Hydrophilic Interaction Liquid Chromatography Research Articles

A Novel Strategy for the Detection of Semicarbazide in Crustaceans by Modified QuEChERS Coupled with Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

Semicarbazide (SEM), a metabolite of nitrofurazone (NFZ), is widely used to detect the illegal application of NFZ in crustaceans. The conventional detection method involves chemical derivatization combined with reversed-phase liquid chromatography–tandem mass spectrometry (RPLC-MS/MS), which is both complex and time-consuming. To address this limitation, a more efficient approach was developed for SEM detection. This study introduces a modified QuEChERS pretreatment method coupled with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) for detecting SEM in crustaceans. The proposed method is simple, fast, and highly accurate, making it universally applicable for SEM detection in crustaceans. Additionally, the method was applied to investigate NFZ metabolism in Macrobrachium rosenbergii with a kinetic model. The findings suggested a plausible mechanism for the absorption of NFZ and its subsequent transfer from meat to the shell. In conclusion, this study provides a simple and rapid technique for SEM detection in crustaceans with immense application value.

Development and Validation of Targeted Metabolomics Methods Using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) for the Quantification of 235 Plasma Metabolites

Plasma contains metabolites with diverse physicochemical properties, ranging from highly polar to highly apolar, and concentrations spanning at least nine orders of magnitude. Plasma metabolome analysis is valuable for monitoring health and evaluating medical interventions but is challenging due to the metabolome’s diversity and complexity. This study aims to develop and validate targeted LC-MS/MS methods for quantifying 235 mammalian metabolites from 17 compound classes in porcine plasma without prior derivatization. Utilizing reversed-phase and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry, each analyte is identified and quantified using two selected reaction monitoring (SRM) transitions. Fast polarity switching and scheduled SRM enhance the metabolome coverage and throughput, enabling the analysis of one sample in about 40 min. A simple “dilute and shoot” sample preparation protocol was employed, with samples injected at two dilution levels to align metabolite concentrations within calibration curve ranges. Validation in porcine plasma included assessments of carryover, linearity, detection and quantification limits, repeatability and recovery. The method was further applied to plasma samples from various animal species, demonstrating its applicability to human and animal studies. This study establishes two robust LC-MS/MS methods for comprehensive porcine plasma metabolome quantification, advancing large-scale targeted metabolomics in biomedical research.

Ion suppression correction and normalization for non-targeted metabolomics

Ion suppression is a major problem in mass spectrometry (MS)-based metabolomics; it can dramatically decrease measurement accuracy, precision, and sensitivity. Here we report a method, the IROA TruQuant Workflow, that uses a stable isotope-labeled internal standard (IROA-IS) library plus companion algorithms to: 1) measure and correct for ion suppression, and 2) perform Dual MSTUS normalization of MS metabolomic data. We evaluate the method across ion chromatography (IC), hydrophilic interaction liquid chromatography (HILIC), and reversed-phase liquid chromatography (RPLC)-MS systems in both positive and negative ionization modes, with clean and unclean ion sources, and across different biological matrices. Across the broad range of conditions tested, all detected metabolites exhibit ion suppression ranging from 1% to >90% and coefficients of variation ranging from 1% to 20%, but the Workflow and companion algorithms are highly effective at nulling out that suppression and error. To demonstrate a routine application of the Workflow, we employ the Workflow to study ovarian cancer cell response to the enzyme-drug L-asparaginase (ASNase). The IROA-normalized data reveal significant alterations in peptide metabolism, which have not been reported previously. Overall, the Workflow corrects ion suppression across diverse analytical conditions and produces robust normalization of non-targeted metabolomic data.

