Metabolic Fingerprint in Childhood Acute Lymphoblastic Leukemia.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy. Despite high cure rates, several questions remain regarding predisposition, response to treatment, and prognosis of the disease. The role of intermediary metabolism in the individualized mechanistic pathways of the disease is unclear. We have hypothesized that children with any (sub)type of ALL have a distinct metabolomic fingerprint at diagnosis when compared: (i) to a control group; (ii) to children with a different (sub)type of ALL; (iii) to the end of the induction treatment. MATERIALS AND METHODS: In this prospective case-control study (NCT03035344), plasma and urinary metabolites were analyzed in 34 children with ALL before the beginning (D0) and at the end of the induction treatment (D33). Their metabolic fingerprint was defined by targeted analysis of 106 metabolites and compared to that of an equal number of matched controls. Multivariate and univariate statistical analyses were performed using SIMCAP and scripts under the R programming language. RESULTS: Metabolomic analysis showed distinct changes in patients with ALL compared to controls on both D0 and D33. The metabolomic fingerprint within the patient group differed significantly between common B-ALL and pre-B ALL and between D0 and D33, reflecting the effect of treatment. We have further identified the major components of this metabolic dysregulation, indicating shifts in fatty acid synthesis, transfer and oxidation, in amino acid and glycerophospholipid metabolism, and in the glutaminolysis/TCA cycle. CONCLUSIONS: The disease type and time point-specific metabolic alterations observed in pediatric ALL are of particular interest as they may offer potential for the discovery of new prognostic biomarkers and therapeutic targets.

Detection of 26 Drugs of Abuse and Metabolites in Quantitative Dried Blood Spots by Liquid Chromatography-Mass Spectrometry.

A method was developed for the determination of 26 drugs of abuse from different classes, including illicit drugs in quantitative dried blood spots (qDBSs), with the aim to provide a convenient method for drug testing by using only 10 µL of capillary blood. A satisfactory limit of quantification (LOQ) of 2.5 ng/mL for 9 of the compounds and 5 ng/mL for 17 of the compounds and a limit of detection (LOD) of 0.75 ng/mL for 9 of the compounds and 1.5 ng/mL for 17 of the compounds were achieved for all analytes. Reversed-phase liquid chromatography was applied on a C18 column coupled to MS, providing selective detections with both +ESI and -ESI modes. Extraction from the qDBS was performed using AcN-MeOH, 1:1 (v/v), with recovery ranging from 84.6% to 106%, while no significant effect of the hematocrit was observed. The studied drugs of abuse were found to be stable over five days under three different storage conditions (at ambient temperature 21 °C, at -20 °C, and at 35 °C), thus offering a highly attractive approach for drug screening by minimally invasive sampling for individuals that could find application in forensic toxicology analysis.

Establishing a framework for best practices for quality assurance and quality control in untargeted metabolomics.

BACKGROUND: Quality assurance (QA) and quality control (QC) practices are key tenets that facilitate study and data quality across all applications of untargeted metabolomics. These important practices will strengthen this field and accelerate its success. The Best Practices Working Group (WG) within the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) focuses on community use of QA/QC practices and protocols and aims to identify, catalogue, harmonize, and disseminate current best practices in untargeted metabolomics through community-driven activities. AIM OF REVIEW: A present goal of the Best Practices WG is to develop a working strategy, or roadmap, that guides the actions of practitioners and progress in the field. The framework in which mQACC operates promotes the harmonization and dissemination of current best QA/QC practice guidance and encourages widespread adoption of these essential QA/QC activities for liquid chromatography-mass spectrometry. KEY SCIENTIFIC CONCEPTS OF REVIEW: Community engagement and QA/QC information gathering activities have been occurring through conference workshops, virtual and in-person interactive forum discussions, and community surveys. Seven principal QC stages prioritized by internal discussions of the Best Practices WG have received participant input, feedback and discussion. We outline these stages, each involving a multitude of activities, as the framework for identifying QA/QC best practices. The ultimate planned product of these endeavors is a "living guidance" document of current QA/QC best practices for untargeted metabolomics that will grow and change with the evolution of the field.

Dried urine spot (DUS) applied for sampling prior to the accurate HILIC-MS/MS determination of 14 amino acids.

