Preliminary investigation of the application of on-line membrane extraction of trifluoroacetic acid as an aid to improvement of negative ion electrospray mass spectrometry data.

We have recently investigated the biodegradation of a number of acidic aromatic compounds that give excellent chromatography using trifluoroacetic acid (TFA) based HPLC methods. Unfortunately HPLC methods using TFA are not usually compatible with detection by negative ion mass spectrometry as TFA suppresses ionisation of the analyte during the electrospray process. We present a preliminary investigation of the use of an anion-exchange micro-membrane suppressor to remove TFA on-line post column with the aim of improvement of mass spectral data using an aromatic acid as an example, Thus LC-MS using a TFA based HPLC method with negative ion mass spectral detection is shown to be possible with good sensitivity.

Analysis of process impurities in the basic drug SB-253149 using capillary electrophoresis and on-line mass spectrometric detection.

Capillary electrophoresis with on-line electrospray ionisation mass spectrometry (CE/ESI-MS) has been used to identify process impurities in a batch of the anti-atherosclerotic drug, SB-253149. The impurities were separated from the main drug compound by capillary electrophoresis (CE) using an ammonium formate buffer at low pH in an untreated fused silica capillary. The CE method was initially developed using UV as the detection mode and then later structural elucidation work was achieved using an ion trap mass spectrometer. To maintain peak resolution and peak shape when the CE system was coupled to the mass spectrometer, a modified capillary cassette linked to a coaxial sheath flow electrospray ionisation (ESI) interface was used. By performing MS/MS experiments in conjunction with chemical knowledge of the reactivities of SB-253149, it was possible to propose molecular structures for impurities detected in the batch of SB-253149. The results from this study revealed that most of the process impurities in SB-253149 were dimeric derivatives of the parent molecule as well as trace levels of the starting material. This type of information was vital in process control and optimisation for the synthetic route for this drug.

ATP-Citrate lyase as a target for hypolipidemic intervention. 2. Synthesis and evaluation of (3R,5S)-omega-substituted-3-carboxy-3, 5-dihydroxyalkanoic acids and their gamma-lactone prodrugs as inhibitors of the enzyme in vitro and in vivo.

A series of (3R,5S)-omega-substituted-3-carboxy-3, 5-dihydroxyalkanoic acids have been synthesized and evaluated as inhibitors of the recombinant human form of ATP-citrate lyase. The best of these have Ki's in the 200-1000 nM range. As the corresponding thermodynamically favored gamma-lactone prodrugs, a number of compounds are able to inhibit cholesterol and fatty acid synthesis in HepG2 cells and reduce plasma triglyceride levels in vivo. The best of these, compound 77, is able to induce clear hypocholesterolemic and hypotriglyceridaemic responses when administered orally to rat and dog. These results provide evidence to support the hypothesis that compounds which inhibit ATP-citrate lyase have the potential to be a novel class of hypolipidemic agent, which possess combined hypocholesterolemic and hypotriglyceridemic activities.

Minimizing cationization effects in the analysis of complex mixtures of oligosaccharides.

We outline simple methodology for the rapid and selective analysis of 2-aminoacridone (2-AMAC) derivatised oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. This involves the addition of small amounts of lithium chloride to the matrix before evaporation of solvent and crystallization. Signals mainly attributed to proton, sodium and potassium adducts are suppressed to a great extent, and a single signal due to (M + Li)+ is observed. This technique is rapid and is most useful for the direct analysis of complex glycan mixtures, after derivatization with 2-aminoacridone and without separation of the individual components.

High-performance liquid chromatographic analysis of complex N-linked glycans derivatized with 2-aminoacridone.

2-Aminoacridone (2-AMAC) has been used to derivatize mixtures of N-linked oligosaccharides released from alpha(1)-acid glycoprotein and immunoglobulin G. In each case, the HPLC profile obtained for the derivatized glycans was compared to that obtained after digestion with sialidase and a two-enzyme array system made up of sialidase and alpha-fucosidase, prior to derivatization by 2-AMAC. These studies are rapid and provide a wealth of preliminary information about the degree of sialylation and core fucosylation in the corresponding parent glycans. Moreover, collection of glycans from one single injection has provided enough material for molecular weight determination by MALDI-MS analysis. In this study we have also carried out limited MS-MS studies on enriched fractions of 2-AMAC-glycans using a nanospray orthogonal quadrupole time-of-flight mass spectrometer.

