. Functional studies demonstrated that RTXM83 is similar to the reference product regarding the three known mechanisms of action of rituximab: CDC, ADCC, and apoptosis induction. Binding affinities to CD20, complement component C1q, and different FcRs were also equivalent. RTXM83 is similar to its reference product in all critical quality attributes.

Metabolism of Milk Oligosaccharides in Preterm Pigs Sensitive to Necrotizing Enterocolitis.

Human milk oligosaccharides (HMO) are major components of breast milk that may have local effects in the gastrointestinal tract and systemic functions after being absorbed, both depending on their metabolism. Using preterm pigs, we investigated the metabolic fate of HMO in three experiments with two different HMO blends. In addition, we examined effects on the colonic microbiota in the presence or absence of necrotizing enterocolitis (NEC). Thus, preterm pigs (n = 112) were fed formula without or with HMO supplementation (5–10) g/L of a mixture of 4 (4-HMO) or >25 HMO (25-HMO) for 5 (Experiment 1 and 2) or 11 days (Experiment 3). Individual HMO were quantified in colon contents and urine using MALDI-TOF-MS (matrix-assisted laser desorption ionization mass spectrometry) and HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection). Microbial colonization was analyzed by sequencing of 16S rRNA gene tags. Intestinal permeability was measured by lactulose to mannitol ratio in urine. HMO supplemented to formula were detected in urine and colon contents in preterm piglets after 5 and 11 days in all three experiments. The amount of HMO excreted via the gut or the kidneys showed large individual variations. Microbial diversity in the colon changed from high levels of Firmicutes (dominated by Clostridium) at day 5 (Exp 2) to high levels of Proteobacteria dominated by Helicobacter and Campylobacter at day 11 (Exp 3). Colonic microbiota composition as well as HMO excretion pattern varied greatly among piglets. Interestingly, the 5-day supplementation of the complex 25-HMO blend led to low concentrations of 3-fucosyllactose (FL) and lacto-N-fucopentaose (LNFP) I in colonic contents, indicating a preferred utilization of these two HMO. Although the interpretation of the data from our piglet study is difficult due to the large individual variation, the presence of Bifidobacteria, although low in total numbers, was correlated with total HMO contents, and specifically with 2′FL levels in colonic content. However, early supplementation of formula with HMO did not affect NEC incidence.

Hydrothermal conversion of wood, organosolv, and chlorite pulps

In this work, beech wood has been submitted to fractionation and hydrothermal liquefaction in order to produce fermentable carbohydrates. Fractionation either by ethanol organosolv or by sodium chlorite/acetic acid (SC/AA) treatment allowed obtaining delignification yields of 50 and 91 wt.% respectively. The recovered pulps were then submitted to liquefaction in hot-compressed water (HCW). Three temperatures were studied: 180, 200, and 220 °C during 2 h. The same experiments were conducted on untreated beech and model cellulose (Avicel PH-101). Product yields and total carbohydrates were determined. The liquefaction products, i.e., solid residue, water-soluble compounds, and permanent gas, were analyzed respectively by X-ray diffraction (XRD), high-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC-PAD), and gas chromatography (GC). SC/AA treatment allowed a high recovery of hemicelluloses and for this reason, xylose content was significantly higher than for the organosolv pulp. The maximum yields of total carbohydrates (based on the initial carbohydrates in wood), found when the liquefaction temperature was 220 °C, increased from 9.0 wt.% for the raw beech to 13.5 and 33.7 wt.% for the organosolv and SC/AA pulps respectively.

Simultaneous Quantification of L-arginine and Monosaccharides during Fermentation: An Advanced Chromatography Approach.

Increasing demand for L-arginine by the food and pharmaceutical industries has sparked the search for sustainable ways of producing it. Microbial fermentation offers a suitable alternative; however, monitoring of arginine production and carbon source uptake during fermentation, requires simple and reliable quantitative methods compatible with the fermentation medium. Two methods for the simultaneous quantification of arginine and glucose or xylose are described here: high-performance anion-exchange chromatography coupled to integrated pulsed amperometric detection (HPAEC-IPAD) and reversed-phase ultra-high-performance liquid chromatography combined with charged aerosol detection (RP-UHPLC-CAD). Both were thoroughly validated in a lysogeny broth, a minimal medium, and a complex medium containing corn steep liquor. HPAEC-IPAD displayed an excellent specificity, accuracy, and precision for arginine, glucose, and xylose in minimal medium and lysogeny broth, whereas specificity and accuracy for arginine were somewhat lower in medium containing corn steep liquor. RP-UHPLC-CAD exhibited high accuracy and precision, and enabled successful monitoring of arginine and glucose or xylose in all media. The present study describes the first successful application of the above chromatographic methods for the determination and monitoring of L-arginine amounts during its fermentative production by a genetically modified Escherichia coli strain cultivated in various growth media.

