Hydrophilic Interaction Liquid Chromatography Separation 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.

Characterization of synthetic guide ribonucleic acids through hydrophilic interaction chromatography coupled with mass spectrometry

In this study, we aimed to develop a hydrophilic interaction liquid chromatography (HILIC) method for the analysis of single guide ribonucleic acid (sgRNA), a critical reagent used in CRISPR genome editing. Our results showed that effective profiling of sgRNA can be achieved by suppressing the surface charge of the stationary phase in HILIC. We identified hydrogen bonding as the primary retention mechanism with potential weak partitioning in HILIC separation of large oligonucleotides like 100-mer sgRNA. Moreover, we demonstrated that direct coupling of HILIC with mass spectrometry (MS) allows the intact mass analysis of sgRNA and its impurities with minimal adduct present. Finally, we characterized the post peak shown in the low temperature HILIC and identified it as sgRNA aggregates. Our findings provide valuable insight into the characterization of sgRNA and highlight the potential of HILIC-MS as a powerful analytical tool for relatively large oligonucleotide analysis.

Simultaneous analysis of highly polar and multi-residue-type pesticides by heart-cutting 2D-LC-MS

Liquid chromatography-mass spectrometry (LC-MS) is currently the gold-standard technique for the analysis of non-volatile small organic molecules. However, one-dimensional liquid chromatography (1D-LC) cannot efficiently deal with mixtures of analytes with different physicochemical properties and, thus, specific chromatographic behaviour. As an alternative, this work proposes a two-dimensional liquid chromatography/high-resolution mass spectrometry (2D-LC-HRMS) approach for the simultaneous analysis of compounds with different polarities. It is based on the combination of hydrophilic interaction liquid chromatography (HILIC) in the first dimension (1D) and reversed-phase chromatography (RPLC) in the second dimension (2D), employing the heart-cutting methodology. The coupling between 1D and 2D was performed by a multiple heart-cutting (MHC) interface equipped with an active solvent modulation (ASM) valve. The aim of the study was the development of a 2D-LC methodology able to (i) acquire the 1D and 2D content by MS in a single analytical run, avoiding the loss of information caused by the MHC algorithm for filling the sampling loops; (ii) overcome the breakthrough problem caused by solvent incompatibility, modifying the 2D gradient during the ASM phase for this purpose. To evaluate the 2D-LC approach, pesticide residue analysis was proposed, selecting 20 pesticides covering a wide range of polarities (log Kow from −3.2 to 4.3) and including some of the so-called single residue method pesticides because of the difficulty of including them in 1D-LC multi-residue methods with satisfactory chromatographic resolution. The proposed strategy was to transfer in a single cut the void volume from the HILIC separation (consisting of the nonpolar pesticides) to the 2D for analysis under RPLC conditions. The developed assembly was assessed in a vegetable matrix (tomato) employing a hybrid QuEChERS/QuPPe sample treatment based on acetonitrile and methanol extraction. The proposed setup may be extended for 2D-LC applications where it is essential to acquire the entire content of both dimensions in a single data file just by coupling a selection valve to the MHC interface.

Miniaturized Two-Dimensional Heart Cutting for LC–MS-Based Metabolomics

Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics usually combines hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) chromatography to cover a wide range of metabolomes, requiring both significant sample consumption and analysis time for separate workflows. We developed an integrated workflow enabling the coverage of both polar and nonpolar metabolites with only one injection of the sample for each ionization mode using heart-cutting trapping to combine HILIC and RP separations. This approach enables the trapping of some compounds eluted from the first chromatographic dimension for separation later in the second dimension. In our case, we applied heart-cutting to non-retained metabolites in the first dimension. For that purpose, two independent miniaturized one-dimensional HILIC and RP methods were developed by optimizing the chromatographic and ionization conditions using columns with an inner diameter of 1 mm. They were then merged into one two-dimensional micro LC-MS method by optimization of the trapping conditions. Equilibration of the HILIC column during elution on the RP column and vice versa reduced the overall analysis time, and the multidimensionality allows us to avoid signal measurements during the solvent front. To demonstrate the benefits of this approach to metabolomics, it was applied to the analysis of the human plasma standard reference material SRM 1950, enabling the detection of hundreds of metabolites without the significant loss of some of them while requiring an injection volume of only 0.5 μL.

