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.     

Separation of monosaccharides by hydrophilic interaction chromatography with evaporative light scattering detection

Hydrophilic interaction liquid chromatography (HILIC) was used to separate monosaccharides that are common in N-linked oligosaccharides in glycoproteins and other compounds. A TSKgel Amide-80 column was eluted with 82% acetonitrile, in 5 mM ammonium formate (pH 5.5). Column temperature was 60 degrees C and evaporative light scattering was used for detection (ELSD). With this method, L-fucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetylneuraminic acid, and D-glucuronic acid were separated, with detection limits of 0.3-0.5 microg for each monosaccharide, and intermediate precisions were 3-6% RSD (n=6).     

Fingerprint analysis of Ligusticum chuanxiong using hydrophilic interaction chromatography and reversed-phase liquid chromatography

Fingerprint analysis is considered one of the most powerful approaches to quality control in traditional Chinese medicines (TCMs). In this study, a binary chromatographic fingerprint analysis was developed using hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) to gain more chemical information about polar compounds and weakly polar compounds. This method was used to construct a chromatographic fingerprint of Ligusticum chuanxiong. The two chromatographic methods demonstrated good precision, reproducibility, and stability, with relative standard deviations of <2% for retention time and 7% for peak area for both HILIC and RPLC separations. Data from the analysis of 14 samples by HILIC and RPLC were processed with similarity analysis, with correlation coefficients and congruence coefficients. This binary fingerprint analysis, using two chromatographic modes, is a powerful tool for characterizing the quality of samples, and can be used for the comprehensive quality control of TCMs.     

Measurement of phospholipids by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry: the determination of choline containing compounds in foods

A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) method using multiple scan modes was developed to separate and quantify 11 compounds and lipid classes including acetylcholine (AcCho), betaine (Bet), choline (Cho), glycerophosphocholine (GPC), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphocholine (PCho) and sphingomyelin (SM). This includes all of the major choline-containing compounds found in foods. The method offers advantages over other LC methods since HILIC chromatography is readily compatible with electrospray ionization and results in higher sensitivity and improved peak shapes. The LC-MS/MS method allows quantification of all choline-containing compounds in a single run. Tests of method suitability indicated linear ranges of approximately 0.25-25 μg/ml for PI and PE, 0.5-50 μg/ml for PC, 0.05-5 μg/ml for SM and LPC, 0.5-25 μg/ml for LPE, 0.02-5 μg/ml for Cho, and 0.08-8 μg/ml for Bet, respectively. Accuracies of 83-105% with precisions of 1.6-13.2% RSD were achieved for standards over a wide range of concentrations, demonstrating that this method will be suitable for food analysis. 8 polar lipid classes were found in a lipid extract of egg yolk and different species of the same class were differentiated based on their molecular weights and fragment ion information. PC and PE were found to be the most abundant lipid classes consisting of 71% and 18% of the total phospholipids in egg yolk.     

The retention behaviour of polar compounds on zirconia based stationary phases under hydrophilic interaction liquid chromatography conditions

The most separations in HILIC mode are performed on silica-based supports. Nevertheless, recently published results have indicated that the metal oxides stationary phases also possess the ability to interact with hydrophilic compounds under HILIC conditions. This paper primarily describes the retention behaviour of model hydrophilic analytes (4-aminobenzene sulfonic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, 3,4-diaminobenzoic acid, 3-aminophenol and 3-nitrophenol) on the polybutadine modified zirconia in HILIC. The results were simultaneously compared with a bare zirconia and a silica-based HILIC phase. The mobile phase strength, pH and the column temperature were systematically modified to assess their impact on the retention of model compounds. It was found that the retention of our model hydrophilic analytes on both zirconia phases was mainly governed by adsorption while on the silica-based HILIC phase partitioning was primarily involved. The ability of ligand-exchange interactions of zirconia surface with a carboxylic moiety influenced substantially the response of carboxylic acids on the elevated temperature as well as to the change of the mobile phase pH in contrast to the silica phase. However, no or negligible ligand-exchange interactions were observed for sulfanilic acid. The results of this study clearly demonstrated the ability of modified zirconia phase to retain polar acidic compounds under HILIC conditions, which might substantially enlarge the application area of the zirconia-based stationary phases.     

