Current state of hydrophilic interaction liquid chromatography of oligonucleotides.

There has been a significant increase in the use of hydrophilic interaction liquid chromatography (HILIC) to separate oligonucleotides. This rise in the use of HILIC has correlated to the increasing success of oligonucleotides as therapeutic treatments and reagents in biomedical research. As more scientists need to routinely analyze oligonucleotides in addition to small molecules, peptides, and proteins using the same analytical instruments, it becomes difficult to use traditional types of analyses such as ion-pair reversed-phase chromatography. This increased use has led to new approaches that have improved the utility of HILIC to the point where it has become a legitimate alternative approach to ion-pair reversed-phase chromatography. This review highlights recent advances in HILIC separations of oligonucleotides with a focus on the underlying mechanisms of action. While HILIC has made significant gains in performance, there still remain challenges, which if properly addressed will continue to propel this approach forward.

Top-down mass spectrometry analysis of capsid proteins of recombinant adeno-associated virus using multiple ion activations and proton transfer charge reduction.

Adeno-associated viruses (AAVs) are common vectors for emerging gene therapies due to their lack of pathogenicity in humans. Here, we present our investigation of the viral proteins (i.e., VP1, VP2, and VP3) of the capsid of AAVs via top-down mass spectrometry (MS). These proteins, ranging from 59 to 81 kDa, were chromatographically separated using hydrophilic interaction liquid chromatography and characterized in the gas-phase by high-resolution Orbitrap Fourier transform MS. Complementary ion dissociation methods were utilized to improve the overall sequence coverage. By reducing the overlap of product ion signals via proton transfer charge reduction on the Orbitrap Ascend BioPharma Tribrid mass spectrometer, the sequence coverage of each VP was significantly increased, reaching up to ∼40% in the case of VP3. These results showcase the improvements in the sequencing of proteins >30 kDa that can be achieved by manipulating product ions via gas-phase reactions to obtain easy-to-interpret fragmentation mass spectra.

Developing Method for Minor Acidic O-Glycan Analysis in Mucin and Cancer Cell Samples.

Minor acidic glycans, such as sulfated and phosphorylated glycans, constitute only a small fraction of biological glycome, making their analysis a considerable challenge. In this study, we developed a technique to analyze minor acidic O-glycans in biological samples. First, efficient reaction conditions for the release of O-glycans from the proteins were determined. Next, a high-throughput method was established for the recovery of minor acidic glycans using NH2 spin columns. The performance of the established method was evaluated using mucin samples, and sulfated O-glycans were successfully detected in bovine submaxillary gland mucin and porcine stomach mucin. We also analyzed the minor acidic O-glycans in cultured cancer cells. In addition to trifucosylated sulfated O-glycans and disulfated O-glycans, sulfated O-glycans with KDN were detected in LS174T cells. The relative amount of sulfated glycans in LS174T cells was almost 10-fold higher than that in the other cells. Moreover, a large polylactosamine-type sulfated O-glycan with a molecular weight >3500 was detected in MKN45 cells. Interestingly, phosphorylated ribose, possibly bound to serine/threonine, was observed in all the cells used in this study. Thus, our established analytical method allows for the analysis of minor acidic O-glycans that cannot be detected using existing glycomics methods.

Peptide retention time prediction for hydrophilic interaction liquid chromatography at acidic pH in formic-acid based eluents.

