Simultaneous determination of canonical purine metabolism using a newly developed HILIC-MS/MS in cultured cells.

Purine metabolism acts as the core role in human metabolic network. It offers purine metabolites as raw material for building blocks in cell survival and proliferation. Purine metabolites are the most abundant metabolic substrates in organisms. There are few reports to simultaneously quantify canonical purine metabolism in cells. A novel hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS/MS) method was developed to simultaneously determine purines profile in biological samples. Chromatographic separation was achieved using a HILIC (Waters Xbridge™ Amide) column. Different optimizing chromatographic conditions and mass spectrometric parameters were tested in order to provide the best separation and the lowest limit of quantification (LLOQ) values for targeted metabolites. The validation was evaluated according to the Food and Drug Administration guidelines. The limit of determination (LOD) and the LOQ values were in the range of 0.02-8.33 ng mL-1 and 0.1-24.5 ng mL-1, respectively. All calibration curves displayed good linear relationship of with excellent correlation coefficient (r) ranging from 0.9943 to 0.9999. Both intra-day and inter-day variability were below 15 %, respectively. Trueness, expressed as relative error, was always within ±15 %. In addition, no derivatization procedure and ion-pair reagents are in need. The innovated approach demonstrates high sensitivity, strong specificity, and good repeatability, making it suitable for absolute quantitative studies of canonical purine metabolism in cultured cells.

Determination of Choline-Containing Compounds in Rice Bran Fermented with Aspergillus oryzae Using Liquid Chromatography/Tandem Mass Spectrometry.

Choline-containing compounds are essential nutrients for human activity, as they are involved in many biological processes, including cell membrane organization, methyl group donation, neurotransmission, signal transduction, lipid transport, and metabolism. These compounds are normally obtained from food. Fermented brown rice and rice bran with Aspergillus oryzae (FBRA) is a fermented food product derived from rice and rice ingredients. FBRA exhibits a multitude of functional properties with respect to the health sciences. This study has a particular focus on choline-containing compounds. We first developed a simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis method for seven choline-containing compounds. The method was subsequently applied to FBRA and its ingredients. Hydrophilic interaction chromatography (HILIC) and selected reaction monitoring were employed for the simultaneous analysis of seven choline-containing compounds. MS ion source conditions were optimized in positive ion mode, and the product ions derived from the choline group were obtained through MS/MS optimization. Under optimized HILIC conditions, the peaks exhibited good shape without peak tailing. Calibration curves demonstrated high linearity across a 300- to 10,000-fold concentration range. The application of the method to FBRA and other ingredients revealed significant differences between food with and without fermentation. In particular, betaine and α-glycerophosphocholine were found to be highest in FBRA and brown rice malt, respectively. The results indicated that the fermentation processing of rice ingredients results in alterations to the choline-containing compounds present in foods. The developed HILIC/MS/MS method proved to be a valuable tool for elucidating the composition of choline-containing compounds in foods.

Review of the occurrence, treatment technologies, and detection methods for saxitoxins in freshwaters.

The increasing occurrence of saxitoxins in freshwaters is becoming a concern for water treatment facilities owing to its structural properties which make it resistant to oxidation at pH < 8. Hence, it is crucial to be able to monitor these toxins in surface and drinking water to protect public health. This review aims to outline the current state of knowledge related to the occurrence of saxitoxins in freshwaters and its removal strategies and provide a critical assessment of the detection methods to provide a basis for further development. Temperature and nutrient content are some of the factors that influence the production of saxitoxins in surface waters. A high dose of sodium hypochlorite with sufficient contact time or activated carbon has been shown to efficiently remove extracellular saxitoxins to meet the drinking water guidelines. While HILIC-MS has proven to be a powerful technology for more sensitive and reliable detection of saxitoxin and variants after solid phase extraction, ELISA is cost-effective and easy to use and is used by Ohio EPA for surveillance with a limit of detection of 0.015 µg/L. However, there is a need for the development of cost-effective and sensitive techniques that can quantify the variants of saxitoxin.