A coordinated high-performance liquid chromatographic, capillary electrophoretic, and mass spectrometric approach for the analysis of oligosaccharide mixtures derivatized with 2-aminoacridone.

Glycans derivatized with 2-aminoacridone have been analyzed consecutively by reverse-phase high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MECC). The 2-aminoacridone derivatizing agent used in the present study is highly hydrophobic and is well separated from the glycan derivatives in both separation techniques, ensuring that excess reagent does not interfere with the oligosaccharide analysis. The methodology outlined uses the high resolving power of capillary electrophoresis to determine the heterogeneity of samples after collection and preconcentration by HPLC. Collected glycan samples are submitted for mass spectrometric analysis to determine molecular weight. This methodology has been applied to linear oligosaccharides derived from dextran and to N-linked mannose-rich glycans from ribonuclease B.

Simple differentiation between core-fucosylated and nonfucosylated glycans by capillary electrophoresis.

Core-fucosylated glycans, derivatized with 2-aminoacridone, consistently migrate slower than the corresponding oligosaccharides which lack this fucose residue, using the micellar electrophoretic capillary chromatography conditions outlined in this study. alpha-Fucosidase digestion of glycans followed by CE analysis has allowed facile differentiation of these two classes of oligosaccharides and this methodology has been applied to obtain preliminary information on the carbohydrate content from two glycoproteins, a monoclonal IgG antibody and the soluble complement receptor type 1 (sCR1).

Enhanced resolution in the fingerprinting of short-chain oligonucleotides using the dodecasodium salt of phytic acid in capillary electrophoresis.

The inclusion of the dodecasodium salt of phytic acid in separation buffers has been found to improve considerably the separation of a number of synthetic short-chain oligonucleotides. A combination of the simplicity and the improved resolving power of this methodology allows the facile analysis of mixtures of such compounds. The presence of a large concentration of sodium ions reduces electroosmotic mobility due to more efficient shielding of negative charges on the inner surface of the capillary wall. This, in turn, leads to a more gradual fall in potential between this surface and the center of the capillary.

Improved peptide mapping using phytic acid as ion-pairing buffer additive in capillary electrophoresis.

By digestion of the highly basic polypeptide aprotinin or bovine pancreatic trypsin inhibitor (BPTI) with endoproteinase Lys-C after unfolding, reduction and pyridylethylation, five fragments are obtained. These fragments are separated by free solution capillary electrophoresis using a phosphate buffer at neutral pH. The effect of the ion-pairing buffer additive phytic acid on the separation was investigated. It is shown that phytic acid through ion-pair formation influences the mobility of only those peptide fragments having a net positive charge at the pH of the separation buffer. The affinity of phytic acid to the peptides correlates with their isoelectric point and the charge to mass ratios. Hence, by changing the concentration of phytic acid, it is possible to manipulate the migration order and the separation of the peptides.

Fingerprinting of glycans as their 2-aminoacridone derivatives by capillary electrophoresis and laser-induced fluorescence.

Capillary electrophoresis and laser-induced fluorescence detection have been used to fingerprint the 2-aminoacridone derivatives of complex glycans released from bovine fetuin and human IgG monoclonal antibodies. The utility of this method in distinguishing between N- and O-linked oligosaccharides and in determining the presence of sialic acid residues in glycan mixtures at an early stage of analysis has been demonstrated.

Effective ion-pairing for the separation of basic proteins in capillary electrophoresis.

The addition of the sodium salt of phytic acid to the separation buffer (pH's 6.0-9.5) has allowed the analysis of a number of basic proteins (pI's > 9) by capillary electrophoresis. The method of analysis is simple and leads to considerable improvement in peak shape. Some very basic proteins, totally adsorbed onto the capillary fused silica surfaces in the presence of buffer only, can be analysed as sharp signals when this polyanionic species is included in the running electrolyte. These improvements in analysis are thought to arise as a result of the suppression of coulombic interactions between these positively charged proteins (ion-paired to phytic acid) and the negatively charged silanol groups on the inner wall of the capillary.

High resolution and rapid analysis of branched oligosaccharides by capillary electrophoresis.