Peptide separation selectivity was evaluated for hydrophilic interaction liquid chromatography (HILIC) ZIC-HILIC, ZIC-cHILIC, and XBridge Amide sorbents using formic acid as eluent additive (pH 2.7). Sequence-specific retention prediction algorithms were trained using retention datasets of ∼30,000 peptides for each column. Our retention models were able to attain ∼0.98 R2-value and yielded retention coefficients that can be probed to understand peptide-stationary phase interaction. Overall, the hydrophilicity for these columns decreased when the mobile phase changed pH from 4.5 to 2.7, when using 0.1 % formic acid in the mobile phase. The acidic residues became protonated, and the resultant hydrophilic interaction is dampened at the lower pH, leaving only the basic residues as the primary hydrophilic interactors. Hence, peptides of increasing charge have higher retention. In this comparison between the three columns, ZIC-HILIC has the highest chromatographic resolution between groups of peptides of different charge. From the position-dependent retention coefficients for ZIC-HILIC at pH 2.7, we found that the amino acids at the terminal positions of the peptide modulate the basicity of the N-terminal amino group or the C-terminal Arg/Lys for tryptic peptides. With respect to the separation orthogonality between HILIC and acidic pH RPLC for two dimensional separations, the orthogonality values were lower at pH 2.7 than operating HILIC at pH 4.5 for the first dimension. We also demonstrate that ZIC-HILIC was able to distinguish citrullinated and deamidated peptides based on predicted retention values.

Chromatographic evaluation and application of nitrogenous heterocyclic ring-bonded stationary phase in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) based on polar stationary phases has vital research significance in the separation of polar compounds. Numerous HILIC stationary phases with different structures have been developed, which do not have universal properties and broad selectivity, making it a challenge to select the suitable column based on the properties of the samples. Consequently, it is particularly important to develop a bonded phase capable of separating a wide variety of samples, while having enhanced retention, improved selectivity, symmetric peak shape and good stability. Herein, a novel nitrogen-containing heterocyclic bonded phase with multiple functionalities, such as thioether, amino and hydroxyl groups (named AMTA) was employed as HILIC stationary phase. Detailed chromatographic evaluations were carried out, and the results showed that it was superior to other hydrophilic chromatographic columns in terms of selectivity, peak shapes and practical sample separation. Lastly, it has been verified that AMTA exhibited high orthogonality with the XBridge C18 column of reversed-phase liquid chromatography (RPLC) mode. In summary, we anticipate our assay to be instructive to other researchers in developing the HILIC stationary phase.

Effect of anionic groups in zwitterionic hydrophilic stationary phases on their chromatographic characteristics.

The structure of zwitterion has great impact on the separation properties of zwitterionic hydrophilic stationary phases. To better understand the role of anionic groups of zwitterions, a novel carboxybetaine-based zwitterionic monolithic column was first prepared through thermo-initiated copolymerization of functional monomer (3-acrylamidopropyl)-dimethyl-(2-carboxymethyl) ammonium (CBAA) and crosslinker ethylene dimethacrylate (EDMA) within 100 µm ID capillary. The optimal poly(CBAA-co-EDMA) monolithic column exhibited satisfactory mechanical and chemical stability, good repeatability, high column efficiency (96,000 plates/m), and excellent separation performance for different classes of polar compounds (i.e., phenols, monophosphate nucleotides, urea and allantoin). A comparative study was then performed among three zwitterionic hydrophilic stationary phases containing different anionic groups, i.e. poly(CBAA-co-EDMA) (carboxybetaine), poly(2-{2-(methacryloyloxy) ethyldimethylammonium}ethyl n-butyl phosphate-co-EDMA) (phosphocholine), and poly(N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl) ammonium betaine-co-EDMA) (sulfobetaine) using benzoic acid derivatives, amine compounds, nucleobases and nucleosides as model analytes. The carboxybetaine-based monolithic column exhibited much higher positive zeta-potential and hydrophilicity, which endows it with a stronger retention capacity for acidic and neutral compounds, but sulfobetaine-based monolithic column exhibited much higher selectivity and retention capacity for the amines. Moreover, their enrichment efficiencies for N-glycopeptides were also evaluated based on their different hydrophilicity, and it was observed that the poly(CBAA-co-EDMA) monolithic material captured 4-8 times more N-glycopeptides compared to the other two materials.

Expanding the structural resolution of glycosylation microheterogeneity in therapeutic proteins by salt-free hydrophilic interaction liquid chromatography tandem mass spectrometry.