Urine amino acid analysis has proven valuable for an array of clinical or nutritional studies. However, transportation of liquid urine sample shows certain disadvantages, such as possible leakage, need for cold chain and thus higher costs for their transport. Utilization of dried urine spots (DUS) can offer an interesting alternative. In the present study, a method was developed for the determination of 14 amino acids in DUS including the testing of in-house collection device and drying of the sample before analysis. Normal filter paper was tested as the means for sample collection. Absorption and extraction experiments were performed on 3 different types of filter paper, with 3 different extraction solvents and two different solvent volumes. The solvents used were mixtures of common analytical solvents (methanol, water, acetonitrile) using total volumes of 1 mL and 1.5 mL. Finally, 1 mL of acetonitrile: methanol: water 40:40:20 (v/v/v) was chosen as the optimal system. Analysis was performed on a UHPLC-MS system, using stable isotope labeled internal standards. Method validation included the study of limits of detection (LOD) and quantification (LOQ), linearity ranges, precision, matrix effect, extraction recovery, precision, and stability for each analyte. The obtained results were satisfactory, thus enabling application of the proposed method as an alternative to the analysis of liquid urine. Further utilization of DUS can offer advantages by enabling patient centric sampling even in long distances far from the analytical laboratories.

Determination of plastic polyester oligomers in real samples and their bioeffects.

Plastics are ubiquitously, becoming part of our everyday life. Recently, the issue of human exposure to micro- and nanoplastic particles and potentially resulting toxicological consequences has been broached, triggered by the discovery of microplastics in foodstuff and dietary exposure via contaminated food and beverages. Within this EU-FORA fellowship project, a determination and quantification of plastic polyester plastics oligomers in food samples was performed to assess exposure at these categories of 'nanoplastics', evaluating them as potential contaminants or as indicators and marker compounds of the exposure to specific nanoplastics/microplastics from polyesters, such as polyethylene terephthalate (PET) and polybutylene terephthalate (PBT). UHPLC-TOF-MS/MS analysis has been set-up for 10 PET and PBT oligomers and analysis has been performed in foods and drinks. Moreover, the project focused also on the effects of these oligomers in in vitro and ex vivo experiments. These data would be combined with EFSA Comprehensive Food Consumption Database, for the exposure and risk assessment of these 'Nanoplastics'.

Current Practices in LC-MS Untargeted Metabolomics: A Scoping Review on the Use of Pooled Quality Control Samples.

Untargeted metabolomics is an analytical approach with numerous applications serving as an effective metabolic phenotyping platform to characterize small molecules within a biological system. Data quality can be challenging to evaluate and demonstrate in metabolomics experiments. This has driven the use of pooled quality control (QC) samples for monitoring and, if necessary, correcting for analytical variance introduced during sample preparation and data acquisition stages. Described herein is a scoping literature review detailing the use of pooled QC samples in published untargeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics studies. A literature query was performed, the list of papers was filtered, and suitable articles were randomly sampled. In total, 109 papers were each reviewed by at least five reviewers, answering predefined questions surrounding the use of pooled quality control samples. The results of the review indicate that use of pooled QC samples has been relatively widely adopted by the metabolomics community and that it is used at a similar frequency across biological taxa and sample types in both small- and large-scale studies. However, while many studies generated and analyzed pooled QC samples, relatively few reported the use of pooled QC samples to improve data quality. This demonstrates a clear opportunity for the field to more frequently utilize pooled QC samples for quality reporting, feature filtering, analytical drift correction, and metabolite annotation. Additionally, our survey approach enabled us to assess the ambiguity in the reporting of the methods used to describe the generation and use of pooled QC samples. This analysis indicates that many details of the QC framework are missing or unclear, limiting the reader's ability to determine which QC steps have been taken. Collectively, these results capture the current state of pooled QC sample usage and highlight existing strengths and deficiencies as they are applied in untargeted LC-MS metabolomics.

Volumetric Absorptive Microsampling in the Analysis of Endogenous Metabolites.

