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Study design A pilot study for the analysis of the metabolome of the cell fraction of human milk was conducted. The study included fifteen HM samples collected at different postnatal ages and labeled as colostrum (n=1,1-3 days), transitional (n=3, 4-14 days) or mature (n=11, >14 days) milk. The cellular fraction was studied with microscopy and staining techniques, and their metabolic fingerprint was analyzed by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC–QqTOF-MS) in the positive and negative electrospray ionization modes. Sample processing & metabolomic analysis A 15 mL aliquot of fresh HM was centrifuged during 20 min at 800 x g and 4 °C. The supernatant was discarded, and the pellet was resuspended in PBS and centrifuged during 5 min at 800 x g and 4 °C. The washing step was repeated. For cell lysis and metabolite extraction, 300 µL of lysis solution (i.e., CH3OH:H2O 3:1, v/v) containing the internal standards (IS) L-tryptophan-D5 (≥99%), caffeine-D9 (≥99%), and L-phenylalanine-D5 (≥99%) at 0.25 μM each were added to the cell pellet. The mixture was transferred to a microcentrifuge tube placed on ice and 200 µL of the lysis solution were additionally employed to ensure quantitative transfer of cells. After three freeze-thaw cycles, samples were stored at -80 °C until further processing. After thawing, the samples were centrifuged at 15493 x g and 4 °C for 5 minutes. The supernatant was transferred to a microcentrifuge tube and evaporated under vacuum at 30 °C on a miVac centrifugal vacuum concentrator (Genevac LTD, Ipswich, UK). Dry residues were resuspended in 70 µL of H2O:CH3CN (85:15 v/v, 0.1% v/v HCOOH) and homogenized, followed by a centrifugation at 15493 x g and 4 °C for 5 minutes to remove cell debris. Supernatants were transferred to microcentrifuge tubes and stored at -80 °C until analysis. A pooled quality control (QC) sample was prepared by mixing 10 µL of each sample extract. A blank extract was prepared following the same procedure as described for samples, but without cells. In addition, the supernatant obtained from the second washing step of one sample was processed as described for samples and analyzed. Metabolomic analysis of HM cell extracts was carried out by UPLC–QqTOF-MS as described elsewhere [1]. Briefly, separations were performed on a SynergiTM Hydro-RP 80 Å LC C18 column (150 × 2 mm, 4 μm, Phenomenex, Torrance, USA) in a 1290 Infinity UPLC system running the following binary gradient with solvent A (H2O, 0.1% v/v HCOOH) and solvent B (CH3CN, 0.1% v/v HCOOH) as mobile phase components: 1% B for 2 min, linear gradient from 1 to 80% B in 8 min, from 80 to 98% B in 0.1 min, 98% B for 1.9 min, return to initial conditions in 0.1 min, and column equilibration with 1% B during 2.9 min. The flow rate was set to 0.4 mL min-1 and column and autosampler temperatures were set at 40 and 4 °C, respectively. For MS detection an Agilent 6550 Spectrometer iFunnel quadrupole time-of-flight (QqTOF) MS system was used operating in ESI+ and ESI- modes. Full scan MS data was acquired between 70 and 1500 m/z using the following ionization parameters: gas T, 200 ⁰C; drying gas, 14 L min-1; nebulizer, 30 psi; sheath gas T, 350 ⁰C; sheath gas flow, 10 L min-1. Mass reference standards were introduced into the source for automatic MS spectra recalibration during analysis via a reference sprayer valve using the 149.02332 (background contaminant), 121.050873 (purine), and 922.009798 (HP-0921) m/z as references in ESI+, and 119.036 (purine) and 980.0163 ([HP-0921 + CH3COOH-H]-) m/z in ESI-. MS2 data were acquired using iterated data dependent acquisition as described elsewhere [2] using centroid mode at a rate of 5 Hz in the extended