Hydrophilic Interaction Liquid Chromatography Mode Research Articles

Retention behavior of nucleosides and nucleobases on a 3 μm undecylenic acid-functionalized silica column in per aqueous liquid chromatography and hydrophilic interaction liquid chromatography separation modes

A 3 μm undecylenic acid-functionalized stationary phase (UAS) was prepared for the separation of nucleosides and nucleobases using per aqueous liquid chromatography (PALC) and hydrophilic interaction liquid chromatography (HILIC). The retention behaviors of nucleosides and nucleobases in PALC and HILIC modes were explored by adjusting parameters such as water content, buffer concentration, pH of the mobile phase and column temperature. The experimental data and separation chromatogram demonstrated that PALC could provide retention comparable to that of HILIC for nucleosides and nucleobases. Comparative studies using diluted adenosine solutions evaluated theoretical plates and peak shape for the same retention factors (between 0.25 and 5.0) in PALC and HILIC. There was no buffer component in the mobile phases used to operate the comparisons. HILIC mode is more efficient for adenosine than PALC mode at low retention factors. It's the exact opposite phenomenon for high retention factors. It is proposed that the mass transfer of adenosine between the UAS, the water-rich layer and the ACN-rich mobile phase in HILIC is relatively slow. Given the significant use of toxic ACN in HILIC, PALC emerges as a safer and more effective alternative for separating nucleosides and nucleobases.

IgG glycopeptide enrichment using hydrophilic interaction chromatography-based solid-phase extraction on anaminopropyl column.

The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which havenot yet beenconsidered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.

Reverse phase, ion exchange, HILIC and mix-mode chromatography for the determination of metformin and evogliptin in human plasma and pharmaceutical formulations

Separation and quantification of highly polar metformin (MTF) alone or in combination with dipeptidyl peptidase-4 inhibitors, such as evogliptin (EVG), sitagliptin, saxagliptin, vildagliptin, linagliptin and teneligliptin, specifically from human plasma and pharmaceutical products is still difficult in mostly preferred ODS based RP-HPLC techniques. Since, owing to weak retention of MTF in ODS, it elutes together with the biological fluid components and drug excipients. Therefore, in this study, alternative to ODS based RP-HPLC, comparatively less known analytical techniques like strong cation exchange chromatography (SCX-3), hydrophilic interaction liquid chromatography (HILIC) and mix-mode chromatography (MMC) were comprehensively evaluated for their potentials and limitations in simultaneous quantification of newly approved EVG and MTF combination (Valera-M). In results, prolonged application of SCX-3, exhibits irreproducible and irreversible retention of MTF and EVG. While in HILIC with Cyano column, excessive acetonitrile as eluent developed the precipitation of MTF and column back pressure. Comparatively, the Acclaimed® mix-mode HILIC-1, demonstrated much promising and reproducible results for MTF and EVG separation and importantly, it can be used either reverse phase or HILIC mode. Considering the overall benefits of Acclaimed® mix-mode HILIC-1, it was used in estimation of MTF and EVG from human plasma and pharmaceutical formulations.

Application of Normal-Phase Silica Column in Hydrophilic Interaction Liquid Chromatography Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples via Liquid Chromatography-Tandem Mass Spectrometry.

In neonatal screening, amino acids have a significant diagnostic role. Determination of their values may identify abnormal conditions. Early diagnosis and continuous monitoring of amino acid disorders results in a better disease outcome. An easy and simple LC-MS/MS method was developed for the quantitation of underivatized amino acids. Amino acids were separated using a normal-phase HPLC column having a totally porous silica stationary phase and using classical reversed-phase eluents. Mass spectrometry in multiple reaction monitoring mode was used for the analysis, providing high selectivity and sensitivity. A standard addition calibration model was applied for quantitation using only one isotope-labeled internal standard for all amino acids. Five calibration points were used for quantitation, and the method was successfully validated. The slopes of the calibration curves of the individual amino acids in parallel measurements were found to be similar. Since the measured slopes were reproducible, one serum sample could represent every series of serum samples of a given day. The method was tested on human serum samples and adequate results were obtained. This new method can be easily applied in clinical laboratories.

