Hydrophilic Interaction Liquid Chromatography Research Articles

Glycan-Tailored Glycoproteomic Analysis Reveals Serine is the Sole Residue Subjected to O-Linked Glycosylation in Acinetobacter baumannii.

Protein glycosylation is a ubiquitous process observed across all domains of life. Within the human pathogen Acinetobacter baumannii, O-linked glycosylation is required for virulence; however, the targets and conservation of glycosylation events remain poorly defined. In this work, we expand our understanding of the breadth and site specificity of glycosylation within A. baumannii by demonstrating the value of strain specific glycan electron-transfer/higher-energy collision dissociation (EThcD) triggering for bacterial glycoproteomics. By coupling tailored EThcD-triggering regimes to complementary glycopeptide enrichment approaches, we assessed the observable glycoproteome of three A. baumannii strains (ATCC19606, BAL062, and D1279779). Combining glycopeptide enrichment techniques including ion mobility (FAIMS), metal oxide affinity chromatography (titanium dioxide), and hydrophilic interaction liquid chromatography (ZIC-HILIC), as well as the use of multiple proteases (trypsin, GluC, pepsin, and thermolysis), we expand the known A. baumannii glycoproteome to 33 unique glycoproteins containing 42 glycosylation sites. We demonstrate that serine is the sole residue subjected to glycosylation with the substitution of serine for threonine abolishing glycosylation in model glycoproteins. An A. baumannii pan-genome built from 576 reference genomes identified that serine glycosylation sites are highly conserved. Combined this work expands our knowledge of the conservation and site specificity of A. baumannii O-linked glycosylation.

Addressing a major interference in the quantification of psilocin in mouse plasma: Development of a validated liquid chromatography tandem mass spectrometry method

Psilocybin is a psychedelic compound found in some hallucinogenic “magic mushrooms”. Psilocin is the active metabolite of Psilocybin, and it is the subject of several studies for the treatment of psychological disorders, such as anxiety, depression, and post-traumatic stress disorder. As such, the pharmacokinetic properties of psilocin should be evaluated to ensure its safety and efficacy as part of the drug development process. Based on the previously published studies, reversed-phase liquid chromatography (LC) was tested for psilocin quantification. The analysis, however, showed a major interference in mouse plasma that was not, to the best of our knowledge, reported previously. We, therefore, aimed to identify and separate the interference, using various chromatographic columns, mobile phase conditions, and mass spectrometers (MS) instruments. Chromatographic separation was achieved on an ultra high performance liquid chromatography (UHPLC) system, and a quadrupole-linear ion trap equipped with an electrospray ionization (ESI) source was used in positive ion mode with multiple reaction monitoring (MRM). Several chromatographic conditions and column chemistries, including C-18 and Phenyl-hexyl were initially tested, and failed to separate the interference. Exact mass measurement and MS/MS analysis were used to determine the structure of the interfering compound, which was confirmed to be tryptophan. Using the identified structure of the interfering compound, a fast and reliable hydrophilic interaction liquid chromatography (HILIC)-MS/MS method was developed and validated, that was capable of separating psilocin from the interference while achieving a 0.5 ng/ml lower limit of quantification (LLOQ). The validated method was successfully applied to a pharmacokinetic study where psilocin was orally administered to C57BL/6 mouse subjects. Psilocin concentration in all the analyzed mouse plasma samples was successfully determined.