A revisit to the retention mechanism of hydrophilic interaction liquid chromatography using model organic compounds

In this work, a revisit to the retention mechanism of HILIC was attempted to point out critical factors that contribute to the chromatographic regime as well as to bring out subtle details of the relative contribution of partitioning and surface adsorption. In this vein, the retention behaviour of a set of water-soluble vitamins (WSVs) and toluene on three silica based columns was evaluated under varying chromatographic conditions. The data obtained were associated with the hydration degree of the stationary phases and the ability of the organic solvents to disrupt the formation of the water-enriched layer. Moreover, the elution behaviour of toluene at different buffer salt concentrations in the mobile phase, confirmed the preferential partition of salt ions into the stagnant layer, as ACN content was increased. The results from the fitting of partitioning and surface adsorption models indicated differences in the contribution of the two retention mechanisms to both neutral and charged compounds. The occurrence of surface adsorption and the retentivity differences for neutral WSVs depend on the hydration degree and the hydrogen bonding properties of the solutes and the column surface, respectively. For charged solutes experiencing electrostatic repulsion, the contribution of the adsorption mechanism at highly organic mobile phases, emanates from both the weak effect of buffer salt ions on the electrostatic interaction and the strong effect of hydrophilic interactions. On the other hand, the chromatographic retention of electrostatically attracted solutes indicates that the surface adsorption dominates, even at mobile phases rich in water.     

Development and validation of a hydrophilic interaction liquid chromatographic method for determination of aromatic amines in environmental water

A simple, precise, and accurate hydrophilic interaction liquid chromatographic (HILIC) method has been developed for the determination of five aromatic amines in environmental water samples. Chromatography was carried out on a bare silica column, using a mixture of acetonitrile and a buffer of NaH₂PO₄–H₃PO₄ (pH 1.5, containing 10 mM NaH₂PO₄) (85:15, v/v) as a mobile phase at a flow rate of 1 mL min⁻¹. Aromatic amines were detected by UV absorbance at 254 nm. The linear range of amines was good (r² > 0.998) and limit of detection (LOD) within 0.02–0.2 mg L⁻¹ (S/N = 3). The retention mechanism for the analytes under the optimum conditions was determined to be a combination of adsorption, partition and ionic interactions. The proposed method was applied to the environmental water samples. Aromatic amines were isolated from aqueous samples using solid-phase extraction (SPE) with Oasis HLB cartridges. Recoveries of greater than 75% with precision (RSD) less than 12% were obtained at amine concentrations of 5–50 μg L⁻¹ from 100 mL river water and influents from a wastewater treatment plant (WWTP). The present HILIC technique proved to be a viable method for the analysis of aromatic amines in the environmental water samples.     

Separation of carboxylates by hydrophilic interaction liquid chromatography on titania

Hydrophilic interaction liquid chromatography (HILIC) can be performed on titania. To better understand the retention mechanisms on titania, a series of model carboxylates were used. Increasing acetonitrile above 60% dramatically increased the retention and efficiency for carboxylates. The effect of buffer type, buffer concentration, buffer pH and column temperature were also studied. Multiple retention mechanisms are operative on titania, and whether electrostatic repulsion, ligand exchange or HILIC dominates retention and separation depends on the eluent conditions. Guidelines for separations on titania are: (1) higher %ACN most improves retention and efficiency; (2) higher salt concentration increases retention; (3) elution strength is in the order acetate ? malate < methyl phosphonate ? phosphate; (4) electrostatic repulsion (ERLIC) is more operative at low %ACN than high %ACN. A bare titania column (150 mm × 4.6 mm I.D., 5 μm) was used for the separation of diphenylacetate, 4-nitrobenzoate, benzoate, 4-aminobenzoate, 4-hydroxybenzoate, phthalate, 3-aminophthalate, 1,3,5-benzenetricarboxylate, 1,2,4-benzenetricarboxylate, 1,2,4,5-benzenetetracarboxylate, benzenepentabenzoate and mellitate under HILIC conditions based on these guidelines, with efficiencies of 2800–55,000 plates/m.     