Glycosylation affects the safety and efficacy of therapeutic proteins and is often considered a critical quality attribute (CQA). Therefore, it is important to identify and quantify glycans during drug development. Glycosylation is a highly complex post-translational modification (PTM) due to its structural heterogeneity, i.e. glycosylation site occupancy, glycan compositions, modifications, and isomers. Current analytical tools compromise either structural resolution or site specificity. Hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry (HILIC-FLR-MS) is the gold standard for structural analysis of released glycans, but lacks information on site specificity and occupation. However, HILIC-FLR-MS often uses salt in the solvent, which impairs analysis robustness and sensitivity. Site-specific glycosylation analysis via glycopeptides, upon proteolytic digestion, is commonly performed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), but provides only compositional and limited structural glycan information. In this study, we introduce a salt-free, glycopeptide-based HILIC-tandem mass spectrometry (HILIC-MS/MS) method that provides glycan identification, glycan isomer separation and site-specific information simultaneously. Moreover, HILIC-MS/MS demonstrated comparable relative quantification results as released glycan HILIC-FLR-MS. Further, our new method improves the retention of hydrophilic peptides, allowing simultaneous analysis of important CQAs such as deamidation in antibodies. The developed method offers a valuable tool to streamline the site-specific glycosylation analysis of glycoproteins, which is particularly important for the expanding landscape of novel therapeutic formats in the biopharmaceutical industry.

Highly sensitive two-dimensional profiling of N-linked glycans by hydrophilic interaction liquid chromatography and dual stacking capillary gel electrophoresis.

BACKGROUND: N-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required. RESULTS: In this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R2 > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates. SIGNIFICANCE AND NOVELTY: In conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.

Hydrophilic materials based on hydrogel polymers for enrichment of glycopeptides from serum of colorectal cancer patients.

In this work, a composite hydrogel material consisting of chitosan-based composite hydrogel was prepared by a simple and rapid synthetic method and will be named three-dimensional (3D)-IL-COF-1@CS hydrogel. Possessing a stable 3D network structure and outstanding hydrophilicity, the novel hydrogel is capable of capturing glycopeptides. The 3D-IL-COF-1@CS hydrogel showed good sensitivity (0.1 fmol/µL) and selectivity (1:2000). In addition, 19 glycopeptides were captured in standard samples. In the analysis of human serum, 148 glycopeptides assigned to 72 glycoproteins were assayed in the serum of normal individuals, and 245 glycopeptides corresponding to 100 glycoproteins were found in the serum of colorectal cancer (CRC) patients. More importantly, several functional programs based on Gene Ontology analysis supported molecular biological processes that may be relevant to the pathogenesis of CRC, including aging, fibrinogen complex, and arylesterase activity. The low cost, simplicity, rapid synthesis, and good enrichment performance have a great future in glycoproteomics analysis and related diseases.

Quantitative analysis of the impurities in Etimicin using hydrophilic interaction liquid chromatography coupled with charged aerosol detector.

Etimicin is a typical aminoglycoside antibiotic (AG). High performance liquid chromatography-evaporation light scattering detector (HPLC-ELSD) method is a commonly used method for determining impurities in Etimicin. However, due to the poor reproducibility, low sensitivity and narrow linear range of the ELSD, high-throughput quantitative analysis of impurities in Etimicin currently poses a challenge. In this study, a sensitive method using hydrophilic interaction liquid chromatography coupled with charged aerosol detector (HILIC-CAD) was developed for the analysis of the impurities in Etimicin. The liquid phase conditions for determination impurities in Etimicin were optimized using Box Behnken design (BBD) and response surface methodology (RSM), resulting in satisfactory separation and optimal CAD output signal. We also studied the influence of CAD parameters on the signal-to-noise ratio and linearity of Etimicin and its impurities. This method has also been proven to be effective in separating impurities from two other typical AGs, Isepamicin and Amikacin. In the method validation, the coefficient of determination (R2) of Etimicin, Isepamicin and Amikacin and their impurities were all greater than 0.999, within the range of 0.5-50 µg/mL. The average recoveries of the impurities of three typical AGs were 99.03 %-101.22 %, RSDs all were less than 2.5 % for intra-day and inter-day precision, with good precision and accuracy. The developed HILIC-CAD quantification method was sensitive, accurate and highly selective for quantitative analysis of impurities in the AGs without need ion-pairing reagents, which is ensure the public medication safety. The method is first reported application of HILIC-CAD method for quantitative analysis of the impurities in AGs.

