Hydrophilic Liquid Chromatography Research Articles

Fast and Sensitive HILIC-ESI-MS Technique for the Screening of Perflourooctane Sulphonate in Environmental Water

Perflourooctane sulphonate(PFOS) is in use for several decades as surfactants in a wide variety of industrials and consumer products. Potential health risk of PFOS include developmental toxicity, cancer and bioaccumulation. In the present study a fast and sensitive liquid chromatography mass spectrometry(LCMS) method has been developed using high peroformance hydrophilic interactive liquid chromatography(HILIC) based stationary phase and electrospary ionization(ESI) mass spectrometry(MS). The limit of detection and quantification of PFOS was 0.1 µg/L and 0.2 µg/L respectively and response was linear up to 1 µg/L with correlation coefficient of 0.9999. Water samples obtained from different environmental sources were spiked with 0.2 µg/L of PFOS and subjected to preconcentration on a solid phase extraction catridge prior to LCMS analysis showed excellent recoveries ranging within 83.7 to 101.0%.The method was validated with respect to linearity, accuracy and precision using one way analysis of variance(ANOVA) and regression statistics.

Direct determination of underivatized amino acids from Ginkgo biloba leaves by using hydrophilic interaction ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry

Ginkgo biloba leaf extract has been widely used in dietary supplements and more recently in some foods and beverages. In addition to the well-known flavonol glycosides and terpene lactones, G. biloba leaves are also rich in amino acids. To determine the content of free amino acids, a reliable method has been established by using hydrophilic interaction ultra-HPLC coupled with ESI-MS. 20 free amino acids were simultaneously determined without derivatization in 12 min. The proposed method was fully validated in terms of linearity, sensitivity, repeatability, as well as recovery. Furthermore, the principal component analysis was applied to different G. biloba leaves collected in November (after fruit harvest season), which revealed that the samples from different production areas exhibited regional disparity in different clusters in accordance with their various hydrophilic interaction chromatograms coupled with mass profiles. The established approach could be helpful for evaluation of the potential values as dietary supplements and the quality control of G. biloba leaves, which might also be utilized for the investigation of other medicinal herbs containing amino acids.

The analysis of the human plasma N-glycome in endometriosis patients

ObjectiveAnalysis of the plasma N-glycome in endometriosis patients compared with controls. Study designIn a case-control study, blood samples were collected from patients who underwent either diagnostic or operative laparoscopy between 2008 and 2011 in the Semmelweis University, Budapest, I. Department of Obstetrics and Gynaecology. From these patients, 92 with endometriosis (30 stage I–II and 62 stage III–IV, including altogether 18 deep infiltrating cases) and 62 controls were selected for glycan analysis. After release, plasma N-glycans were subjected to hydrophilic interaction high performance liquid chromatography, which resulted in 19 chromatographic glycan peaks (GP). The abundances of the GPs were compared between the study groups. For statistical analysis a non-parametric test, the Mann–Whitney-U test, was used. ResultsWe found a statistically significant decrease of GP1 and increase of GP14, GP17 and GP18 in endometriosis patients. The latter peaks consist of glycans which play a role in inflammatory processes and malignancy. We also found significant differences in GP2, GP4, GP6, and GP9 between controls and the different endometriosis stage groups. The observed alterations in GP2, GP4 and GP6 may be related to altered glycosylation and remodelling of the glycan branches of the IgG molecule. The alterations of GP9 are presumably associated with changes of transferrin glycosylation. Furthermore we detected a highly significant decrease of GP1 in patients with deep infiltrating endometriosis compared with controls. ConclusionsThis is the first analysis of the plasma N-glycome in endometriosis. The observed changes in GP14, GP17 and GP18 and in GP2, GP4, GP6 and GP9 provide new aspects to the pathophysiology of the disease and the alterations of the GP1 may serve as a new potential marker in the future.

Hydrophilic interaction ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry for determination of nucleotides, nucleosides and nucleobases in Ziziphus plants