Quantification of amino acids secreted by yeast cells by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Monitoring the levels of amino acids (AAs) in biological cell cultures provides key information to understand the regulation of cell growth and metabolism. Saccharomyces cerevisiae can naturally excrete AAs, making accurate detection and determination of amino acid levels within the cultivation medium pivotal for gaining insights into this still poorly known process. Given that most AAs lack ultraviolet (UV) chromophores or fluorophores necessary for UV and fluorescence detection, derivatization is commonly utilized to enhance amino acid detectability via UV absorption. Unfortunately, this can lead to drawbacks such as derivative instability, labor intensiveness, and poor reproducibility. Hence, this study aimed to develop an accurate and stable hydrophilic interaction liquid chromatography-tandem mass spectrometry analytical method for the separation of all 20 AAs within a short 17-min run time. The method provides satisfactory linearity and sensitivity for all analytes. The method has been validated for intra- and inter-day precision, accuracy, recovery, matrix effect, and stability. It has been successfully applied to quantify 20 AAs in samples of yeast cultivation medium. This endeavor seeks to enhance our comprehension of amino acid profiles in the context of cell growth and metabolism within yeast cultivation media.

A novel halloysite nanotubes-based hybrid monolith for in-tube solid-phase microextraction of polar cationic pesticides.

The accurate determination of polar cationic pesticides in food poses a challenge due to their high polarity and trace levels in complex matrices. This study hypothesized that the use of halloysite nanotubes (HNTs) can significantly enhance the extraction efficiency and sensitivity of these analytes because of their rich hydroxyl groups and cation exchange sites. Therefore, we chemically incorporated HNTs with organic polymer monoliths for in-tube solid-phase microextraction (SPME). This novel hybrid monolith extended service life, improved adsorption capacity, and exhibited excellent extraction performance for polar cationic pesticides. Based on these advancements, a robust and sensitive in-tube SPME-HILIC-MS/MS method was constructed to determine trace levels of polar cationic pesticides in complex food matrices. The method achieved limits of detection of 1.9, 2.1, and 0.1 µg/kg for maleic hydrazide, amitrole, and cyromazine, respectively. The spiked recoveries in five food samples ranged from 80.2 to 100.8%, with relative standard deviations below 10.7%.

Highly sensitive and accurate measurement of underivatized phosphoenolpyruvate in plasma and serum via EDTA-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate quantification of PEP in plasma or serum poses a significant challenge owing to its strong polarity and metal affinity. In this study, a sensitive method for the direct determination of PEP in plasma and serum based on ethylenediaminetetraacetic acid (EDTA)-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed. Superior chromatographic retention and peak shapes were achieved using a zwitterionic stationary-phase HILIC column with a metal-inert inner surface. Efficient dechelation of PEP-metal complexes in serum/plasma samples was achieved through the introduction of EDTA, resulting in a significant enhancement of the PEP signal. A PEP isotopically labelled standard was employed as a surrogate analyte for the determination of endogenous PEP, and validation assessments proved the sensitivity, selectivity, and reproducibility of this method. The method was applied to the comparative quantification of PEP in plasma and serum samples from mice and rats, as well as in HepG2 cells, HEK293T cells, and erythrocytes; the results confirmed its applicability in PEP-related biomedical research. The developed method can quantify PEP in diverse biological matrices, providing a feasible opportunity to investigate the role of PEP in relevant biomedical research.

New analytical methods focusing on polar metabolite analysis in mass spectrometry and NMR-based metabolomics.

Following in the footsteps of genomics and proteomics, metabolomics has revolutionised the way we investigate and understand biological systems. Rapid development in the last 25 years has been driven largely by technical innovations in mass spectrometry and nuclear magnetic resonance spectroscopy. However, despite the modest size of metabolomes relative to proteomes and genomes, methodological capabilities for robust, comprehensive metabolite analysis remain a major challenge. Therefore, development of new methods and techniques remains vital for progress in the field. Here, we review developments in LC-MS, GC-MS and NMR methods in the last few years that have enhanced quantitative and comprehensive metabolome coverage, highlighting the techniques involved, their technical capabilities, relative performance, and potential impact.

