Hydrophilic interaction 10 microm I.D. porous layer open tubular columns for ultratrace glycan analysis by liquid chromatography-mass spectrometry

The sensitivity of glycan analysis using nano-liquid chromatography interfaced with electrospray ionization mass spectrometry (ESI-MS) increases with the decrease of the mobile phase flow rate, accompanied by reduced ion suppression. In this study, we describe the preparation and performance of high efficiency 10 microm I.D. amine-bonded poly(vinylbenzyl chloride-divinylbenzene) hydrophilic interaction (HILIC) porous layer open tubular (PLOT) columns operated at 20 nL/min for the separation and analysis of glycan mixtures. HILIC-PLOT columns with a uniform porous polymer layer were reproducibly prepared ( approximately 4% RSD in retention time from column-to-column) via in situ polymerization, followed by one step modification with ethylenediamine. When coupled on-line with negative ESI-MS, low detection limits (0.3fmol) for a 3-sialyl-tetrasaccharide were achieved using a 2.5mx10 microm I.D. HILIC-PLOT column. A dextran ladder standard was used to evaluate the performance of the column, and high efficiency separation was achieved with detection of the dextrans up to G22 from approximately 50 fmol amounts injected. As an example of the high sensitivity of the column, MS(6) characterization of glycan structures was possible from the injection of 10 fmol of a neutral and sialylated glycan. As another example of high sensitivity LC-MS analysis of 3 ng of a PNGase F digest of ovalbumin allowed 28 N-linked glycans to be confidently identified from a single analysis. High quality MS/MS spectra for each ovalbumin glycan were acquired and manually interpreted for structure analysis. The HILIC-PLOT column is a very promising approach for LC-MS analysis of glycans at the ultratrace level.     

Oligosaccharide relative quantitation using isotope tagging and normal-phase liquid chromatography/mass spectrometry

The growing interest in the conversion of plant biomass into biofuels has recently highlighted the lack of analytical techniques that are able to profile the fine structures of plant cell-wall polysaccharides. Here we present a new liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) platform called Oligosaccharide Quantitation using Isotope Tagging (OliQuIT) developed for profiling the oligosaccharides derived from glycosyl hydrolase digestion of polysaccharides. The method is demonstrated using different arabinoxylan-derived oligosaccharide samples, which are reductively aminated with either the light (12C6) or heavy (13C6) form of aniline. The complex oligosaccharide mixtures are analysed by capillary normal-phase (NP)-LC and ESI-MS. Importantly, arabinoxylan oligosaccharide isomers are separated by NP-LC and their relative abundance in different samples can be determined from the intensities of ions labeled with the different isotopes. OliQuIT will be of use in multiple applications, including screening for plant varieties with improved saccharification properties, characterizing glycosyl hydrolase specificities and analysing plant glycosyl transferase mutants.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Effects of pH in reversed-phase liquid chromatography

The effects of concomitant variations in pH and organic modifier concentration on retention, efficiency and peak symmetry are considered for reversed-phase liquid chromatography (RPLC) on octadecyl-modified silica (ODS) columns. A number of factors are discussed, which make the systematic exploitation of pH effects in RPLC more complicated than the optimization of solvent composition. If the pH is varied, a second factor (usually the concentration of organic modifier) will need to be varied simultaneously to maintain retention (capacity factors) in the optimum range. When pH is considered as a parameter in RPLC, not only its effects on retention, but also the variations in efficiency (plate count) and peak shape (asymmetry) need to be considered. These parameters turn out to vary drastically between individual solutes and between different experimental conditions. The results of a study involving a number of acidic, basic and neutral solutes, two different ODS columns and mixtures of either methanol or acetonitrile with aqueous buffers are reported. In the earlier part of the study, using methanol as the organic modifier, reproducible data for retention, peak width and peak symmetry were obtained and these data are reported. In the later part of the study, using acetonitrile, a gradual change in retention as a function of time was observed, this effect coinciding with a decrease in column efficiency. It is concluded that ODS columns are subject to considerable degradation during studies in which the pH is varied. Although this effect can be described mathematically, the preferred solution is thought to be the use of pH-stable columns.     