Finding the best column for polar basic analytes across reversed-phase and hydrophilic interaction liquid chromatography

Finding the best column for polar basic analytes across reversed-phase and hydrophilic interaction liquid chromatography

Complete Polar Lipid Profile of Kefir Beverage by Hydrophilic Interaction Liquid Chromatography with HRMS and Tandem Mass Spectrometry

Kefir, a fermented milk product produced using kefir grains, is a symbiotic consortium of bacteria and yeasts responsible for driving the fermentation process. In this study, an in-depth analysis of kefir’s lipid profile was conducted, with a focus on its phospholipid (PL) content, employing liquid chromatography with high-resolution mass spectrometry (LC-HRMS). Nearly 300 distinct polar lipids were identified through hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization (ESI) and Fourier-transform orbital-trap MS and linear ion-trap tandem MS/MS. The identified lipids included phosphatidylcholines (PCs), lyso-phosphatidylcholines (LPCs), phosphatidylethanolamines (PEs) and lyso-phosphatidylethanolamines (LPEs), phosphatidylserines (PSs), phosphatidylglycerols (PGs), and phosphatidylinositols (PIs). The presence of lysyl-phosphatidylglycerols (LyPGs) was identified as a key finding, marking a lipid class characteristic of Gram-positive bacterial membranes. This discovery highlights the role of viable bacteria in kefir and underscores its probiotic potential. The structural details of minor glycolipids (GLs) and glycosphingolipids (GSLs) were further elucidated, enriching the understanding of kefir’s lipid complexity. Fatty acyl (FA) composition was characterized using reversed-phase LC coupled with tandem MS. A mild epoxidation reaction with meta-chloroperoxybenzoic acid (m-CPBA) was performed to pinpoint double-bond positions in FAs. The dominant fatty acids were identified as C18:3, C18:2, C18:1, C18:0 (stearic acid), C16:0 (palmitic acid), and significant levels of C14:0 (myristic acid). Additionally, two isomers of FA 18:1 were distinguished: ∆9-cis (oleic acid) and ∆11-trans (vaccenic acid). These isomers were identified using diagnostic ion pairs, retention times, and accurate m/z values. This study provides an unprecedented level of detail on the lipid profile of kefir, shedding light on its complex composition and potential nutritional benefits.

HILIC-DAD Method for Simultaneous Determination of Acid and Basic Drugs: Application to the Quantitation of Ibuprofen, Atenolol, and Salbutamol in Urine After Solid-Phase Extraction

A simple method has been developed for the simultaneous analysis of ibuprofen (acid drug), and salbutamol and atenolol (basic drugs) in urine samples at concentrations of 0.40 µg·mL−1. Simultaneous chromatographic separation has been possible using hydrophilic interaction liquid chromatography (Kinetex HILIC® column (2.1 mm × 150 mm, 2.6 μm particle size diameter and 100 Å pore size) combined with gradient elution by employing a mixture of acetonitrile–acetate buffer 5 mM at pH 6 (from 95:5 to 75:25 (v/v)) as the mobile phase. Detection was performed at 227 and 275 nm. The simultaneous preconcentration and cleaning of the sample has been possible by solid-phase extraction using the HLB ExtraBond® polymeric-type sorbent (which is a pyrrolidone-modified divinylbenzene polystyrene type). It has provided recoveries between (63 ± 9)% for salbutamol, (74 ± 8)% for ibuprofen, and (96 ± 9)% for atenolol in 10 mL of synthetic urine containing 4.0 μg of each of the drugs analyzed. The detection limits were 0.025 µg·mL−1 for ibuprofen, µg·mL−1 for salbutamol, and 0.007 µg·mL−1 for atenolol. The detection limits obtained allow the evaluation of the free forms of ibuprofen, atenolol, and salbutamol at the excreted concentration levels at the therapeutic doses usually administered. The coefficients of variation between days were in the range 4.5–10.9%.

The Glycosylation of Serum IgG Antibodies in Post-COVID-19 and Post-Vaccination Patients.

The signature of human serum IgG glycosylation is critical in the defense against pathogens. Alterations of IgG N-glycome were associated with COVID-19 (Coronavirus disease 2019) severity, although knowledge on the response to vaccination is limited. IgG N-glycome was analyzed in this study in post-COVID-19 and post-vaccination patients to reveal potential glycosylation-based alterations using hydrophilic interaction liquid chromatography (HILIC-UPLC) with fluorescence (FLR) and mass-spectrometric (MS) detection. IgG antibodies were purified from serum samples through protein G affinity chromatography followed by PNGase F digestion-based deglycosylation. The released glycans were fluorescently derivatized by procainamide labeling and purified via solid-phase extraction. Higher levels of sialylation and afucosylation were identified in post-COVID-19 patients, which was further expanded by vaccination, but only in those who were previously SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) infected.