The fluorophore 2-aminoacridone has been used to label a number of branched oligosaccharides previously released from various glycoproteins. Complex glycans were derivatized via their reducing end with this fluorophore by a Schiff's base mechanism followed by reduction to a secondary amine using sodium cyanoborohydride. This process of derivatization was carried out efficiently and in a nonselective manner over a period of 30 min at 90 degrees C. The resulting derivatives were separated with high resolution by capillary electrophoresis using borate buffer, containing taurodeoxycholate, as the separation buffer. The method described does not require the removal of sialic acid residues prior to derivatization, so that treatment of glycans with N-acetyl neuraminidase provided useful and additional structural information. Attempts have also been made to relate the electrophoretic mobility of branched oligosaccharides with their molecular volume.

Micellar electrokinetic chromatographic studies in D2O based buffer solutions.

The addition of deuterium oxide or organic solvents to surfactant-based electrolytes is an effective method for improving resolution in micellar electrokinetic capillary chromatography (MECC). Buffers containing phosphate, boric acid and sodium dodecyl sulphate at pH or pD 7 in the presence and absence of deuterated and non-deuterated solvents were used to separate some hydrophobic compounds covering a range of octanol-water partition coefficients (log P). Significant improvements in resolution of the more hydrophobic compounds were achieved by using mixtures of heavy water and deuterated methanol, instead of water and methanol. Quantitative structure-retention relationships were also derived to provide a better understanding of the intermolecular interactions that contribute to the separation of analytes in MECC. Multiple regression analysis has shown that capacity factors were significantly related to both log P and molecular refractivity. The latter is a measure of the molecular volume of analytes and is related to their ability to undergo dispersive interactions with the micellar components.

Improved resolution of tryptic digest fragments from haemoglobin variants using phytic acid in free zone capillary electrophoresis.

Capillary electrophoresis can be applied to the rapid characterization of tryptic digests of proteins. The addition of phytic acid to the separation buffer was found to improve resolution considerably when the technique was applied to differentiate between tryptic digests derived from variant haemoglobins. Moreover, analysis time was of the order of 15 min, which is considerably shorter than that obtained using gradient reversed-phase high-performance liquid chromatography or two-dimensional paper chromatography-electrophoresis.

The effect of phytic acid on the resolution of peptides and proteins in capillary electrophoresis.

Studies are reported on the effect of the sodium salt of phytic acid on the resolution of peptides and proteins. Improved separation in the case of peptides is shown to be due to ion-ion pairing interactions between the positively charged peptides and the phytic acid polyanionic species. The improved peak shapes related to the proteins can be interpreted in terms of the sample preconcentration due to injection of analytes from a water medium to one of high ionic strength.

Micellar electrokinetic capillary chromatography of amoxycillin and related molecules.

A micellar electrokinetic capillary chromatographic method has been developed for the qualitative assay of amoxycillin and its degradation products and clavulanic acid. Together with amoxycillin the latter acid is an important constituent in the antibiotic Augmentin. The analytical procedure is fast and analytes can be identified both from their migration times and from changes in migration time observed either at different pH values or in electropherograms run in H2O and D2O based buffers of the same acidity.

Analytical and micropreparative capillary electrophoresis of the peptides from calcitonin.

The capillary electrophoresis (CE) of peptide fragments from the tryptic digest of salmon calcitonin and elcatonin has been carried out in H2O- and D2O-based buffer solutions. Analysis in heavy water was found to be superior to that in H2O especially at a pH or pD of 7.93. From a single CE run on elcatonin digest we were also able to harvest three pure cleavage peptides in sufficient quantity to determine each amino acid residue by protein sequencing. The order of elution from CE agreed with that predicted on the basis of net charge calculated for each peptide.

Separation by capillary electrophoresis followed by dynamic elution.

In this study we have explored the behaviour of peptides after capillary electrophoresis (CE) followed by elution under pressure. The use of D2O- rather than H2O-based buffer solutions appears to restrict the diffusion of peptides after CE, resulting in little loss of resolution when peptides are eluted by dynamic flow. In this paper we present results showing that a simple two-step process, involving CE at a low voltage, switching off the power supply, and connecting the fused capillary at the anode end to a syringe pump for dynamic flow, can retain separation characteristics and can be used for the isolation of picomole quantities of peptides for sequence determination.