Development and validation of an HILIC/MS/MS method for determination of nusinersen in rabbit plasma.

A hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC/MS/MS) method was developed and validated for the quantitative analysis of the fully phosphorothioate modified oligonucleotide nusinersen. HILIC/MS/MS method is more robust and compatible with mass spectrometry than ion pair reversed-phase liquid chromatography-tandem mass spectrometry (IP-RP-LC/MS/MS). Various types and concentrations of additives and different pH of mobile phase affected the mass spectrometry response, chromatographic peak shape and retention of nusinersen. The optimized extraction method of nusinersen employs hydrophilic-lipophilic balance solid phase extraction, with a recovery of up to 80 %. Chromatographic quantification was performed using a gradient system on an amide column and the mobile phase consisted of ammonium acetate, acetonitrile and water in a certain proportion. The fully phosphorothioate modified nusinersen can obtain a high mass spectrometry response by providing greater peak symmetry and high ionization efficiency in a high-pH mobile phase. Moreover, the significant carry over interference was observed at the pH 6.3 of the mobile phase. Adjusting the pH value up to 10, and the carry over interference disappeared. The lower limit of quantitation of this developed HILIC/MS/MS assay was 30.0 ng/mL and the method was systematic methodology validated. This HILIC/MS/MS method provides an attractive and robust alternative for the quantitative analysis of nusinersen and was applied in the pharmacokinetic study of nusinersen in rabbits.

Quantification of Trimethylamine-N-Oxide and Trimethylamine in Fish Oils for Human Consumption.

Supplementing fish oil is one of the strategies to reduce the risk of cardiovascular disease, the leading cause of death around the world. Contradictorily, fish oil may also contain trimethylamine-N-oxide, a recently emerged risk factor for cardiovascular disease, as well as one of its precursors, trimethylamine. A method suitable for routine quantification of trimethylamine-N-oxide and trimethylamine in fish oil with a quick and easy liquid extraction without derivatization has been developed. Liquid chromatography with tandem mass spectrometry detection was employed along with a zwitterionic hydrophilic interaction liquid chromatography column and a gradient elution with eluents containing 50 mmol/L of ammonium formate. An internal standard (triethylamine) was used for quantification by mass spectrometry with an external calibration. The assay proved high linearity in the ranges of 10 to 100 ng/mL and 100 to 1000 ng/mL for trimethylamine-N-oxide and trimethylamine, respectively. The lowest limit of quantification was determined to be 100 µg/kg for trimethylamine and 10 µg/kg for trimethylamine-N-oxide, with the limit of detection at 5 µg/kg and 0.25 µg/kg, respectively. Accuracy ranged from 106-119%. Precision was below 7% the relative standard deviation for both analytes. The method was successfully applied for the determination of trimethylamine-N-oxide and trimethylamine contents in nine commercially available liquid fish oils and three commercially available fish oil capsules, showing that trimethylamine and trimethylamine-N-oxide are not present in highly refined fish oils.

Development of a Cation Exchange SPE-HILIC-MS/MS Method for the Determination of Ningnanmycin Residues in Tea and Chrysanthemum.

Ningnanmycin is a widely used antibiotic in agricultural production that effectively controls fungal and viral diseases in tea trees and chrysanthemums. The polarity characteristic of ningnanmycin has posed limitations on the development of robust detection methods, thereby hindering effective monitoring and control measures. By combining cation exchange solid phase extraction (SPE) with hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS), we have effectively tackled the issue pertaining to the separation and retention of ningnanmycin. The average recoveries of ningnanmycin in green tea, black tea, and chrysanthemum were 77.3-82.0%, 80.1-81.5%, and 74.0-80.0%, respectively. The intraday and interday relative standard deviations (RSDs) were below and equal to 7.7%. Good linearity was observed in the concentration range of 1-1000 µg/L (R2 > 0.998). The limits of detection (LODs) ranged from 1.1 µg/kg to 7.1 µg/kg, and the limits of quantification (LOQs) ranged from 3.6 µg/kg to 23.7 µg/kg for ningnanmycin. These results indicate the good accuracy, repeatability, reproducibility, and sensitivity of the method. It is suitable for detecting ningnanmycin in tea and chrysanthemum.

