Hydrophilic interaction chromatography/electrospray mass spectrometry analysis of carbohydrate-related metabolites from Arabidopsis thaliana leaf tissue

This work describes the development and application of an on-line liquid chromatography/mass spectrometry (LC/MS) method using hydrophilic interaction chromatography (HILIC) coupled to negative ion mode electrospray ionisation ion trap mass spectrometry (ESI-MS) for the analysis of highly polar carbohydrate-related metabolites commonly found in plants, ranging from reducing and non-reducing sugars and sugar alcohols to sugar phosphates. Using this method, separation and detection of a mixture of eight authentic standard compounds containing glucose (Glc), sucrose (Suc), raffinose, verbascose, mannitol, maltitol, glucose-6-phosphate (Glc6P) and trehalose-6-phosphate (Tre6P) were achieved in less than 15 min. The method is rapid, robust, selective, and sensitive, with limits of detection (LODs) ranging from 0.2 microM obtained for neutral sugars, to 1.0 microM obtained for sugar alcohols, and 2.0 microM obtained for negatively charged sugar phosphates. We have studied the negative ion collision-induced dissociation (CID) fragmentation behaviour of the non-reducing raffinose family oligosaccharides (RFOs) raffinose, stachyose, and verbascose. Mainly Bi and Ci glycosidic and Ai cross-ring structurally informative cleavages are observed. We have applied this HILIC/ESI-MS method for the analysis of Arabidopsis thaliana wild-type Columbia-0 (Col-0) and its starchless phosphoglucomutase mutant (pgm1) leaf extracts. The method was used to quantify Glc, Suc, raffinose, and Glc6P in A. thaliana extracts. Data obtained using this HILIC/ESI-MS method were compared with those obtained using a comparable porous graphitic carbon-based LC/ESI-MS method.     

硅胶色谱柱的亲水作用保留机理及其影响因素

亲水作用色谱（HILIC）是替代反相色谱（RPLC）分离强极性及亲水性化合物的另一色谱模式,其分离机理与RPLC有很大不同,具有和RPLC互补的选择性。在HILIC模式中,采用正相色谱（NPLC）中的极性固定相及含高浓度有机溶剂（通常为乙腈）的水溶液为流动相。硅胶是开发最早、研究最为深入及应用最为广泛的HILIC固定相,本文介绍了硅胶色谱柱的HILIC保留机理,详细概述了操作条件如硅胶柱类型、流动相组成及柱温对HILIC分离的影响,并对硅胶填料色谱柱的HILIC模式的发展方向与应用前景进行了展望。     

The application of novel 1.7 microm ethylene bridged hybrid particles for hydrophilic interaction chromatography

An un-derivatized 1.7 microm ethylene bridged hybrid (BEH) particle was evaluated for its utility in retaining polar species in hydrophilic interaction chromatography (HILIC), and was compared to a 3 microm un-derivatized silica material. Retentivity as a function of mobile phase pH, polar modifier and ACN content was examined. Also, the efficiency of the two particle substrates was compared by plotting HETP vs. linear velocity. Improved chemical resistance of the un-derivatized BEH particle was compared to un-derivatized silica at pH 5, demonstrating no performance deterioration over the course of 2000 injections for the BEH particle, while the silica particle deteriorated rapidly after 800 injections. Lastly, ESI-MS sensitivity as a function of particle size and separation mode was demonstrated. A 2.2 to 4.7-times higher ESI-MS response was observed on the 1.7 microm particle compared to the 3 microm particle, whereas a 5.6 to 8.8-times higher ESI-MS response was observed using HILIC as when compared to traditional RP chromatography.     

Hydrophilic interaction liquid chromatography in the separation of a moderately lipophilic drug from its highly polar metabolites--the cardioprotectant dexrazoxane as a model case

This paper presents a systematic study of the retention behavior of a model bisdioxopiperazine drug, dexrazoxane (DEX) and its three polar metabolites (two single open-ring intermediates-B and C and an EDTA-like active compound ADR-925) on different stationary phases intended for hydrophilic interaction liquid chromatography (HILIC). The main aim was to estimate advantages and limitations of HILIC in the simultaneous analysis of a moderately lipophilic parent drug and its highly polar metabolites, including positional isomers, under MS compatible conditions. The study involved two bare silica columns (Ascentic Express HILIC, Atlantis HILIC) and two stationary phases with distinct zwitterionic properties (Obelisc N and ZIC HILIC). The chromatographic conditions (mobile phase strength and pH, column temperature) were systematically modified to assess their impact on retention and separation of the studied compounds. It was found that the bare silica phases were unable to separate the positional isomers (intermediates B and C), whereas both columns with zwitterionic properties (Obelisc N and ZIC HILIC) were able to separate these structurally very similar compounds. However, only ZIC HILIC phase allowed appropriate separation of DEX and all its metabolites to a base line within a single run. A mobile phase composed of a mixture of ammonium formate (0.5 mM) and acetonitrile (25:75, v/v) was suggested as optimal for the simultaneous analysis of DEX and its metabolites on ZIC HILIC. Thereafter, HILIC-LC-MS analysis of DEX and all its metabolites was performed for the first time to obtain basic data about the applicability of the suggested chromatographic conditions. Hence, this study demonstrates that HILIC could be a viable solution for the challenging analysis of moderately polar parent drug along with its highly polar metabolites including the ability to separate structurally very similar compounds, such as positional isomers.     