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.

Preparation of three structurally similar stationary phases with different ionizable terminal groups and evaluation of their retention performances under multiple modes in high performance liquid chromatography

Three structurally similar silane reagents with different terminal groups were prepared and bonded to silica to obtain three structurally similar stationary phases (Sil-Ph-COOH, Sil-Phe and Sil-Ph-NH2). The prepared stationary phases were characterized through elemental analysis (EA) and Fourier Transform Infrared Spectroscopy (FT-IR). These three stationary phases provided acceptable retention repeatability (relative standard deviations between 0.08% and 0.13%) and high column efficiency (7.3 × 104 plates/m for uridine on Sil-Phe). The retention behavior of the three columns was investigated under different chromatographic conditions including different mobile phase ratio, salt concentration, pH etc. The retention mechanisms were explored by linear solvation energy relationships and Van't Hoff plots. Applications in separation under reversed phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and ion exchange chromatography (IEC) mode were investigated. The results showed that the retention capacity of the stationary phases with different terminal groups to the analytes is very different, especially for carboxylic acids, because the surface charges of amino groups and carboxyl groups under weakly acidic conditions produce different electrostatic effects with dissociated carboxylic acids. Finally, the Sil-Phe column was employed to detect ibuprofen extracted from pharmaceutical ibuprofen capsules and vitamins extracted from vitamin tablets.

One-pot synthesis of a poly(styrene-acrylic acid) copolymer-modified silica stationary phase and its applications in mixed-mode liquid chromatography

As modified ligands with a wide range of sources, abundant functional groups, and good biocompatibility, polymers have been widely used in the development of silica-based chromatographic stationary phases. In this study, a poly(styrene-acrylic acid) copolymer-modified silica stationary phase (SiO2@P(St-b-AA)) was prepared via one-pot free-radical polymerization. In this stationary phase, styrene and acrylic acid were used as functional repeating units for polymerization and vinyltrimethoxylsilane (VTMS) was used as a silane coupling agent to link the copolymer and silica. Various characterization methods, such as Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), N2 adsorption-desorption analysis, and Zeta potential analysis, confirmed the successful preparation of the SiO2@P(St-b-AA) stationary phase, which had a well-maintained uniform spherical and mesoporous structure. The retention mechanisms and separation performance of the SiO2@P(St-b-AA) stationary phase in multiple separation modes were then evaluated. Hydrophobic and hydrophilic analytes as well as ionic compounds were selected as probes for different separation modes, and changes in the retention of the analytes under various chromatographic conditions, including different methanol or acetonitrile contents and buffer pH values, were investigated. In reversed-phase liquid chromatography (RPLC) mode, the retention factors of alkyl benzenes and polycyclic aromatic hydrocarbons (PAHs) on the stationary phase decreased with increasing methanol content in the mobile phase. This finding could be attributed to the hydrophobic and π-π interactions between the benzene ring and analytes. The retention changes of alkyl benzenes and PAHs revealed that the SiO2@P(St-b-AA) stationary phase, similar to the C18 stationary phase, exhibited a typical reversed-phase retention behavior. In hydrophilic interaction liquid chromatography (HILIC) mode, as the acetonitrile content increased, the retention factors of hydrophilic analytes gradually increased, and a typical hydrophilic interaction retention mechanism was inferred. In addition to hydrophilic interaction, the stationary phase also demonstrated hydrogen-bonding and electrostatic interactions with the analytes. Compared with the C18 and Amide stationary phases prepared by our groups, the SiO2@P(St-b-AA) stationary phase exhibited excellent separation performance for the model analytes in the RPLC and HILIC modes. Owing to the presence of charged carboxylic acid groups in the SiO2@P(St-b-AA) stationary phase, exploring its retention mechanism in ionic exchange chromatography (IEC) mode is of great importance. The effect of the mobile phase pH on the retention time of organic bases and acids was further studied to explore the electrostatic interaction between the stationary phase and charged analytes. The results revealed that the stationary phase has weak cation exchange ability toward organic bases and electrostatically repels organic acids. Moreover, the retention of organic bases and acids on the stationary phase was influenced by the analyte structure and mobile phase. Thus, the SiO2@P(St-b-AA) stationary phase could provide multiple interactions, as demonstrated by the separation modes described above. The SiO2@P(St-b-AA) stationary phase showed excellent performance and reproducibility in the separation of mixed samples with different polar components, indicating that it has promising application potential in mixed-mode liquid chromatography. Further investigation of the proposed method confirmed its repeatability and stability. In summary, this study not only described a novel stationary phase that could be used in RPLC, HILIC, and IEC modes but also presented a facile "one-pot" preparation approach that could provide a new route for the development of novel polymer-modified silica stationary phases.

