Hydrophilic Interaction Liquid Chromatography Research Articles

A high-efficiency integrated strategy for the targeted discovery of novel barrigenol-type triterpenoid saponins from the shell of Xanthoceras sorbifolium Bunge by offline two-dimensional chromatography-Orbitrap mass spectrometry, neutral loss data acquisition, and predicted natural product screening

Natural products are considered as potential sources of leading compounds and play an important role in drug discovery. The liquid chromatography-mass spectrometry (LC-MS) technique is a powerful tool for compound-guided isolation from natural products. In this study, a high-efficiency integrated strategy was adopted to improve the new leading compounds discovery, including offline two-dimensional (2D) LC to extend the peak capacities, target neutral loss (NL) data-dependent acquisition (DDA) for barrigenol-type triterpenoids saponins and automatic screening through predicted natural product screening (PNPS) in TraceFinder. To validate the integrated strategy, the shell of Xanthoceras sorbifolium Bunge (XSB) was taken as a case. An offline 2D-LC system was constructed with hydrophilic interaction chromatography (HILIC) and reversed phase (RP) C18 column, and orthogonality of 0.66 and peak capacity of 3494. The 2D-LC system improved chromatographic baseline separation and peak resolution. Full MS/all ion fragmentation (AIF)/NL dd-MS2 DDA was employed for the detection of the barrigenol saponins. PNPS strategy was adopted and markedly extended the screening coverage. The combined strategy showed about 5 times improvement in the screening capability. The PNPS screening process, using TraceFinder software, discovered a total of 752 barrigenol saponins from the shell of XSB, including 707 potentially new barrigenol saponins, accounting for 94.02%. The feasibility of the strategy was also confirmed by the isolation of two novel barrigenol saponins, the structures of which were unambiguously identified using nuclear magnetic resonance (NMR). Furthermore, this strategy could also be applied to rapidly discover new bioactive constituents from other herbal medicines or other natural sources, especially the barrigenol saponins constituents.

Hydrophilic Interaction Liquid Chromatography at Subzero Temperature for Hydrogen-Deuterium Exchange Mass Spectrometry.

Chromatographic separations at subzero temperature significantly improve the precision of back-exchange-corrected hydrogen-deuterium exchange mass spectrometry (HDX-MS) determinations. Our previously reported dual-enzyme HDX-MS analysis instrument used reversed phase liquid chromatography (RPLC) at -30 °C, but high backpressures limited flow rates and required materials and equipment rated for very high pressures. Here, we report the design and performance of a dual-enzyme HDX-MS analysis instrument comprising a RPLC trap column and a hydrophilic interaction liquid chromatography (HILIC) analytical column in a two-dimensional RPLC-HILIC configuration at subzero temperature. During operation at -30 °C, the HILIC column manifests greatly reduced backpressure, which enables faster analytical flow rates and the use of materials rated for lower maximum pressures. The average peptide eluted from a HILIC column during a 40 min gradient at -30 °C contained ≈13% more deuterium than peptides eluted from a tandem RPLC-RPLC apparatus using a conventional 8 min gradient at 0 °C. A subset of peptides eluted from the HILIC apparatus contained ≈24% more deuterium.

Circulating choline and phosphocholine measurement by a hydrophilic interaction liquid chromatography–tandem mass spectrometry

BackgroundGiven the growing interest in studying the role of choline and phosphocholine in the development and progression of tumor pathology, in this study we describe the development and validation of a fast and robust method for the simultaneous analysis of choline and phosphocholine in human plasma. MethodsCholine and phosphocholine quantification in human plasma was obtained using a hydrophilic interaction liquid chromatography–tandem mass spectrometry technique. Assay performance parameters were evaluated using EMA guidelines. ResultsCalibration curve ranged from 0.60 to 38.40 μmol/L (R2 = 0.999) and 0.08–5.43 μmol/L (R2 = 0.998) for choline and phosphocholine, respectively. The Limit Of Detection of the method was 0.06 μmol/L for choline and 0.04 μmol/L for phosphocholine. The coefficient of variation range for intra-assay precision is 2.2–4.1 % (choline) and 3.2–15 % (phosphocholine), and the inter-assay precision range is < 1–6.5 % (choline) and 6.2–20 % (phosphocholine). The accuracy of the method was below the ±20 % benchmarks at all the metabolites concentration levels. In-house plasma pool of apparently healthy adults was tested, and a mean concentration of 15.97 μmol/L for Choline and 0.34 μmol/L for Phosphocholine was quantified. ConclusionsThe developed method shows good reliability in quantifying Choline and Phosphocholine in human plasma for clinical purposes.

