Hydrophilic Interaction Chromatography Separation Research Articles

A detailed evaluation of the advantages and limitations of online RP-LC×HILIC compared to HILIC×RP-LC for phenolic analysis

The combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) has proved effective in the LC × LC analysis of polyphenols due to the high degree of orthogonality associated with these separation modes for various classes of phenolic compounds. However, despite the growing number of such applications, HILIC is almost exclusively used as the first dimension (1D) separation mode, and RP-LC in the second dimension (2D). This is somewhat surprising in light of the potential advantages of swapping these separation modes. In this contribution, we present a detailed evaluation of the potential of online RP-LC × HILIC-MS for the analysis of phenolic compounds, comparing the performance of this system to the more established HILIC × RP-LC-MS configuration.Method development was performed using a predictive optimisation program, and fixed solvent modulation was employed to combat the solvent incompatibility between HILIC and RP-LC mobile phases. Red wine, rooibos tea, Protea and chestnut phenolic extracts containing a large diversity of phenolic compound classes were analysed by both HILIC × RP-LC- and RP-LC × HILIC-MS in order to compare the separation performance.Overall, the kinetic performance of HILIC × RP-LC was found to be clearly superior, with higher peak capacities and better resolution obtained for the majority of samples compared to RP-LC × HILIC analyses using similar column dimensions. Dilution of the 1D solvent combined with large volume injections proved insufficient to focus especially phenolic acids in the 2D HILIC separation, which resulted in severe 2D peak distortion for these compounds, and negatively impacted on method performance. On the other hand, a noteworthy improvement in the sensitivity of RP-LC × HILIC-MS analyses was observed due to higher ESI-MS response for the 2D HILIC mobile phase and greater sample loading capacity of the 1D RP-LC column, brought on by the high solubility of phenolic samples in aqueous solutions. As a result, a significantly higher number of compounds were detected in the RP-LC × HILIC-MS separations. These findings point to the potential advantage of RP-LC × HILIC as a complementary configuration to HILIC × RP-LC for phenolic analysis.

Analysis of chemical constituents in classical prescription Danggui Buxue Decoction based on RP-Q-TOF-MS and HILIC-Q-TOF-MS

The chemical constituents of classical prescription Danggui Buxue Decoction were analyzed by reversed-phase(RP) chromatography and hydrophilic interaction chromatography(HILIC) coupled with quadrupole time-of-flight mass spectrometry. RP separation of Danggui Buxue Decoction was performed on ACQUITY UPLC HSS T3(2.1 mm×100 mm, 1.8 μm), while HILIC separation was on Waters BEH Amide(2.1 mm×100 mm, 1.7 μm). Mass spectrometry(MS) data were acquired in both negative and positive ion modes. Chemical constituents of Astragali Radix and Angelicae Sinensis Radix were searched from Reaxys and thus the in-house library was established. MS data were further analyzed by MassLynx 4.1 combined with in-house library, HMDB, Reaxys, and comparison with reference substances. In conclusion, a total of 154 compounds were identified and characterized: 16 saponins, 44 flavonoids, 10 phthalides, 7 phenylpropanoids, 15 bases and the corresponding nucleosides, 30 oligosaccharides, and 32 other compounds. Among them, 65 compounds were detected by HILIC-MS/MS. This study provides experimental evidences for the material basis research, quality control, and preparation development of Danggui Buxue Decoction and a reference method for comprehensive characterization of Chinese medicine decoctions typified by classical prescriptions.

Poly(acrylamide-co-N,N'-methylenebisacrylamide) Monoliths for High-Peak-Capacity Hydrophilic-Interaction Chromatography-High-Resolution Mass Spectrometry of Intact Proteins at Low Trifluoroacetic Acid Content.

In this study, we optimized a polymerization mixture to synthesize poly(acrylamide-co-N,N′-methylenebisacrylamide) monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of cross-linker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy (SEM). Further characterization of the monolithic structure revealed a permeable (Kf between 2.5 × 10–15 and 1.40 × 10–13 m2) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 μm core–shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA), the peak capacities of the two columns were similar (30 and 34 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained similar separation performance and selectivity (peak capacity 23), whereas the packed column showed greatly reduced performance (peak capacity 12), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA, the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.