One-step fabrication of dipeptide-based bifunctional polymer for individual enrichment of glycopeptides and phosphopeptides from serum.

A dipeptide-based bifunctional material immobilized with Ti4+ (denoted as APE-MBA-VPA-Ti4+) was developed using precipitation polymerization. This polymer combines hydrophilic interaction liquid chromatography (HILIC) and immobilized metal affinity chromatography (IMAC) enrichment strategies, allowing for the individual and simultaneous enrichment of glycopeptides and phosphopeptides. It demonstrated high sensitivity (0.1 fmol µL-1 for glycopeptides, 0.005 fmol µL-1 for phosphopeptides), strong selectivity (molar ratio HRP: BSA = 1:1000, ß-casein: BSA = 1:2500), consistent reusability (10 cycles) and satisfactory recovery rate (93.5 ± 1.8 % for glycopeptides, 91.6 ± 0.6 % for phosphopeptides) in the individual enrichment. Utilizing nano LC-MS/MS technology, the serum of liver cancer patients was analyzed after enrichment individually, resulting in the successful capture of 333 glycopeptides covering 262 glycosylation sites, corresponding to 131 glycoproteins, as well as 67 phosphopeptides covering 57 phosphorylation sites, related to 48 phosphoproteins. In comparison, the serum of normal healthy individuals yielded a total of 283 glycopeptides covering 244 glycosylation sites corresponding to 126 glycoproteins, as well as 66 phosphopeptides covering 56 phosphorylation sites related to 37 phosphoproteins. Label-free quantification identified 10 differentially expressed glycoproteins and 8 differentially expressed phosphoproteins in the serum of liver cancer patients. Among them, glycoproteins (HP, BCHE, AGT, C3, and PROC) and phosphoproteins (ZYX, GOLM1, GP1BB, CLU, and TNXB) showed upregulation and displayed potential as biomarkers for liver cancer.

[Applications of high performance liquid chromatography-mass spectrometry in proteomics].

Proteomics profiling plays an important role in biomedical studies. Proteomics studies are much more complicated than genome research, mainly because of the complexity and diversity of proteomic samples. High performance liquid chromatography-mass spectrometry (HPLC-MS) is a fundamental tool in proteomics research owing to its high speed, resolution, and sensitivity. Proteomics research targets from the peptides and individual proteins to larger protein complexes, the molecular weight of which gradually increases, leading to sustained increases in structural and compositional complexity and alterations in molecular properties. Therefore, the selection of various separation strategies and stationary-phase parameters is crucial when dealing with the different targets in proteomics research for in-depth proteomics analysis. This article provides an overview of commonly used chromatographic-separation strategies in the laboratory, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC), as well as their applications and selectivity in the context of various biomacromolecules. At present, no single chromatographic or electrophoretic technology features the peak capacity required to resolve such complex mixtures into individual components. Multidimensional liquid chromatography (MDLC), which combines different orthogonal separation modes with MS, plays an important role in proteomics research. In the MDLC strategy, IEC, together with RPLC, remains the most widely used separation mode in proteomics analysis; other chromatographic methods are also frequently used for peptide/protein fractionation. MDLC technologies and their applications in a variety of proteomics analyses have undergone great development. Two strategies in MDLC separation systems are mainly used in proteomics profiling: the "bottom-up" approach and the "top-down" approach. The "shotgun" method is a typical "bottom-up" strategy that is based on the RPLC or MDLC separation of whole-protein-sample digests coupled with MS; it is an excellent technique for identifying a large number of proteins. "Top-down" analysis is based on the separation of intact proteins and provides their detailed molecular information; thus, this technique may be advantageous for analyzing the post-translational modifications (PTMs) of proteins. In this paper, the "bottom-up" "top-down" and protein-protein interaction (PPI) analyses of proteome samples are briefly reviewed. The diverse combinations of different chromatographic modes used to set up MDLC systems are described, and compatibility issues between mobile phases and analytes, between mobile phases and MS, and between mobile phases in different separation modes in multidimensional chromatography are analyzed. Novel developments in MDLC techniques, such as high-abundance protein depletion and chromatography arrays, are further discussed. In this review, the solutions proposed by researchers when encountering compatibility issues are emphasized. Moreover, the applications of HPLC-MS combined with various sample pretreatment methods in the study of exosomal and single-cell proteomics are examined. During exosome isolation, the combined use of ultracentrifugation and SEC can yield exosomes of higher purity. The use of SEC with ultra-large-pore-size packing materials (200 nm) enables the isolation of exosomal subgroups, and proteomics studies have revealed significant differences in protein composition and function between these subgroups. In the field of single-cell proteomics, researchers have addressed challenges related to reducing sample processing volumes, preventing sample loss, and avoiding contamination during sample preparation. Innovative methods and improvements, such as the utilization of capillaries for sample processing and microchips as platforms to minimize the contact area of the droplets, have been proposed. The integration of these techniques with HPLC-MS shows some progress. In summary, this article focuses on the recent advances in HPLC-MS technology for proteomics analysis and provides a comprehensive reference for future research in the field of proteomics.