Unravelling the brain metabolome: A review of liquid chromatography - mass spectrometry strategies for extracellular brain metabolomics

The analysis of the brain extracellular metabolome is of interest for numerous subdomains within neuroscience. Not only does it provide information about normal physiological functions, it is even more of interest for biomarker discovery and target discovery in disease. The extracellular analysis of the brain is particularly interesting as it provides information about the release of mediators in the brain extracellular fluid to look at cellular signaling and metabolic pathways through the release, diffusion and re-uptake of neurochemicals. In vivo samples are obtained through microdialysis, cerebral open-flow microperfusion or solid-phase microextraction. The analytes of potential interest are typically low in concentration and can have a wide range of physicochemical properties. Liquid chromatography coupled to mass spectrometry has proven its usefulness in brain metabolomics. It allows sensitive and specific analysis of low sample volumes, obtained through different approaches. Several strategies for the analysis of the extracellular fluid have been proposed. The most widely used approaches apply sample derivatization, specific stationary phases and/or hydrophilic interaction liquid chromatography. Miniaturization of these methods allows an even higher sensitivity. The development of chiral metabolomics is indispensable, as it allows to compare the enantiomeric ratio of compounds and provides even more challenges. Some limitations continue to exist for the previously developed methods and the development of new, more sensitive methods remains needed. This review provides an overview of the methods developed for sampling and liquid chromatography-mass spectrometry analysis of the extracellular metabolome.

LC-MS/MS Quantitation of HILIC-Enriched N-glycopeptides Derived from Low-Abundance Serum Glycoproteins in Patients with Narcolepsy Type 1.

Glycoproteomic analysis is always challenging because of low abundance and complex site-specific heterogeneity. Glycoproteins are involved in various biological processes such as cell signaling, adhesion, and cell-cell communication and may serve as potential biomarkers when analyzing different diseases. Here, we investigate glycoproteins in narcolepsy type 1 (NT1) disease, a form of narcolepsy characterized by cataplexy-the sudden onset of muscle paralysis that is typically triggered by intense emotions. In this study, 27 human blood serum samples were analyzed, 16 from NT1 patients and 11 from healthy individuals serving as controls. We quantified hydrophilic interaction liquid chromatography (HILIC)-enriched glycopeptides from low-abundance serum samples of controls and NT1 patients via LC-MS/MS. Twenty-eight unique N-glycopeptides showed significant changes between the two studied groups. The sialylated N-glycopeptide structures LPTQNITFQTESSVAEQEAEFQSPK HexNAc6, Hex3, Neu5Ac2 (derived from the ITIH4 protein) and the structure IVLDPSGSMNIYLVLDGSDSIGASNFTGAK HexNAc5, Hex4, Fuc1 (derived from the CFB protein), with p values of 0.008 and 0.01, respectively, were elevated in NT1 samples compared with controls. In addition, the N-glycopeptide protein sources Ceruloplasmin, Complement factor B, and ITH4 were observed to play an important role in the complement activation and acute-phase response signaling pathways. This may explain the possible association between the biomarkers and pathophysiological effects.

Simultaneous Analysis of Hydrophobic Atractylenolides, Atractylon and Hydrophilic Sugars in Bai-Zhu Using a High-Performance Liquid Chromatography Column Tandem Technique.