Influence of ion-pairing reagents on the separation of intact glycoproteins using hydrophilic-interaction liquid chromatography - high-resolution mass spectrometry

Hydrophilic-interaction liquid chromatography (HILIC) of intact proteins offers high-resolution separations of glycoforms of glycoproteins differing in the number of (neutral) glycans. However, to obtain efficient separations it is essential that the positively charged sites of the proteins are shielded by acidic (negative) ion-pair reagents (IPRs), so as to enhance the contribution of the hydroxyl groups of the (neutral) sugars in the glycoprotein. Here, we studied the influence of various IPRs that differ in physico-chemical properties, such as hydrophobicity and acidity, on the capillary-scale HILIC separation of intact (glyco)proteins. We evaluated the use of fluoroacetic acid (MFA), difluoroacetic acid (DFA), trifluoroacetic acid (TFA), and heptafluorobutyric acid (HFBA) as diluents for sample preparation, as solvents for sample loading on a reversed-phase trap prior to the HILIC separation, and as mobile-phase components for HILIC and HILIC-MS. To reduce the contribution of ion-exchange interaction with the (silica-based) stationary phase, we used an acrylamide-based monolithic column. We studied the influence of the different IPRs on each step of the separation of a mixture of proteins of different size and hydrophilicity and on the separation of the five glycoforms of ribonuclease B. The content of IPR in the sample was shown not to affect the separation and the MS detection. However, a low content of TFA and DFA in the mobile phase is favourable, as it reduces adduct formation and leads to higher signal intensity. The optimized HILIC conditions successfully resolved nine major glycoforms groups of a ∼40 kDa glycoprotein horseradish peroxidase (HRP), as an example of a complex glycoprotein.

Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection

The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.

Making high salt concentrations for optimal chromatography compatible with electrospray ionization mass spectrometry using an ion exchange membrane suppressor: Analysis of biomarkers for transporter protein inhibition as a case study

This study reports on the potential of using ion-exchange suppressor technology in liquid chromatography—electrospray ionization mass spectrometry workflows. The aim was to use high salt concentrations to improve separation performance, while overcoming the resulting significant ion suppression during electrospray ionization. As a case study, we apply suppressor technology to the hydrophilic interaction liquid chromatography separation and detection of taurine and glycochenodeoxycholate sulfate, endogenous biomarkers for organic anion transporter protein inhibition. The desired chromatographic selectivity was achieved applying 100 mM ion-pairing agent, while competing ions negatively affecting MS sensitivity were actively removed post-column from the solvent via a charged partially permeable membrane and replaced with protons, resulting in an up to 10-fold increase in detection sensitivity.

Easy-to-Use HPLC Method to Measure Intracellular Ascorbic Acid Levels in Human Peripheral Blood Mononuclear Cells and in Plasma

Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible method to measure AA status in the human body. Serum AA concentrations do not correlate well with tissue levels, but AA levels in leukocytes do. However, a standard method for clinical application is lacking. This present study describes a method to measure AA in the peripheral blood mononuclear cells (PBMCs) with hydrophilic interaction liquid chromatography (HILIC). The method can also be used in plasma and other leukocyte subsets. The measurements of AA in PBMCs and plasma were performed with HPLC with HILIC separation and UV detection. The sample preparation involved the isolation of PBMCs and lysis and precipitation with acetonitrile. European Medicine Agency guidelines for bioanalytic method validation were followed for the evaluation. A highly precise execution of the method was found with intra- and inter-assay variations at a maximum of 7.8%. In 40 healthy donors, a mean intracellular AA concentration of 7.9 microgram/108 cells was found in PBMCs. A correlation between plasma and PBMC AA concentration was not present (r = 0.22). In conclusion, we developed a convenient, reliable, and reproducible method for the quantitative determination of AA within PBMCs and plasma from human blood.

Grafting copolymer brushes on polyhedral oligomeric silsesquioxanes silsesquioxane-decorated silica stationary phase for hydrophilic interaction liquid chromatography

A strategy is proposed to develop a stationary phase for hydrophilic interaction liquid chromatography (HILIC) using the synergistic effect of polyhedral oligomeric silsesquioxane (POSS) and copolymer brushes. Octahedral octa-aminopropylsisesquioxane (8NH2-POSS) was first bound to silica gel, followed by bromination to form a cubic initiator. Then, using acrylamide (AM) and dihydroxypropyl methacrylate (DPMA) as mixed monomers, surface initiated-atom transfer radical polymerization was conducted to prepare a stationary phase comprising cubic copolymer brushes with amide and diol groups. The characterization of the stationary phase confirmed the successful synthesis of Sil-NH2-POSS/Poly(AM-co-DPMA). The chromatographic properties were investigated using nucleosides, organic acids and β-agonists to find that our designed column has superior hydrophilic property, better separation performance compared with classical HILIC columns consisting of diol- or amino-modified silica. The systematic investigation of the retention mechanism and separation selectivity using various types of polar compounds revealed that Sil-NH2-POSS/Poly(AM-co-DPMA) follows a mixed-mode retention composed of HILIC and electrostatic interactions. Besides, it exhibits good column efficiency and stability. The role of 8NH2-POSS in the separation was evaluated by comparing the performance of Sil-NH2-POSS/Poly(AM-co-DPMA) and poly(AM-co-DPMA)-modified silica without 8NH2-POSS. In conclusion, our designed based on POSS and hydrophilic copolymer brushes can contribute to the development of HILIC separation materials with enhanced performance.