Measurement of atmospheric amines and aminoamides by column adsorption/extraction and hydrophilic liquid chromatography-electrospray-tandem mass spectrometry.

Sampling and chromatography-mass spectrometry methods were investigated to measure atmospheric amines and aminoamides. Amines and their amide derivatives play significant roles in new particle formation (NPF) in the atmosphere, especially diamines and aminoamides have higher NPF potentials compared to monoamines. For amine sampling, silica gel tube collection and formic acid extraction gave good overall recoveries (>93 ± 8%) for mono-, di-, tri-, tetramines, and aminoamides. Two chromatography methods were subjected to analyze the extracted amines. One involved direct analysis using hydrophilic interaction liquid chromatography with carboxyl or diol group functioned separation column (carboxyl-HILIC or diol-HILIC), and the other utilized derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and subsequent reversed-phase chromatography (HPLC). Separated amines were detected by electrospray ionization and tandem mass spectrometry in both cases. DBD-F-HPLC method provided good sensitivity for mono- and all polyamines (limit of detection (LOD) < 4.6 nM, relative standard deviation (RSD) for 100 nM < 9.2%). However, aminoamides could not be detected by DBD-F-HPLC. Carboxyl-HILIC provided good sensitivities for mono- and diamines and aminoamides (LOD < 1.6 nM, RSD < 4.8%). Forest air measurement was performed and data obtained by carboxyl-HILIC and DBD-F-HPLC showed good agreement for 1,3-diaminopropane, 1,4-diaminobutane (putrescine) and 1,5-diaminopentane (cadaverine) (R2 = 0.9215-0.9739, n = 7-14). Carboxyl-HILIC method was the best for the amine analysis, and combination with silica gel tube sampling provides atmospheric monitoring available. The developed method can be used not only to study atmospheric chemistry of diamines and aminoamides but also to analyze flavor/odor of foods, flowers and wastes.

Exploring separation mechanisms and lipophilicity in hydrophilic interaction chromatography conditions by thin-layer chromatography of anesthetics and adjuvant drugs as polar model compounds.

The chromatographic behavior of the selected compounds was studied under conditions of hydrophilic interaction liquid chromatography (HILIC). The effect of mobile phase composition on the retention in different chromatographic systems was systematically examined using high-performance thin-layer chromatography. The sorbents of different polarity and adsorption characteristics were selected and mixtures of water and organic solvents of various compositions, from pure water to pure organic solvent were used as mobile phases. Increasing the amount of water in the mobile phase leads to a conversion of the separation mechanism, and the retention curves have a characteristic "U" shape. The conversion between the adsorption and partition mechanisms is most likely continuous and depends on the chemical nature of separated substances, the stationary phase as well as on organic component of the mobile phase. Silica gel can be considered the most suitable stationary phase for the systematic investigation of the chromatographic behavior of the test compounds, whereas acetonitrile was the most suitable solvent. The obtained results contribute to the understanding of the dominant separation mechanism, the type, and the intensity of the interactions between separated substances with both stationary and mobile phases. Besides, the lipophilicity parameters obtained under HILIC conditions were evaluated and correlated with the calculated values.