Unveiling TMP Process Water Potential As an Industrial Sourcing of Valuable Lignin–Carbohydrate Complexes toward Zero-Waste Biorefineries

The development of zero-waste biorefineries is a promising yet challenging pathway toward a sustainable bioeconomy. Deep insights on the composition of biorefineries side streams along with the development of green technologies are mandatory to achieve such a goal. In this Article, a sustainable pilot-scale membrane process was used to recover valuable organic matter dissolved in the main side stream of a thermomechanical pulping (TMP) plant. A great number of analytical tools were combined to get an extensive characterization of the generated fraction, including inductively coupled plasma atomic emission spectroscopy (ICP-AES), high-performance ion chromatography coupled with pulse amperometric detection (HPIC-PAD), Fourier transform infrared (FTIR), size-exclusion chromatography (SEC), and C-13 and 2D heteronuclear single quantum correlation (HSQC) NMR. The results revealed the presence of a considerable amount of native lignin-carbohydrate complexes (LCCs) in the fraction representing 40% of the dry weight. The LCCs are composed of partially acetylated galactoglucomannans (GGMs) covalently linked to low condensed and ) beta-O-4-rich lignin through phenyl glycosidic bonds (25.5 bonds/100 aromatic rings (ar)) and characterized by a relatively low molecular weight around 7 kDa. Such complexes present an interesting potential for valorization with unique properties resulting from the combination of their aromatic and sugar moieties. Different applications are discussed in regards to the generated data in this work as an industrial sourcing of LCC is reported.

Processing of inulin-enriched apple juice using high-intensity ultrasound technology

The effects of high-intensity ultrasound (HIUS) processing on chemical composition of inulin-enriched apple juice were evaluated at different specific energy levels (0, 1, 4, 7 and 10 kJ/g). The functional beverage was characterized with respect to fructooligosaccharides (FOS) and total sugars content. In addition, the inulin chemical profile was evaluated. The analysis of sugars and fructo-oligosaccharides in the functional juice was performed by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Application of ion chromatography with pulsed amperometric detection for the determination of trace cyanide in biological samples, including breast milk

Cyanide (CN) is the biomarker of exposure to the components of tobacco smoke, although its presence in biological samples is also due to the consumption of products containing cyanogenic glycosides. In this work, we determine the concentration of the free cyanide in urine, saliva and breast milk matrices, using ion chromatography with pulsed amperometric detection (IC-PAD). IC-PAD is an emerging method, with only few documented applications in urine and saliva, and the presented determination in breast milk is its first published report for any method. The biological samples, which were obtained from women staying in a maternity ward, showed cyanide concentrations spanning 1.82–98.47 μg L−1. Under the optimized chromatographic conditions, the IC-PAD system exhibited satisfactory repeatability (R < 3%, n = 3) and good linearity in the range of 1–100 μg L−1. Thus, it proved to be an effective tool for monitoring trace cyanide concentration in a series of human body fluid matrices, including breast milk. This last matrix is especially important due to the possible effect on infant health related to the mothers' smoking habits.

Characterization of sugar composition in Chinese royal jelly by ion chromatography with pulsed amperometric detection

An ion chromatography coupled with pulsed amperometric detection (IC-PAD) method was developed for the determination of sugars in royal jelly (RJ). The method enables quantification of 19 sugars with higher sensitivity than HPLC method. Good linearity was observed in the range of 0.02–500 mg L–1 with coefficients of determination (R2) ≥ 0.9994. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 1.0–110 μg L–1 and 3.7–380 μg L–1, respectively. The average recoveries ranged from 91.0% to 108.0%. This simple method can be an effective alternative to GC method, which requires complicated purification and derivatization steps. It was used for the analysis of 100 Chinese RJ samples from three different areas and the results of analyzed sugars were found to be in agreement with those reported in literatures. The sugar profile results have suggested that some minor sugars are better markers for discrimination in relation to geographical origin.