Corona charged aerosol detector non-uniform response factors of purified alcohol ethoxylated homologues using liquid chromatography.

Surfactants are used in various applications: cosmetics, pharmaceuticals, petrochemicals, environmental, etc. Many of these compounds are polydisperse, and because of this intrinsic polydispersity, it is essential to have a universal detector with a uniform response to quantify them in a simple way. Indeed, Charged Aerosol Detector (CAD) was presented as a universal detector with a uniform response. Thus, in the present study, the CAD response, in a High-Performance Liquid Chromatography - CAD configuration (HPLCCAD), was evaluated using purified alcohol ethoxylated surfactants. A semi-preparative liquid chromatography step using a Hydrophilic interaction chromatography (HILIC) bare silica column (150mm, 4.6mm, 2.6µm) was implemented to prepare eleven homologues of BrijC10, a nonionic surfactant. These homologues differed only by the number of ethylene oxide units. BrijC10 homologues were analyzed by HPLCCAD, using a HILIC bare silica column (150mm, 2.1mm, 2.6µm) to determine the HPLCCAD response factors of purified homologues. From the calibration curves (from 100 to 500mg.kg-1), their response factors were estimated: differences in response factors were observed and a maximum difference in response factors of 3.6 was obtained. Thus, it could be concluded that CAD hyphenated to HILIC separation did not present a uniform response for this homologue's distribution.

Method for evaluation of ionic interactions in liquid chromatography

In this work we utilized basic and acidic analytes to investigate the ionic interaction participation in retention behavior of selected reversed-phase and polar columns. The test analytes included nitrate, benzenesulfonate and trimethylphenylammonium ions. The fully aqueous mobile phase comprising 10 mM dichloroacetic acid buffered with ammonia solution to desirable pH was used for retention experiments. Developed method was utilized to study the ionic interactions of stationary phases in pH range between 2.5 and 9.0. We demonstrate that selected sorbents used for reversed-phase and hydrophilic interaction chromatography separations exhibit cation- or anion-exchange interactions. We compare the results to novel Atlantis PREMIER BEH C18 AX mixed-mode column that combines reversed-phase and anion-exchange interaction modes. We evaluated the relative retention strength of selected columns for anionic and cationic analytes.

Comprehensive analysis of tara tannins by reversed-phase and hydrophilic interaction chromatography coupled to ion mobility and high-resolution mass spectrometry.

Reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) methods hyphenated to diode array detection and ion mobility (IM) high-resolution mass spectrometry (HR-MS) were used for the analysis of gallic acid derivatives and gallotannins in a commercial tara extract. UV spectra combined with low and high-collision energy mass spectral data and known RP-LC elution orders allowed the identification of 45 isomeric gallic acid derivatives and gallotannins. The synergy between IM and UV data was found to provide a simple means to determine the number of depsidic bonds and thus to distinguish between positional isomers. IM also facilitated the assignment of individual isomeric species between HILIC and RP-LC separations. For the hydrolysable tannins present in tara, RP-LC provided superior resolution and specificity compared to HILIC. The results reported in this paper highlight the utility of IM in combination with optimised complementary chromatographic separations and HR-MS for the detailed qualitative analysis of hydrolysable tannins in complex mixtures of these compounds. Graphical abstract.

Evaluating the Adsorbed Water Layer on Polar Stationary Phases for Hydrophilic Interaction Chromatography (HILIC)

The water-rich liquid layer immobilized on the surface of the polar stationary phases is critical to the retention of polar compounds in hydrophilic interaction chromatography (HILIC). Although the presence of the adsorbed water layer has been investigated and confirmed by multiple techniques, there is a lack of quantitative measures that can be easily determined and linked to chromatographic parameters. This study proposes a simple measure termed volume ratio (the ratio of the adsorbed water layer volume and the mobile phase volume) that can be easily determined using toluene elution volume. The volume ratio values measured using the proposed method indicate that the volume of the adsorbed water layer varies in a wide range in the stationary phases commonly used in HILIC separation. It was observed that the volume ratio increases with the acetonitrile content and ammonium acetate concentration in the mobile phase. In addition, increasing the column temperature had the effect of reducing the volume ratio and diminishing the adsorbed water layer.