Applications of chromatography in glycomics

Glycomics, an emerging "omics" technology that was developed after genomics and proteomics, is a discipline that studies the composition, structure, and functions of glycomes in cells, tissues, and organisms. Glycomics plays key roles in understanding the laws of major life activities, disease prevention and treatment, and drug quality control and development. At present, the structural analysis of glycans relies mainly on mass spectrometry. However, glycans have low abundance in biological samples. In addition, factors such as variable monosaccharide compositions, differences in glycosidic bond positions and modes, diverse branching structures, contribute to the complexity of the compositions and structures of glycans, posing great challenges to glycomics research. Liquid chromatography can effectively remove matrix interferences and enhance glycan separation to improve the mass spectrometric response of glycans. Thus, liquid chromatography and liquid chromatography coupled with mass spectrometry are important technical tools that have been actively applied to solve these problems; these technologies play indispensable roles in glycomics research. Different studies have highlighted similarities and differences in the applications of various types of liquid chromatography, which also reflects the versatility and flexibility of this technology. In this review, we first discuss the enrichment methods for glycans and their applications in glycomics research from the perspective of chromatographic separation mechanisms. We then compare the advantages and disadvantages of these methods. Some glycan-enrichment modes include affinity, hydrophilic interactions, size exclusion, and porous graphitized carbon adsorption. A number of newly developed materials exhibit excellent glycan-enrichment ability. We enumerate the separation mechanisms of reversed-phase high performance liquid chromatography (RP-HPLC), high performance anion-exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC), and porous graphitic carbon (PGC) chromatography in the separation and analysis of glycans, and describe the applications of these methods in the separation of glycans, glycoconjugates, and glyco-derivatives. Among these methods, HILIC and PGC chromatography are the most widely used, whereas HPAEC and RP-HPLC are less commonly used. The HILIC and RP-HPLC modes are often used for the separation of derived glycans. The ionization efficiency and detectability of glycans are significantly improved after derivatization. However, the derivatization process is relatively cumbersome, and byproducts inevitably affect the accuracy and completeness of the detection results. HPAEC and PGC chromatography exhibit good separation effects on nonderivative glycans, but issues related to the detection integrity of low-abundance glycans owing to their poor detection effect continue to persist. Therefore, the appropriate analytical method for a specific sample or target analyte or mutual verification must be selected. Finally, we highlight the research progress in various chromatographic methods coupled with mass spectrometry for glycomics analysis. Significant progress has been made in glycomics research in recent years owing to advancements in the development of chromatographic separation techniques. However, several significant challenges remain. As the development of novel separation materials and methods continues, chromatographic techniques may be expected to play a critical role in future glycomics research.

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.

Mass spectrometric characterization of aminophospholipids containing N-(2-hydroxyethyl)glycine in kombu algae extracts.

1,2-Diacyl-sn-glycero-3-phospho-O-[N-(2-hydroxyethyl)glycines] (PHEGs) are a class of rare aminophospholipids found specifically in brown algae, including kombu seaweed. Despite their potential importance in algal physiology, a comprehensive mass spectrometry (MS) characterization, useful to understand their biological behaviour, is still lacking. To establish the structural regiochemical features of PHEGs, we employed hydrophilic interaction liquid chromatography (HILIC). Following separation, the isolated band of PHEGs was analyzed using MS techniques. This included multistage tandem MS experiments, performed in both positive and negative electrospray ionization modes at low and high resolution. By comparing MS/MS and MS3 spectra acquired in negative ion mode, the regiochemical rules for PHEG identification were established. The most abundant PHEG species in kombu seaweed, from both Laminaria ochroleuca (European Atlantic) and Laminaria longissima (Japan), was identified as PHEG 20:4/20:4. Less abundant species included PHEG 20:4/20:5 and hydroxylated forms of both PHEG 20:4/20:4 (i.e. 40:8;O) and 20:4/20:5 (40:9;O). The presence of a lyso PHEG 20:4 was consistently detected but at very low levels. This study employed MS analysis to elucidate the regiochemical patterns of PHEGs in kombu seaweed. We identified PHEG 20:4/20:4 as the dominant species, along with several less abundant variants, including hydroxylated forms. These findings provide valuable insights into the potential roles and metabolism of PHEGs in brown algae, paving the way for further investigation into their biological functions.