Volumetric absorptive microsampling (VAMS) has arisen as a relevant tool in biological analysis, offering simplified sampling procedures and enhanced stability. Most of the attention VAMS has received in the past decade has been from pharmaceutical research, with most of the published work employing VAMS targeting drugs or other exogenous compounds, such as toxins and pollutants. However, biomarker analysis by employing blood microsampling has high promise. Herein, a comprehensive review on the applicability of VAMS devices for the analysis of endogenous metabolites/biomarkers was performed. The study presents a full overview of the analysis process, incorporating all the steps in sample treatment and validation parameters. Overall, VAMS devices have proven to be reliable tools for the analysis of endogenous analytes with biological importance, often offering improved analyte stability in comparison with blood under ambient conditions as well as a convenient and straightforward sample acquisition model.

Analysis of Migrant Cyclic PET Oligomers in Olive Oil and Food Simulants Using UHPLC-qTOF-MS.

Oligomers are a particular category of non-intentionally added substances (NIAS) that may be present in food contact materials (FCMs), such as polyethylene terephthalate (PET), and consequently migrate into foods. Here, an ultra-high-pressure liquid chromatography quadruple time-of-flight mass spectrometry (UHPLC-qTOF-MS) method was developed for the analysis of 1st series cyclic PET oligomers in virgin olive oil (VOO) following a QuEChERS clean-up protocol. Oligomer migration was evaluated with two different migration experiments using bottles from virgin and recycled PET: one with VOO samples stored in household conditions for a year and one using the food simulant D2 (95% v/v ethanol in water) at 60 °C for 10 days. Calibration curves were constructed with fortified VOO samples, with the LOQs ranging from 10 to 50 µg L-1 and the recoveries ranging from 86.6 to 113.0%. Results showed no migration of PET oligomers in VOO. However, in the simulated study, significant amounts of all oligomers were detected, with the migration of cyclic PET trimers from recycled bottles being the most abundant. Additional substances were tentatively identified as linear derivatives of PET oligomers. Again, open trimer structures in recycled bottles gave the most significant signals.

Analysis of oligomers to assess exposure to microplastics from foods. A perspective.

There is an emerging interest in evaluating the presence of microplastic (MP) and nanoplastic (NP) residues in food. Despite their potential threat to human health, there is still a need for harmonized methods to evaluate and quantify their presence. Incomplete polymerization may occur during the production of plastic. Conversely, oligomers are formed during chemical, mechanical, or enzymatic depolymerization. Oligomers are a few nanometers in size. Recent advances in analytical chemistry have enabled the quantification and identification of these oligomers in various complex biological matrices. Therefore, we propose that the specific nanosized oligomers can be considered markers for the presence of MPs/NPs. This advance may facilitate a broader perspective for the assessment of MPs/NPs exposure, leading to the evaluation of food safety and associated risks to humans.

Untargeted screening and in silico toxicity assessment of semi- and non-volatile compounds migrating from polysaccharide-based food contact materials.

The chemical safety of representative polysaccharide films made with pea starch, organocatalytic acetylated pea starch and pectin was investigated at different migration conditions (20 °C/10 days, 70 °C/2 h) using two official simulants signifying hydrophilic (simulant A, 10% ethanol) or lipophilic (simulant D1, 50% ethanol) foods. Migrating semi-volatile and non-volatile compounds were identified and semi-quantified by ultra-high performance liquid chromatography-trap ion mobility time-of-flight mass spectrometry (UHPLC-TIMS-TOF-MS/MS), whereas their toxicity was evaluated by in silico models based on qualitative structure activity (QSAR). Physicochemical analysis revealed polymer wash-off into the simulants. Migration testing at 70 °C for 2 h using simulant D1 resulted in detectable concentrations of glycerol (≤72.1 mg/kg), monoacetylated maltose (≤6.5 mg/kg), and dibutyl phthalate (DBP) (≤0.5 mg/kg, compliant with the existing legislative migration limits) in samples containing acetylated starch. Migrating 3-ß-galactopyranosyl glucose (≤8.9 mg/kg) and 2,5-diketo-d-gluconic acid (≤4.9 mg/kg) were detected at 20 °C/10 days. In-silico toxicity emphasized no significant toxicity and categorized organocatalytic acetylated pea starch of no safety concern.

Metabolic Fingerprinting of Muscat of Alexandria Grape Musts during Industrial Alcoholic Fermentation Using HS-SPME and Liquid Injection with TMS Derivatization GC-MS Methods.