Analysis of Monosaccharides from Arabidopsis Seed Mucilage and Whole Seeds Using HPAEC-PAD

Arabidopsis seed coat epidermal cells deposit a significant quantity of mucilage, composed of the cell wall components pectin, hemicellulose, and cellulose, into the apoplast during development. When mature seeds are hydrated, mucilage extrudes to form a gelatinous capsule around the seed. Determining the monosaccharide composition of both extruded mucilage and whole seeds is an essential technique for characterizing seed coat developmental processes and mutants with altered mucilage composition. This protocol covers growth of plants to produce seeds suitable for analysis, extraction of extruded mucilage using water and sodium carbonate (used for mutants with impaired mucilage release), and extraction of alcohol insoluble residue (AIR) from whole seeds. The prepared polysaccharides are then hydrolyzed using sulfuric acid, which hydrolyses all polysaccharides including cellulose. Sensitive and reproducible quantification of the resulting monosaccharides is achieved using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Determination of glufosinate, aminomethylphosphonic acid, and glyphosate residues in farmland soil by post-column addition of alkali using high performance anion exchange chromatography-pulse amperometric detection

A chromatographic method was developed for the determination of glufosinate (GLUF), aminomethylphosphonic acid (AMPA), and glyphosate (GLY) in farmland soil by the post-column addition of alkali using high performance anion exchange chromatography-pulse amperometric detection. Samples were extracted with 2 mmol/L sodium hydroxide solution, then filtered through a 0.22 μm membrane, and purified by a IC-C18 column and IC-Na column. Three target compounds and coexisting ions in the filtered solution were separated on an IonPacAS11-HC anion exchange column (250 mm×4 mm), and were detected by the post-column addition of alkali using pulse amperometric detection. Results showed that GLUF and GLY had good linearity in the range of 20.0-1000 μg/L, and AMPA had good linearity in the range of 5.0-400 μg/L, with correlation coefficients above 0.999. The limits of detection of GLUF, AMPA, and GLY were 0.08, 0.02, and 0.04 mg/kg, respectively, and the recoveries were in the range of 80.2%-106% with RSDs of 0.7%-5.0% (n=6). The method provides strong anti-interference, high sensitivity, and accuracy, and can be adopted simply and quickly; therefore, it is suitable for detecting the residues of GLUF, AMPA, and GLY in farmland soil.

Wheat Gluten Amino Acid Analysis by High-Performance Anion-Exchange Chromatography with Integrated Pulsed Amperometric Detection.

The present chapter describes an accurate and user-friendly method for determining amino acid composition of wheat gluten proteins and their gliadin and glutenin fractions. The method consists of hydrolysis of the peptide bonds in 6.0M hydrochloric acid (HCl) solution at 110°C for 24h, followed by evaporation of the acid and separation of the free amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD). In contrast to conventional methods, the analysis requires neither pre- or post-column derivatization nor a time-consuming oxidation or derivatization step prior to hydrolysis. Correction factors account for incomplete release of Val and Ile even after hydrolysis for 24h and for losses of Ser during evaporation. Gradient conditions including an extra eluent allow multiple sequential sample analyses without risk of Glu accumulation on the anion-exchange column which otherwise would result from high Gln levels in gluten proteins.

Simultaneous determination of twenty amino acids and six carbohydrates in soy sauce by ion chromatography with integrated pulsed amperometric detection

A method of gradient elution ion chromatography with integrated pulsed amperometric detection for simultaneous determination of twenty amino acids and six carbohydrates in soy sauce was established. The effects of column temperature, pH value of solution, and standing time were studied, and then the suitable gradient elution conditions were found for the determination. In the conditions that the flow rate is 0.25 mL/min, pH value of solution is in the range of 5.2-6.7, temperature of column is 35℃, the linear relationships of the 26 components are good and the correlation coefficients are not less than 0.995. Except glutamine, leucine, isoleucine and methionine, the limits of detection (S/N=3) of the other 22 components are less than 0.03 mg/L. The recoveries of the 26 components in soy sauce are 84.2%-109% at the spiked levels of 0.20, 0.50, 2.00 mg/L, and the relative standard deviations (RSDs) are in the range of 2.7%-7.8%. The method is efficient, easy, sensitive, and accurate for simultaneous determination of amino acids and carbohydrates in soy sauce, and can provide an effective research technique for adulterated soy sauce.