Green HPLC Enantioseparation of Chemopreventive Chiral Isothiocyanates Homologs on an Immobilized Chiral Stationary Phase Based on Amylose tris-[(S)-α-Methylbenzylcarbamate

Sulforaphane is a chiral phytochemical with chemopreventive properties. The presence of a stereogenic sulfur atom is responsible for the chirality of the natural isothiocyanate. The key role of sulfur chirality in biological activity is underscored by studies of the efficacy of individual enantiomers as chemoprotective agents. The predominant native (R) enantiomer is active, whereas the (S) antipode is inactive or has little or no biological activity. Here we provide an enantioselective high-performance liquid chromatography (HPLC) protocol for the direct and complete resolution of sulforaphane and its chiral natural homologs with different aliphatic chain lengths between the sulfinyl sulfur and isothiocyanate group, namely iberin, alyssin, and hesperin. The chromatographic separations were carried out on the immobilized-type CHIRALPAK IH-3 chiral stationary phase with amylose tris-[(S)-methylbenzylcarbamate] as a chiral selector. The effects of different mobile phases consisting of pure alcoholic solvents and hydroalcoholic mixtures on enantiomer retention and enantioselectivity were carefully investigated. Simple and environmentally friendly enantioselective conditions for the resolution of all chiral ITCs were found. In particular, pure ethanol and highly aqueous mobile phases gave excellent enantioseparations. The retention factors of the enantiomers were recorded as the water content in the aqueous-organic modifier (methanol, ethanol, or acetonitrile) mobile phases progressively varied. U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography retention mechanism on the CHIRALPAK IH-3 chiral stationary phase. Finally, experimental chiroptical studies performed in ethanol solution showed that the (R) enantiomers were eluted before the (S) counterpart under all eluent conditions investigated.

A singular chromatographic column breakthrough: Achieving full polarity range separations with the epoxy propanol molecular cage bonded silica stationary phase

The epoxy propanol molecular cage bonded silica stationary phase, RCC3-GLD@silica, synthesized through the ring-opening reaction of secondary amine with epoxy propanol using RCC3-R as the scaffold unit, was successfully prepared as confirmed by infrared spectroscopy, thermogravimetric analysis, and nitrogen adsorption-desorption characterization. This stationary phase demonstrated excellent separation performance in both reversed-phase and hydrophilic chromatography modes, effectively separating a wide variety of compounds including alkylbenzenes, polycyclic aromatic hydrocarbons, phenols, anilines, sulfonamides, nucleosides, amino acids, sugars, and acids. The development of RCC3-GLD@silica benefits from the synergistic effects of its hydrophobic and hydrophilic actions, as evidenced by the U-shaped characteristic of the retention factor for nucleoside compounds with changes in the aqueous content of the mobile phase, further confirming the simultaneous presence of reversed-phase and hydrophilic chromatography mechanisms. Not only did this stationary phase successfully separate 33 compounds in reversed-phase chromatography mode, but it also separated 54 compounds in hydrophilic interaction chromatography mode, showcasing its broad separation capability from weakly polar to strongly polar compounds on a single chromatographic column. This indicates a wide application prospect in the field of chromatographic analysis.

Pillar Array Mixer for Postcolumn Derivatization Integrated into Liquid Chromatography-Based Microfluidic Device.

The chemical derivatization of target analytes can enhance the sensitivity and selectivity of separation-based methods for metabolite analysis using microfluidic devices. However, the development of chromatography-based microfluidic devices with integrated derivatization units is challenging. In this study, a novel derivatization unit with a pillar array (PA)-based mixing channel was developed for postcolumn derivatization during on-chip liquid chromatography (LC). The PA mixer enhanced mixing between the derivatization reagents and analytes in the transverse direction, while preventing analyte dispersion in the flow direction. After the concept was confirmed using computational fluid dynamics analysis, microfluidic devices with a LC column and PA mixer were fabricated on a 20 × 20 mm silicon plate. Fluid experiments were performed using a PA mixer with a pillar size of 5 or 10 μm or a hollow-channel mixer, which revealed that the PA mixer enhanced transverse mixing without increasing the width of the analyte peak. Moreover, the developed device enabled the analysis of three amino acids within 40 s by separation via hydrophilic interaction chromatography followed by postcolumn fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde and fluorescence detection. Our results demonstrate the potential of integrated derivatization units for the development of micrototal analysis systems for use in bioanalysis.

The Impact of Protein Glycosylation on the Identification of Patients with Pediatric Appendicitis.