dynamic range mode (2 GHz), a collision energy set to 20 V, medium isolation window (~4 amu), MS2 fragmentation with automated selection of five precursor ions per cycle, and an exclusion window of 0.15 min after two consecutive selections of the same precursor. UPLC-MS data acquisition was carried out employing MassHunter Workstation (version B.07.00) from Agilent. The analytical batch included an initial system suitability check (2 μM IS solution) followed by 9 QCs replicates for system conditioning and MS2 data acquisition. HM sample extracts were injected in random order. The QC was injected once at the beginning, twice at the end, and during the batch for the assessment of instrumental performance [3]. The blank extract was injected twice and the blank extract from cell supernatant once at the end of the measurement sequence to identify signals from other than biological origin, and possible carryover [4]. Data description From de ESI+ and ESI- analyses: 21 .mzXML files including MS1 data of 15 samples, 4 QCs and 2 blanks 9 .ms2 files including MS/MS data of QC injections 9 .mgf files including MS/MS data of QC injections .xlsx file with metadata (results from the cytochemical and immunocytochemical characterization of HM cells have been included), {"references":["Ten-Doménech I, Ramos-Garcia V, Moreno-Torres M, et al (2022) The effect of Holder pasteurization on the lipid and metabolite composition of human milk. Food Chem 132581. https://doi.org/10.1016/j.foodchem.2022.132581","Ten-Doménech I, Martínez-Sena T, Moreno-Torres M, et al (2020) Comparing Targeted vs. Untargeted MS2 Data-Dependent Acquisition for Peak Annotation in LC–MS Metabolomics. Metabolites 10:126. https://doi.org/10.3390/metabo10040126","Broadhurst D, Goodacre R, Reinke SN, et al (2018) Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies. Metabolomics Off J Metabolomic Soc 14:72. https://doi.org/10.1007/s11306-018-1367-3","Martínez-Sena T, Luongo G, Sanjuan-Herráez D, et al (2019) Monitoring of system conditioning after blank injections in untargeted UPLC-MS metabolomic analysis. Sci Rep 9:1–9. https://doi.org/10.1038/s41598-019-46371-w"]}

Transposition of the great arteries (TGA), a congenital heart disease characterized by hypoxemia and abnormal circulation, is responsible for brain alterations and long-term neurodevelopmental delay. Atrial septostomy causes a sudden increase in arterial blood oxygenation leading to complex metabolic changes. We studied the metabolic switch in infants with TGA after atrial septostomy in order to shed light on the effect of the changes in cerebral oxygenation. Blood samples of 9newborns with TGA were collected 5 minutes before and at several time points after the intervention (i.e. 5 min, 6 h, 24 h, 48 h, and 72 h). Plasma samples were measured employing Ultra-Performance-Liquid-Chromatography quadrupole-time-of-flight-Mass-Spectrometry (UPLC-qTOFMS) operating in positive and negative electrospray mode. Peak tables were extracted using the XCMS online platform and within-batch effect was corrected employing quality control samples and support vector regression. After data clean-up and MS/MS-based annotation, statistical analysis was performed employing Principal Component Analysis (PCA), Pearson correlations, and network analysis (mummichog algorithm, and Metaboanalyst). We observed correlation of metabolic features with cerebral tissue oxygen extraction (FTOE) and affected pathways with significant differences included pentose phosphate pathway (P-value = 0. 00002), pentose and glucuronate interconversions (P-value = 0. 003), ascorbate and aldarate metabolism (P-value = 0. 013), inositol phosphate metabolism (P-value = 0. 03), and primary bile acid biosynthesis (P-value = 0. 04).