Structure analysis of β-glucan in barley and effects of wheat β-glucanase

In this study, β-glucan in samples of sifted flour from six barley varieties was sequentially extracted with water and NaOH obtaining three fractions: water-extractable (WE), NaOH- extractable (NaE) and residual (Res). β-Glucan isolates were incubated with lichenase and oligomers released were analysed with high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). A higher ratio of 3-O-β-cellobiosyl-D-glucose (DP3) to 3-O-β-cellotriosyl-D-glucose (DP4) is reported in WE, NaE and Res fractions in variety SLU 7 (shrunken endosperm) compared to the other varieties, suggesting a more packed β-glucan structure. Water-extractable and water-unextractable β-glucan fractions were incubated with wheat extract and effects of wheat β-glucanase on the structure was assessed after lichenase digestion and analysis with HPAEC-PAD. Findings suggest that wheat β-glucanase manifests a selective hydrolysis towards β-glucan with lower DP3/DP4 ratio and that β-glucan of SLU 7 is composed of a population with higher DP3/DP4 ratio and hence with a more tighten structure which may be more resistant to enzymic action. Overall, the results obtained are of interest to characterise barleys differing in starch and dietary fibre composition, specifically shrunken endosperm barley SLU 7 in the perspective of its inclusion in the production of β-glucan enriched foods.

Differential activity of lytic polysaccharide monooxygenases on celluloses of different crystallinity. Effectiveness in the sustainable production of cellulose nanofibrils

A series of cellulosic substrates has been produced, treated with lytic polysaccharide monooxygenase (LPMO) from Streptomyces ambofaciens (SamLPMO10C), and analyzed by high performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The activity of the bacterial LPMO showed high variability depending on the origin and degree of crystallinity of the substrate. Additionally, we tested the effectiveness of SamLPMO10C in the nanofibrillation of flax, a high crystalline agricultural fiber, as a single pretreatment or in combination with cellulases. All pretreatments were followed by a mechanical defibrillation by high-pressure homogenization (HPH) to obtain cellulose nanofibrils (NFC). The combined LPMO-cellulase treatment showed higher fibrillation yield, optical transmittance and carboxylate content than control reactions. Therefore, it could be explored as a promising green alternative to reduce the energy consumption in the production of NFC. To our knowledge, this is the first study reporting the effect of a bacterial LPMO in nanocellulose production.

Label-free targeted LC-ESI-MS2 analysis of human milk oligosaccharides (HMOS) and related human milk groups with enhanced structural selectivity.

Human milk (HM) supports the healthy development of neonates and exerts many of its beneficial effects via contained free human milk oligosaccharides (HMOS). These HMOS exhibit a complexity and structural diversity that pose a significant analytical challenge. A detailed characterization of HMOS is essential as every individual structure may have a different function/activity. Certain HMOS isomers may even fundamentally differ in their biological function, and especially their characterization by LC or LC-MS is often impaired by co-elution phenomena. Thus, more efficient analytical methodologies with enhanced structural selectivity are required. Therefore, we developed a negative ion mode LC-ESI-MS2 approach featuring straightforward sample preparation, environmentally friendly EtOH gradient elution, and enhanced, semiquantitative characterization of distinct native HMOS by multiple reaction monitoring (MRM). Our MRM-LC-MS setup takes advantage of highly selective, glycan configuration-dependent collision-induced dissociation (CID) fragments to identify individual neutral and acidic HMOS. Notably, many human milk oligosaccharide isomers could be distinguished in a retention time-independent manner. This contrasts with other contemporary MRM approaches relying on rather unspecific MRM transitions. Our method was used to determine the most abundant human milk tri-, tetra-, penta-, and hexaoses semiquantitatively in a single LC-MS assay. Detected HMO structures included fucosyllactoses (e.g., 2'-FL), lacto-N-difucotetraose (LDFT), lacto-N-tetraoses (LNTs), lacto-N-fucopentaoses (e.g., LNFP I, LNFP II and III), lacto-N-difucohexaoses (LNDFHs) as well as sialyllactoses (SLs) and tentatively assigned blood group A and B tetrasaccharides from which correct human milk type assignment could be also demonstrated. Correctness of milk typing was validated for milk groups I-IV by high pressure anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (HPAEC-PAD). Graphical Abstract ᅟ.

Methods for the determination of levoglucosan and other sugar anhydrides as biomass burning tracers in environmental samples - A review.