Inception and development of a LC-MS/MS assay for the multiplexed quantitation of nine human drug transporter biomarkers.

Background: It has become common practice to assess solute carrier transporter (SLC)-mediated drug-drug interactions (DDIs) by quantitating various individual endogenous compounds as biomarkers in human plasma and urine. The goal of this work was to develop biomarker multiplex assays that could be utilized during first in human studies to support the simultaneous assessment of clinical DDI risk across various SLCs. Methodology: Hydrophilic interaction chromatography-MS/MS methods were developed, and validations were performed. Results: The multiplex assays were applied to a first in human study. Placebo/reference subject biomarker data were consistent with single assay in-house and published data. Conclusion: This work demonstrates the utility of these multiplex methods to support the concurrent evaluation of clinical DDI risk across various SLCs.

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 dimethylated nucleosides in serum from colorectal cancer patients by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

RNA modifications play a crucial regulatory role in a variety of biological processes and are closely related to numerous diseases, including cancer. The diversity of metabolites in serum makes it a favored biofluid for biomarkers discovery. In this work, a robust and accurate hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) approach was established for simultaneous determination of dimethylated nucleosides in human serum. Using the established method, we were able to accurately quantify the concentrations of N6-2'-O-dimethyladenosine (m6Am), N2,N2-dimethylguanosine (m2,2G), and 5,2'-O-dimethyluridine (m5Um) in serum samples from 53 healthy controls, 57 advanced colorectal adenoma patients, and 39 colorectal cancer (CRC) patients. The results showed that, compared with healthy controls and advanced colorectal adenoma patients, the concentrations of m6Am and m2,2G were increased in CRC patients, while the concentration of m5Um was decreased in CRC patients. These results indicate that these three dimethylated nucleosides could be potential biomarkers for early detection of colorectal cancer. Interestingly, the level of m5Um was gradually decreased from healthy controls to advanced colorectal adenoma patients to CRC patients, indicating m5Um could also be used to evaluate the level of malignancy of colorectal tumor. In addition, this study will contribute to the investigation on the regulatory mechanisms of RNA dimethylation in the onset and development of colorectal cancer.

The Quantitative Measurement of Peptidoglycan Components Obtained from Acidic Hydrolysis in Gram-Positive and Gram-Negative Bacteria via Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry.

The high throughput in genome sequencing and metabolic model (MM) reconstruction has democratised bioinformatics approaches such as flux balance analysis. Fluxes' prediction accuracy greatly relates to the deepness of the MM curation for a specific organism starting from the cell composition. One component is the cell wall, which is a functional barrier (cell shape, exchanges) with the environment. The bacterial cell wall (BCW), including its thickness, structure, and composition, has been extensively studied in Escherichia coli but poorly described for other organisms. The peptidoglycan (PG) layer composing the BCW is usually thinner in Gram- bacteria than in Gram+ bacteria. In both bacteria groups, PG is a polymeric mesh-like structure of amino acids and sugars, including N-acetylglucosamine, N-acetylmuramic acid, and amino acids. In this study, we propose a high-throughput method to characterise and quantify PG in Gram-positive and Gram-negative bacteria using acidic hydrolysis and hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS). The method showed a relatively short time frame (11 min analytical run), low inter- and intraday variability (3.2% and 4%, respectively), and high sensitivity and selectivity (limits of quantification in the sub mg/L range). The method was successfully applied on two Gram-negative bacteria (Escherichia coli K12 MG1655, Bacteroides thetaiotaomicron DSM 2079) and one Gram-positive bacterium (Streptococcus salivarius ssp. thermophilus DSM20259). The PG concentration ranged from 1.6% w/w to 14% w/w of the dry cell weight. The results were in good correlation with previously published results. With further development, the PG concentration provided by this newly developed method could reinforce the curation of MM.