Hydrophilic interaction liquid chromatography/electrospray mass spectrometry determination of acyclovir in pregnant rat plasma and tissues

Reversed-phase chromatography is the most common means of separation for small drug molecules. However, polar drugs may suffer from poor retention and peak shape in reversed-phase high-performance liquid chromatography (RP-HPLC). Hydrophilic interaction liquid chromatography (HILIC) provides a viable alternative to RP-HPLC and is an excellent way to separate polar compounds. This paper describes a HILIC/ESI-MS/MS assay for the determination of acyclovir from rat plasma, amniotic fluid, placental tissue, and fetal tissue. The isocratic separation utilizes an underivatized silica column with an acetonitrile/formate buffer mobile phase (80:20). The method is validated over a range of 50 ng/mL to 50 micro g/mL with % error and % relative standard deviation of <15% over 3 days. All samples are prepared by acetonitrile protein precipitation, which yields high recovery (>84% for acyclovir). This assay can be applied to the pharmacokinetic study of the placental transfer of acyclovir.     

Stationary phases for hydrophilic interaction chromatography, their characterization and implementation into multidimensional chromatography concepts

Hydrophilic interaction chromatography (HILIC) is becoming increasingly popular for separation of polar samples on polar columns in aqueous-organic mobile phases rich in organic solvents (usually ACN). Silica gel with decreased surface concentration of silanol groups, or with chemically bonded amino-, amido-, cyano-, carbamate-, diol-, polyol-, or zwitterionic sulfobetaine ligands are used as the stationary phases for HILIC separations, in addition to the original poly(2-sulphoethyl aspartamide) strong cation-exchange HILIC material. The type of the stationary and the composition of the mobile phase play important roles in the mixed-mode HILIC retention mechanism and can be flexibly tuned to suit specific separation problems. Because of excellent mobile phase compatibility and complementary selectivity to RP chromatography, HILIC is ideally suited for highly orthogonal 2-D LC-LC separations of complex samples containing polar compounds, such as peptides, proteins, oligosaccharides, drugs, metabolites and natural compounds. This review attempts to present an overview of the HILIC separation systems, possibilities for their characterization and emerging HILIC applications in 2-D off-line and on-line LC-LC separations of various samples, in combination with RP and other separation modes.     

亲水作用色谱及其在碱性药物分析中的应用

亲水作用色谱是采用极性固定相、高含量极性有机溶剂-水相缓冲液为流动相的一种分离技术,它能有效地保留反相色谱中保留弱或不保留的强极性碱性药物。本文综述了以未键合硅胶为固定相的亲水作用色谱分离碱性药物的机理、特点及其应用的最新进展。     

亲水作用色谱及其在环境分析中的应用进展

亲水作用色谱(HILIC)是一种分离极性和亲水性化合物的液相色谱模式,其作为反相液相色谱(RPLC)的重要补充,近年来越来越受到各个领域的关注和重视。这不只是因为强极性化合物的分离问题在各个领域引起了重视,而且因为与RPLC比较,HILIC具有流动相组成黏度低、色谱柱渗透性好、与质谱联用的灵敏度高及反压较低等优势。本文简要概述了HILIC的发展历程、特点及保留机理,重点介绍了HILIC用于环境分析的最新进展,评述了HILIC及RPLC用于污染物分析的优缺点,并指出了HILIC用于环境分析的未来发展趋势。     

Separation of phospholipid classes by hydrophilic interaction chromatography detected by electrospray ionization mass spectrometry

A new isocratic separation method was developed for separation of phospholipid (PL) classes based on a silica hydrophilic interaction liquid chromatography (HILIC) column with electrospray ionization (ESI) mass spectrometric detection. Although HILIC is typically used for polar compounds, also amphiphilic molecules like phospholipids can be separated very well. Compared to normal-phase (NP) chromatography, which is usually used for PL class separation, HILIC has the advantage to use on-line ESI-MS detection because its eluents are ESI compatible. Furthermore, this HILIC method is isocratic and hence less time consuming than most (gradient) NP HPLC methods. A chromatographic baseline separation of a standard mixture containing phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC) was achieved within a total run time of 17 min using a mobile phase consisting of acetonitrile, methanol and ammonium acetate 10 mM. The new method was subsequently tested on phospholipid fractions of a body fluid (human blood plasma) and a tissue extract (swine brain) whereby it achieved nearly the same baseline separation of the PL classes. The detected classes in both cases were PE, PC, SM and LPC.     