Comprehensive untargeted polar metabolite analysis using solvent switching liquid chromatography tandem mass spectrometry.

Metabolomics analyses enable the examination and identification of endogenous biochemical reaction products, revealing information on the metabolic pathways and processes active within a living cell or organism. Determination of metabolic shifts can provide important information on a treatment or disease. Unlike other omics fields that typically have analytes of the same chemical class with common building blocks, those that fall under the nomenclature of metabolites encompass a wide array of different compounds with very diverse physiochemical properties. Development of a comprehensive metabolomic pipeline therefore can be a troublesome and complicated process for the analyst. Often single liquid chromatography-mass spectrometry methods on unfractionated samples are carried out in order to be time-efficient, however this could potentially produce data with a low number of identifiable metabolites. In the present studies, we developed a comprehensive polar metabolomics pipeline for cell-based metabolomics. SH-SY5Y neuroblastoma cells were selected as the sample matrix for method development since they are one of the most widely used cell lines for human neurotoxicity studies. This was accomplished by investigating and optimising different mass spectrometry source and chromatographic conditions to enhance the signal of polar metabolites. Optimised hydrophilic interaction liquid chromatography (HILIC) based metabolomic methods at different pH values were examined in positive, negative, and polarity switching modes to determine which combination yielded the highest number of confidently identified metabolites. Additionally, the use of sequentially running two methods was also compared to determine the degree of overlap and whether there is merit in running two separate methods on one sample. It was determined that solvent switching between two optimised methods, acidic chromatographic conditions in positive mode and basic chromatographic conditions in negative mode, yielded the highest number of unique identifiable metabolites. This could be run in a single analytical batch due to the large pH range of the column. A quick switch method in-between each method allowed both conditioning the column and preparation of the MS source conditions for the sequential method.

Intact N-glycopeptide analysis of human platelets reveals a Glycostructure important for platelet function.

Glycosylation is an important posttranslational modification in platelets, and the glycosylation pattern is critical for platelet function. To date, the exploration of the roles of various glycoforms in specific platelet functions is largely lacking. In this study, a global analysis of intact N-glycopeptides in human platelets was performed to map all the glycopeptides, glycosites and glycans of platelets. The glycopeptides were enriched by the ZIC- hydrophilic interaction chromatography method and then analyzed by Liquid Chromatography-Tandem Mass Spectrometry analysis. A total of 1,425 intact glycopeptides belonging to 190 N-glycoproteins from human platelets were identified. Moreover, 358 glycans modified 328 glycosites from those glycoproteins. Functional analysis revealed that these glycoproteins are involved mainly in processes and pathways related to platelet adhesion. Among the proteins in these adhesion-related annotations, von Willebrand factor, thrombospondin 1and glycoprotein V were found to contain a possible Lewis y structure, and this finding was further verified by immunoprecipitation assays. As a blood group-related antigen, Lewis y was previously reported to exist in human platelets, but its function remains unclear. Since the glycosylation of von Willebrand factor, thrombospondin 1 and glycoprotein V is involved in platelet-collagen adhesion, the importance of Lewis y on platelet function was evaluated by adhesion assays, which demonstrated that the blockade of Lewis y on platelets decreased the adhesion of platelets to collagen I under both static and flow conditions.

Altered Pattern of Serum N-Glycome in Subarachnoid Hemorrhage and Cerebral Vasospasm.