An analytical method was established using high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) with -C18 and -NH2 column tandem for the simultaneous determination of hydrophobic atractylenolide I, II, III, atractylone and hydrophilic compounds glucose, fructose and sucrose in the dried rhizome of Atractylodes macrocephala Koidz (a natural raw material for health foods, Bai-Zhu aka. in Chinese). The method combines the different separation capabilities of reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography. It can provides a new choice for the simultaneous determination of hydrophilic and hydrophobic compounds in traditional Chinese medicines and health foods. It provided a reference method for the quality control of Bai-Zhu. The results showed that the linear correlation coefficients of the established column tandem chromatographic method were all greater than 0.9990, the relative standard deviation was 0.1-2.8%, and the average recovery was 96.7-103.1%. The contents of atractylenolide I, II, III, atractylone, fructose, glucose, and sucrose in 17 batches of Baizhu were 172.3-759.8 μg/g, 201.4-612.8 μg/g, 160.3-534.2 μg/g, 541.4-8723.1 μg/g, 6.9-89.7 mg/g, 0.7-7.9 mg/g, and 1.2-21.0 mg/g, respectively.

A Single-Run HPLC-MS Multiplex Assay for Therapeutic Drug Monitoring of Relevant First- and Second-Line Antibiotics in the Treatment of Drug-Resistant Tuberculosis.

The treatment of drug-resistant Mycobacterium tuberculosis relies on complex antibiotic therapy. Inadequate antibiotic exposure can lead to treatment failure, acquired drug resistance, and an increased risk of adverse events. Therapeutic drug monitoring (TDM) can be used to optimize the antibiotic exposure. Therefore, we aimed to develop a single-run multiplex assay using high-performance liquid chromatography-mass spectrometry (HPLC-MS) for TDM of patients with multidrug-resistant, pre-extensively drug-resistant and extensively drug-resistant tuberculosis. A target profile for sufficient performance, based on the intended clinical application, was established and the assay was developed accordingly. Antibiotics were analyzed on a zwitterionic hydrophilic interaction liquid chromatography column and a triple quadrupole mass spectrometer using stable isotope-labeled internal standards. The assay was sufficiently sensitive to monitor drug concentrations over five half-lives for rifampicin, rifabutin, levofloxacin, moxifloxacin, bedaquiline, linezolid, clofazimine, terizidone/cycloserine, ethambutol, delamanid, pyrazinamide, meropenem, prothionamide, and para-amino salicylic acid (PAS). Accuracy and precision were sufficient to support clinical decision making (≤±15% in clinical samples and ±20-25% in spiked samples, with 80% of future measured concentrations predicted to fall within ±40% of nominal concentrations). The method was applied in the TDM of two patients with complex drug-resistant tuberculosis. All relevant antibiotics from their regimens could be quantified and high-dose therapy was initiated, followed by microbiological conversion. In conclusion, we developed a multiplex assay that enables TDM of the relevant first- and second-line anti-tuberculosis medicines in a single run and was able to show its applicability in TDM of two drug-resistant tuberculosis patients.

Ion-pair reversed-phase and hydrophilic interaction chromatography methods for analysis of phosphorothioate oligonucleotides

In this study we investigated the separation of a 25 mer fully phosphorothioated oligonucleotide from its truncated n-1 (24 mer) species and selected phosphodiester 25 mer impurities using ion-pair reversed-phase chromatography. The hydrophobicity of ion-pairing agents (alkylamines) impacts n-1 separation selectivity. 25 mer impurities with single and double phosphodiester bonds eluted prior to the parent phosphorothioate oligonucleotide in the same region as 24 mer impurities, which complicated the chromatographic separation. An alternative technique, hydrophilic interaction chromatography, provided a different retention pattern; 24 mer n-1 impurities eluted prior to the 25 mer, while the phosphodiester impurities eluted after the full-length oligonucleotide. This enabled an improved chromatographic separation of the truncated and phosphodiester impurities from the phosphorothioate oligonucleotide of interest.