In this study, a rapid and sensitive analytical method was developed for the determination of 20 nucleobases, nucleosides and nucleotides in Ziziphus plants at trace levels by using hydrophilic interaction ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HILIC–UHPLC-TQ-MS/MS) in multiple-reaction monitoring (MRM) mode. Under the optimized chromatographic conditions, good separation for 20 target compounds were obtained on a UHPLC Amide column with sub-2μm particles within 10min. The overall LODs and LOQs were between 0.11–3.12ngmL−1 and 0.29–12.48ngmL−1 for the 20 analytes, respectively. It is the first report about simultaneous analysis of nucleobases, nucleosides and nucleotides in medicinal plants using HILIC–UHPLC-TQ-MS/MS method, which affords good linearity, precision, repeatability and accuracy. The developed method was successfully applied to Ziziphus plant (Z. jujuba, Z. jujuba var. spinosa and Z. mauritiana) samples. The analysis showed that the fruits and leaves of Ziziphus plants are rich in nucleosides and nucleobases as well as nucleotides, and could be selected as the healthy food resources. Our results in present study suggest that HILIC–UHPLC-TQ-MS/MS method could be employed as a useful tool for quality assessment of the samples from the Ziziphus plants as well as other medicinal plants or food samples using nucleotides, nucleosides and nucleobases as markers.

“Dilute & Shoot” approach for rapid determination of trace amounts of nicotine in zero-level e-liquids by reversed phase liquid chromatography and hydrophilic interactions liquid chromatography coupled with tandem mass spectrometry-electrospray ionization

Two analytical procedures are proposed where HILIC and RPLC techniques are coupled with tandem mass spectrometry detection for rapid determination of trace amounts of nicotine in zero-level liquids for electronic cigarettes. Samples are prepared on the basis of the approach “dilute & shoot” which makes this important step quick and not complicated. The chromatographic separation was carried out on a Zorbax XDB column (RPLC method) and Ascentis Si column (HILIC mode). Within-run precisions (CVs) measured at three concentration levels were as follows: 0.73%, 0.98% and 1.44% for RPLC method and 1.39%, 1.44% and 0.57% (HILIC mode). Between-run CVs were as follows: 1.94%, 1.02% and 1.22% for RPLC mode and 1.49%, 1.20% and 1.22% for HILIC mode. The detection limits of RPLC and HILIC modes were 4.08 and 3.90ng/mL respectively. The proposed procedures are rapid, not complicated, sensitive and are suitable for fast determination of trace amounts of nicotine in zero-level liquids for electronic cigarettes.

Facile preparation of organic-silica hybrid monolith for capillary hydrophilic liquid chromatography based on “thiol-ene” click chemistry

In this work, a one-step approach to facile preparation of organic–inorganic hybrid monoliths was successfully developed. After vinyl-end organic monomers and azobisisobutyronitrile (AIBN) were mixed with hydrolyzed tetramethoxysilane (TMOS) and 3-mercaptopropyltrimethoxysilane (MPTMS), the homogeneous mixture was introduced into a fused-silica capillary for simultaneous polycondensation and “thiol-ene” click reaction to form the organic-silica hybrid monoliths. By employing this strategy, two types of organic-silica hybrid monoliths with positively charged quaternary ammonium and amide groups were prepared, respectively. The functional groups were successfully introduced onto the monoliths during the sol–gel process with “thiol-ene” click reaction, which was demonstrated by ζ-potential assessment, energy dispersive X-ray spectroscopy (EDX), and Fourier transform infrared (FT-IR) spectroscopy. The porous structure of the prepared monolithic columns was examined by scanning electron microscopy (SEM), nitrogen adsorption–desorption measurement, and mercury intrusion porosimetry. These results indicate the prepared organic-silica hybrid monoliths possess homogeneous column bed, large specific surface area, good mechanical stability, and excellent permeability. The prepared monolithic columns were then applied for anion-exchange/hydrophilic interaction liquid chromatography. Different types of analytes, including benzoic acids, inorganic ions, nucleosides, and nucleotides, were well separated with high column efficiency around 80,000–130,000plates/m. Taken together, we present a facile and universal strategy to prepare organic-silica hybrid monoliths with a variety of organic monomers using one-step approach.

A Fast and Sensitive Method for the Simultaneous Determination and Quantification of Six Anionic Surfactants in Surface Water using HILIC-ESI-MS Technique

The hydrophobic hydrocarbon chain and the polar sulfate group confer surfactant properties and enable them to beused as anionic surfactants. Anionic surfactants (AS) are known for their adverse impact on environment, particularly on aquaticecosystem. In the present study a fast, sensitive and selective method for the determination and subsequent quantification of sixanionic surfactants was developed using hydrophilic interactive liquid chromatography (HILIC) coupled to a electrospray ionization(ESI) mass spectrometer (MS), in the concentration range 15-20 μg/L. The capability of the method was establishedusing regression analysis and ANOVA. The method performance was evaluated by analyzing real time surface water spiked with1-dodecyl hydrogen sulfate at 15 μg/L. Combined efficiency of solid phase extraction and MS detection established recovery of89% in presence of natural matrix. These results point out that HILIC coupled to multistage MS procedures can be a powerfultechnique for environmental applications concerning the screening of polar contaminants.