Overloading study of ionized compounds in hydrophilic interaction chromatography

The overloading behaviour of charged acidic (acetylsalicylic acid and nicotinic acid) and basic (creatinine and 1-ethyl-2,3-dimethylimidazolium) solutes was investigated on two stationary phases (bare silica and amidebonded silica) in the hydrophilic interaction chromatography separation mode at three mobile phase pH values (3.0, 5.0 and 7.0). On the bare silica both cationic solutes showed the increased peak tailing as the sample load increased. However, on the amide phase they exhibited a quite different overloading behaviour. The peak shapes for creatinine showed the increased tailing whereas for 1-ethyl-2,3-dimethylimidazolium peak fronting occurs with the sample load. Anionic solutes on both phases showed the increased peak fronting as the sample load increased. In neutral and slightly acidic mobile phases the loadability of the bare silica phase was much higher for bases than for acids. In addition, the loading capacity for the bases increases with pH whereas an opposite trend was observed for the acidic compounds. The amide phase gave similar loading capacity values for both types of solutes and its loadability is less sensitive to pH changes, compared to that of bare silica. The obtained results indicate that ionic interactions with dissociated surface silanols play an important role in the overloading behaviour of charged solutes.

Interaction of toluene with polar stationary phases under conditions typical of hydrophilic interaction chromatography probed by saturation transfer difference nuclear magnetic resonance spectroscopy

Toluene has been used as void volume (zero retention) marker since the inception of hydrophilic interaction chromatography (HILIC), based on the assumption that its hydrophobicity should prevent it from interacting with stationary phases envisioned to be covered by relatively thick layers of water. Recent work has shown that the void volumes of partly water-swollen HILIC phases are not identical to the volumes probed by toluene, yet the compound is still ubiquitously used as void volume marker. As part of our investigations of the retention mechanisms in HILIC, we probed the extent to which toluene is capable of penetrating into the water-enriched layer and to interact with the functional groups of three commercially available hydrophilic and polar stationary phases with different charge properties and water-retaining abilities, using saturation transfer difference 1H nuclear magnetic resonance (STD-NMR) spectroscopy at high resolution magic angle spinning (HR-MAS) conditions. The test solutions were 1000 ppm of toluene in deuterated acetonitrile and water mixtures, with and without addition of ammonium acetate, in order to mimic a set of conditions typically encountered in HILIC separations. Interactions between toluene and the functional groups on the stationary phases were probed by equilibrating the phases with these eluent mimics and measuring the transfer of magnetization from stationary phase protons to the protons of toluene. Our results show that toluene is indeed capable of traversing the water-enriched layers of all the three tested phases and of interacting with protons that are tightly associated with the stationary phases.

Preparation of attapulgite nanoparticles-based hybrid monolithic column with covalent bond for hydrophilic interaction liquid chromatography

In current study, an attapulgite nanoparticles-based hybrid monolith was prepared by crosslinking 3-trimethoxysilylpropyl methacrylate modified attapulgite nanoparticles with acrylamide and N, N′-methylenebisacrylamide. The crosslinking of attapulgite into the hybrid monolithic matrix has notable increased the column efficiency of adenosine comparing with the neat one without attapulgite. The resulting hybrid monoliths showed good permeability and good mechanical stability. It was further applied for separation of polar compounds by hydrophilic interaction chromatography (HILIC). The key factors affecting the separation efficiency of the developed hybrid monoliths (i.e. acetonitrile content, salt concentration and pH in the mobile phase) have been completely investigated. The column efficiency was up to 147,613 plates/m for the HILIC separation of aspirin. Good repeatability of retention time was achieved, with relative standard deviations for run-to-run (n = 3), column-to-column (n = 3) and batch-to-batch (n = 3) in the range of 1.08–1.45%, 2.44–3.41% and 2.15–4.96%, respectively. We propose that the attapulgite nanoparticles-based hybrid monolith would provide a promising stationary phase for hydrophilic interaction chromatography.