Muscat of Alexandria is one of the most aromatic grape cultivars, with a characteristic floral and fruity aroma, producing popular appellation of origin wines. The winemaking process is a critical factor contributing to the quality of the final product, so the aim of this work was to study metabolomic changes during the fermentation of grape musts at the industrial level from 11 tanks, 2 vintages, and 3 wineries of Limnos Island. A Headspace Solid-Phase Microextraction (HS-SPME) and a liquid injection with Trimethylsilyl (TMS) derivatization Gas Chromatography-Mass Spectrometry (GC-MS) methods were applied for the profiling of the main volatile and non-volatile polar metabolites originating from grapes or produced during winemaking, resulting in the identification of 109 and 69 metabolites, respectively. Multivariate statistical analysis models revealed the differentiation between the four examined time points during fermentation, and the most statistically significant metabolites were investigated by biomarker assessment, while their trends were presented with boxplots. Whilst the majority of compounds (ethyl esters, alcohols, acids, aldehydes, sugar alcohols) showed an upward trend, fermentable sugars, amino acids, and C6-compounds were decreased. Terpenes presented stable behavior, with the exception of terpenols, which were increased at the beginning and were then decreased after the 5th day of fermentation.

Optimization of Carob Products Preparation for Targeted LC-MS/MS Metabolomics Analysis.

Carob (Ceratonia siliqua) is an exceptional source of significant bioactive compounds with great economic importance in the Mediterranean region, where it is widely cultivated. Carob fruit is used for the production of a variety of products and commodities such as powder, syrup, coffee, flour, cakes, and beverages. There is growing evidence of the beneficial effects of carob and the products made from it on a range of health problems. Therefore, metabolomics could be used to explore the nutrient-rich compounds of carob. Sample preparation is a crucial step in metabolomics-based analysis and has a great impact on the quality of the data obtained. Herein, sample preparation of carob syrup and powder was optimized, to enable highly efficient metabolomics-based HILIC-MS/MS analysis. Pooled powder and syrup samples were extracted under different conditions by adjusting pH, solvent type, and sample weight to solvent volume ratio (Wc/Vs). The metabolomics profiles obtained were evaluated using the established criteria of total area and number of maxima. It was observed that the Wc/Vs ratio of 1:2 resulted in the highest number of metabolites, regardless of solvent type or pH. Aqueous acetonitrile with a Wc/Vs ratio of 1:2 satisfied all established criteria for both carob syrup and powder samples. However, when the pH was adjusted, basic aqueous propanol 1:2 Wc/Vs and acidic aqueous acetonitrile 1:2 Wc/Vs provided the best results for syrup and powder, respectively. We strongly believe that the current study could support the standardization of the metabolomics sample preparation process to enable more efficient LC-MS/MS carob analysis.

Untargeted UHPLC-TOF/MS Lipidomic Analysis for the Investigation of Egg Yolks after Xylanase Supplementation of the Diet of Laying Hens.

Xylanase supplementation of diets is used to enhance nutrient digestibility in monogastrics which lack necessary enzymes for non-starch polysaccharide degradation. The effects of enzymatic treatment in the nutritional value of the feed are typically not comprehensively studied. Though the fundamental effects of xylanase on performance are well studied, limited data is available on the complex interactions between xylanase supplementation and hen physiology; therefore, the aim of this study was to develop a new, simple UPLC-TOF/MS lipidomics method for the analysis of hen egg yolks after supplementation with different amounts of xylanase. Sample preparation for the extraction of lipids was optimized and different sample preparation modes and solvent mixtures were tested. Optimal results for the extraction of total lipids were obtained by using the solvent mixture MTBE: MeOH (5:1, v/v). Multivariate statistical analysis of the signals of hundreds of lipids in positive and negative ionisation modes highlighted differences in several egg yolk lipid species-classes. Four lipid species-classes, phosphatidylcholines (PC and PC O), phosphatidylethanolamines (PE and PE O), phosphatidylinositols (PI), and fatty acids (FA), were among those contributing to the separation of the experimental groups (control-treated) in negative ionisation mode. In positive ionisation mode, principal beneficial lipid compounds such as phosphatidylcholines (PC and PC O), phosphatidylethanolamines (PE and PE O), triacylglycerols (TG), diacylglycerols (DG), and ceramides (Cer) were found to be increased in treated groups. Overall, supplementation of laying hens' diets with xylanase significantly changed the lipid profile of egg yolks compared to the control diet. The association between the lipid profiles of egg yolks and hens' diets, as well as the underlying mechanisms, require further investigation. These findings are of practical significance for the food industry.