Hydrophilic interaction ultra performance liquid chromatography retention prediction under gradient elution

The development and application of new separation mechanisms such as hydrophilic interaction chromatography (HILIC) is of high importance for the simultaneous analysis of polar molecules such as primary metabolites. However the retention mechanism in HILIC is not fully understood and as a result retention prediction tools are not at hand for this chromatographic approach. In the present report we study the utility of a simple algorithm, based on a simple linear and/or a simple logarithmic retention model, for retention prediction in HILIC gradient separation of a mixture of 23 selected compounds including (poly)amines, amino acids, saccharides, and other molecules. Utilizing two types of gradient elution programs with or without an isocratic part, retention data were collected in order to build prediction models. Starting from at least three gradient runs the prediction of analyte retention was very satisfactory for all gradient programs tested, providing useful evidence of the value of such retention time prediction methodologies.

Detection of allantoin in clinical samples using hydrophilic liquid chromatography with stable isotope dilution negative ion tandem mass spectrometry

Allantoin is the major oxidation product of urate in humans and is a potential biomarker of oxidative stress. Several methods are used to measure allantoin in biological samples but they have inherent issues that can include lack of specificity and sensitivity, difficulty in sample preparation, or artefactual generation of allantoin. We have developed a method for measuring allantoin using hydrophilic liquid chromatography with stable isotope dilution tandem mass spectrometry (HILIC–MS/MS). It was validated for measuring allantoin in plasma, synovial fluid and urine from human subjects. The limit of quantification was determined to be 10fmol and the assay displayed excellent linearity for the wide range of concentrations found in clinical samples. Relative standard deviations were <5% for between-day and <7% for within-day variation. Accuracy was between 100% and 104%. Concentrations of allantoin in plasma of healthy controls (2.0μM; interquartile range 1.4–3.6μM, n=35) was significantly lower (p<0.001) than that in plasma from patients with rheumatoid arthritis (3.7μM; IQR 3.0–5.6μM, n=43) and in synovial fluid of patients with gout (3.3μM; IQR 2.8–5.8μM, n=10). This newer HILIC–MS/MS method is a simple and highly sensitive assay for detection of allantoin. It can be used to assess the level of oxidative stress in human pathologies.

Quantitative profiling of polar primary metabolites using hydrophilic interaction ultrahigh performance liquid chromatography–tandem mass spectrometry

A hydrophilic interaction liquid chromatography (HILIC)-MS/MS profiling method was developed for the efficient separation and quantification of small polar molecules, mostly primary metabolites. The method was based on an ultrahigh performance liquid chromatography (UHPLC) separation system coupled with ESI mass spectrometry on a triple quadrupole mass spectrometer, operating in both positive and negative ionisation mode using rapid polarity switching. With the developed method quantitation of 135 compounds belonging in four major classes of polar compounds (sugars, aminoacids, organic acids and amines) was achieved in a single run of 30min. The method was applied to grape extracts from different varieties and provided information on primary metabolite content. Multivariate statistical analysis was applied using the concentrations found, with the aim of investigating the differences in metabolite profiles. Classification of grapes according to their skin colour was carried out using principle component analysis based on the concentration variation of a number of the metabolites studied.

Preparation and characterization of methacrylate-based monolith for capillary hydrophilic interaction chromatography

In current study, poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EDMA) monolith was successfully prepared for capillary hydrophilic liquid chromatography (cHILIC). The polymerization was optimized by changing the ratio of MAA to EDMA, the type and amount of porogen. The characterization indicated that “hydrophilic” monolithic column possessed homogeneous column bed, good permeability and narrow pore size distribution. Under HILIC mode, the “hydrophilic” monolith prepared with PEG and DMSO showed stronger hydrophilicity than the “hydrophobic” monolith prepared with dodecanol and toluene. Finally, the “hydrophilic” monolith was applied in the separation of tryptic digests of bovine serum albumin (BSA) with cHILIC-ESI-qTOF–MS system. Our results revealed that 49 peptides were identified with 50% sequence coverage under HILIC mode, which was much better than the peptides identified using particulate-packed commercial column with RPLC-ESI-qTOF–MS system or “hydrophobic” monolith with cHILIC-ESI-qTOF–MS system. Taken together, the “hydrophilic” monolithic column prepared in current study, demonstrated the excellent chromatographic performance on the separation of complex samples, which offers the potential application of the monolith on proteomics study.