Selectivity tuning of serially coupled columns in high performance liquid chromatography

The tuning of selectivity by changing the flow rate has been investigated in HPLC: two columns with different retention characteristics were coupled in series via a T-piece and the relative retention of components chromatographed on the system were changed by varying the individual flow rates in the coupled columns. The flow rate alteration was performed by adding a second flow after the first column. The flow rate ratio necessary for optimum resolution can be easily calculated on the basis of the capacity factors measured on the individual columns. The performance of this method for adjusting selectivity has been demonstrated by using different column combinations to separate several mixtures containing chlorophenols, nitroaromatic compounds, and aromatic hydrocarbons.     

Packed column supercritical fluid chromatography with light-scattering detection. II. Retention behaviour of squalane and glucose with mixed mobile phases

Summary Evaporative light scattering detectors can be used to detect organic substances without chromophoric groups in packed column supercritical fluid chromatography (SFC). A detector of this type has been used to detect squalane and glucose after SFC with various packed columns and binary mobile phases. In this study, the amount of organic modifier in carbon dioxide/modifier mixtures was varied. The results give further insight into the mechanisms that influence retention behaviour in packed column separations with super- and subcritical mobile phases. Squalane is an ideal non-polar test solute which shows long retention times on non-polar columns while its elution can be accelerated by non-polar modifiers in carbon dioxide. Glucose is an extremely polar solute containing hydroxyl groups. Elution of this sugar can be improved with polar modifiers. Column packings with polar end groups lead to high capacity ratios and long retention times for glucose. Most columns used in this study contained silica-based packing materials. For purposes of comparison, a polymeric packing (HEMA RP-18) was also employed.     

Liquid chromatography/electrospray time-of-flight mass spectrometry for the characterisation of cyclic phosphazenes

Cyclic phosphazenes with different substituents were synthesised and investigated by liquid chromatography (LC) and electrospray ionisation mass spectrometry (ESI-MS). Hexachlorocyclotriphosphazene was functionalised with aliphatic substituents as alcohols and amines, leading to product mixtures, which were subsequently analysed. In contrast to classical methods of structural analysis such as nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, which are restricted to pure compounds, these complex mixtures can favourably be analysed by means of LC/ESI-MS. The main products could be separated from by-products and, moreover, all the components of the unknown mixture were unambiguously identified by accurate mass measurements. For all compounds with different side-chain ratios, remaining chlorine atoms or hydroxyl groups and even for spiro or ansa products, molecular structures could be suggested.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

Comparison of direct infusion and on-line liquid chromatography/electrospray ionization mass spectrometry for the analysis of nucleic acids

The applicability of ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) and direct infusion/ESI-MS to the characterization of nucleic acid mixtures was evaluated by the analysis of the reaction products obtained from solid-phase synthesis of a 39-mer oligonucleotide. IP-RP-HPLC/ESI-MS was performed using 200 microm i.d. capillary columns packed with octadecylated, micropellicular poly(styrene-divinylbenzene) particles and applying gradients of acetonitrile in 50 mM triethylammonium bicarbonate (TEAB). Three different solvent systems were utilized for direct infusion/ESI-MS with removal of metal cations by on-line cation exchange: (1) 10 mM triethylamine (TEA) in 50% aqueous acetonitrile, (2) 2.2 mM TEA, 400 mM hexafluoro-2-propanol (HFIP) in 20% aqueous methanol and (3) 50 mM TEAB in 10% aqueous acetonitrile. Owing to its separation capability, the highest selectivity and specificity were achieved with IP-RP-HPLC/ESI-MS, which, apart form the 39-mer target sequence, allowed the identification of two isobutyryl-protected target sequences and a 10-mer and 20-mer failure sequence. Direct infusion/ESI-MS with TEA-acetonitrile or TEA-HFIP-methanol as solvent revealed signals for the 39-mer in the m/z range 700-1600. The presence of derivatives containing one, two, three and four isobutyryl groups indicated that the hydrolysis of the protecting groups after solid-phase synthesis was not complete. Failure sequences could not be identified by direct infusion/ESI-MS under conditions favoring multiple charging of the analytes owing to the high chemical background and coincidental overlapping of m/z signals. However, efficient charge state reduction upon addition of carbonic acid to the electrosprayed solvent shifted the signals of the 39-mer and derivatives to m/z values >2400 and allowed the detection of seven different failure sequences, ranging from the 8-mer to the 23-mer, in the mixture.     