Discrimination of sulfated isomers of chondroitin sulfate disaccharides by HILIC-MS.

Chondroitin sulfate (CS) glycosaminoglycans are biologically active sulfated polysaccharides that pose an analytical challenge for their structural analysis and functional evaluation. In this study, we developed a hydrophilic interaction liquid chromatography separation method and its on-line coupling to mass spectrometry (MS) allowing efficient differentiation and sensitive detection of mono-, di-, and trisulfated CS disaccharides and their positional isomers, without requiring prior derivatization. The composition of the mobile phase in terms of pH and concentration showed great influence on the chromatographic separation and was varied to allow the distinction of each CS without signal overlap for a total analysis time of 25min. This methodology was applied to determine the disaccharide composition of biological reaction media resulting from various enzymatic transformations of CS, such as enzymatic desulfation of CS disaccharides by a CS 4-O-endosulfatase, and depolymerization of the CS endocan by chondroitinase lyase ABC.

Simple and efficient preparation of high-purity trehalulose from the waste syrup of isomaltulose production using solid-phase extraction followed by hydrophilic interaction chromatography.

A simple and efficient method was developed for the preparation of high-purity trehalulose from the waste syrup of isomaltulose production. The waste syrup was pre-treated with C18 solid-phase extraction, where 98% decolorization and 97% reducing sugar recovery were obtained, followed by hydrophilic interaction liquid chromatography separation on a cysteine-bonded zwitterionic column. Under optimized conditions, trehalulose was separated from isomaltulose isomer and prepared on a semi-preparative scale with >99% purity. The structure of the prepared trehalulose was subsequently confirmed by nuclear magnetic resonance, and three tautomers of trehalulose (α-D-glucosylpyranosyl-1,1-β-D-fructopyranose, α-D-glucosylpyranosyl-1,1-β-D-fructofuranose, and α-D-glucosylpyranosyl-1,1-α-D-fructofuranose) were detected and completely characterized by 13 C NMR spectroscopy for the first time in this study. The tautomerization of α-D and β-D type transition was observed by hydrophilic interaction liquid chromatography on an AdvanceBio Glycan Mapping column, with smaller particle size (2.7 μm). Furthermore, the prepared trehalulose was applied as a standard for trehalulose quantification during the sucrose conversion by Klebsiella sp. LX3. The combination of solid-phase extraction and hydrophilic interaction liquid chromatography offers a new avenue for the preparation of sugar isomers from complex natural or fermentation products.

Analytical characterization of DNA and RNA oligonucleotides by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Liquid chromatography-mass spectrometry has been widely implemented as a powerful tool for providing in-depth characterization of nucleic acid therapeutic modalities, such as anti-sense oligonucleotides and small interfering RNAs (siRNAs). In this study, we developed a generic hydrophilic interaction liquid chromatography (HILIC) hyphenated with tandem mass spectrometry method in the absence of ion-pairing reagents and demonstrated its capability as an attractive and robust alternative for oligonucleotide and siRNA analysis. HILIC separation of mixtures of unmodified and fully phosphorothioate-modified DNA oligonucleotides and their synthetic 3’ exonuclease-digested metabolites were also assessed. High-resolution mass spectrometric (HRMS) analysis was used to determine the deconvoluted masses of oligonucleotide and siRNA standards and their impurities. To enable unbiased sequence characterization with tandem mass spectrometry (MS/MS), we also optimized higher-energy C-trap dissociation (HCD) on improving the sequence coverage of DNA and RNA oligonucleotides. Lastly, we evaluated on-column sensitivity for a phosphorothioate oligonucleotide by performing targeted analysis with either targeted selected ion monitoring (tSIM) or parallel reaction monitoring (PRM). Higher on-column sensitivity of 13 ng, equivalent to 2.0 pmol, of a phosphorothioate oligonucleotide was achieved by tSIM analysis as compared to PRM analysis.

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.