Method optimization of hydrophilic interaction chromatography separation of nucleotides using design of experiment approaches I: Comparison of several zwitterionic columns

A systematic method in hydrophilic interaction chromatography (HILIC) was developed for the separation of four monophosphate nucleotides using design of experiment (DOE) approaches. Three HPLC parameters, the buffer concentration (ammonium acetate concentration), gradient time, and temperature, were evaluated within the quality design framework, and the effects on chromatographic parameters were investigated. Four zwitterionic columns (ZIC-HILIC, ZIC-cHILIC, NUCLEODUR HILIC, and PC HILIC) were used to separate four nucleotides, and the HPLC conditions for each column were successfully optimized, although PC HILIC did not give peaks that were suitable for optimization. In addition, it was proved that optimized HPLC conditions differed from column to column even when the same types of zwitterionic sulfobetaine-functionalized columns were applied. This tendency was explained by differences in the separation characteristics of each column, the thickness of the water-enriched layer on the surface of the silica supports, and the pH. DOE for development of the HPLC method provides an effective explanation of the interactions among the variable parameters, especially in HILIC mode. Finally, a robust analytical method could be established by setting the optimum parameters. Among the employed columns, ZIC-cHILIC provided the widest range of suitable analytical conditions. NUCLEODUR HILIC was difficult to build a robust analytical method since the elution order of cytidine monophosphate and guanosine monophosphate was reversed.

Predictive Liquid Chromatography of Peptides Based on Hydrophilic Interactions for Mass Spectrometry-Based Proteomics

High-performance liquid chromatography (HPLC) is widely used for separation of complex peptide mixtures before mass spectrometry-based proteome analysis. In this analysis, reversed phase HPLC (RPHPLC) using non-polar stationary phases such as surface-modified silica containing alkyl groups (e.g., C18) is typically employed. Because of the high heterogeneity of proteomic samples, multidimensional separation approaches gained increasing attention recently to tackle this complexity and extremely high range of concentrations. In two-dimensional liquid chromatography, hydrophilic interaction chromatography (HILIC) is often a method of choice for combination with RP-HPLC because it uses reversed-phase type eluents and allows efficient separation of polar peptides. Due to the high degree of orthogonality in this two-dimensional separation space, it is tempting to develop approaches for predicting peptide retention times for HILIC-based separations similar to the ones for RP-HPLC. Recent successful efforts in this area were focused on developing retention coefficient (RC)-based approaches. Herein, we explored the feasibility of using a statistical thermodynamic model for prediction of peptide retention times in HILIC separations and determined the phenomenological parameters of the model for a bare silica column. The performance of the developed model was tested using HPLC-MS analysis of a set of synthetic peptides, as well as a tryptic peptide mixture.

Untargeted saliva metabonomics study of breast cancer based on ultra performance liquid chromatography coupled to mass spectrometry with HILIC and RPLC separations

Breast cancer (BC) is not only the most frequently diagnosed cancer, but also the leading cause of cancer death among women worldwide. This study aimed to screen the potential salivary biomarkers for breast cancer diagnosis, staging, and biomarker discovery. For the first time, a UPLC-MS based method along with multivariate data analysis, was proposed for the global saliva metabonomics analysis and diagnosis of BC, which used both hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) separations and operated in both positive (ESI+) and negative (ESI−) ionization modes. On account of different polarities of endogenous metabolites, this method was established to overcome the boundedness of a single chromatographic approach. As a result, 18 potential metabolites for diagnosing BC were identified. A nonparametric Mann-Whitney U test, heat map, and the receiver operating characteristic (ROC) were exploited to analyze the data with the purpose of evaluating the predictive power of the 18 biomarkers. Significant differences (P<0.05) were disclosed in terms of the levels of the 18 potential biomarkers between BC patients and healthy controls (HC). Among the 18 biomarkers, three up-regulated metabolites, LysoPC (18:1), LysoPC (22:6) and MG (0:0/14:0/0:0) displayed the area under the curve (AUC) values of 0.920, 0.920 and 0.929, respectively, indicating the high accuracy of this method to predict BC. In this study, an integrated metabonomics analysis in human saliva for identifying potential biomarkers to diagnose and stage BC was successfully eastablished, which was non-invasive, reliable, low-cost, and simple. The HILIC was demonstrated to be essential for a comprehensive saliva metabonomics profiling as well as RPLC separation. This saliva metabonomics technique may provide new insight into the discovery and identification of diagnostic biomarkers for BC.