Hydrophilic interaction chromatography

Separation of polar compounds on polar stationary phases with partly aqueous eluents is by no means a new separation mode in LC. The first HPLC applications were published more than 30 years ago, and were for a long time mostly confined to carbohydrate analysis. In the early 1990s new phases started to emerge, and the practice was given a name, hydrophilic interaction chromatography (HILIC). Although the use of this separation mode has been relatively limited, we have seen a sudden increase in popularity over the last few years, promoted by the need to analyze polar compounds in increasingly complex mixtures. Another reason for the increase in popularity is the widespread use of MS coupled to LC. The partly aqueous eluents high in ACN with a limited need of adding salt is almost ideal for ESI. The applications now encompass most categories of polar compounds, charged as well as uncharged, although HILIC is particularly well suited for solutes lacking charge where coulombic interactions cannot be used to mediate retention. The review attempts to summarize the ongoing discussion on the separation mechanism and gives an overview of the stationary phases used and the applications addressed with this separation mode in LC.     

亲水作用色谱填料的研究进展

亲水作用色谱是一种新型的色谱分离模式.此类色谱模式集反相色谱的经济廉价与正相色谱的优点于一体,有效补充了反相色谱的不足.简单介绍实验室中合成的新型亲水色谱固定相.     

Separation efficiencies in hydrophilic interaction chromatography

Hydrophilic interaction chromatography (HILIC) is important for the separation of highly polar substances including biologically active compounds, such as pharmaceutical drugs, neurotransmitters, nucleosides, nucleotides, amino acids, peptides, proteins, oligosaccharides, carbohydrates, etc. In the HILIC mode separation, aqueous organic solvents are used as mobile phases on more polar stationary phases that consist of bare silica, and silica phases modified with amino, amide, zwitterionic functional group, polyols including saccharides and other polar groups. This review discusses the column efficiency of HILIC materials in relation to solute and stationary phase structures, as well as comparisons between particle-packed and monolithic columns. In addition, a literature review consisting of 2006-2007 data is included, as a follow up to the excellent review by Hemstr?m and Irgum.     

亲水作用色谱固定相及其在中药分离中的应用

亲水作用色谱(HILIC)作为一种分离极性化合物的液相色谱模式,近年来越来越受到关注和重视。一方面是因为强极性化合物的分离问题引起了各个研究领域的重视,如药物分析、代谢组学、蛋白质组学等研究领域都不同程度地涉及强极性化合物的分离问题;另一方面是由于HILIC具有流动相组成简单、分离效率较高、与质谱兼容以及反压较低等优势。固定相是HILIC发展和应用的基础,本文主要从固定相分子结构的角度对HILIC固定相的结构特征、保留特性以及应用概况等进行了综述。对传统正相色谱固定相用于HILIC以及专门设计的HILIC固定相进行了介绍,评述了各自的优缺点和应用概况;对近年来HILIC固定相在中药分离中的应用进行了介绍;并对HILIC固定相的发展进行了展望。     

Hydrophilic interaction liquid chromatography with alcohol as a weak eluent

There has been a significant increase of interest in polar compound separation by hydrophilic interaction liquid chromatography (HILIC), in which acetonitrile is mostly used as a weak eluent. Although replacing acetonitrile with alcohols as organic modifiers has been previously reported, the separation mechanism was poorly understood. In this paper we explored the separation mechanism through the method development for the analysis of the trace amounts of polar and basic hydrazines, which were genotoxic in nature. Separation parameters such as the type and concentration of alcohol, acid modifier, and buffer in mobile phase as well as the choice of stationary phase and column temperature were studied. The data indicated that both electrostatic and hydrophilic interactions contributed to the retention and separation of the hydrazines. The results presented here provide insight into the adjustment of the retention and separation of analytes in HILIC mode with alcohol as a weak eluent. The optimized HILIC method coupled with chemiluminescent nitrogen detection (CLND) is simple and sensitive (reporting limit at 0.02%) and was applied to simultaneous analysis of hydrazine and 1,1-dimethylhydrazine in a pharmaceutical intermediate.     