Calibration for quantitative Fc-glycosylation analysis of therapeutic IgG1-type monoclonal antibodies by using glycopeptide standards

Fc-glycosylation has crucial impact on the efficacy and safety of IgG-type therapeutic monoclonal antibodies (mAbs). In order to enhance the performance of MS-based bottom-up quantitation strategy, a library of glycopeptide standards containing 26 common IgG1-type Fc-glycoforms has been constructed via modified two-dimensional hydrophilic interaction liquid chromatography (HILIC) purification. Taking advantage of the acquired glycopeptide standards, calibrated quantitation strategy for Fc-glycosylation analysis of mAbs was established and evaluated on the basis of three LC-MS-based methods, including HILIC-MRM (multiple reaction monitoring), HILIC-SIM (selected ion monitor) and RPLC-SIM. Molar concentrations of eleven individual Fc-glycoforms (0.03 ± 0.001–13.77 ± 0.64 nmol mg−1) as well as degree of fucosylation (75.44–97.04%), galactosylation (3.39–49.47%) and mannosylation (1.12–21.22%) in six IgG1-type mAbs were achieved. In addition, Fc-glycosylation site occupancy was also determined from 98.05% to 99.83%. Compared with traditional MS-based quantitation via peak area normalization, the quantitation accuracy and precision of the calibrated strategy had been remarkably improved, especially when combining with HILIC separation. In addition, the transferability of calibrated quantitation as assessed by using MRM-based method had also been significantly enhanced on different instruments from different laboratories. This calibrated quantitation strategy using glycopeptide standards as calibrators will be useful for Fc-glycosylation analysis of IgG1-type mAbs with multiple glycosylation sites.

Hydrophilic Interaction Liquid Chromatography to Characterize Nutraceuticals and Food Supplements Based on Flavanols and Related Compounds

Purified extracts from different types of berries and medicinal plants are increasingly used as raw materials for the production of nutraceuticals and dietary supplements, mainly due to their high content in bioactive substances. This is, for instance, the case of phenolic compounds such as flavonoids, which exhibit a wide range of beneficial properties, including antioxidant, anti-inflammatory, antineoplastic and antimicrobial activities. This paper tackles the characterization of several kinds of nutraceuticals based on hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection (FLD). The study focused on the determination of flavanols and related compounds such as condensed tannins. Analytes were recovered by solvent extraction using methanol:water:hydrochloric acid (70:29:1 v:v:v) as the extraction solvent under sonication for 30 min at 55 °C. Experimental design with response surface methodology was used to optimize the HILIC separation to achieve good resolution of the main components, using acetonitrile:acetic acid (99:1 v/v) (solvent A) and methanol:water:acetic acid, (95:3:2 v/v/v) (solvent B) as the components of the mobile phase. For the assessment of the elution gradient, factors under study were solvent B percentage and gradient time. The best conditions were achieved with 10% solvent B as the initial percentage and 30 min of linear gradient to reach 25% solvent B. Principal component analysis and partial least square-discriminant analysis were used to characterize and compare the compositional features of dietary supplements based on both targeted and non-targeted approaches. Results revealed that the sample distribution relied on the oligomeric nature of descriptors.

A Protocol for Untargeted Metabolomic Analysis: From Sample Preparation to Data Processing.

Untargeted metabolomics has rapidly become a profiling method of choice in many areas of research, including mitochondrial biology. Most commonly, untargeted metabolomics is performed with liquid chromatography/mass spectrometry because it enables measurement of a relatively wide range of physiochemically diverse molecules. Specifically, to assess energy pathways that are associated with mitochondrial metabolism, hydrophilic interaction liquid chromatography (HILIC) is often applied before analysis with a high-resolution accurate mass instrument. The workflow produces large, complex data files that are impractical to analyze manually. Here, we present a protocol to perform untargeted metabolomics on biofluids such as plasma, urine, and cerebral spinal fluid with a HILIC separation and an Orbitrap mass spectrometer. Our protocol describes each step of the analysis in detail, from preparation of solvents for chromatography to selecting parameters during data processing.

Complementary approach for analysis of phospholipids by liquid chromatography hyphenated to elemental and molecular mass spectrometry.

Phospholipids are one of the most important lipid categories with multiple functions in biological systems. Their analysis can contribute to a better understanding of metabolomic and kinetic processes in living cells. Comprehensive methods based on liquid chromatography coupled to mass spectrometry are available for phospholipid identification and quantification. However, quantification of phospholipids using electrospray ionization-mass spectrometry with internal standards is still challenging due to several reasons. In particular, the detector response of phospholipid species differs with variation of the head group as well as the fatty acid chain length and double bond number. Inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) provides an alternative approach for their absolute quantification with universal detector response for phosphorus independent of its chemical form and proportional to its quantity. Therefore, a quantification method based on compound-independent calibration using hydrophilic interaction liquid chromatography (HILIC) coupled to ICP-MS/MS was developed. An inverse gradient system was implemented for constant mobile phase composition after HILIC separation, which provides steady plasma ionization conditions. Isobaric phosphorus interferences were decreased by using the oxygen reaction mode of the triple quadrupole based ICP-MS/MS instrument. Complementary molecular information was obtained by ESI-high-resolution MS and MS/MS. The applicability of this approach was demonstrated in a proof of concept by complementary analysis of a total lipid extract of baker's yeast.