The estimation of steatosis in a liver graft is mandatory prior to liver transplantation, as the risk of graft failure increases with the level of infiltrated fat. However, the assessment of liver steatosis before transplantation is typically based on a qualitative or semi-quantitative characterization by visual inspection and palpation, and histological analysis. Thus, there is an unmet need for transplantation surgeons to have access to a diagnostic tool enabling an in situ fast classification of grafts prior extraction. In this study, we have assessed an Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopic method compatible with the requirements of an operation room, for the evaluation of the lipid contents in human livers. A set of 20 human liver biopsies obtained from organs intended for transplantation were analyzed by expert pathologists, ATR-FTIR spectroscopy, lipid biochemical analysis, and UPLC-ESI(+/-)TOFMS for lipidomic profiling. Comparative analysis of multi-source data showed strong correlations between ATR-FTIR, clinical and lipidomic information. Results show that ATR-FTIR captures a global picture of the lipid composition of the liver, along with information for the quantification of the TAGs content in liver biopsies. Although the methodology performance needs to be further validated, results support the applicability of ATR-FTIR for the in situ determination of the grade of liver steatosis at the operation room as a fast, quantitative method, alternative to the qualitative and subjective pathological examination This data set includes the LC-MS annotated peak table, ATR-FTIR and clinical data, and MATLAB scripts used for data analysis. SM_ATRFTIR.xlxs: ATR-FTIR spectra from dry-film and tissue (liver) samples SM_LCMS.xlsx: Annotated peak table from the lipidomic analysis of tissue (liver) samples by UPLC-ESI(+/-)-TOFMS plot_idclasses3dso.m: MATLAB function to reproduce the distribution of lipid classes or subclasses in the data set object Liver_IRMS.mat: MATLAB file including the ATR-FTIR, LC-MS, reference and clinical information used in the study. Liver_IRMS.mlx (and Liver_IRMS.pdf): MATLAB .mlx file with the code required to reproduce results included in the study.

The dataset includes the annotated features from the peak table generated using XCMS software and UPLC-TOFMS data acquired from the analysis of plasma samples from 47 quiescent UC and CD patients (23 fatigued, 24 non-fatigued), for the identification of a metabolic phenotype associated to fatigue (SM_peak_table.csv). It also includes the network of protein-protein and protein-metabolite interactions inferred by PIUMet (SM_output_network.html) using a set of 180 annotated features selected as discriminant between fatigue and non-fatigued by univariate (t-test) or multivariate (PLSDA) analysis. UC: ulcerative colitis CD: Crohn disease PIUMet: L. Pirhaji, P. Milani, M. Leidl, T. Curran, J. Avila-Pacheco, C.B. Clish, F.M. White, A. Saghatelian, E. Fraenkel, Revealing disease-associated pathways by network integration of untargeted metabolomics, Nat. Methods, 13 (2016) 770–776.

Fetal life evolves in a hypoxic environment. Changes in the oxygen content in utero caused by conditions such as pre-eclampsia or type I diabetes or by oxygen supplementation to the mother lead to increased free radical production and correlate with perinatal outcomes. In the fetal-to-neonatal transition asphyxia is characterized by intermittent periods of hypoxia ischemia that may evolve to hypoxic ischemic encephalopathy associated with neurocognitive, motor, and neurosensorial impairment. Free radicals generated upon reoxygenation may notably increase brain damage. Hence, clinical trials have shown that the use of 100% oxygen given with positive pressure in the airways of the newborn infant during resuscitation causes more oxidative stress than using air, and increases mortality. Preterm infants are endowed with an immature lung and antioxidant system. Clinical stabilization of preterm infants after birth frequently requires positive pressure ventilation with a gas admixture that contains oxygen to achieve a normal heart rate and arterial oxygen saturation. In randomized controlled trials the use high oxygen concentrations (90% to 100%) has caused more oxidative stress and clinical complications that the use of lower oxygen concentrations (30–60%). A correlation between the amount of oxygen received during resuscitation and the level of biomarkers of oxidative stress and clinical outcomes was established. Thus, based on clinical outcomes and analytical results of oxidative stress biomarkers relevant changes were introduced in the resuscitation policies. However, it should be underscored that analysis of oxidative stress biomarkers in biofluids has only been used in experimental and clinical research but not in clinical routine. The complexity of the technical procedures, lack of automation, and cost of these determinations have hindered the routine use of biomarkers in the clinical setting. Overcoming these technical and economical difficulties constitutes a challenge for the immediate future since accurate evaluation of oxidative stress would contribute to improve the quality of care of our neonatal patients., Graphical abstract fx1, Highlights • Fetal life elapses in a low oxygen environment. • Hypoxia and Hyperoxia after birth cause oxidative stress and free radical derived conditions. • Asphyxia causes acute and long-term complications. • Preterm infants are susceptible to free radical associated diseases. • The use of lower oxygen to stabilize preterm infants improves clinical outcome. • Oxidative stress biomarkers correlate with clinical outcomes.