Retention and selectivity of stationary phases for hydrophilic interaction chromatography

More and more polar stationary phases have become available for the separation of small polar compounds in the past decade as hydrophilic interaction chromatography (HILIC) continues to find applications in new fields (e.g., metabolomics and proteomics). Bare silica phases remain popular, especially in the bio-analytical area. A wide range of functional groups (e.g., amino, amide, diol, sulfobetaine, and triazole) have been employed as polar stationary phases for HILIC separation. This review provides a survey of the popular stationary phases commercially available and discusses the retention and selectivity characteristics of the polar stationary phases in HILIC. The purpose of the review is not to provide a comprehensive overview of literature reports, but rather focuses on findings that demonstrate retention and selectivity of the polar stationary phases in HILIC.     

Hydrophilic interaction chromatography coupled to nuclear magnetic resonance spectroscopy and mass spectroscopy--a new approach for the separation and identification of extremely polar analytes in bodyfluids

A method for the unambiguous identification of highly polar molecules based on the separation on a silica gel column run in hydrophilic interaction chromatography (HILIC) mode followed by mass spectroscopic (MS) analysis and subsequent measurement by nuclear magnetic resonance (NMR) spectroscopy is described. Polar neutral, acidic and basic compounds of small molecular size usually not retained on reversed phase stationary phases can be separated and unequivocally identified by means of MS and NMR spectroscopy. The method is applied to exemplify the identification of the endogenous metabolite trigonelline and the polar antibiotic amoxicilline in human urine.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

Separation of polar mushroom toxins by mixed-mode hydrophilic and ionic interaction liquid chromatography-electrospray ionization-mass spectrometry

Reversed-phase liquid chromatography (RPLC) is commonly used to analyze nonvolatile contaminants and naturally occurring toxins in foods. However, polar compounds, such as hydrophilic polypeptides and quaternary ammonium salts, are often not satisfactorily separated by RPLC and present a challenge for analytical scientists. In this study, hydrophilic interaction liquid chromatography (HILIC), on an amide-based stationary phase in combination with electrospray ionization (ESI) tandem mass spectrometry (MS-MS), is successfully employed to simultaneously separate polar mushroom toxins, including amanitins and phallotoxins, which are cyclic oligopeptides and muscarine, a quaternary ammonium compound, in mushrooms. The sensitivity of different ionization modes is studied, and the positive ionization mode is found to provide a more sensitive and effective tool for the unambiguous identification of the concerned polar toxins because of their characteristic fragmentation patterns. The properties of the mobile phase are also found to have significant impacts on the separation. At a high acetonitrile (ACN) concentration, hydrophilic interaction dominates, and all analytes under study demonstrate a much higher affinity with the stationary phase. The addition of methanol (MeOH) as a modifier could further enhance the HILIC separation for amanitins, phallotoxins, and muscarine. Valley-to-valley separation is achieved upon the optimatizatiqn of the mobile phase (comprising of ACN, MeOH, and ammonium formate buffer at pH approximately 3.5) and the solvent gradient. HILIC coupled with ESI-MS-MS is demonstrated to be a novel technique for the simultaneous separation and confirmatory analysis of the concerned polar toxins by providing an environment of solubility and retention that could not be achieved through the use of RPLC.     

Hydrophilic interaction chromatography coupled to electrospray mass spectrometry for the separation of peptides and protein digests

In this study, the analysis of a peptide set, chosen for their differences in hydrophilicity, and the tryptic digests of bovine cytochrome c and β-lactoglobulin by hydrophilic interaction chromatography–electrospray ionisation mass spectrometry (HILIC–ESI-MS) is demonstrated. Two different types of HILIC phases, i.e., an amide- and an amino-modified silica-based phase, packed into narrow bore or capillary columns, were investigated with separations conducted under either low pH or neutral pH conditions. The separation performance of the two HILIC columns with respect to peak efficiency and selectivity have been documented under these different mobile phase conditions, and the results compared with the performance of a conventional capillary reversed-phase C18 column of similar dimensions. It was found that very good separation of the peptide set could be achieved by using the amide-modified silica column over a broad pH range. Moreover, with the protein digest samples, excellent separation of the tryptic digests was obtained with the amide-modified HILIC column under neutral pH conditions. Compared to the conventional reversed-phase C18 separations, the use of these HILIC columns not only provided complementary separation selectivity, but also offered the capability to identify unique peptides using tandem HILIC–mass spectrometric techniques. These studies therefore highlight the potential of HILIC procedures for future proteomic applications.