The impact of pharmaceutical pollutants on daphnids - A metabolomic approach.

Pharmaceuticals have been classified as emerging contaminants in the aquatic ecosystem, mainly due to their increased use and improper disposal. A significant range of pharmaceutical compounds and their metabolites have been globally detected in surface waters and pose detrimental effects to non-target organisms. Monitoring pharmaceutical water pollution relies on the analytical approaches for their detection, however, such approaches are limited by their sensitivity limit and coverage of the wide range pharmaceutical compounds. This lack of realism in risk assessment is bypassed with effect-based methods, which are complemented by chemical screening and impact modelling, and are able to provide mechanistic insight for pollution. Focusing on the freshwater ecosystem, in this study we evaluated the acute effects on daphnids for three distinct groups of pharmaceuticals; antibiotics, estrogens, and a range of commonly encountered environmentally relevant pharmaceutical pollutants. Combining several endpoints such as mortality, biochemical (enzyme activities) and holistic (metabolomics) we discovered distinct patterns in biological responses. In this study, changes in enzymes of metabolism e.g. phosphatases and lipase, as well as the detoxification enzyme, glutathione-S-transferase, were recorded following acute exposure to the selected pharmaceuticals. A targeted analysis of the hydrophilic profile of daphnids revealed mainly the up-regulation of metabolites following metformin, gabapentin, amoxicillin, trimethoprim and ß-estradiol. Whereas gemfibrozil, sulfamethoxazole and oestrone exposure resulted in the down-regulation of majority of metabolites.

Ceramides biomarkers determination in quantitative dried blood spots by UHPLC-MS/MS.

A method was developed for the analysis of four ceramide species; namely C16:0, C18:0, C24:0 and C24:1 in quantitative Dried Blood Samples (qDBS) by LC-MS/MS and validated with the aim to give prominence to an interesting application of at-home blood microsampling for health monitoring. Ceramides, being key-role metabolites implicated in regulation of diverse cellular processes have been considered as emerging biomarkers for different disease states, such as cardiovascular diseases, type 2 diabetes and others. Here, Capitainer device was utilized to provide accurate and consistent volumes of samples, ideal for accurate determinations. The method requires a 10 µL sample offering duplicate analysis by device, is quick and enables the sample collection by distance as it was proved that ceramides under study were stable at various conditions, including RT. Intra and inter-day accuracy of the determination were estimated between 87.6% - 113% and 90.6% -113%, respectively, while intra- and inter-day precision were calculated from 0.2% to 9.9% %RSD and 0.1% - 8.0% %RSD, respectively. The data acquired by ten healthy individuals indicated that circulating ceramides are at higher levels in whole blood taken from the fingertip in comparison to the reported values in plasma or serum.

Ensuring Fact-Based Metabolite Identification in Liquid Chromatography-Mass Spectrometry-Based Metabolomics.

Metabolite identification represents a major bottleneck in contemporary metabolomics research and a step where critical errors may occur and pass unnoticed. This is especially the case for studies employing liquid chromatography-mass spectrometry technology, where there is increased concern on the validity of the proposed identities. In the present perspective article, we describe the issue and categorize the errors into two types: identities that show poor biological plausibility and identities that do not comply with chromatographic data and thus to physicochemical properties (usually hydrophobicity/hydrophilicity) of the proposed molecule. We discuss the problem, present characteristic examples, and propose measures to improve the situation.

Liquid chromatography-mass spectrometry metabolite library for metabolomics: Evaluating column suitability using a scoring approach.

Untargeted metabolomic studies require an extensive set of analyte (metabolic) information to be obtained from each analyzed sample. Thus, highly selective, and efficient analytical methodologies together with reversed-phase (RP) or hydrophilic interaction liquid chromatography (HILIC) are usually applied in these approaches. Here, we present a performance comparison of five different chromatographic columns (C18, C8, RP Amide, zicHILIC, OH5 HILIC phases) to evaluate their sufficiency of analysis for a large analyte library, consisting of 817 authentic standards. By taking into account experimental chromatographic parameters (i.e. retention time, peak tailing and asymmetry, FWHM, signal-to-noise ratio and peak area and intensity), the proposed column scoring approach provides a simple criterion that may assist analysis in the select of a stationary phase for those metabolites of interest. RPLC methods offered better results regarding metabolic library coverage, while the zicHILIC stationary phase delivered a bigger number of properly eluted compounds. This study demonstrates the importance of choosing the most suitable configuration for the analysis of different metabolic classes.