Background: Subarachnoid hemorrhage is a serious condition caused by ruptured intracranial aneurysms, resulting in severe disability mainly in young adults. Cerebral vasospasm is one of the most common complication of subarachnoid hemorrhage; thus, active prevention is key to improve the prognosis. The glycosylation of proteins is a critical quality attribute which is reportedly altered in patients diagnosed with acute ischemic stroke. In this study, we examined the N-glycosylation profile of serum glycoproteins in patients with subarachnoid hemorrhage without vasospasm compared to patients with vasospasm. Methods: The serum N-glycans were released by PNGase F (Peptide: N-glycosidase F) digestion and subsequently labeled by procainamide via reductive amination. The samples were analyzed by hydrophilic-interaction liquid chromatography after solid-phase extraction-based sample purification. Results: Besides the glycosylation pattern, we also investigated the biomarkers following subarachnoid hemorrhage. Multiple statistical analyses were performed in order to find significant differences and identify potential prediction factors of cerebral vasospasm. Significant differences were identified such as higher sialylation on bi-, tri-, and tetra-antennary structures in patients with subarachnoid hemorrhage and cerebral vasospasm. Conclusions: Our results suggest that glycosylation analysis can improve the identification of patients with cerebral vasospasm in combination with laboratory parameters.

Development of Citric-Acid-Modified Cellulose Monolith for Enriching Glycopeptides.

Prior to mass spectrometry (MS) analysis, pretreatment of low-abundance glycopeptides is vital for identifying protein glycosylation. In this study, we fabricated an environmentally friendly citric-acid-modified cellulose monolith (CCM) characterized by a coral-like porous structure and high-density hydrophilic groups using a thermally induced phase separation (TIPS) method. The CCM production leverages biomass resources, specifically cellulose and citric acid, utilizing TIPS to synthesize continuous porous materials through a straightforward heating and cooling process of polymer solutions. We demonstrated the efficacy of CCM as a hydrophilic interaction liquid chromatography (HILIC) medium for the efficient enrichment of glycopeptides. It exhibited remarkable selectivity in enriching glycopeptides from trypsin-digested immunoglobulin G (IgG), serving as a model protein, even in the presence of a significant amount of non-glycopeptide contaminants from bovine serum albumin (BSA) at a ratio of BSA/IgG of 1000/1. Additionally, CCM showed a low detection limit (0.25 fmol μL-1) and commendable reusability in glycopeptide enrichment, successfully enriching 35 glycopeptides from IgG. Additionally, 641 unique N-glycosylation sites of 698 unique glycopeptides from 393 glycosylated proteins were identified from the triplicate analysis of 900 μg of human hepatocellular carcinoma tissue. Therefore, CCM holds significant promise as an eco-friendly stationary phase for hydrophilic interaction liquid chromatography aimed at glycopeptide enrichment.

Facile Fabrication of Monodisperse Vinyl Hybrid Core-Shell Silica Microsphere with Short Range Radial Channel in bi-phase System.

The development of monodisperse hybrid silica microspheres with highly regular pore structure and uniform distribution of functional groups have significant value in the biomolecular separation field. In this work, the short range ordered pore channels are precisely constructed onto the non-porous silica microsphere surface by a bi-phase assembly method, and the cylindrical silica channel introduced a plethora of vinyl groups by "one-pot" co-condensation to form vinyl hybrid silica shell. As hydrophilic interaction chromatography (HILIC) stationary phase, the vinyl hybrid core-shell silica microsphere is simply modified with zwitterion glutathione (SiO2@SiO2-GSH), in which the HILIC enrichment process is significantly shortened due to its specific porous characteristics. Most importantly, SiO2@SiO2-GSH microsphere can enrich 2186 N-glycopeptides from the rat liver protein digest within 2min, which mapped to 806 glycoproteins. Compared with HILIC enrichment result within 1h, the glycoproteins and glycopeptides overlap are 88.3% and 79.1%, performing excellent reproducibility. The short range ordered channels onto the silica microsphere surface exhibit excellent mass transfer efficiency, so the developed bi-phase assembly method is expected to design more advanced hybrid silica materials for other urgently fields.

Protocol for Oligonucleotides Characterization Using Hydrophilic Interaction Chromatography.