Preparation and characterization of mixed-mode hydrophilic stationary phase based on triple hydrophilic functionalized moieties and its application to traditional Chinese herbal compound

In this paper, the surface modification of spherical silica was sequentially grafted with functional groups including imidazole, amide and sulfonic acid. The novel surface polymerization type stationary phases (SPs) for hydrophilic interaction liquid chromatography (HILIC) were obtained in three procedures. Elemental analysis, Fourier transform infrared spectroscopy, transmission scanning electron microscopy, Brunauer Emmett Teller and Barrett Joyner Halenda were carried out for the characterization of the SPs. The bonding rate of the functionalized group (1-acrylamide propyl sulfonic) was calculated to be 2.02 μmol/m2. Positive outcomes were attained for the surface grafting of the SPs. The effect of various chromatographic conditions on the retention time of various analytes was investigated on high performance liquid chromatography. The dual retention mechanism of hydrophilic interaction and ion exchange mechanism of the prepared SPs was confirmed. The polymeric HILIC SPs had more abundant functionalized groups on the surface, which provided superior hydrophilic properties, ion exchange and thus higher selectivity. Compared with the C18 column, the resolution of the HILIC column was better. The HILIC SPs were used for the separation of sulfonamides and flavonoid natural components, and had achieved better baseline separations. In addition, the HILIC mode was used for the separation of the extract of the Fructus Ligustri Lucidi, in which the quality markers had satisfactory peak shape and baseline separation. In conclusion, the prepared surface polymeric SPs were competent for the analysis of polar components and applicable to complex samples.

Efficient strategies for preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa.

Efficient strategies for the preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa using preparative high-performance liquid chromatography combined with appropriate pretreatment technologies were developed. Four fractions (Fr.1-1, Fr.1-2, Fr.1-3, and Fr.2-1) were firstly isolated from the crude extract of Hedyotis diffusa by column chromatography with C18, resin, and silica gel materials, respectively. Then, corresponding separation strategies were developed according to the polarity and chemical constituents. High-polar compounds of Fr.1-1 were purified by hydrophilic reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode. The combination of C18 and phenyl columns realized the complementary separation of iridoid glycosides in Fr.1-2. Meanwhile, the improved selectivity caused by the change of organic solvent in the mobile phase was utilized to realize the purification of flavonoid glycosides in Fr.1-3 and Fr. 2-1. Finally, 27 compounds (purity>95%) mainly involving nine iridoid glycosides and five flavonoid glycosides were obtained. A complete strategy was established for the separation of a complex sample with a wide polarity range, to jointly solve the problems of enrichment of target components and separation of structural analogs.