The identification of pediatric appendicitis is challenging due to the lack of specific markers thereby several factors are included in the diagnostic process such as abdominal pain, ultrasonography and altered laboratory parameters (C reactive protein, absolute neutrophil cell number and white blood cell number). The glycosylation pattern of serum N-glycome was analyzed in this study of 38 controls and 40 patients with pediatric appendicitis. The glycans were released by enzymatic deglycosylation followed by fluorescent labeling and solid-phase extraction. The prepared samples were analyzed by hydrophilic interaction liquid chromatography with fluorescence and mass-spectrometric detection. The generated data were analyzed by multiple statistical tests involving the most important laboratory parameters as well. Significant differences associated with the examined patient groups were revealed suggesting the potential use of glycosylation analysis supporting the detection of pediatric appendicitis.

Unravelling the Link between Oligonucleotide Structure and Diastereomer Separation in Hydrophilic Interaction Chromatography.

Therapeutic oligonucleotides (ONs) commonly incorporate phosphorothioate (PS) modifications. These introduce chiral centers and generate ON diastereomers. The increasing number of ONs undergoing clinical trials and reaching the market has led to a growing interest to better characterize the ON diastereomer composition, especially for small interfering ribonucleic acids (siRNAs). In this study, and for the first time, we identify higher-order structures as the major cause of ON diastereomer separation in hydrophilic interaction chromatography (HILIC). We have used conformational predictions and melting profiles of several representative full-length ONs to first analyze ON folding and then run mass spectrometry and HILIC to underpin the link between their folding and diastereomer separation. On top, we show how one can either enhance or suppress diastereomer separation depending on chromatographic settings, such as column temperature, pore size, stationary phase, mobile-phase ionic strength, and organic modifier. This work will significantly facilitate future HILIC-based characterization of PS-containing ONs; e.g., enabling monitoring of batch-to-batch diastereomer distributions in full-length siRNAs, a complex task that is now for the first time shown as possible on this delicate class of therapeutic double-stranded ONs.

Solving the retention time repeatability problem of hydrophilic interaction liquid chromatography

Hydrophilic interaction (liquid) chromatography (HILIC) has become the first choice LC mode for the separation of hydrophilic analytes. Numerous studies reported the poor retention time repeatability of HILIC. The problem was often ascribed to slow equilibration and insufficient re-equilibration time to establish the sensitive semi-immobilized water layer at the interface of the polar stationary phase and the bulk mobile phase. In this study, we compare retention time repeatability in HILIC for borosilicate glass and PFA (co-polymer of tetrafluoroethylene and perfluoroalkoxyethylene) solvent bottles. During this study, we observed peak patterns shifting towards higher retention times (for metabolites and peptides) and lower retention times (oligonucleotide sample) with ongoing analysis time when standard borosilicate glass bottles were used as solvent reservoirs. It was hypothesized that release of ions (sodium, potassium, borate, etc.) from the borosilicate glass bottles leads to alterations (thickness and electrostatic screening effects) in the semi-immobilized water layer which is adsorbed to the polar stationary phase surface under acetonitrile-rich eluents in HILIC with concomitant shifts in retention. When PFA solvent bottles were employed instead of borosilicate glass, retention time repeatability was greatly improved and changed from average 8.4 % RSD for the tested metabolites with borosilicate glass bottles to 0.14 % RSD for the PFA solvent bottles (30 injections over 12 h). Similar improvements were observed for peptides and oligonucleotides. This simple solution to the retention time repeatability problem in HILIC might contribute to a better acceptance of HILIC, especially in fields like targeted and untargeted metabolomics, peptide and oligonucleotide analysis.

Ammonium bicarbonate buffers combined with hybrid surface technology columns improve the peak shape of strongly tailing lipids

BackgroundLipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column. ResultsThe combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M − H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation. SignificanceIn conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.

Hyphenation of Liquid Chromatography and Trapped Ion Mobility - Mass Spectrometry for Characterization of Isomeric Phosphatidylethanolamines with Focus on N-Acylated Species.