Method development and validation for optimised separation of salicylic, acetyl salicylic and ascorbic acid in pharmaceutical formulations by hydrophilic interaction chromatography and response surface methodology

This paper introduces a design of experiments (DOE) approach for method optimisation in hydrophilic interaction chromatography (HILIC). An optimisation strategy for the separation of acetylsalicylic acid, its major impurity salicylic acid and ascorbic acid in pharmaceutical formulations by HILIC is presented, with the aid of response surface methodology (RSM) and Derringer's desirability function. A Box-Behnken experimental design was used to build the mathematical models and then to choose the significant parameters for the optimisation by simultaneously taking both resolution and retention time as the responses. The refined model had a satisfactory coefficient (R2>0.92, n=27). The four independent variables studied simultaneously were: acetonitrile content of the mobile phase, pH and concentration of buffer and column temperature each at three levels. Of these, the concentration of buffer and its cross-product with pH had a significant, positive influence on all studied responses. For the test compounds, the best separation conditions were: acetonitrile/22 mM ammonium acetate, pH 4.4 (82:18, v/v) as the mobile phase and column temperature of 28°C. The methodology also captured the interaction between variables which enabled exploration of the retention mechanism involved. It would be inferred that the retention is governed by a compromise between hydrophilic partitioning and ionic interaction. The optimised method was further validated according to the ICH guidelines with respect to linearity and range, precision, accuracy, specificity and sensitivity. The robustness of the method was also determined and confirmed by overlying counter plots of responses which were derived from the experimental design utilised for method optimisation.     

Application of a new zwitterionic hydrophilic interaction chromatography column for determination of paralytic shellfish poisoning toxins

A novel method for paralytic shellfish poisoning (PSP) toxins which is based on the chromatographic separation of the toxins using a zwitterionic (ZIC) hydrophilic interaction chromatography (HILIC) column is presented. Efficient retention of the polar PSP toxins on the ZIC-HILIC column allowed their selective and sensitive determination by the application of mass spectrometric (MS/MS) detection or as derivatives after oxidation prior to fluorescence detection (FD). Low buffer concentrations and the omission of ion-pair reagents decreased the limits of detection (LODs) by MS/MS analysis and showed a good linearity for both methods of detection. This method can be applied for the qualitative and quantitative determination of PSP toxins in various types of phytoplankton, and for the routine analysis of seafood.     

Determination of aromatic amines in aqueous extracts of polyurethane foam using hydrophilic interaction liquid chromatography and mass spectrometry

A method is presented for the determination of aromatic amines in aqueous extracts of polyurethane (PUR) foam. The method is based on the extraction of PUR foam using aqueous acetic acid (0.1%, w/v) followed by determination of extracted aromatic amines using hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS) with positive electrospray ionisation. The injections of volumes up to 5 μL of aqueous solutions were made possible by on-column focusing with partially filled loop injections. The fragmentation patterns for 2,4- and 2,6-toluene diamine (TDA) and 4,4′-methylene dianiline (MDA) were clarified by performing a hydrogen-deuterium exchange study.TDA and MDA were determined using trideuterated 2,4- and 2,6-TDA and dideuterated 4,4′-MDA as internal standards. Linear calibration graphs were obtained over the range 0.025-0.5 μg mL⁻¹ with correlation coefficients >0.996 and the instrumental detection limit for each compound was <50 fmol. The stability of the amines was influenced by the matrix, so their concentrations decreased over time.Agreement was observed between the results of analyses of PUR foam extracts by HILIC-MS/MS and results obtained by ethyl chloroformate derivatisation and reversed phase (RP) liquid chromatography-mass spectrometry (LC-MS/MS).TDA was observed to be unstable in extracts of foam but not in pure solutions.     

Efficiency of the same neat silica column in hydrophilic interaction chromatography and per aqueous liquid chromatography