Zwitterionic polymer-terminated porous silica stationary phases for highly selective separation in hydrophilic interaction chromatography

We described two novel zwitterionic polymer-terminated porous silica stationary phases containing the same pyridinium cation and anions of different side chains (carboxylate and phosphonate groups) for use in hydrophilic interaction liquid chromatography (HILIC). These two novel columns were prepared by polymerizing 4-vinylpyridine and grafting it onto a silica surface, followed by quaternization reaction with 3-bromopropionic acid (Sil-VPC24) and (3-bromopropyl) phosphonic acid (Sil-VPP24), which possess positively charged pyridinium groups, and negatively charged carboxylate and phosphonate groups, respectively. The products obtained were verified through relevant characterization techniques such as elemental analysis, Fourier-transform infrared spectroscopy, thermogravimetric analysis, Zeta potential analysis, and Brunauer–Emmett–Teller analysis. The retention properties and mechanisms of different types of compounds (neutral, cationic, and anionic) on the two zwitterionic-modified silica stationary phases were studied by varying the buffer salt concentration and pH of the eluent. The separation of phenol and aromatic acids, disubstituted benzene isomers, sulfonamide drugs, as well as nucleosides/nucleobases were investigated on the two packed novel columns and a commercial zwitterionic column in identical HILIC mode, ensuring a thorough comparison between both novel columns and with a commercial standard. The results illustrated that various compounds could be separated up to various efficiencies based on the mechanism of hydrophilic interaction-based retention between the solutes and the two zwitterionic polymer stationary phases. The Sil-VPP24 column demonstrated the best separation performance out of the three, as well as flexible selectivity and excellent resolution. Both novel columns exhibited excellent stability and chromatographic repeatability for the separation of seven nucleosides and bases.

Ionic liquid-functionalized dendrimer grafted silica for mixed-mode chromatographic separation and online solid-phase extraction

The efficient sample pretreatment and high-performance chromatographic separation are essential for an accurate analysis of the complex sample. Finding a material that can apply as both sorbent for sample preparation and stationary phase for chromatography is greatly important for analytical chemistry. With a large number of active sites and designable properties, the dendrimers displayed potential performance for sample preparation and chromatographic separation. In order to improve its selectivity restricted by the limited type of functional groups (amide, ethylidene, amino groups), ionic liquid (1,1′-(1,4-butyricdiyl)bis(1-aminopropyl imidazolium)dibromide) were introduced to functionalize dendrimers. With porous silica as the base, ionic liquid-functionalized dendrimer grafted silica (SiO2@G1.5-IL) was obtained and characterized by X-ray photoelectron spectroscopy, elemental analyzer, as well as specific surface and porosity analyzer. The material was used as stationary phase for mixed-mode chromatography and sorbent for SPE, respectively. Under reversed-phase liquid chromatography (RPLC), it successfully separated some hydrophobic analytes (five polycyclic aromatic hydrocarbons (PAHs), two bisphenols, diphenylpropane, and diphenylmethane), by utilizing hydrophobic, π-stacking and hydrogen bonding interactions. Based on the hydrophilic effect, it could separate hydrophilic analytes (nucleosides and alkaloids), which coincident with hydrophilic interaction liquid chromatography (HILIC). And it exhibited better separation capacity than NH2@SiO2 and SiO2@G1.5 under the above two modes. Furthermore, the thermodynamic parameters (ΔH, ΔS, and ΔG) of the above types of analytes on the SiO2@G1.5-IL column were also investigated in detail, revealing the controlling factors during the chromatographic separation. And the relative standard deviations (RSDs, 0.03–2.95%, n = 10) of the repeatability of chromatographic separation were satisfactory. After that, SiO2@G1.5-IL-based SPE column was online combined with high-performance liquid chromatography to investigate the extraction selectivity for three types of hydrophobic organic pollutants (PAHs, estrogens, and bisphenols), and compared to SiO2 and C18 columns. According to their extraction performance, PAHs were selected as the target analytes, and the online analytical method was established. The limits of detection were as low as 0.001 μg L-1 due to the high enrichment factor reach to 3869, limits of quantity were 0.003–0.17 μg L-1, linear ranges were from 0.003 to 15.0 µg L-1, the adsorption capacity was 16.10 μg g−1, as well as RSDs were 0.5–3.7%, 0.6–6.7%, and 4.2–9.0% for intraday, interday tests and reproducibility. Finally, it was applied to analyze the environmental water samples (tap water, snow water, and rainwater), and satisfactory results were obtained (spiked recoveries were 86.8%-120.9%). Therefore, SiO2@G1.5-IL was a powerful material that can be used for the chromatographic separation of both hydrophobic and hydrophilic samples by RPLC or HILIC mode, as well as the efficient online extraction of hydrophobic targets prior to C18 column-based high-performance liquid chromatography.