In prior research, hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) has demonstrated applicability for characterizing regioisomers in lipidomics studies, including phosphatidylglycerols (PG) and bis(monoacyl)glycerophosphates (BMP). However, there are other lipid regioisomers, such as phosphatidylethanolamines (PE) and lyso-N-acyl-PE (LNAPE), that have not been studied as extensively. Therefore, hyphenated mass spectrometric methods are needed to investigate PE and LNAPE regioisomers individually. The asymmetric structure of LNAPE favors isomeric species, which can result in coelution and chimeric MS/MS spectra. One way to address the challenge of chimeric MS/MS spectra is through mobility-resolved fragmentation using trapped ion mobility spectrometry (TIMS). Therefore, we developed a multidimensional HILIC-TIMS-MS/MS approach for the structural characterization of isomeric phosphatidylethanolamines in both negative and positive ionization modes. The study revealed the complementary fragmentation pattern and ion mobility behavior of LNAPE in both ionization modes, which was confirmed by a self-synthesized LNAPE standard. With this knowledge, a distinction of regioisomeric PE and LNAPE was achieved in human plasma samples. Furthermore, regioisomeric LNAPE species containing at least one unsaturated fatty acid were noted to exhibit a change in collision cross-section in positive ionization mode, leading to a lipid characterization with respect to fatty acyl positional level. Similar mobility behavior was also observed for the biological LNAPE precursor N-acyl-PE (NAPE). Application of this approach to plasma and cereal samples demonstrated its effectiveness in regioisomeric LNAPE and NAPE species' elucidation.

Effect of Mobile Phase pH and Counterion Concentration on Retention and Selectivity for a Diol Column in Hydrophilic Interaction Liquid Chromatography

This work focuses on the effects of the mobile phase pH and the counterion concentration in buffer solution on retention in hydrophilic interaction liquid chromatography (HILIC) mode. Analytes with various acid-base properties and a silica-based Diol stationary phase were used. Retention and separation selectivity changes with ionization of the analytes and the adsorbent’s groups were discussed. It was demonstrated that the Diol phase behaved as a cation exchanger at pH 5.76 because of its residual dissociated silanols, while the phase provided almost no charge at lower pH (2.85). Separation efficiency and asymmetry factors for ionized compounds were also affected by the changes of mobile phase pH and counterion concentration. Separation conditions for the mixture of analytes of various acid-base properties were established.

Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

Investigation of Retention Mechanism of B2 and B3 in Home-Made ZIC-HILIC Columns and Its Application for The Simultaneous Estimation in Multivitamin Tablets

In the present study, the retention behavior of two vitamins (B2 and B3) was investigated using a zwitterionic hydrophilic interaction liquid chromatography technique (ZIC-HILIC) on two home-made columns, and two analytical methods for these vitamins were established as a result. Several chromatographic parameters were carefully investigated and optimized. The best separation was achieved on two new home-made columns (ZIC-1 and ZIC-3) employing acetonitrile/sodium acetate buffer as the mobile phase (40 mM, pH 4.75). The target vitamins were detected at 254 nm. In the ZIC-1 and ZIC-3 columns, the linear ranges of the target vitamins (B2 and B3) were 0.06-11.0 and 0.08-7.0 µg/mL, respectively. For both target vitamins, the detection and quantitation limits of the suggested methods were 0.011-0.055 and 0.033-0.166 µg/mL, respectively. They also showed high precision (RSD ≤ 0.93), linearity (r2 ≥ 0.9988), and recovery (between 99.0 and 101.71%). The suggested procedures have been validated and tested on multivitamin tablets, proving that they are suitable for analyzing food supplements for these tablets, and acceptable results were obtained for the study.

Hydrophilic Interaction Liquid Chromatography for the Analysis of Pharmaceutical Formulations

Abstract: For a long time, Reversed-Phase Liquid Chromatography (RPLC) was the most dominant technique for the analysis of pharmaceutical compounds, but with poor efficiency in the separation of small polar molecules. From the efforts to solve the problem of insufficient retention of these molecules, during the last decades, a mode of liquid chromatography named Hydrophilic Interaction Liquid Chromatography (HILIC) has experienced vast expansion. It is based on the use of a highly hydrophilic stationary phase along with an aqueous mobile phase with high organic modifier content. In this review, the characteristics of stationary and mobile phases used in HILIC are described, and corresponding separation mechanisms are discussed. An overview of recently published papers dealing with the application of HILIC in analyzing pharmaceuticals in biological and non-biological samples is provided. Besides, the application of HILIC systems in the determination of the physicochemical properties of compounds is described.