Novel stationary phases based on epoxidized polystyrene-divinylbenzene for three modes of liquid chromatography

Mixed-mode stationary phases based on epoxidized copolymer of styrene and divinylbenzene have been obtained by amination with methylamine, further alkylation with 1,4-butanediol diglycidyl ether and opening the terminal oxirane rings with dimethylethanolamine. To evaluate the effect of the number of anchor amino and diol groups on the degree of hydrophilization of resins, the quantity of reagents was varied. Polymerization of glycidol in the functional layer at an increased pH of the reaction medium was carried out for additional shielding of the substrate. It was found that increasing the number of anchor amino groups is promising for increasing hydrophilicity in suppressed ion chromatography and hydrophilic interaction liquid chromatography modes, while polymerization of glycidol increases the degree of substrate shielding. The applicability of the most hydrophilic adsorbent in three HPLC modes was demonstrated. Novel mixed-mode stationary phase allows the separation of six nucleosides and nitrogenous bases by hydrophilic interaction liquid chromatography, 7 alkylbenzenes by reversed phase liquid chromatography, and 20 organic and inorganic anions by suppressed ion chromatography.

Stereochemistry of aminoacylated cardiolipins and phosphatidylglycerols from bacteria.

Hydrophilic interaction liquid chromatography (HILIC) connected with electrospray high-resolution tandem mass spectrometry (MS) was used for the analysis of unusual amino acid (AA) substituted phosphatidylglycerols (PG) and cardiolipins (CL) in mesophilic and thermophilic bacteria. Individual peaks from the lipid class separation by HILIC were isolated and hydrolyzed to determine the absolute configuration of the aminoacyl side chain. The configuration of the aminoacyl side chain was assigned by indirect liquid chromatography (LC) enantiomer separation after the hydrolysis of the aminoacylated (aminoacyl) lipids using N-(4-nitrophenoxycarbonyl)-l-phenylalanine 2-methoxyethyl ester as chiral derivatizing agent and reversed phase LC-MS for analysis. When two chromatographic methods were combined, less common AAs, such as d-allo-Ile and d-allo-Thr, were identified. The taxonomic classification of bacteria showed that bacteria of the family Bacillaceae (Bacillus and Geobacillus) produce branched-chain AAs, that is, d-allo-Ile, d-Ile, and d-Leu. These AAs were present only in the genera Bacillus and Geobacillus and not in Alicyclobacillus acidoterrestris (family Alicyclobacillaceae). On the contrary, hydroxy AAs, that is, l- and d-Thr, and l- and d-allo-Thr, were identified as aminoacyl-PG and aminoacyl-CL in A. acidoterrestris and were not present in the genera Bacillus and Geobacillus. Therefore, the complete analysis made it possible to identify the stereochemistry of AAs in aminoacyl PGs and CLs and use this fact for chemotaxonomy.

Development and Validation of LC-MS/MS Method for Determination of Cytisine in Human Serum and Saliva.

Cytisine (CYT) is a quinolizidine alkaloid used for nicotine addiction treatment. Recent clinical trial data regarding cytisine confirm its high effectiveness and safety as a smoking cessation treatment. CYT's popularity is growing due to its increased availability and licensing in more countries worldwide. This increased use by smokers has also resulted in an urgent need for continued drug research, including developing appropriate analytical methods for analyzing the drug in biological samples. In this study, a simple, fast, and reliable method combining hydrophilic interaction liquid chromatography and electrospray ionization quadrupole time of flight mass spectrometry (HILIC/ESI-QTOF-MS) for the determination of CYT in human serum and saliva was developed and validated. This was undertaken after the previous pre-treatment of the sample using solid-phase extraction (SPE). A hydrophilic interaction liquid chromatography (HILIC) column with a silica stationary phase was used for chromatographic analysis. In a linear gradient, the mobile phase consisted of acetonitrile (ACN) and formate buffer at pH 4.0. The proposed method was fully validated and demonstrated its sensitivity, selectivity, precision, and accuracy. The method was successfully applied to determine CYT in serum and, for the first time, in saliva. The findings indicate that saliva could be a promising non-invasive alternative to measure the free concentration of CYT.