The dependencies on the mobile phase flow velocity of the efficiency of a column packed with shell particles of neat porous silica (Halo) was measured under two different sets of experimental conditions. These conditions corresponded to the retention mechanisms of per   aqueous liquid chromatography (PALC) at low acetonitrile concentrations and of hydrophilic interaction chromatography (HILIC) at high acetonitrile concentrations. The results are compared. Small amounts of a diluted solution of caffeine were injected in order to record the chromatograms under strictly linear conditions. These efficiencies were measured in both water-rich (PALC retention mechanism) and acetonitrile-rich (HILIC mechanism) mobile phases for the same retention factors, between 0.25 and 2.5. The mobile phases were mixtures of acetonitrile and water containing neither supporting salt nor buffer component. At low retention factors, the efficiency of caffeine is better in the PALC than in the HILIC mode. For k^′=0.5$k^{\prime }=0.5$, the minimum reduced height equivalent to a theoretical plate (HETP) is close to 2.5 in PALC while it exceeds 5 in HILIC. The converse is true for high retention factors. For k^′>2.5$k^{\prime }>2.5$, the HETP is lower in HILIC than in PALC, because the major contribution to band broadening and peak tailing in this latter mode originates from the heterogeneous thermodynamics of retention and eventually restricts column performance in PALC. Most interestingly, the reduced HETP measured in HILIC for caffeine never falls below 4. This suggests that the mass transfer of caffeine between the multilayer adsorbed phase (due to the interactions of the strong solvent and the silanol groups) and the acetonitrile-rich bulk eluent is slow.     

Off-line comprehensive 2-dimensional hydrophilic interaction × reversed phase liquid chromatography analysis of procyanidins

The development of an off-line comprehensive 2-dimensional liquid chromatography (2-D-LC) method for the analysis of procyanidins is reported. In the first dimension, oligomeric procyanidins were separated according to molecular weight by hydrophilic interaction chromatography (HILIC), while reversed phase LC was employed in the second dimension to separate oligomers based on hydrophobicity. Fluorescence, UV and electrospray ionisation mass spectrometry (ESI-MS) were employed for identification purposes. The combination of these orthogonal separation methods is shown to represent a significant improvement compared to 1-dimensional methods for the analysis of complex high molecular weight procyanidin fractions, by simultaneously providing isomeric and molecular weight information. The low correlation (r² < 0.2100) between the two LC modes afforded a practical peak capacity in excess of 2300 for the optimal off-line method. The applicability of the method is demonstrated for the analysis of phenolic extracts of apple and cocoa.     

Structure elucidation of highly polar basic degradants by on-line hydrogen/deuterium exchange hydrophilic interaction chromatography coupled to tandem mass spectrometry

A hydrophilic interaction liquid chromatography (HILIC) method was used to separate a commonly used pharmaceutical starting material, 4-aminomethylpyridine (4-AMP), and its degradants. The structures of the major degradants were characterized and elucidated without prior isolation by accurate mass measurement, MS/MS analysis and on-line hydrogen/deuterium (H/D) exchange experiments. The mass spectra obtained from H/D exchange experiments are particularly useful to differentiate structural isomers, to elucidate the fragmentation pathways, and to aid in structure elucidation in the absence of MS/MS fragmentation information. The impact of deuterium oxide and temperature on HILIC separation has also been explored here. The integration of H/D exchange with HILIC has been described here for the first time and has been demonstrated to be a powerful structure elucidation tool via the study of degradants in 4-AMP.     

Fluorine-18 and medical imaging: Radiopharmaceuticals for positron emission tomography

Fluorine presents among its radioactive isotopes fluorine-18, that decays with a 109 min half-life and a β⁺ emission, allowing external detection of the two coincident γ photons obtained after annihilation. Production techniques (medical cyclotron), radiochemical reactions for isotope incorporation in radiopharmaceuticals and development of specific detection cameras (positron emission tomographs) allowed development of a vast investigation field in medical imaging.Applications of PET in oncology ([¹⁸F]fluorodeoxyglucose, FDG) largely improved detection and management of cancers; tracer molecules labelled with fluorine-18 also allow fruitful researches in molecular imaging.     

Column-switching reversed phase-hydrophilic interaction liquid chromatography/tandem mass spectrometry method for determination of free estrogens and their conjugates in river water