Epigenetic modulation of the HeLa cell membrane N-glycome

BackgroundEpigenetic changes play a role in all major events during tumorigenesis and changes in glycan structures are hallmarks of virtually every cancer. Also, proper N-glycosylation of membrane receptors is important in cell to cell and cell–environment communication. To study how modulation of epigenetic information can affect N-glycan expression we analyzed effects of epigenetic inhibitors on HeLa cell membrane N-glycome. MethodsHeLa cells were treated with DNA methylation (zebularin and 5-aza-2-deoxycytidine) and histone deacetylation (trichostatin A and Na-butyrate) inhibitors. The effects on HeLa cell membrane N-glycome were analyzed by hydrophilic interaction high performance liquid chromatography (HILIC). ResultsEach of the four epigenetic inhibitors induced changes in the expression of HeLa cell membrane N-glycans that were seen either as an increase or a decrease of individual glycans in the total N-glycome. Compared to DNA methylation inhibitors, histone deacetylation inhibitors showed more moderate changes, probably due to their higher gene target selectivity. ConclusionsThe results clearly show that composition of HeLa cell membrane N-glycome can be specifically altered by epigenetic inhibitors. General significanceGlycans on the cell membrane are essential elements of tumor cell's metastatic potential and are also an entry point for nearly all pathogenic microorganisms. Since epigenetic inhibitors used in this work are registered drugs, our results provide a new line of research in the application of these drugs as anticancer and antimicrobial agents. This article is part of a Special Issue entitled Glycoproteomics.

High Throughput Isolation and Glycosylation Analysis of IgG–Variability and Heritability of the IgG Glycome in Three Isolated Human Populations

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.

Future Developments for Triple Quadrupole Mass Spectrometers: Not Just Hardware

BioanalysisVol. 3, No. 7 EditorialFuture developments for triple quadrupole mass spectrometers: not just hardwarePatrick BennettPatrick BennettThermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134, USA. Search for more papers by this authorEmail the corresponding author at patrick.bennett@thermofisher.comPublished Online:31 Mar 2011https://doi.org/10.4155/bio.11.41AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: future innovationhigh resolutionmatrix effectssystem specializationtriple quadrupoleFiguresReferencesRelatedDetailsCited ByQuantitative profiling of polar primary metabolites using hydrophilic interaction ultrahigh performance liquid chromatography–tandem mass spectrometryJournal of Chromatography A, Vol. 125910years of MS instrumental developments – Impact on LC–MS/MS in clinical chemistryJournal of Chromatography B, Vol. 883-884 Vol. 3, No. 7 Follow us on social media for the latest updates Metrics Downloaded 503 times History Published online 31 March 2011 Published in print April 2011 Information© Future Science LtdKeywordsfuture innovationhigh resolutionmatrix effectssystem specializationtriple quadrupoleFinancial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry

We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol–chloroform, hydrolyzed using 88% formic acid at 140 °C, spiked with cytosine-2,4- 13C 15N 2 as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC–MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5 pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1–50 and 1–100 ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70–4.09% and 0.60–4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC–MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation.

Human Plasma Glycome in Attention-Deficit Hyperactivity Disorder and Autism Spectrum Disorders

Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction.

Validated hydrophilic interaction LC-MS/MS method for determination of 2-pyrrolidinone residue: applied to a depletion study of 2-pyrrolidinone in swine liver

A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for determination of 2-pyrrolidinone in swine liver was developed and validated. After the fortification of 2-pyrrolidinone-d(6) as the internal standard, 2-pyrrolidinone in swine liver was extracted by acetonitrile, and the supernatant was led through a C18 + WAX mixed-mode solid phase extraction (SPE) cartridge. Furthermore, the eluate was adjusted to pH 5.0 and then led through a strong cationic exchange SPE cartridge. 2-Pyrrolidinone and 2-pyrrolidinone-d(6) were concentrated and eluted by acetonitrile containing 2% ammonium hydroxide. The final eluate was acidified and then injected for hydrophilic interaction LC-MS/MS analysis. Mass spectrometry detection was carried using positive turbo-ion spray ionization mode. The multiple reaction monitoring transitions were 86 → 69 for 2-pyrrolidinone and 92 → 75 for 2-pyrrolidinone-d(6). The C18 + WAX mixed-mode SPE cleanup greatly prevented the rapid contamination of mass spectrometer. The further SCX SPE cleanup thoroughly eliminated the absolute matrix effect. Solvent calibration standards could be readily used for quantitative analysis of 2-pyrrolidinone with excellent precision and accuracy. Endogenous levels of 2-pyrrolidinone in some blank matrices was readily determined. Full recoveries were readily achieved by the optimize extraction protocol, and thus the role of 2-pyrrolidinone-d(6) was to just compensate the variation of the injections. The detection limit was 5 ng g(-1) swine liver. The validated method was applied to a depletion study of 2-pyrrolidinone in swine liver following intramuscular administration of a drug 2-pyrrolidinone formulation. The matrix effect from tissue samples usually represented a technical challenge for LC-MS/MS analysis, and a very small molecule such as 2-pyrrolidinone also represented a technical barrier for LC-MS/MS analysis. However, the extraction protocol developed in the present study reached the best outcome: zero matrix effect and full recovery.