Untargeted metabolomics analysis on kidney tissues from mice reveals potential hypoxia biomarkers

Chronic hypoxia may have a huge impact on the cardiovascular and renal systems. Advancements in microscopy, metabolomics, and bioinformatics provide opportunities to identify new biomarkers. In this study, we aimed at elucidating the metabolic alterations in kidney tissues induced by chronic hypoxia using untargeted metabolomic analyses. Reverse phase ultrahigh performance liquid chromatography-mass spectroscopy/mass spectroscopy (RP–UPLC–MS/MS) and hydrophilic interaction liquid chromatography (HILIC)–UPLC–MS/MS methods with positive and negative ion mode electrospray ionization were used for metabolic profiling. The metabolomic profiling revealed an increase in metabolites related to carnitine synthesis and purine metabolism. Additionally, there was a notable increase in bilirubin. Heme, N-acetyl-l-aspartic acid, thyroxine, and 3-beta-Hydroxy-5-cholestenoate were found to be significantly downregulated. 3-beta-Hydroxy-5-cholestenoate was downregulated more significantly in male than female kidneys. Trichome Staining also showed remarkable kidney fibrosis in mice subjected to chronic hypoxia. Our study offers potential intracellular metabolite signatures for hypoxic kidneys.

A comprehensive evaluation of a polymeric zwitterionic hydrophilic monolith for nucleotide separation.

Rapid and effective separation of nucleotides (NTs) and their derivatives is crucial for studying their physiological functions. In this work, we comprehensively evaluated the separation ability of a zwitterionic hydrophilic monolith, i.e., poly(N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammonium betaine-co-N,N'-methylenebisacrylamide) (poly(SPP-co-MBA)) for NTs analysis, including its selectivity, chemical stability under extremely basic condition and compatibility with hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (HILIC-MS). The poly(SPP-co-MBA) monolith exhibited excellent chemical stability, as evidenced by the low relative standard deviation of retention time (0.16-1.05%) after 4000 consecutive injections over one month under strong alkaline elution condition (pH 10). After optimizing the separation conditions, including buffer pH and concentration, organic solvent content and column temperature, four nucleoside triphosphates, five nucleoside diphosphates and five nucleoside monophosphates were baseline separated within 7min. Additionally, the mixtures containing one nucleoside and its corresponding mono-, di-, and triphosphates were baseline separated within only 3min, respectively. It is good HILIC-MS compatibility was also confirmed by the satisfactory peak shape and high response of nine NTs. Overall, the proposed poly(SPP-co-MBA) monolith exhibited good mechanical stability and compatibility of HILIC-MS, making it a promising technique for NTs analysis.

Human Cerebrospinal Fluid Sample Preparation and Annotation for Integrated Lipidomics and Metabolomics Profiling Studies

Cerebrospinal fluid (CSF) is a metabolically diverse biofluid and a key specimen for exploring biochemical changes in neurodegenerative diseases. Detecting lipid species in CSF using mass spectrometry (MS)-based techniques remains challenging because lipids are highly complex in structure, and their concentrations span over a broad dynamic range. This work aimed to develop a robust lipidomics and metabolomics method based on commonly used two-phase extraction systems from human CSF samples. Prioritizing lipid detection, biphasic extraction methods, Folch, Bligh and Dyer (B&D), Matyash, and acidified Folch and B&D (aFolch and aB&D) were compared using 150 μL of human CSF samples for the simultaneous extraction of lipids and metabolites with a wide range of polarity. Multiple chromatographical separation approaches, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), and gas chromatography (GC), were utilized to characterize human CSF metabolome. The aB&D method was found as the most reproducible technique (RSD < 15%) for lipid extraction. The aB&D and B&D yielded the highest peak intensities for targeted lipid internal standards and displayed superior extracting power for major endogenous lipid classes. A total of 674 unique metabolites with a wide polarity range were annotated in CSF using, combining RPLC-MS/MS lipidomics (n = 219), HILIC-MS/MS (n = 304), and GC-quadrupole time of flight (QTOF) MS (n = 151). Overall, our findings show that the aB&D extraction method provided suitable lipid coverage, reproducibility, and extraction efficiency for global lipidomics profiling of human CSF samples. In combination with RPLC-MS/MS lipidomics, complementary screening approaches enabled a comprehensive metabolite signature that can be employed in an array of clinical studies.Graphical abstract

Untargeted Metabolomic Study of Lung Cancer Patients after Surgery with Curative Intent.