The coupling of liquid chromatography (LC) to infrared (IR) spectrometry is an interesting analytical tool, because high resolution provided by LC is combined with the non-destructive and molecular specific information of IR spectrometry. However, the potential of on-line LC-IR employing a flow cell interface, characterized by its technical simplicity, cannot yet be fully exploited due to the limited sensitivity of IR detection and difficulties in on-line measurements arising from the dominating absorption of most of the commonly used mobile phase components. Accordingly, the objective of this Thesis was to promote the use of the on-line hyphenation of LC and IR spectroscopy through the development of instrumental and chemometric tools to overcome existing limitations, thus increasing the applicability of this technique. Chapter 2 describes innovations in the field of isocratic separation. A method for the determination of lecithin and soybean oil in dietary supplements using on-line Gel Permeation Chromatography – Attenuated Total Reflectance – Infrared (GPC-ATR-IR) was developed including a simple procedure to select the optimum wavenumber used for the extraction of characteristic elution profiles. For the monitoring of polymerized triglycerides in deep-frying oil, an approach employing on-line GPC-IR spectrometry and the Science Based Calibration (SBC) multivariate approach for chromatogram-extraction were reported. An on-line LC-IR procedure for the determination of glycolic acid in cosmetics employing the rapid scan data acquisition mode was proposed. Chapter 3 deals with different methods for background correction in on-line LC-IR using reference spectra matrices (RSM). The determination of the critical eluent composition for polyethylene glycols using methanol as organic modifier was discussed employing the absorbance ratio as identification parameter for background correction. For the determination of fructose, glucose, sucrose and maltose in beverages employing acetonitrile as organic modifier, again the absorbance ratio was used as identification parameter. A Partial Least Squares (PLS) procedure as well as the application of point-to-point matching algorithms for automated background correction were discussed for background correction in acetonitrile:water gradients using different examples of applications. Chapter 4 deals with the compensation of the background contribution employing column-wise techniques. Polynomial regressions modelling the background absorbance at each wavenumber throughout the gradient run were applied for acetonitrile:water mobile phase gradients. The use of cubic smoothing splines could be demonstrated for background correction under reversed phase gradient conditions employing different alcohols as organic modifiers. In Chapter 5, background correction based on factor analysis and reference spectra matrices was discussed. The contribution of the background absorption to overall LC-IR signals could be compensated using two approaches based on Principal Component Analysis (PCA) and simple-to-use interactive self modeling analysis (SIMPLISMA), obtaining good results in acetonitrile:water systems. Subsequently, multivariate curve resolution-alternating least squares (MCR-ALS) was applied improving peak and spectral resolution and eliminating remaining signal variation due to background absorption and detector drift. The chemometric extraction of ‘analyte-specific’ chromatograms in on-line LC-IR was discussed in Chapter 6. Obtained results confirm that the SBC method is particularly well suited for recovering ‘analyte-specific’ signals from on-line LC-IR chromatograms. The last chapter describes instrumental developments in on-line LC-IR. On-line IR detection in gradient capillary LC using micromachined nanoliter-flow cells was discussed. Four model compounds were separated and identified using an acetonitrile:H2O gradient with limits of detection in the concentration range of 35-94 ng μL-1, representing an increase in mass sensitivity by a factor of approximately 30 as compared to standard LC systems. The last chapter also focuses on the development of LC with on-line dual Quantum Cascade Laser (QC-laser) detection. Compared to state-of-the-art FTIR spectrometers, the use of the developed QC-laser based system provided a significant improvement in both, sensitivity and data acquisition frequency., The coupling of liquid chromatography (LC) to infrared (IR) spectrometry