Distinguishing α/ß-linkages and linkage positions of disaccharides in galactooligosaccharides through mass fragmentation and liquid retention behaviour.

Galactooligosaccharides (GOS) are important prebiotics with function closely related to their structure. However, a comprehensive overview of the structure-function relationship is still limited due to the challenge in characterizing multiple isomers in GOS. This study presents a strategy of combining both hydrophilic interaction liquid chromatography (HILIC) retention time and tandem mass spectrometry (MS/MS) fragmentation pattern to distinguish α/ß-linkages and linkage positions of disaccharide isomers in GOS through HILIC-MS/MS analysis. The results indicated that the ratio of m/z 203.0524 to m/z 365.1054 could distinguish α/ß-linkages, while the ratios of m/z 347.0947 to m/z 365.1054, m/z 245.0642 to m/z 365.1054 and HILIC retention time could distinguish (1 â†’ 2), (1 â†’ 3), (1 â†’ 4) and (1 â†’ 6) linkages. The above rules enabled effective characterization of disaccharides in GOS-containing food samples, including milk powder, rice flour, drink, yogurt. This method can be used in the quality control of GOS and future research on the structure-specific health effects of GOS.

Lipidomics reveals the reshaping of the mitochondrial phospholipid profile in cells lacking OPA1 and mitofusins.

Depletion or mutations of key proteins for mitochondrial fusion, like optic atrophy 1 (OPA1) and mitofusins 1 and 2 (Mfn 1 and 2), are known to significantly impact the mitochondrial ultrastructure, suggesting alterations of their membranes' lipid profiles. In order to make an insight into this issue, we used hydrophilic interaction liquid chromatography coupled with electrospray ionization-high resolution MS to investigate the mitochondrial phospholipid (PL) profile of mouse embryonic fibroblasts knocked out for OPA1 and Mfn1/2 genes. One hundred sixty-seven different sum compositions were recognized for the four major PL classes of mitochondria, namely phosphatidylcholines (PCs, 63), phosphatidylethanolamines (55), phosphatidylinositols (21), and cardiolipins (28). A slight decrease in the cardiolipin/PC ratio was found for Mfn1/2-knockout mitochondria. Principal component analysis and hierarchical cluster analysis were subsequently used to further process hydrophilic interaction liquid chromatography-ESI-MS data. A progressive decrease in the incidence of alk(en)yl/acyl species in PC and phosphatidylethanolamine classes and a general increase in the incidence of unsaturated acyl chains across all the investigated PL classes was inferred in OPA1 and Mfn1/2 knockouts compared to WT mouse embryonic fibroblasts. These findings suggest a reshaping of the PL profile consistent with the changes observed in the mitochondrial ultrastructure when fusion proteins are absent. Based on the existing knowledge on the metabolism of mitochondrial phospholipids, we propose that fusion proteins, especially Mfns, might influence the PL transfer between the mitochondria and the endoplasmic reticulum, likely in the context of mitochondria-associated membranes.

Retention mechanisms of amitriptyline and its impurities on amide, amino, diol, and silica columns in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) has been widely applied to challenging analysis in biomedical and pharmaceutical fields, bridging the gap between normal-phase high-performance liquid chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). This paper comprehensively explores the retention mechanisms of amitriptyline and its impurities A, B, C, D, F, and G on amide, amino, diol, and silica columns. Dual HILIC/RP-HPLC retention mechanisms were developed, and transitional points between HILIC and RP-HPLC mechanisms were calculated on amide, diol, and silica columns. Adsorption and partition contributions to overall retention mechanisms were evaluated using Python software in HILIC and RP-HPLC regions. The cation exchange mechanism dominates overall retention for ionized analytes in the silica column (R2 > 0.995), whereas the retention of ionized analytes increases with pH. Impacts of acetonitrile content, buffer ionic strength, and pH, along with their interactions on the retention of ionized analytes in the silica column, were determined using the chemometric approach. Acetonitrile content showed the most significant impact on the retention mechanisms. These findings highlight that a detailed investigation into retention mechanisms provides notable insights into factors influencing analyte retention and separation, promising valuable guidance for future analysis.