Lung cancer (LC) is a leading cause of mortality, claiming more than 1.8 million deaths per year worldwide. Surgery is one of the most effective treatments when the disease is in its early stages. The study of metabolic alterations after surgical intervention with curative intent could be used to assess the response to treatment or the detection of cancer recurrence. In this study, we have evaluated the metabolomic profile of serum samples (n = 110) from preoperative (PRE) and postoperative (POST) LC patients collected at two different time points (1 month, A; 3-6 months, B) with respect to healthy people. An untargeted metabolomic platform based on reversed phase (RP) and hydrophilic interaction chromatography (HILIC), using ultra-high performance liquid chromatography (UHPLC) and mass spectrometry (MS), was applied (MassIVE ID MSV000092213). Twenty-two altered metabolites were annotated by comparing all the different studied groups. DG(14,0/22:1), stearamide, proline, and E,e-carotene-3,3'-dione were found altered in PRE, and their levels returned to those of a baseline control group 3-6 months after surgery. Furthermore, 3-galactosyllactose levels remained altered after intervention in some patients. This study provides unique insights into the metabolic profiles of LC patients after surgery at two different time points by combining complementary analytical methods.

Analysis of glycosylated cardiolipins from thermophilic bacteria using GC–MS and LC–ESI–MS/MS methods

Unusual glucose-substituted cardiolipins (Glcx-CLs) in three genera of thermophilic bacteria, having more than one glycosidically linked glucose to the hydroxyl of the central glycerol of Glcx-CLs were identified for the first time in thermophilic bacteria of the genera Geobacillus, Meiothermus, and Thermus. The number of glucoses reached up to five units. The structure of glycosidically linked oligosaccharides was determined based on shotgun analysis MS (electrospray high-resolution tandem mass spectrometry), partially methylated alditol acetates were identified by GC–MS, both electron ionization (EI) and positive chemical ionization (PCI), hydrophilic interaction liquid chromatography (HILIC) separation and identification of CLs glycosides by high resolution MS–ESI, and digestion by specific glycosidases.

Silica-Based Stationary Phase with Surface Bound N-Acetyl-glucosamine for Hydrophilic Interaction Liquid Chromatography.

A hydrophilic silica-based stationary phase with surface bound N-acetylglucosamine (GlcNAc-silica) was prepared in house and characterized physically via Fourier transform infrared (FTIR) analysis and thermogravimetric analysis (TGA) and chromatographically over a wide range of mobile phase compositions. While both FTIR and TGA confirmed the attachment of the GlcNAc ligands to the silica surface, the chromatographic evaluation of GlcNAc-silica with polar and slightly polar standard solutes (e.g., sugars, nucleic acid fragments, phenolic, and benzoic acid derivatives) yielded the typical hydrophilic interaction liquid chromatography (HILIC) behaviors in the sense that retention increased with increases in solute polarity and the organic content (i.e., acetonitrile) of the hydro-organic mobile phase (i.e., ACN-rich mobile phase). Sugars derivatized with 1-naphthylamine (1-NA) and 2-aminoanthrcene (2-AA) such as xylose, glucose, and short chains maltooligosaccharides constituted the most polar species for HILIC retention evaluation, and in addition, the maltooligosaccharides offered a polar homologous series for gauging the hydrophilicity of GlcNAc-silica in analogy with alkylbenzene homologous series and other nonpolar homologues for evaluating the hydrophobicity of non-polar stationary phases. On the other hand, the benzoic acid and phenolic acid derivatives were the probe solutes for evaluating the HILIC retention dependence of ionizable solutes on the pH of the mobile phase. Similarly, the nucleobase and nucleoside weak basic solutes as well as some typical cyclic nucleotide acidic solutes allowed for the examination of the dependence of solute retention on the pH of the mobile as well as the polarity of the species.