Oligonucleotides (ONs) are an increasingly popular category of molecules in the pharmaceutical landscape, particularly attractive for the treatment of genetic and rare diseases. However, analyzing these molecules presents significant challenges, due to their highly hydrophilic nature, multiple negative charges, and the presence of closely related impurities resulting from the complex solid-phase synthesis process. Ion pairing reverse-phase liquid chromatography (IP-RPLC) is the preferred technique for ONs analysis but is not ideal for mass spectrometry (MS) coupling. Consequently, there is a growing interest in exploring alternative strategies with hydrophilic interaction chromatography (HILIC) emerging as one of the most promising options. As HILIC is not yet fully established for the analysis of ONs, we have prepared this protocol paper to facilitate entry into this field. It not only provides best practices, opportunities, and potential advantages but also caveats and other important considerations for using HILIC to characterize ONs. The paper addresses the selection of stationary and mobile phases, optimization of gradient conditions, MS coupling, and key aspects to consider when manipulating ON samples. We hope this protocol will help establish HILIC as a more universal solution for ONs analysis.

Hydrophilic interaction chromatography coupled to high resolution mass spectrometry (HILIC-LC-HRMS): An approach to study natural peptides in Viperidae snake venom.

Although proteins in snake venoms have been extensively studied and characterized, low-mass molecules remain relatively unexplored, mainly due to their low abundance, secondary role in envenomation, and some analytical technique limitations. However, these small molecules can provide new important data related to venom toxins' molecular structure, functions, and evolutionary relationships. This research aimed to characterize molecules below 10 kDa in the venoms of snakes from the Viperidae families (Bothrops, Agkistrodon, and Bitis) and compare two chromatographic approaches: reverse-phase chromatography (RP), a classic technique, and hydrophilic interaction liquid chromatography (HILIC), an alternative technique, both coupled with high-resolution mass spectrometry (HRMS). The results showed that the separation of the HILIC column provided a more efficient evenly distributed ion profile than RP, contributing to a 25.6% increase in the sequences identified. Homologous sequences for Bradykinin-potentiating peptides (BPPs) and fragments of major venom proteins, possibly cryptids, were found. In addition, BPP 13a, peptides rich in histidine and glycine (pHpG), and spacer sequences were identified in all snakes analyzed, especially with HILIC separation, suggesting that these sequences may be conserved within Viperidae. These findings indicate that the use of the HILIC column, compared to RP, is a promising approach for characterizing peptides in snake venom obtained by the ultrafiltration process. It contributes to the study of these still poorly understood molecules and is also a good option for studying other complex protein/peptide mixtures.

Stationary phase effects in hydrophilic interaction liquid chromatographic separation of oligonucleotides.

The use of liquid chromatography coupled with mass spectrometry (LC-MS) for the characterization of oligonucleotides and nucleic acids is a powerful analytical method. Recently, hydrophilic interaction chromatography (HILIC) has been proposed as a reasonable alternative to ion-pair reversed phase separations of oligonucleotides prior to MS. A wide variety of HILIC stationary phase surface chemistries are currently available. Although their selectivity can be considerably different, few studies have compared these chemistries for LC-MS analysis of oligonucleotides. We evaluated ten different HILIC column chemistries to understand their capabilities for separating a variety of oligonucleotides. In general, we found that most columns were ineffective at separating larger (n > 15-mer) oligonucleotides under the mobile phase and gradient conditions evaluated here. However, several stationary phases were found to be effective for separating smaller oligonucleotides such as endonuclease digestion products. Given that early eluting oligonucleotides were found to be compatible with standard electrospray ionization conditions, several different HILIC stationary phase options are available for LC-MS studies of smaller oligonucleotides including those generated in RNA modification mapping experiments.

HILIC-MS/MS Multi-targeted Method for Metabolomics Applications.

Metabolomics aims at identification and quantitation of key end point metabolites, basically polar, in order to study changes in biochemical activities in response to pathophysiological stimuli or genetic modifications. Targeted profiling assays enjoying a growing popularity over the last years with LC-MS/MS as a powerful tool for development of such (semi-)quantitative methods for a large number of metabolites. Here we describe a method for absolute quantitation of ca. 100 metabolites belonging to key metabolite classes such as sugars, amino acids, nucleotides, organic acids, and amines with a hydrophilic interaction liquid chromatography (HILIC) system comprised with ultra (high) performance liquid chromatography (UHPLC) with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes.