Development of a targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry based lipidomics platform applied to a coronavirus disease severity study.

The importance of lipids seen in studies of metabolism, cancer, the recent COVID-19 pandemic and other diseases has brought the field of lipidomics to the forefront of clinical research. Quantitative and comprehensive analysis is required to understand biological interactions among lipid species. However, lipidomic analysis is often challenging due to the various compositional structures, diverse physicochemical properties, and wide dynamic range of concentrations of lipids in biological systems. To study the comprehensive lipidome, a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)-based screening method with 1200 lipid features across 19 (sub)classes, including both nonpolar and polar lipids, has been developed. HILIC-MS/MS was selected due to its class separation property and fatty acyl chain level information. 3D models of class chromatographic retention behavior were established and evaluations of cross-class and within-class interferences were performed to avoid over-reporting these features. This targeted HILIC-MS/MS method was fully validated, with acceptable analytical parameters in terms of linearity, precision, reproducibility, and recovery. The accurate quantitation of 608 lipid species in the SRM 1950 NIST plasma was achieved using multi-internal standards per class and post-hoc correction, extending current databases by providing lipid concentrations resolved at fatty acyl chain level. The overall correlation coefficients (R2) of measured concentrations with values from literature range from 0.64 to 0.84. The applicability of the developed targeted lipidomics method was demonstrated by discovering 520 differential lipid features related to COVID-19 severity. This high coverage and targeted approach will aid in future investigations of the lipidome in various disease contexts.

A comparison of hydrophilic interaction liquid chromatography and capillary electrophoresis for the metabolomics analysis of human serum.

Cationic, anionic, zwitterionic and, partially polar metabolites are very important constituents of blood serum. Several of these metabolites underpin the core metabolism of cells (e.g., Krebs cycle, urea cycle, proteins synthesis, etc.), while others might be considered ancillary but still important to grasp the status of any organism through blood serum analysis. Due to its wide chemical diversity, modern metabolomics analysis of serum is still struggling to provide a complete and comprehensive picture of the polar metabolome, due to the limitations of each specific analytical method. In this study, two metabolomics-based analytical methods using the most successful techniques for polar compounds separation in human serum samples, namely hydrophilic interaction liquid chromatography (HILIC) and capillary electrophoresis (CE), are evaluated, both coupled to a high-resolution time-of-flight mass spectrometer via electrospray ionization (ESI-Q-TOF-MS). The performance of the two methods have been compared using five terms of comparison, three of which are specific to metabolomics, such as (1) compounds' detectability (2) Pezzatti score (Pezzatti et al. 2018), (3) intra-day precision (repeatability), (4) ease of automatic analysis of the data (through a common deconvolution alignment and extrapolation software, MS-DIAL, and (5) time & cost analysis. From this study, HILIC-MS proved to be a better tool for polar metabolome analysis, while CE-MS helped identify some interesting variables that gave it interest in completing metabolome coverage in metabolomics studies. Finally, in this framework, MS-DIAL demonstrates for the first time its ability to process CE data for metabolomics, although it is not designed for it.

Mass spectrometry of the white adipose metabolome in a hibernating mammal reveals seasonal changes in alternate fuels and carnitine derivatives.