Polymethacrylate monolithic and hybrid particle-monolithic columns for reversed-phase and hydrophilic interaction capillary liquid chromatography

We prepared hybrid particle-monolithic polymethacrylate columns for micro-HPLC by in situ polymerization in fused silica capillaries pre-packed with 3–5 μm C₁₈ and aminopropyl silica bonded particles, using polymerization mixtures based on laurylmethacrylate–ethylene dimethacrylate (co)polymers for the reversed-phase (RP) mode and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl) zwitterionic (co)polymers for the hydrophilic interaction (HILIC) mode. The hybrid particle-monolithic columns showed reduced porosity and hold-up volumes, approximately 2–2.5 times lower in comparison to the pure monolithic columns prepared in the whole volume of empty capillaries. The elution volumes of sample compounds are also generally lower in comparison to packed or pure monolithic columns. The efficiency and permeability of the hybrid columns are intermediate in between the properties of the reference pure monolithic and particle-packed columns. The chemistries of the embedded solid particles and of the interparticle monolithic moiety in the hybrid capillary columns contribute to the retention to various degrees, affecting the selectivity of separation. Some hybrid columns provided improved separations of proteins in comparison to the reference particle-packed columns in the reversed-phase mode. Zwitterionic hybrid particle-monolithic columns show dual mode retention HILIC/RP behaviour depending on the composition of the mobile phase and allow separations of polar compounds such as phenolic acids in the HILIC mode at lower concentrations of acetonitrile and, often in shorter analysis time in comparison to particle-packed and full-volume monolithic columns.     

Blending bulk dissolution and gas-liquid-adsorption with the mixing of two liquid stationary phases to increase selectivity

Summary It is shown that the blending of some retention effects can make the resolution of complex mixtures on short classical columns as effective as on capillary columns.     

Comparison of atmospheric pressure chemical ionization and electrospray ionization mass spectrometry for the detection of lignans from sesame seeds

In sesame seeds, high concentrations of lignans are present. When these lignans are fermented in the human colon, a range of structurally different lignans is formed. A good liquid chromatography/mass spectrometry (LC/MS) protocol for the analysis of lignans in complex mixtures is lacking. In order to develop such a protocol, electrospray ionization (ESI)-MS and atmospheric pressure chemical ionization (APCI)-MS, both in the positive and negative ionization mode, were compared. An extract from defatted sesame meal was analyzed by APCI-MS and ESI-MS, before and after deglucosylation. APCI-MS was found to be a more generic method than ESI-MS because lignans, especially sesamolin, sesamin and pinoresinol, were better detected by APCI-MS than by ESI-MS. Positive and negative ionization modes had to be combined in order to detect all lignans in a bacterial culture grown on aglyconic, acid-treated lignans from sesame oil and defatted sesame meal. Lignans with methylenedioxy-bridged furanofuran structures mostly lack phenolic hydroxyl groups and were, therefore, optimally detected in positive ionization mode. Dibenzylbutadiene lignans, which were formed during fermentation, carry hydroxyl groups and were better detected in negative ionization mode.     

Profiling of N-linked oligosaccharides using phenylhydrazine derivatization and mass spectrometry

N-linked oligosaccharide standards obtained from commercial sources were derivatized with phenylhydrazine (PHN) and analyzed by on-line reversed-phase high performance liquid chromatography (HPLC)/electrospray ionization mass spectrometry (ESI-MS). This procedure was then applied to mixtures of N-glycans enzymatically released from hen ovalbumin. Under ESI-MS conditions, phenylhydrazones of asialylated oligosaccharide standards and ovalbumin glycans produced mainly [M + 2H]2+ molecular ions at low cone voltage values, while minimal fragmentation was observed. Reversed-phase HPLC/ESI-MS total and selected ion chromatograms obtained for derivatized N-glycans from ovalbumin showed partial but useful separation. Overall glycan profiles obtained by ESI-MS were compared with results obtained by matrix-assisted laser desorption/ionization (MALDI)-MS. Qualitatively, profiles were similar from one technique to the other in terms of relative abundance of glycans versus composition. Post-source decay (PSD) analysis of the [M + Na]+ ions of PHN-glycans showed dominant B, C and internal B/Y, C/Y cleavages. These patterns were helpful in relating fragmentation to proposed structures. Cross-ring cleavage fragment ions (A-type) were also observed in most cases. The PHN derivatization method is fast and simple. It produces abundant parent ions in both MALDI-MS and ESI-MS, while avoiding the presence of salt contaminants during the labeling procedure.     