Determination of Nε-(1-Carboxymethyl)-L-Lysine in brewing soy sauce by ultrahigh performance liquid chromatography coupled with tandem mass spectrometry

A method for the determination of Nε-(1-Carboxymethyl)-L-Lysine (CML) in brewing soy sauce was developed using ultrahigh performance liquid chromatography-tandem mass spectrometry. A mixed cation exchange solid phase extraction cartridge was used for purification, while gradient elution of a hydrophilic interaction chromatography column was performed with a 0.1% formic acid aqueous solution-acetonitrile. The adding standard recovery rate of this method ranged from 86.94 to 92.61%, and the relative standard deviation (RSD) was between 3.69 and 6.18%. The limit of detection (LOD) was 0.038 ng/mL, and the limit of quantification was 0.127 ng/mL. The RSD values of the repeatability test and the parallel test were 3.44 and 6.56%, respectively. Using this method, the CML contents determined in eight brewing soy sauce samples were 3.16 ± 0.18 – 15.10 ± 1.19 µg/mL. This method is simple, accurate, sensitive, and precise, and it is an effective method for monitoring the CML content in brewing soy sauce.

One-step preparation of a biphasic monolithic separation media for enriching and purifying lupeol in medicine plants

In this study, a monolith consisted of two materials with different separation modes were prepared by a one-step method at both ends of a column. Acrylic acid and 2-hydroxyethyl methacrylate (HEMA) were used as monomers for hydrophilic and hydrophobic parts, respectively. The total pore volumes and porosities of hydrophilic and hydrophobic materials were 3.1098 mL/g and 75.2535 %, 4.4380 mL/g and 72.5317 %, respectively. The prepared monolithic columns had excellent mechanical strength and good permeability, and can bear high flow rates up to 5 mL/min. The biphasic monolith functioned in two retention modes: hydrophilic interaction chromatography (HILIC) and hydrophobic interaction chromatography (HIC), and the dual-mode were realized by switching the ratio of acetonitrile in the mobile phase. The biphasic monolithic column was used as solid phase extraction (SPE) adsorbent for on-line enrichment and purification of lupeol in Chinese herbal medicines (CHM), which showed good adsorption performance and excellent reproducibility. The spiked recoveries were from 88.52 % to 93.68 % under the optimal experimental conditions; the repeatability could reach 100 times. The method showed good linearity in the range of 0.13 ∼ 2.00 × 103 μg /mL with a correlation coefficient of r = 0.9991; the limit of detection (LOD) was 0.023 μg/mL and the limit of quantification (LOQ) was 0.076 μg/mL; the intra-day and inter-day precisions were both less than 1.05 %.

Recent advances in carbon dots-based chromatographic separation materials

Chromatographic column is the core of chromatographic separation, and chromatographic separation material is considered the soul of the chromatographic column. The type and characteristics of the chromatographic separation material directly determine the separation mode and performance of chromatographic columns. The development and preparation of separation materials with novel structures and good separation performance is an ongoing hotspot in chromatography research. Given rapid developments in nanoscience and technology, nanomaterials with unique surface functional groups and large specific surface areas have attracted extensive attention and great interest from researchers in the field of separation science. Carbon dots (CDs), a new type of zero-dimensional fluorescent carbon nanomaterials, have been widely used in bioimaging, light-emitting diodes, sensing, catalysis, drug delivery, and other applications since they were first reported in 2004. These nanomaterials present several advantages over other types of separation materials, including a simple preparation process, low toxicity, easy functionalization, excellent biocompatibility, and photobleaching resistance. In addition, compared with traditional carbon nanomaterials such as graphene and carbon nanotubes, CDs have abundant surface functional groups, nanoscale sizes, and moderate adsorption properties. Hence, when CDs-based new materials are applied as a stationary phase for column chromatography, they can provide rich reaction sites and ensure the uniformity of the chromatographic packing process. The use of CDs can effectively avoid the peak-tailing phenomenon caused by the strong interaction of large π-conjugated systems with some analytes and improve the efficiency of the chromatographic column. As such, these nanomaterials show good application prospects in the field of chromatographic separation. In this review, the development history, classification, and synthesis strategies of CDs are briefly described. We then focus on the development of CDs-based chromatographic separation materials by systematically reviewing the recent advances in the use of CDs-based materials as a stationary phase for high-performance liquid chromatography (including hydrophilic interaction, reversed-phase, mixed-mode, and chiral chromatography), gas chromatography, and capillary electrochromatography, with special emphasis on the preparation methods and applications of various stationary phases. Finally, the development prospects of CDs and future research efforts on these materials are also analyzed and discussed. This review can provide guidance on the rational design and application of new CDs-based chromatographic separation materials.