Analysis, Stability and Distribution of Pharmaceuticals and Drugs of Abuse over a Period of One Year in Formalin-Fixed Liver and Formalin Solutions.

The present study reports a thorough research on the stability of drugs of abuse and pharmaceuticals over a time period of 12 months. Fixed-liver tissues and formalin solutions where the tissues were preserved were analyzed using an ultra high performance liquid chromatography--tandem mass spectrometry method that has been developed and validated for this purpose. The method monitors 84 drugs in a 13-minute run. The concentrations of the drugs found were compared with their concentrations determined in the fresh liver tissues in a previous study. In the study, 14 cases with forensic interest were included with the main objective of the analysis and the study of the stability and the distribution of drugs of abuse and pharmaceuticals in the human liver and the formalin solution during preservation. The results showed that the number of detected compounds in the first month was significantly lower than the compounds found in fresh tissues. The effect of formalin was catalytic, and few substances could be detected. Specifically, out of the 86 positive detections of the monitored substances in the fresh tissues (in which 25 different substances were found), only 32 (37%) remained detectable 3 months after, 20 (23%) 6 months after and 15 (17%) 12 months after.

Blood Metabolomics May Discriminate a Sub-Group of Patients with First Demyelinating Episode in the Context of RRMS with Increased Disability and MRI Characteristics Indicative of Poor Prognosis.

Biomarker research across the health-to-disease continuum is being increasingly applied. We applied blood-based metabolomics in order to identify patient clusters with a first demyelinating episode, and explored the prognostic potential of the method by thoroughly characterizing each cluster in terms of clinical, laboratory and MRI markers of established prognostic potential for Multiple Sclerosis (MS). Recruitment consisted of 11 patients with Clinically Isolated Syndrome (CIS), 37 patients with a first demyelinating episode in the context of Relapsing-Remitting MS (RRMS) and 11 control participants. Blood-based metabolomics and hierarchical clustering analysis (HCL) were applied. Constructed OPLS-DA models illustrated a discrimination between patients with CIS and the controls (p = 0.0014), as well as between patients with RRMS and the controls (p = 1 × 10−5). Hierarchical clustering analysis (HCL) for patients with RRMS identified three clusters. RRMS-patients-cluster-3 exhibited higher mean cell numbers in the Cerebro-spinal Fluid (CSF) compared to patients with CIS (18.17 ± 6.3 vs. 1.09 ± 0.41, p = 0.004). Mean glucose CSF/serum ratio and infratentorial lesion burden significantly differed across CIS- and HCL-derived RRMS-patient clusters (F = 14.95, p < 0.001 and F = 6.087, p = 0.002, respectively), mainly due to increased mean values for patients with RRMS-cluster-3. HCL discriminated a cluster of patients with a first demyelinating episode in the context of RRMS with increased disability, laboratory findings linked with increased pathology burden and MRI markers of poor prognosis.

A Prospective, Case-Control Study of Serum Metabolomics in Neonates with Late-Onset Sepsis and Necrotizing Enterocolitis.

Late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) are major causes of neonatal morbidity and mortality. In this prospective, case-control study, we evaluated the metabolic profile of neonates with LOS and NEC. Blood samples were collected from 15 septic neonates and 17 neonates with NEC at the clinical suspicion of the specific diseases. Sixteen gestational and postnatal age-matched neonates without sepsis/NEC served as controls. Serum metabolic profiles were assessed using liquid chromatography-quadrupole time-of-flight mass spectrometry. Metabolomic analysis revealed significant differences in the metabolic profile of neonates with LOS or NEC compared to controls. More specifically, a number of molecules possibly identified as phosphatidylcholines or lysophosphatidylcholines were found to be significantly reduced both in neonates with LOS and those with NEC compared to controls. Additionally, L-carnitine could efficiently discriminate NEC cases from controls. The results of the current study suggest that certain phospholipids and their derivatives could possibly be used as biomarkers for the early detection of LOS and NEC.