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

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

Metabolic plasma signatures in thoracic aortic aneurysm associated to tricuspid and bicuspid aortic valves

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III with co-funding from the ERDF and Comunidad de Madrid Background/Introduction Thoracic aortic aneurysm (TAA) develops asymptomatic with life-threatening consequences and its diagnosis is usually fortuitous. TAA is mostly idiopathic, although its prevalence is significantly higher in subjects with bicuspid aortic valve (BAV) for unknown reasons. We hypothesize that it is essential to investigate idiopathic TAA by differentiating between BAV and tricuspid aortic valve (TAV) patients for personalized diagnosis and treatment. Purpose We aimed to identify plasma metabolic profiles of aortic dilatation in idiopathic TAA, differentiating between subjects with BAV or TAV. Methods Plasma samples from 41 idiopathic TAA patients and 57 subjects without aortic dilatation (control, C) were collected and classified according to aortic valve type (BAV or TAV). Full metabolomics coverage was carried out by 1H RMN and LC-MS/MS untargeted analysis, including reverse phase and hydrophilic interaction chromatography and positive and negative electrospray ionization modes. Significant variation in metabolite levels were considered if FDR-adjusted p-value <0.05. Results Our analyses highlighted a metabolic signature specific for BAV and TAV subjects. BAV patients presented a marked glycerophospholipid metabolism alteration involving different lysophospholipids (LPAs, LPCs and LPEs) and phospholipids (PCs, PEs, PAs and PIs). Particularly, LPCs and LPEs were increased in BAV-TAA vs BAV-C, being an indicator of stimulation of glucose utilization through glycolysis and suppression of fatty acid biosynthesis. In contrast, carnitine-related metabolism was the most altered pathway observed in TAV subjects. In particular, we detected an increment in plasma acylcarnitines in TAV-TAA compared to TAV-C suggesting an altered β-oxidation of fatty acids and mitochondrial overload. Conclusions Idiopathic TAA development is differentially reflected in plasma from BAV and TAV patients, supporting an individualized diagnosis valve-dependent and suggesting also differential therapeutic targets.

Chiral Hydroxy Metabolite of Mebendazole: Analytical and Semi-Preparative High-Performance Liquid Chromatography Resolution and Chiroptical Properties.

Mebendazole (MBZ) is a benzimidazole carbamate anthelmintic used worldwide for the treatment and prevention of parasitic disorders in animals and humans. A large number of in vivo and in vitro studies have demonstrated that MBZ also has anticancer activity in multiple types of cancers. After oral administration, the phenylketone moiety of MBZ is rapidly reduced to the hydroxyl group to form the chiral hydroxy metabolite (MBZ-OH). To the best of our knowledge, there is no information in the literature on the stereochemical course of transformation and the anthelmintic and antitumor activity of individual enantiomers of MBZ-OH. In the present study, we describe in detail the direct HPLC resolution of MBZ-OH on a 100 mm × 4.6 mm Chirapak IG-3 column packed with 3 μm silica particles containing amylose (3-chloro-5-methylphenylcarbamate) as a selector. At 25 °C and using pure methanol as the mobile phase, the enantioseparation and resolution factors were 2.38 and 6.13, respectively. These conditions were scaled up at a semi-preparative scale using a 250 mm × 10 mm Chiralpak IG column to isolate multi-milligram amounts of both enantiomeric forms of the chiral metabolite. The chiroptical properties of the collected enantiomers were determined and, through a theoretical study, were related to the more stable conformations of MBZ-OH. The first and second eluted enantiomers were dextrorotatory and levorotatory, respectively, in dimethylformamide solution. Finally, by recording the retention factors of the enantiomers as the water content in the water-acetonitrile mobile phases was progressively varied, U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography retention mechanism on the Chirapak IG-3 chiral stationary phase.