is an interesting analytical tool, because high resolution provided by LC is combined with the non-destructive and molecular specific information of IR spectrometry. However, the potential of on-line LC-IR employing a flow cell interface, characterized by its technical simplicity, cannot yet be fully exploited due to the limited sensitivity of IR detection and difficulties in on-line measurements arising from the dominating absorption of most of the commonly used mobile phase components. Accordingly, the objective of this Thesis was to promote the use of the on-line hyphenation of LC and IR spectroscopy through the development of instrumental and chemometric tools to overcome existing limitations, thus increasing the applicability of this technique. Chapter 2 describes innovations in the field of isocratic separation. A method for the determination of lecithin and soybean oil in dietary supplements using on-line Gel Permeation Chromatography – Attenuated Total Reflectance – Infrared (GPC-ATR-IR) was developed including a simple procedure to select the optimum wavenumber used for the extraction of characteristic elution profiles. For the monitoring of polymerized triglycerides in deep-frying oil, an approach employing on-line GPC-IR spectrometry and the Science Based Calibration (SBC) multivariate approach for chromatogram-extraction were reported. An on-line LC-IR procedure for the determination of glycolic acid in cosmetics employing the rapid scan data acquisition mode was proposed. Chapter 3 deals with different methods for background correction in on-line LC-IR using reference spectra matrices (RSM). The determination of the critical eluent composition for polyethylene glycols using methanol as organic modifier was discussed employing the absorbance ratio as identification parameter for background correction. For the determination of fructose, glucose, sucrose and maltose in beverages employing acetonitrile as organic modifier, again the absorbance ratio was used as identification parameter. A Partial Least Squares (PLS) procedure as well as the application of point-to-point matching algorithms for automated background correction were discussed for background correction in acetonitrile:water gradients using different examples of applications. Chapter 4 deals with the compensation of the background contribution employing column-wise techniques. Polynomial regressions modelling the background absorbance at each wavenumber throughout the gradient run were applied for acetonitrile:water mobile phase J. Kuligowski | NEW INSTRUMENTAL AND CHEMOMETRIC DEVELOPMENTS FOR THE ON-LINE HYPHENATION OF LIQUID CHROMATOGRAPHY AND INFRARED SPECTROSCOPY II gradients. The use of cubic smoothing splines could be demonstrated for background correction under reversed phase gradient conditions employing different alcohols as organic modifiers. In Chapter 5, background correction based on factor analysis and reference spectra matrices was discussed. The contribution of the background absorption to overall LC-IR signals could be compensated using two approaches based on Principal Component Analysis (PCA) and simple-to-use interactive self modeling analysis (SIMPLISMA), obtaining good results in acetonitrile:water systems. Subsequently, multivariate curve resolution-alternating least squares (MCR-ALS) was applied improving peak and spectral resolution and eliminating remaining signal variation due to background absorption and detector drift. The chemometric extraction of ‘analyte-specific’ chromatograms in on-line LC-IR was discussed in Chapter 6. Obtained results confirm that the SBC method is particularly well suited for recovering ‘analyte-specific’ signals from on-line LC-IR chromatograms. The last chapter describes instrumental developments in on-line LC-IR. On-line IR detection in gradient capillary LC using micromachined nanoliter-flow cells was discussed. Four model compounds were separated and identified using an acetonitrile:H2O gradient with limits of detection in the concentration range of 35-94 ng μL-1, representing an increase in mass sensitivity by a factor of approximately 30 as compared to standard LC systems. The last chapter also focuses on the development of LC with on-line dual Quantum Cascade Laser (QC-laser) detection. Compared to state-of-the-art FTIR spectrometers, the use of the developed QC-laser based system provided a significant improvement in both, sensitivity and data acquisition frequency.