Reversed-phase high-performance liquid chromatography of nucleoside analogues. Simultaneous analysis of anomeric D-xylo- and D-lyxofuranonucleosides and some other D-pentofuranonucleosides

Reversed-phase high-performance liquid chromatography using a C18 column was applied to the analysis of reconstituted mixtures of previously synthesized alpha, beta D-xylo- and D-lyxofuranonucleosides as well as a number of commercially available D-ribo- and D-arabinofuranonucleosides. From a detailed study of various parameters (size of support particles, nature and pH of the mobile phase, temperature), optimized conditions were established. Correlations between the retention times and structures of the bases, the orientations of the secondary hydroxyl groups of the sugar moiety and the anomeric configurations of the nucleosides are also reported.     

Characterization of a new methylated Beta-cyclodextrin with a low degree of substitution by electrospray ionization mass spectrometry and liquid chromatography/mass spectrometry

A new methylated beta-cyclodextrin (Me-beta-CD) with a low degree of substitution (DS) was characterized by electrospray ionization mass spectrometry (ESI-MS) and liquid chromatography coupled with ESI-MS (LC/ESI-MS). For ESI-MS analyses, the composition of the infused sample solution was optimized in order to obtain only singly charged ammoniated CDs without fragmentation. The DS value (i.e. the number of methyl groups per glucopyranose unit) was found to be 0.7, which was in accordance with the values previously obtained by other methods. The LC/ESI-MS analysis, derived from a method using evaporative light scattering detection, allowed the study of the substitution isomers of each derivative and appears to be an easy and rapid tool for the accurate characterization of Me-beta-CD mixtures.     

Separation properties of novel and commercial polar stationary phases in hydrophilic interaction and reversed-phase liquid chromatography mode

Four novel nonionic polar stationary phases were synthesised by anchoring first 2-mercaptoethanol and 1-thioglycerol, respectively, onto vinylised silica (ME and TG packings) followed by an on-phase oxidation with excess hydrogen peroxide in aqueous medium which yielded sulphoxide analogues of the embedded sulphide groups, i. e. oxidised 2-mercaptoethanol (MEO) and oxidised 1-thioglycerol (TGO) packings. Chromatographic characteristics of these stationary phases were evaluated comparatively to three commercial so-called 'diol' columns. U-shaped response curves of retention factors of adenosine and guanosine with hydro-organic eluents containing 5-95% v/v ACN as well as noticeable CH(2)-increment selectivity demonstrated multimodal separation capabilities of the developed amphiphilic materials, i. e. columns can be operated both in hydrophilic interaction chromatography (HILIC) and in RP mode. Although the selector ligands were physico-chemically related, considerably differing retention and selectivity patterns were observed in the HILIC mode. Thereby the introduction of additional hydroxyl groups in the chromatographic ligand resulted in selectivity increments that were different from those obtained by sulphur oxidation. For example, a set of five vitamins delivered five different elution orders with the overall seven columns. A close examination of HILIC separations of nucleobases and nucleosides on the developed packings revealed that (i) the amount of ACN in the eluent adopts a pivotal role in adjusting retention, (ii) the linearity of the relationship log (retention factor) versus log (volume fraction of water in the eluent) increases with phase polarity in the range of 5-40% v/v water, (iii) the slopes are higher with solutes having more polar interactive sites, (iv) the van't Hoff plots are linear (range 15-45 degrees C) with negative retention enthalpy values DeltaH (-4.5 to -14.5 kJ/mol) and (v) the -DeltaH values tend to be higher with more polar phases and more polar analytes. Based on these data the HILIC retention mechanism is described to be composed of both partitioning and adsorption processes. Distinct types of polar interactive sites in the chromatographic ligands may generate mixed-mode HILIC separation conditions that may additionally be superimposed by surface silanol contributions.     