We report a column-switching liquid chromatography (LC) tandem mass spectrometry (MS/MS) method for highly sensitive determination of both free estrogens (estrone, estradiol, and estriol) and their conjugates (estrone-3-sulfate, estradiol-3-sulfate, estriol-3-sulfate, estrone-3-glucuronide, estradiol-3-glucuronide, estriol-16-glucuronide, and estriol-3-glucuronide) in river water. This technique combines reversed phase (RP) chromatographic separation of the dansyl chloride derivatized free estrogens and hydrophilic interaction liquid chromatographic (HILIC) separation of the estrogen conjugates with multiple reaction monitoring (MRM). Using this new method, sensitivity increases 100- to 1000-fold for free estrogens and 2- to 10-fold for estrogen conjugates over RPLC-MS/MS alone. Method detection limits (MDL) range from 0.038 to 6.9 ng L⁻¹ with accuracy of 68-105% and precision of 1.7-17%. We successfully used this method to analyze river water samples collected from the North Saskatchewan River at the same location and detected trace concentrations of estrone (0.042 ng L⁻¹) and estrone-3-sulfate (0.84 ng L⁻¹), demonstrating the application of this method for environmental analysis.     

Preparation of chitosan functionalized monolithic silica column for hydrophilic interaction liquid chromatography

A novel cationic hydrophilic interaction monolithic stationary phase based on the chemical modification of carboxymethyl chitosan (CMCH) to the monolithic silica skeleton using carbodiimide as an activation reagent was prepared for performing capillary liquid chromatography. The amino and hydroxy moieties of CMCH functioned as both the ion-exchange sites and polar providers. The performance of the column was studied by the separation of polar acidic compounds. The chitosan functionalized monolithic silica column showed good selectivity for nucleosides, nucleotides, aromatic acids and aliphatic acids. The mechanism for the separation of these compounds was also studied. The results showed that these compounds were separated primarily based on the hydrophilic interaction mechanism.     

Direct determination of glyphosate using hydrophilic interaction chromatography with coulometric detection at copper microelectrode

A simple, rapid, and low-cost coulometric method for direct detection of glyphosate using hydrophilic interaction chromatography is presented. The principle of detection is based on the enhancement of the anodic current of copper microelectrode in the presence of complexing agents, such as glyphosate, with the formation of a soluble Cu(II) complex. Under optimized conditions, the limit of detection (S/R = 3) for glyphosate was 0.1 mg L⁻¹ (0.59 μM) without any preconcentration method. The calibration curve has been found linear in all concentration range tested (from limit of detection to 34 mg L⁻¹) with an excellent correlation coefficient (0.9999). The present method was successfully applied for the determination of glyphosate in fruit juices without any kind of extraction, clean-up, or preconcentration step, with recoveries of 92 and 90% for apple and grape juice, respectively.     

Click novel glycosyl amino acid hydrophilic interaction chromatography stationary phase and its application in enrichment of glycopeptides

A novel glycosyl amino acid hydrophilic interaction chromatography (HILIC) stationary phase was prepared via click chemistry. The key intermediate N₃-glycosyl d-phenylglycine was prepared by a three steps procedure, including selective condensation of amino glucose with N-succinimidyl ester of Boc-d-phenylglycine, deprotection and transformation of amino group to azido group. The structure of all the intermediates and functionalized silica beads were confirmed by ¹H NMR, IR, elemental analysis and ¹³C CP-MAS. The chromatography test showed that this new type of separation material possessed good HILIC properties and glycopeptide enrichment characteristics. Nucleosides and bases could be separated in a simple eluent composition (only acetonitrile in combined with water), and with the same condition, these model compounds could not be separated on the commercial HILIC column (Atlantis). Click glycosyl amino acid thus prepared also showed longer retention and better separation ability in the separation of polar organic acids.     

Determination of uric acid and creatinine in human urine using hydrophilic interaction chromatography

Uric acid is the end-product of purine metabolism and a major antioxidant in humans. The concentrations of uric acid in plasma and urine are associated with various diseases and routinely measured in clinical and biomedical laboratories using enzymatic conversion and colorimetric measurement. In this study a hydrophilic interaction chromatographic (HILIC) method was developed for simultaneous determination of uric acid and creatinine, a biomarker of urine dilution and renal function, in human urine. Urine samples were pretreated by dilution, protein precipitation, centrifugation and filtration. Uric acid and creatinine were separated from other components in urine samples and quantified using HILIC chromatography. A linear relationship between the ratio of the peak area of the standards to that of the internal standard and the concentration of the standards was obtained for both uric acid and creatinine with the square of correlation coefficients >0.999 for both analytes. The detection limits were 0.04 μg/mL for creatinine and 0.06 μg/mL for uric acid. The described HILIC method has proved to be simple, accurate, robust and reliable.