A Novel Approach to the Isolation of Disease Markers: Quantitative Analysis of Amino Acids in Exhaled Breath Condensates from Sleep Apnea Patients with Enhanced Sampling and LC-ESI-MS.

Interest in obstructive sleep apnea is rising due to its neurocognitive and cardiovascular impacts, including systemic hypertension, myocardial infarction, and cerebrovascular events. Obstructive sleep apnea diagnosis can be suggested through symptoms like snoring, daytime sleepiness, and physical signs like increased neck circumference; however, overnight polysomnography is recommended to confirm. Exhaled breath condensate has emerged as a novel, noninvasive technique for biomarker sample collection. It is simple, rapid, repeatable, and suitable for young children and severely ill patients. A direct method using liquid chromatography-mass spectrometry coupled with hydrophilic interaction chromatography was established for quantitative analysis of amino acids in human exhaled breath condensate samples. The separation was performed using XBridge Amide BEH column and a mobile phase of ammonium formate buffer in gradient conditions. The method exhibited a low detection limit (0.08-1.28ng/mL), good linearity (R2 between 0.9909 and 0.9987), and high recoveries (93-101.3%) for 21 studied amino acids with interday RSD of 2.1-7.7%. The LC-MS method was verified and applied to determine amino acids in exhaled breath condensate samples from obstructive sleep apnea patients, offering fast, reliable analysis without derivatization as a noninvasive alternative to standard methods.

An easy analytical method for the determination of L-Canavanine in legumes and pulse-based fish feed

Summary L-Canavanine is a naturally occurring non-protein amino acid found in certain legumes, where it plays a significant role in plant defence acting as an anti-herbivory compound by disrupting the biology of insect herbivores and pulse-based fish feed. In this study, a simple, easy and robust analytical method was developed and validated to monitor the compound accurately and reliable. The developed method includes a simple quick and low-cost extraction procedure using acidified acetonitrile. Chromatographic determination is based on Hydrophilic Interaction Liquid Chromatography coupled to tandem mass spectrometry operating in positive electron spray ionization. The method was successfully validated by assessing accuracy, precision, linearity of response (gradient of the calibration curve) at a range of concentrations 1.25 - 25 μg L−1, matrix effect and limit of quantitation according to European Guidelines. One way analysis of variance (ANOVA) was employed for estimating the contribution to overall uncertainty and finally to the reported value/concentration, of homogenization and sub-sampling step.

Development of a Multianalyte Method for the Determination of Phenolic Compounds in Residues from Beverage Production Using a Cyclofructan-Based Column in HILIC-MS/MS

This study expands and shows the versatility of applications in hydrophilic interaction chromatography coupled with triple quadrupole mass spectrometry (HILIC-MS/MS) using a FRULIC-N column to determine 31 phenolic compounds in beverage industry residues (coffee, apple juice, beer, and wine). Optimization, via Doehlert design, determined the acetonitrile proportion (50-90%) and pH (3.0-7.0) in the mobile phase (MP). A 22 factorial design assessed the concentration of ammonium acetate (10-110 mmol L-1) in the MP and column temperature (20-60 °C). Injection volume (5-45 µL) was univariately optimized. Separations at 60 °C reduced chromatographic run time to less than 11 min. Gradient elution (500 µL min-1) employed an MP comprising acetonitrile and ammonium acetate (110 mmol L-1, pH 7). Validation demonstrated average coefficient of determination (R2 ) 0.9707, limit of detection 0.001-0.503 mg L-1 and quantification 0.004-1.524 mg L-1, precision < 12.2%, recovery 92.4-110.8%. The method was applied to five residues, indicating its viability for determining phenolics in beverage production residues.

Effect of Short-Chain Organic Acids, Cations and Anions on the Retention of Citicoline Under Hydrophilic Interaction Liquid Chromatography Conditions

Effect of Short-Chain Organic Acids, Cations and Anions on the Retention of Citicoline Under Hydrophilic Interaction Liquid Chromatography Conditions