Characterization and quantification of histidine degradation in therapeutic protein formulations by size exclusion-hydrophilic interaction two dimensional-liquid chromatography with stable-isotope labeling mass spectrometry

Two dimensional liquid chromatography (2D-LC) coupling size exclusion (SEC) and hydrophilic interaction chromatography (HILIC) is demonstrated as a useful tool to study polar excipients, such as histidine and its degradant, in protein formulation samples. The SEC-HILIC setup successfully removed interferences from complex sample matrices and enabled accurate mass measurement of the histidine degradation product, which was then determined to be trans-urocanic acid. Because the SEC effluent is a strong solvent for the second dimension HILIC, experimental parameters needed to be carefully chosen, i.e., small transferring loop, fast gradient at high flow rates for the second dimension gradient, in order to mitigate the solvent mismatch and to ensure good peak shapes for HILIC separations. In addition, the generation of trans-urocanic acid was quantified by single heart-cutting SEC-HILIC 2D-LC combined with stable-isotope labeling mass spectrometry. Compared with existing 2D quantification methods, the proposed approach is fast, insensitive to solvent mismatch between dimensions, and tolerant of small retention time shifts in the first dimension. Finally, the first dimension diode array detector was found to be a potential degradation source for photolabile analytes such as trans-urocanic acid.

Analytical Methods for Detonation Residues of Insensitive Munitions

Analytical methods are described for the analysis of post-detonation residues from insensitive munitions. Standard methods were verified or modified to obtain the mass of residues deposited per round. In addition, a rapid chromatographic separation was developed and used to measure the mass of NTO (3-nitro-1,2,4-triazol-5-one), NQ (nitroguanidine) and DNAN (2,4-dinitroanisole). The HILIC (hydrophilic-interaction chromatography) separation described here uses a trifunctionally-bonded amide phase to retain the polar analytes. The eluent is 75/25 v/v acetonitrile/water acidified with acetic acid, which is also suitable for LC/MS applications. Analytical runtime was three minutes. Solid phase extraction and LC/MS conditions are also described.

Hydrophilic interaction liquid chromatography for the separation of acidic agricultural compounds.

Organic acids with very low pKa require extremely low pH conditions to achieve adequate retention in reversed-phase liquid chromatography, but an extremely low pH mobile phase can cause instrument reliability problems and limit the choice of columns. Hydrophilic interaction chromatography is a potential alternative to reversed-phase liquid chromatography for the separation of organic acids using more moderate conditions. However, the hydrophilic interaction chromatography separation mechanism is known to be very complex and involves multiple competing mechanisms. In the present study, a hydrophilic interaction chromatography column packed with bare silica core-shell particles was used as the separation column and six agricultural organic acids were used as model analytes to evaluate the effects of buffer concentration, buffer pH, and temperature on sample loading capacity, selectivity, retention, and repeatability. It was found that using a higher concentration of buffer can lead to a significant improvement in the overall performance and reproducibility of the separation. Investigation of column equilibration time revealed that a very long equilibration time is needed when changing mobile phase conditions in between runs. This limitation needs to be acknowledged in hydrophilic interaction chromatography method development and sufficient equilibration time needs to be allowed in method scouting.

An optimized platform for hydrophilic interaction chromatography-immobilized metal affinity chromatography enables deep coverage of the rat liver phosphoproteome.

While analysis of the phosphoproteome has become an important component of understanding how cells function, it remains a nontrivial task in terms of the number of sample preparation steps and instrument time needed to achieve sufficient depth of coverage to produce meaningful results. We previously described a multidimensional method that uses hydrophilic interaction chromatography (HILIC) followed by Fe(3+) immobilized metal affinity chromatography (IMAC) to reduce complexity, improve selectivity, and increase phosphopeptide identifications. Here we present refinements to our overall protocol that make it simpler and more efficient, while they provide greater coverage of the phosphoproteome. We introduce filter-aided sample prep (FASP) for cell lysis and trypsin digestion. Following HILIC separation, fractions are IMAC enriched using a 96-well filter plate. Finally, enriched samples are analyzed using an LC-MS strategy optimized for the fractionation scheme. The optimized protocol improves protein recovery, simplifies phosphopeptide enrichment, and optimizes instrument time, while it maintains deep coverage of the phosphoproteome. By using the refined protocol, we identified more than 16,000 unique phosphosites from rat liver in a single experiment, which used approximately 1 day of instrument time. All together, we present evidence for 24,485 rat liver phosphosites that represents the deepest coverage of a tissue phosphoproteome to date.