Detection of hazelnut oil in extra‐virgin olive oil by analysis of polar components by micro‐solid phase extraction based on hydrophilic liquid chromatography and MALDI‐ToF mass spectrometry

The oil polar fraction may have a great potential for the characterization of vegetable oils and for the individuation of adulterations. In particular, adulteration of extra-virgin olive oil (EVOO) with hazelnut oil (HO) is one of the most difficult ones to detect due to the similar composition as regards triacylglycerol, total sterol and fatty acid profile. A new micro-solid phase extraction (µ-SPE) procedure based on hydrophilic liquid chromatography (HILIC) micro-columns was developed for the selective extraction and enrichment of polar compounds from EVOO and HO before matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS) analysis. The method permits a simple and fast qualitative analysis of the polar fraction of the oils under study; furthermore, some peaks (such as the m/z ions 496.39, 520.46 and 522.47) were found to be present only in HO, indicating that they could be diagnostic for the presence of HO in EVOO. In order to verify the potential of the method for the individuation of this adulteration, EVOO was progressively adulterated with variable quantities of HO, subjected to the HILIC enrichment and finally to MALDI-ToF-MS analysis; the detection of adulteration was possible up to the level of 5%. Eventually, diagnostic polar compounds were identified as lysophosphatidylcholine (LPC) (16:0/0:0), LPC (18:2/0:0), LPC (18:1/0:0) by means of capillary liquid chromatography-electrospray ionization-quadrupole-ToF-MS (CapLC-ESI-Q-ToF-MS) analysis.

Liquid chromatography–high-resolution electrostatic ion-trap mass spectrometric analysis of GD 3 ganglioside in dairy products

Gangliosides are thought to play a role in modifying the intestinal microflora and promoting neural development in the neonate. Milk is a good source of gangliosides, which are localised in the milkfat globule membrane. Robust quantitative analysis is required to underpin any claims made on ganglioside content in milk products. We have modified and validated our previously published method to quantify the amounts of the two predominant gangliosides classes, GD 3 and GM 3, in a range of bovine dairy products. After extraction and separation using hydrophilic high-performance liquid chromatography, an LTQ-Orbitrap™ mass spectrometer was used to extract and sum the major species of GD 3 and GM 3, generating fully resolved extracted ion current peaks for both standards and samples. The method had good repeatability (coefficient of variation 2–8%) and its results compared favourably with thin layer chromatography, a method that has been used extensively in the literature for the quantification of gangliosides in various sample matrices.

Novel zwitterionic polyphosphorylcholine monolithic column for hydrophilic interaction chromatography

A novel porous zwitterionic monolith was prepared by thermal co-polymerisation of 2-methacryloyloxyethyl phosphorylcholine (MPC) and ethylene glycol dimethacrylate (EDMA) within 100 μm I.D. capillaries. Mercury intrusion porosimetry, scanning electron microscopy (SEM), micro-HPLC (μ-HPLC), elemental analysis and ζ-potential analysis were used to evaluate the monolithic structure. No evidence of swelling or shrinking of the monolith in different polarity solvents was observed. A typical hydrophilic liquid chromatography (HILIC) mechanism was observed at high organic solvent content (acetonitrile >60%). The phosphorylcholine (PC) functionality has both a positively charged quaternary ammonium and a negatively charged phosphate group. For charged analytes, a weak electrostatic interaction was also observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(MPC- co-EDMA) monolithic column. The optimised poly(MPC- co-EDMA) monolith showed very good selectivities for a range of polar test analytes, especially small peptides. This might be ascribed to the good biocompatibility of PC functionality. At low organic solvent content, baseline separation was also observed for a test mixture of seven alkylphenones by a reversed-phase separation mechanism.