Separation of minor steviol glycosides using hydrophilic interaction liquid chromatography (HILIC) and off-line two-dimensional reversed-phase liquid chromatography/HILIC methods

Steviol glycosides (SGs) are increasingly used as noncaloric sweeteners. In this study, hydrophilic interaction liquid chromatography (HILIC) methods and an off-line environmentally friendly two-dimensional reversed-phase liquid chromatography/HILIC (2D-RPLC/HILIC) for the separation of SGs were developed. The retention behavior on the polyacrylamide-based silica (PA) column was investigated by separating twelve SGs and comparing with three HILIC columns (Diol, XAmide and Unitary NH2 columns). Ethanol instead of acetonitrile as the weak eluent in HILIC mode was investigated. The PA column exhibited superior hydrophilicity, higher selectivity and better resolution. Two HILIC-UV methods for separation of SGs on a PA column with water/acetonitrile (LOD, 0.0049–0.013 mg/mL; RSD <3.73%; recovery, 95.02–102.5%) or water/ethanol (LOD, 0.010–0.016 mg/mL; RSD <4.52%; recovery, 91.90–97.76%) as elution system were developed. The preparative separation of minor SGs from stevia extracts was investigated by 2D-RPLC/HILIC using a preparative C18 column with the methanol/water mobile phase in the first dimension and a preparative PA column with the ethanol/water mobile phase in the second dimension. Two SGs were isolated and identified. In summary, safer and cheaper ethanol/water binary eluent system increased the diversity of HILIC retention and improved the solubility of SGs, which is greatly beneficial at a preparative scale in the food industry.

Carbon quantum dots–functionalized silica stationary phase for pharmaceutical analysis by a green liquid chromatography mode

Facing the trends of green chemistry, this work tries to find a novel material for per aqueous liquid chromatography (PALC) aiming to reduce the consumption of hazardous reagents. As a kind of green nanomaterials, the chromatographic performance of carbon quantum dots (CQDs) in PALC was rarely studied. Here, hydrophilic CQDs were prepared by a simple hydrothermal method using citric acid and ethylenediamine as carbon sources. The synthesized CQDs with functional groups of amino, carboxyl, and hydroxyl were decorated on silica gels forming a novel Si-CQDs stationary phase. This Si-CQDs column possesses the typical retention feature of PALC. Compounds with different polarities including hydrophobic pesticides, polar sulfonamides, β-adrenoceptor blockers and agonists, as well as hydrophilic nucleosides and bases obtained satisfactory separation on this Si-CQDs column under PALC mode, even better resolution than in hydrophilic interaction liquid chromatography (HILIC) mode. A mixture of four sulfonamides can be separated within 6min using a mobile phase containing only 5% acetonitrile, and the resolution achieves 2.39, 2.13, and 1.83 with an average column efficiency of 1400. For certain compounds, this Si-CQDs column showed better separation performance than commercial SiO2 column, NH2 column, and C18 column. The retention mechanism includes hydrophobic and electrostatic interactions due to the multifunctional groups of CQDs. This Si-CQDs column achieved the rapid detection of residual sulfonamides in milk with simplified sample pretreatment process and the detection of atenolol in commercial atenolol tablets. The developed Si-CQDs column has great prospects in low-cost and environmentally friendly separation and analysis.