Identification of novel isomeric pectic oligosaccharides using hydrophilic interaction chromatography coupled to traveling-wave ion mobility mass spectrometry

Separation and characterization of complex mixtures of pectic oligosaccharides still remains challenging and often requires the use of multiple analytical techniques, especially when isomeric structures are present. In this work, it is demonstrated that the coupling of hydrophilic interaction chromatography (HILIC) to traveling-wave ion mobility mass spectrometry (TWIMMS) enabled the simultaneous separation and characterization of complex mixtures of various isomeric pectic oligosaccharides. Labeling of oligosaccharides with 3-aminoquinoline (3-AQ) improved MS-ionization efficiency of the oligosaccharides and reduced the complexity of the product ion mass spectra, without losing resolution of the HILIC separation. In addition, labeling enabled quantification of oligosaccharides on molar basis using in-line fluorescence detection. Isomeric structures were distinguished using TWIMMS. The 3-AQ-HILIC–TWIMMS method was used to characterize a series of isomeric sugar beet rhamnogalacturonan I derived oligosaccharides carrying a glucuronic acid substituent. Thereby, some novel structural features were identified for the first time: glucuronic acid was attached to O-3 or to O-2 of galacturonic acid residues and a single galacturonic acid residue within an oligomer could contain both an acetyl group and a glucuronic acid substituent.

HPLC-QTOF-MS Method for Identification and Determination of Bleomycin A2 and B2 Fractions

Bleomycin is a very important drug in oncology used as first-line treatment for many cancers. The development of high-performance liquid chromatography—tandem mass spectrometry method for the separation, identification, and determination of two major structurally related analogs of this antibiotic drug, namely bleomycin A2 and B2, is presented in this work. The proposed method is based on a hydrophilic interaction chromatography (HILIC) stationary phase that enables one to avoid an ion-pairing reagent in the separation system (formerly used in mobile phase for bleomycin) in order to properly separate these highly polar bleomycin analogs. The ion-pairing reagent-free HILIC separation system is suitable for coupling the chromatographic and mass spectrometry stages (for the first time for bleomycin). Some performance parameters, namely, limit of detection (ng/mL-pg/mL), limit of quantification, linearity, precision, and recovery/accuracy, were evaluated showing high reliability, selectivity, and sensitivity of the method. It was successfully applied for the quality drug control determining bleomycin A2 and B2 fractions in commercial pharmaceuticals (Bleomedac infusions). In addition, the high-performance liquid chromatography–quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was able to determine an accurate molecular weight of bleomycin A2 and B2 fractions, confirming an identity/quality of the (commercial) drug. The possibilities of the HPLC-QTOF-MS method to identify bleomycin A2 and B2 in model plasma samples were also demonstrated.

HILIC-MS for metabolomics: An attractive and complementary approach to RPLC-MS.

Hydrophilic interaction chromatography (HILIC) is an emerging separation mode of liquid chromatography (LC). Using highly hydrophilic stationary phases capable of retaining polar/ionic metabolites, and accompany with high organic content mobile phase that offer readily compatibility with mass spectrometry (MS) has made HILIC an attractive complementary tool to the widely used reverse-phase (RP) chromatographic separations in metabolomic studies. The combination of HILIC and RPLC coupled with an MS detector expands the number of detected analytes and provides more comprehensive metabolite coverage than use of only RP chromatography. This review describes the recent applications of HILIC-MS/MS in metabolomic studies, ranging from amino acids, lipids, nucleotides, organic acids, pharmaceuticals, and metabolites of specific nature. The biological systems investigated include microbials, cultured cell line, plants, herbal medicine, urine, and serum as well as tissues from animals and humans. Owing to its unique capability to measure more-polar biomolecules, the HILIC separation technique would no doubt enhance the comprehensiveness of metabolite detection, and add significant value for metabolomic investigations. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:574-600, 2016.