Nowadays, there is a great pressure on finding an alternative source of energy. One such source is biomass combustion. Biomass is any organic matter such as wood, crops, seaweed, and animal wastes that during combustion emits energy but also smoke and solid residue. Biomass burning tracers, such as levoglucosan, mannosan and galactosan, are sugar anhydrides produced during burning of biomass that contain cellulose and hemicellulose. Analysis of environmental samples for tracers is the source of information about the type of biofuel burned. In this article, a literature review of the preparation and determination of biomass burning tracers for environmental samples was presented. The review discusses the preparation of different samples (particulate matter, soils, sediments, biological samples), extraction, derivatization, and determination. Amongst determination methods the most popular was gas chromatography with mass spectrometry but other techniques were also used, such as high-performance liquid chromatography with aerosol charge detection, capillary electrophoresis with pulsed amperometric detection, and ion chromatography with pulsed amperometric detection.

Determination of Stimulants and Diuretics in Dietary Supplements for Weight Loss and Physical Fitness by Ion-pair Chromatography and Pulsed Amperometric Detection (PAD)

Determination of Stimulants and Diuretics in Dietary Supplements for Weight Loss and Physical Fitness by Ion-pair Chromatography and Pulsed Amperometric Detection (PAD)

Chemical Investigation of Major Constituents in Aloe vera Leaves and Several Commercial Aloe Juice Powders.

Background: There are a substantial number of papers in the scientific literature reporting on the chemical composition of the Aloe vera plant. None of these investigations are truly comprehensive nor address the differences in composition that occur through processing variations in fresh leaves and commercially available product forms. Objectives: This work was to analytically examine a range of these forms and compile the findings. Methods: Fresh A. vera leaves and a number of commercial aloe juice powders were investigated for their major chemical constituents. Samples included fresh leaves from China and Mexico, plus commercial powders from different manufacturers made from different plant parts and/or manufacturing processes. The test results include moisture, ash, fiber, protein, lipids, minerals, organic acids, free sugars, and polysaccharides. The analytical methods employed comprise inductively coupled plasma-optical emission spectroscopy for minerals, high-performance anion-exchange chromatography equipped with pulsed amperometric detection for free sugars, HPLC for organic acids, and size exclusion chromatography (SEC)-multi-angle laser light scattering (MALS)-differential refractive index (dRI) for polysaccharide analyses. The absolute MW and MW distribution were determined using MALS measurement. Results: The major constituents of A. vera fresh leaf are fibers, proteins, organic acids, minerals, monosaccharides, and polysaccharides, which accounted for 85-95% of the total composition determined. In the commercial powdered aloe juice samples, four major components-organic acids, minerals, monosaccharides, and polysaccharides-accounted for 78-84% of the total composition. Apart from the four major components, products manufactured by ethanol precipitation contained high amounts of fiber and protein, while the free sugars were removed. In ethanol-precipitated products, the polysaccharide MW was less affected by manufacturing conditions and the concentration of aloe polysaccharides was higher than in products made in the nonethanol manufacturing processes. The overall chemical profiles were found to be consistent, except for the MW and content of polysaccharides in the commercial aloe samples analyzed, which were largely dependent on the types of manufacturing processes employed. Conclusions: This present study provides a comprehensive investigation of the major chemical composition of A. vera leaf and commercially derived products. The use of the SEC combined with MALS and differential RI detectors has proved to be an improved tool for the accurate determination of polysaccharide MW and contents of the various commercially available A. vera products in this study.

Low Diversity of Human Milk Oligosaccharides is Associated with Necrotising Enterocolitis in Extremely Low Birth Weight Infants.

Difference in human milk oligosaccharides (HMO) composition in breast milk may be one explanation why some preterm infants develop necrotizing enterocolitis (NEC) despite being fed exclusively with breast milk. The aim of this study was to measure the concentration of 15 dominant HMOs in breast milk during the neonatal period and investigate how their levels correlated to NEC, sepsis, and growth in extremely low birth weight (ELBW; <1000 g) infants who were exclusively fed with breast milk. Milk was collected from 91 mothers to 106 infants at 14 and 28 days and at postmenstrual week 36. The HMOs were analysed with high-performance anion-exchange chromatography with pulsed amperometric detection. The HMOs diversity and the levels of Lacto-N-difucohexaose I were lower in samples from mothers to NEC cases, as compared to non-NEC cases at all sampling time points. Lacto-N-difucohexaose I is only produced by secretor and Lewis positive mothers. There were also significant but inconsistent associations between 3′-sialyllactose and 6′-sialyllactose and culture-proven sepsis and significant, but weak correlations between several HMOs and growth rate. Our results suggest that the variation in HMO composition in breast milk may be an important factor explaining why exclusively breast milk fed ELBW infants develop NEC.