Preparation and evaluation of two silica-based hydrophilic-hydrophobic and acid-base balanced stationary phases via in-situ surface polymerization

Two new hydrophilic-hydrophobic and acid-base balanced liquid chromatographic stationary phases were obtained by using two long-chain monomers including undecylenic acid (UA) and oleic acid (OA) combined with short-chain 4-vinyl pyridine (Py) in-situ polymerized on silica microspheres surfaces, respectively. These two stationary phases can be used for reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) modes. Hydrophilic, hydrophobic, acidic and basic analytes can be separated on two new stationary phases in RPLC or HILIC mode. Compared with commercial hydrophobic C18 columns and other HILIC columns, two new stationary phases have excellent selectivity and separation performance in the separation of 9 polycyclic aromatic hydrocarbons, 10 aromatic acids, 10 anilines and 5 oxazolidinones, respectively. This novel polymer-functionalized silica design strategy has opened up new ideas for the future of mixed-mode stationary phases with multiple functions and specificity.

A simple generic method for analyzing water sensitive pinacol boronate compounds by hydrophilic interaction liquid chromatography

Pinacol boronates are widely used in the pharmaceutical and biomedical industries as primary building blocks for many chemical reactions, such as Suzuki coupling. However, pinacol boronate esters are often susceptible to hydrolysis and degrade into corresponding boronic acids. This causes a challenge for analyzing these boronates by reversed-phase HPLC (RPLC), where hydrolysis occurs on the column. On the other hand, boronic acids are polar compounds and difficult to be retained in RPLC. Presently, there is a lack of practical and versatile analytical methods that can accurately and simultaneously analyze a wide range of boronate esters and boronic acids. In this work, a simple generic method was developed using hydrophilic interaction liquid chromatography (HILIC) separation to overcome these challenges. More than 40 pinacol boronate compounds in a broad range of diverse structures were screened using ten different HILIC stationary phases. Among all the stationary phases studied, ZIC-cHILIC column demonstrated excellent selectivity and retention of pinacol boronate esters and boronic acids for all of the compounds studied. The high percentage of organic solvent mobile phase in HILIC mode effectively prevents the on-column hydrolysis and provides excellent solubility for boronate ester samples. The compatibility of HILIC mobile phase allows the peaks to be identified directly by mass spectrometry.

CRB-SWATH: A Method for Enhancing Untargeted Precursor Ion Extraction and Automatically Constructing Their Tandem Mass Spectra from SWATH Datasets by Chromatographic Retention Behaviors.

Sequential window acquisition of all theoretical spectra (SWATH) as a typical data-independent acquisition (DIA) strategy is favorable for untargeted metabolomics. It could theoretically acquire product ions of all precursor ions, including precursor ions showing chromatographic peaks of rather poor qualities. However, existing data processing methods present limited capabilities in capturing poor-quality peaks of precursor ions. Thus, although their product ions could be acquired, their precursor ions are absent. Here, we present a new strategy, chromatographic retention behavior-SWATH (CRB-SWATH), that could unbiasedly capture poor-quality peaks and provide high resolutions of multiplexed mass spectroscopy (MS/MS) spectra in SWATH datasets. CRB-SWATH monitors CRBs of SWATH-MS signals under a series of altered elution gradients. As signals of compounds differ from noise by showing CRBs, both the precursor and fragment ions are captured, while ignoring their peak qualities. Moreover, CRB-SWATH offers good chances to resolve highly multiplexed MS/MS spectra in SWATH datasets because precursor ions coeluted in a single elution gradient often present different CRBs. In the untargeted metabolic analysis of Hela cell extracts, CRB-SWATH showed the advantage in exclusively capturing 2645 ions of poor-quality peaks (i.e., tiny peaks, discontinuous ion traces, tailing peaks, zigzag peaks, etc.), accounting for 34.4% of all the untargeted precursor ions extracted. Therein, it is noteworthy that among 2116 negative ions detected in hydrophilic interaction liquid chromatography (HILIC) mode, 1284 poor-quality ion peaks (>60%) were exclusively captured by CRB-SWATH. As CRB-SWATH automatically captures a large sum of true ion peaks of poor qualities, extracts MS/MS spectra of high purities, and provides chromatographic retention behaviors of untargeted metabolites for identification and classification, it could be a useful metabolomics tool for understanding biological phenomena better.