Retention of stevioside polar compounds on a sulfonic acid-functionalized stationary phase

To explore the retention and separation of stevioside polar compounds on a sulfonic acid-functionalized cation exchange column, the effects of different organic solvent-water mobile phases on the retention behavior of polar rebaudioside A (RA) and its analogues on the column were investigated over a wide range of organic solvent contents. The obtained U-shape curves hinted that the retention of the compounds on the same column transitioned from a reversed-phase liquid chromatography (RPLC) mode to a hydrophilic interaction liquid chromatography (HILIC) mode when the water-rich state in the mobile phases changed to an organic solvent-rich state. Under the RPLC mode, no separation of RA from its analogues was observed. The HILIC mode was beneficial to the retention and separation of RA and its analogues. Compared with polar protic solvents, aprotic solvents were more conducive to the retention and separation of the polar compounds based on the HILIC mode in organic solvent-rich mobile phases. Three models were used to evaluate and discuss the HILIC retention and separation of the compounds on the column. In the aprotic solvent-rich mobile phase, the HILIC retention of RA and its analogues was effectively described by a mixed-mode model; in the polar proton solvent-rich mobile phase, the retention of analytes was best described by an linear solvation strength (LSS) model. The content and composition of the organic solvent in the mobile phase were determined to be important influencing factors that regulated the retention time for the RA and its analogues, and even the separation mechanism for HILIC. The present work provides a theoretical basis for guiding one to prepare high-purity RA from its analogues by predicting the retention time.

A new strategy for the preparation of mixed-mode chromatographic stationary phases based on modified dialdehyde cellulose

A new strategy for the preparation of mixed-mode chromatographic stationary phases based on modified dialdehyde cellulose was proposed. Two novel mixed-mode chromatographic stationary phases, dicarboxyl cellulose-modified silica (DCC/SiO2) and (S)-α-phenylethylamine-bonded DCC/SiO2 ((S)-α-PEA/DCC/SiO2), were prepared by utilizing the easy functionalization characteristics of dialdehyde cellulose. The chromatographic evaluation showed that DCC/SiO2 column could be used in hydrophilic interaction liquid chromatography (HILIC) and ion exchange chromatography (IEC) modes, (S)-α-PEA/DCC/SiO2 column could be used in HILIC, IEC and chiral separation modes. The DCC/SiO2 column and (S)-α-PEA/DCC/SiO2 column exhibited excellent chromatographic performance by separating strongly polar compounds, phenylamines and chiral compounds in the above separation modes. The preparation method of modified dialdehyde cellulose-based mixed-mode chromatographic stationary phases was simple, and also provided a new idea for the development of the subsequent novel mixed-mode chromatographic stationary phases.

First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms

The study of glycoproteins is a rapidly growing field, which is not surprising considering that approximately 70% of human proteins are glycosylated and that numerous biological functions are associated to the glycosylation. In this work, our interest focused on the heterodimeric human Chorionic Gonadotropin (hCG) glycoprotein that is the specific hormone of the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. This protein possesses a very high structural heterogeneity, essentially due to the presence of 8 glycosylation sites, but also other types of post-translational modifications. In this study, for the first time, the potential of hydrophilic interaction liquid chromatography (HILIC) was investigated to separate the intact hCG isoforms. Three different HILIC stationary phases were tested using an hCG-based drug as standard, a recombinant hCG. For each stationary phase, the effect of the initial mobile phase composition based on ACN/H2O mixture, the slope of the gradient, the content and nature of the acidic additive (formic acid and trifluoroacetic acid (TFA)), and the addition of a volatile salt (ammonium formate) on the retention and the resolution were studied. The best HILIC separation was obtained with the amide column and a mobile phase composed of water/ACN containing 0.1% of TFA. The repeatability in terms of retention times and peak areas was then assessed. Finally, the method was applied to the analysis of a second hCG-based drug obtained from urine of pregnant women. Both drugs gave chromatograms with more than 10 peaks. However, they were significantly different, which demonstrated the potential of HILIC method for hCG isoform fingerprinting.