Mammalian hibernators undergo substantial changes in metabolic function throughout the seasonal hibernation cycle. We report here the polar metabolomic profile of white adipose tissue isolated from active and hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Polar compounds in white adipose tissue were extracted from five groups representing different timepoints throughout the seasonal activity-torpor cycle and analyzed using hydrophilic interaction liquid chromatography-mass spectrometry in both the positive and negative ion modes. A total of 224 compounds out of 660 features detected after curation were annotated. Unsupervised clustering using principal component analysis revealed discrete clusters representing the different seasonal timepoints throughout hibernation. One-way analysis of variance and feature intensity heatmaps revealed metabolites that varied in abundance between active and torpid timepoints. Pathway analysis compared against the KEGG database demonstrated enrichment of amino acid metabolism, purine metabolism, glycerophospholipid metabolism, and coenzyme A biosynthetic pathways among our identified compounds. Numerous carnitine derivatives and a ketone that serves as an alternate fuel source, beta-hydroxybutyrate (BHB), were among molecules found to be elevated during torpor. Elevated levels of the BHB-carnitine conjugate during torpor suggests the synthesis of beta-hydroxybutyrate in white adipose mitochondria, which may contribute directly to elevated levels of circulating BHB during hibernation.

Capillary electrophoresis-mass spectrometry as a tool for Caenorhabditis elegans metabolomics research.

INTRODUCTION: Polar metabolites in Caenorhabditis elegans (C. elegans) have predominantly been analyzed using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). Capillary electrophoresis coupled to mass spectrometry (CE-MS) represents another complementary analytical platform suitable for polar and charged analytes. OBJECTIVE: We compared CE-MS and HILIC-MS for the analysis of a set of 60 reference standards relevant for C. elegans and specifically investigated the strengths of CE separation. Furthermore, we employed CE-MS as a complementary analytical approach to study polar metabolites in C. elegans samples, particularly in the context of longevity, in order to address a different part of its metabolome. METHOD: We analyzed 60 reference standards as well as metabolite extracts from C. elegans daf-2 loss-of-function mutants and wild-type (WT) samples using HILIC-MS and CE-MS employing a Q-ToF-MS instrument. RESULTS: CE separations showed narrower peak widths and a better linearity of the estimated response function across different concentrations which is linked to less saturation of the MS signals. Additionally, CE exhibited a distinct selectivity in the separation of compounds compared to HILIC-MS, providing complementary information for the analysis of the target compounds. Analysis of C. elegans metabolites of daf-2 mutants and WT samples revealed significant alterations in shared metabolites identified through HILIC-MS, as well as the presence of distinct metabolites. CONCLUSION: CE-MS was successfully applied in C. elegans metabolomics, being able to recover known as well as identify novel putative biomarkers of longevity.

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.

High-sensitivity qualitative and quantitative analysis of human, bovine and goat milk glycosphingolipids using HILIC-MS/MS with internal standards.

Glycosphingolipids (GSLs) in human milk regulate the immune system, support intestinal maturation, and prevent gut pathogens. The structural complexity and low abundance of GSLs limits their systematic analysis. Here, we coupled the use of monosialoganglioside 1-2-amino-N-(2-aminoethyl) benzamide (GM1-AEAB) derivatives as internal standards with HILIC-MS/MS to qualitatively and quantitatively compare GSLs in human, bovine, and goat milk. One neutral glycosphingolipid (GB) and 33 gangliosides were found in human milk, of which 22 were newly detected and three were fucosylated. Five GB and 26 gangliosides were identified in bovine milk, of which 21 were newly discovered. Four GB and 33 gangliosides were detected in goat milk, 23 of them newly reported. GM1 was the main GSL in human milk; whereas disialoganglioside 3 (GD3) and monosialogangloside 3 (GM3) were dominant in bovine and goat milk, respectively; N-acetylneuraminic acid (Neu5Ac) was detected in >88 % of GSLs in bovine and goat milk. N-hydroxyacetylneuraminic acid (Neu5Gc)-modified GSLs were 3.5 times more abundant in goat than in bovine milk; whereas GSLs modified with both Neu5Ac and Neu5Gc were 3 times more abundant in bovine than in goat milk. Given the health benefits of different GSLs, these results will facilitate the development of custom-designed human milk-based infant formula.