Preparation and evaluation of a bacitracin-bonded silica stationary phase for hydrophilic interaction liquid chromatography

New stationary-phase materials are of great interest for use in hydrophilic interaction liquid chromatography (HILIC), a relatively new technique capable of separating highly polar compounds. In this study, a new strategy was established to prepare a novel Sil-Bac stationary phase by bonding bacitracin to silanised silica particles using hexamethylene diisocyanate as a spacer arm. The obtained material was characterised by Fourier transform infrared spectroscopy, Brunauer–Emmett–Teller analysis, transmission electron microscopy (TEM), and elemental analysis. The network layers observed through TEM and change in specific surface area from 315.0820 to 169.6758 m2·g−1 after modification reflected the addition of bacitracin. The chromatographic behaviour of the Sil-Bac column showed that it can be used for HILIC. The influence of the chromatographic conditions on substance retention was explored by varying the acetonitrile content, column temperature, buffer concentration, and pH of the mobile phase. Using cytosine, uracil, nicotinamide, and isonicotinic acid as probes, the Sil-Bac column was demonstrated to have acceptable repeatability and stability, with a relative standard deviation of the retention time in the ranges of 0.05–0.18% for intra-day precision (n = 3) and 0.16–3.28% for inter-day precision (n = 3). Finally, the stationary phase was applied in separating basic nucleosides, water-soluble vitamins, and amino acids. A variety of bases and nucleosides could be separated within 7 min in a mobile phase of ACN/ammonium acetate (pH 3.0) = 90/10 (v/v), T = 30 °C. The Sil-Bac column was also compared with three commercial columns, and the results indicated that the analytes retained on the Sil-Bac column showed better retention and selectivity from an overall view. This novel stationary phase has a good separation ability and selectivity in HILIC mode and provides new ideas for the separation of polar compounds.

Preparation of two amide-bonded stationary phases and comparative evaluation under mixed-mode chromatography.

In this work, the conventional reactions were used to functionalize the silica surface with amide and hydrocarbon chain groups affording two different mixed-mode stationary phases (Sil-amide-C11 and Sil-C12-amide). The prepared stationary phases were analyzed by elemental analysis and thermogravimetric analysis. The retention of benzene, phenol, pyridine, and aniline was investigated and compared with synthesized and commercial columns, and this led to prove the existence of different interactions on the synthesized stationary phases. The mixed-mode stationary phases showed multiple interactions, and different chromatography modes were found under distinct chromatographic conditions. According to the type of amide group (either free or within the hydrocarbon chain), different interactions can be made on the columns. The alkylbenzenes and polycyclic aromatic hydrocarbons, as nonpolar hydrocarbons, were chromatographed under reversed-phase liquid chromatography modes, in which amide groups on the silica could efficiently separate polar analytes under hydrophilic interaction liquid chromatography mode in both prepared stationary phases. The performance of the columns was compared by the separation of the carboxylic acid group and biological samples. The bonding method and the type of amide group showed different interactions leading to different separation and performance.

Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes.

Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.

Prospects of using hyperbranched stationary phase based on poly(styrene-divinylbenzene) in mixed-mode chromatography

Evaluation of the chromatographic properties of covalently bonded hyperbranched stationary phase based on poly(styrene-divinylbenzene) (PS-DVB) and containing zwitterionic fragments in the structure of functional layer was conducted in suppressed ion chromatography (IC), reversed phase high performance liquid chromatography (RP HPLC), and hydrophilic interaction liquid chromatography (HILIC) modes. Besides the possibility of resolving 20 inorganic anions and organic acids using KOH eluent in suppressed IC, prepared resin provided the separation of alkylbenzenes in RP HPLC, water-soluble vitamins, amino acids, and sugars in HILIC mode. Trends in the retention of hydrophobic and polar analytes on the prepared stationary phase indicated the dominating effect of analyte nature on the retention mechanism and proved satisfactory hydrophilization of PS-DVB surface with hyperbranched functional layer for retaining polar compounds. The obtained results revealed good prospects of using hydrophobic PS-DVB substrate for preparing stationary phases for mixed-mode chromatography.