Assessment of Untargeted Metabolomics by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry to Define Breast Cancer Liquid Biopsy-Based Biomarkers in Plasma Samples.

An early diagnosis of cancer is fundamental not only in regard to reducing its mortality rate but also in terms of counteracting the progression of the tumor in the initial stages. Breast cancer (BC) is the most common tumor pathology in women and the second deathliest cancer worldwide, although its survival rate is increasing thanks to improvements in screening programs. However, the most common techniques to detect a breast tumor tend to be time-consuming, unspecific or invasive. Herein, the use of untargeted hydrophilic interaction liquid chromatography-mass spectrometry analysis appears as an analytical technique with potential use for the early detection of biomarkers in liquid biopsies from BC patients. In this research, plasma samples from 134 BC patients were compared with 136 from healthy controls (HC), and multivariate statistical analyses showed a clear separation between four BC phenotypes (LA, LB, HER2, and TN) and the HC group. As a result, we identified two candidate biomarkers that discriminated between the groups under study with a VIP > 1 and an AUC of 0.958. Thus, targeting the specific aberrant metabolic pathways in future studies may allow for better molecular stratification or early detection of the disease.

Determination of Sulfites in Dried Fruits by Paper Spray Ionization Tandem Mass Spectrometry.

Sulfite, a widely used food additive, is subject to regulated labeling. The extraction of sulfite as the stable hydroxymethylsulfonate (HMS) form and its quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been recognized for their good sensitivity, selectivity, and versatility across various food materials. This study aimed to develop a cost-effective and simpler method for sulfite quantitation, while maintaining the superior sensitivity and selectivity of mass spectrometry (MS). To achieve this, we introduced paper spray ionization (PSI), an ambient desorption ionization technique that could achieve the direct measurement of analytes without employing separation. We also employed a novel internal standard (IS) structurally similar to the analyte, replacing the more expensive isotopically labeled IS. Although the PSI-MS/MS method developed in this study exhibited slightly lower analytical performance compared to the conventional LC-MS/MS, it remained effective for sulfite analysis in dried fruits.

Olaris Global Panel (OGP): A Highly Accurate and Reproducible Triple Quadrupole Mass Spectrometry-Based Metabolomics Method for Clinical Biomarker Discovery.

Mass spectrometry (MS)-based clinical metabolomics is very promising for the discovery of new biomarkers and diagnostics. However, poor data accuracy and reproducibility limit its true potential, especially when performing data analysis across multiple sample sets. While high-resolution mass spectrometry has gained considerable popularity for discovery metabolomics, triple quadrupole (QqQ) instruments offer several benefits for the measurement of known metabolites in clinical samples. These benefits include high sensitivity and a wide dynamic range. Here, we present the Olaris Global Panel (OGP), a HILIC LC-QqQ MS method for the comprehensive analysis of ~250 metabolites from all major metabolic pathways in clinical samples. For the development of this method, multiple HILIC columns and mobile phase conditions were compared, the robustness of the leading LC method assessed, and MS acquisition settings optimized for optimal data quality. Next, the effect of U-13C metabolite yeast extract spike-ins was assessed based on data accuracy and precision. The use of these U-13C-metabolites as internal standards improved the goodness of fit to a linear calibration curve from r2 < 0.75 for raw data to >0.90 for most metabolites across the entire clinical concentration range of urine samples. Median within-batch CVs for all metabolite ratios to internal standards were consistently lower than 7% and less than 10% across batches that were acquired over a six-month period. Finally, the robustness of the OGP method, and its ability to identify biomarkers, was confirmed using a large sample set.