Determination of butyltin and phenyltin species by reversed-phase liquid chromatography and fluorimetric detection

Chromatographic separation of monobutyltin (MBT), monophenyltin (MPhT), dibutyltin (DBT), diphenyltin (DPhT), tributyltin (TBT) and triphenyltin (TPhT) was studied using end-capped reversed-phases (RP) and methanol-acetic acid-water or acetronitrile-acetic acid-water mixtures as mobile phases. Several RP columns were evaluated, and the effect of acetic acid, oxalic acid, triethylamine, and organic modifier on peak shape and retention was examined. A method based on gradient elution RPLC and fluorimetric detection is proposed for the determination of DBT, DPhT, TBT and TPhT. The sensitivity of the method makes it suitable for environmental analysis.     

Preparation of glycine-conjugated bile acids and their gas/liquid chromatographic analysis on an aluminum-clad flexible fused silica capillary column.

This paper describes a method for the direct gas/liquid chromatographic (GC) analysis of 46 glycine-conjugated bile acids, which differ from one another in the number, position and configuration of the hydroxyl groups at positions C-2, C-3, C-4, C-6, C-7 and/or C-12. Free bile acids were converted quantitatively on a micro scale to ethyl ester-trimethylsilyl (Et-TMS) and methyl ester-dimethylethylsilyl (Me-DMES) ether derivatives of the corresponding glycine conjugates. The Et-TMS and Me-DMES ethers of the glycine conjugates were chromatographed on an aluminum-clad flexible fused silica capillary column coated with a thin film (0.1 micron) of chemically bonded and cross-linked methylpolysiloxane. Relative retention time (RRT) and methylene unit (MU) values were determined for the 46 compounds and their GC behaviour was discussed. The derivatization procedure and the retention data would be useful for the direct GC identification of unknown glycine-conjugated bile acid mixtures extracted from biological samples.     

Dual parallel electrospray ionization and atmospheric pressure chemical ionization mass spectrometry (MS), MS/MS and MS/MS/MS for the analysis of triacylglycerols and triacylglycerol oxidation products

Two mass spectrometers, in parallel, were employed simultaneously for analysis of triacylglycerols in canola oil, for analysis of triolein oxidation products, and for analysis of triacylglycerol positional isomers separated using reversed-phase high-performance liquid chromatography. A triple quadrupole mass spectrometer was interfaced via an atmospheric pressure chemical ionization (APCI) interface to two reversed-phase liquid chromatographic columns in series. An ion trap mass spectrometer was coupled to the same two columns using an electrospray ionization (ESI) interface, with ammonium formate added as electrolyte. Electrospray ionization mass spectrometry (ESI-MS) under these conditions produced abundant ammonium adduct ions from triacylglycerols, which were then fragmented to produce MS/MS spectra and then fragmented further to produce MS/MS/MS spectra. ESI-MS/MS of the ammoniated adduct ions gave product ion mass spectra which were similar to mass spectra obtained by APCI-MS. ESI-MS/MS produced diacylglycerol fragment ions, and additional fragmentation (MS/MS/MS) produced [RCO](+) (acylium) ions, [RCOO+58](+) ions, and other related ions which allowed assignment of individual acyl chain identities. APCI-MS of triacylglycerol oxidation products produced spectra like those reported previously using APCI-MS. APCI-MS/MS produced ions related to individual fatty acid chains. ESI-MS of triacylglycerol oxidation products produced abundant ammonium adduct ions, even for those molecules which previously produced little or no intact molecular ions under APCI-MS conditions. Fragmentation (MS/MS) of the [M+NH(4)](+) ions produced results similar to those obtained by APCI-MS. Further fragmentation (MS/MS/MS) of the diacylglycerol fragments of oxidation products provided information on the oxidized individual fatty acyl chains. ESI-MS and APCI-MS were found to be complementary techniques, which together contributed to a better understanding of the identities of the products formed by oxidation of triacylglycerols.