Maria T. Matyska

Analysis of hydrophilic metabolites by high-performance liquid chromatography–mass spectrometry using a silica hydride-based stationary phase

A novel silica hydride-based stationary phase was used to evaluate the retention behavior in the aqueous normal-phase (ANP) mode of standards representing three classes of metabolites. The effects on retention behavior of amino acids, carbohydrates and small organic acids were examined by altering the column temperature, and by adding different additives to both the mobile phase and sample solvent. Gradient mode results revealed the repeatability of retention times to be very stable for these compound classes. At both 15 and 30 degrees C, excellent RSD values were obtained with less than 1% variation for over 50 injections of an amino acid mixture. The ability to separate the 19 nonderivatized amino acid standards, organic acids and carbohydrates was demonstrated as well as the potential for this material to separate polar metabolites in complex fluids such as urine.

Synthesis, Characterization and Applications of Hydride-Based Surface Materials for HPLC, HPCE and Electrochromatography

Abstract The chemical modification method based on the silanization of an oxide surface to a hydride intermediate followed by hydrosilation with an organic molecule containing a terminal olefin is reviewed. The resulting bonded organic moiety is attached to the surface via a direct Si-C bond which leads to high stability. The method has been more extensively applied to silica surfaces for the production of stationary phases in HPLC but it can also be used on other oxides such as alumina, zirconia, titania and thoria. More recent applications have been in the modification of the inner wall of fused silica capillaries for HPCE. The bonded moieties possess high stability and useful applications have been developed for the separation of proteins and peptides under a variety of buffer conditions. The same procedure for modifying the inner wall of a fused silica capillary has also been extended to etched surfaces for use in electrochromatography (CEC). This type of CEC has been shown to be applicable to the sep...

Separation of tetracyclines by high-performance capillary electrophoresis and capillary electrochromatography

Separations of various tetracycline mixtures by high-performance capillary electrophoresis (HPCE) and a new form of electrochromatography (CEC) are compared. The new CEC method involves etching the inner wall of the capillary surface with an appropriate reagent (ammonium dihydrogen fluoride) in order to produce a significant increase in surface area. The etched surface is then modified by a silation/hydrosilation reaction sequence to first produce a hydride intermediate which is then further reacted to attach a C18 moiety. The bare and hydride capillaries are tested under HPCE conditions while the C18 capillary functions in the CEC mode. The effects of pH and the presence of an organic modifier (methanol) are also studied. Detection limits ( < 10 micrograms/ml) are comparable to previous HPLC and HPCE results. Resolutions for mixtures which simulate real analytical problems are equal to or better than those reported for separations on polymeric and diol columns by HPLC and in earlier studies by HPCE and MECC.

Separation of proteins and peptides by capillary electrochromatography in diol- and octadecyl-modified etched capillaries

This study involves the evaluation of a capillary electrochromatography method based on etching the inner walls of a fused-silica tube, which is subsequently modified by a silanization/hydrosilation reaction scheme. Two different organic moieties, octadecyl and diol, are attached to the etched capillary wall. The performance of these two columns is compared to a bare capillary using peptide (angiotensins) and protein samples. It is concluded that the etching process increases the surface area of the inner wall sufficiently to induce solute-bonded phase interactions for the capillaries modified with the octadecyl and diol moieties. The separation capabilities of the two modified capillaries are not the same, presumably due to differences in the chemical properties of the two ligands. When compared to a bare capillary where separation is due only to electrophoretic mobility effects, the bonded etched capillaries also exhibit significant differences in separation factors for the same solutes under identical experimental conditions.

Open tubular capillary electrokinetic chromatography in etched fused-silica tubes.

This review describes an open tubular approach to capillary electrochromatography (OTCEC) that first etches the inner surface of the fused-silica tube using ammonium hydrogen diflouride. This process can increase the inner surface area significantly. The new surface is then chemically modified to attach a bonded stationary phase using a silanization/hydrosilation reaction process. The surfaces are characterized spectroscopically by diffuse reflectance infrared Fourier transform and by electroosmotic flow measurements. Applications of OTCEC columns with C18, diol and chiral stationary phases are described.

Analysis of hydrophilic metabolites in physiological fluids by HPLC‐MS using a silica hydride‐based stationary phase

Aqueous normal-phase chromatography was used for the analysis of metabolites in human saliva and urine samples. The column was packed with a silica hydride type separation material. Several gradients were tested with different mobile phase additives in order to produce retention for amino acids, small organic acids, and carbohydrates. Detection was done by TOF MS. In some cases the relative concentration levels of various metabolites in human saliva were compared for normal patients and patients with pancreatic cancer or pancreatitis. The reproducibility of retention of individual metabolites in these complex matrices was tested for several compounds.

Open tubular capillary electrochromatography in etched, chemically modified 20 μm I.D. capillaries

Fused silica capillaries with an I.D. of 20 microns are etched and then chemically modified by the silanization/hydrosilation method to attach an octadecyl moiety for use in electrokinetic chromatography. The etched capillaries after chemical modification are shown to have an anodic electroosmotic flow below pH 4.5. In comparison to bare 20 microns capillaries and unetched but chemically modified 20 microns capillaries, the etched C18 fused silica tubes show better separation of mixtures of lysozymes and cytochrome cs under identical conditions of buffer, pH and applied voltage. It was also demonstrated that this open tubular approach to capillary electrochromatography was amenable to a number of different types of basic compounds ranging in size from typical small amines to biomolecules. As expected, pH is an important variable that must be controlled in order to obtain an optimized separation. Reproducibility studies verify the stability of the silicon-carbon linkage produced in this modification method so that column lifetimes of at least 300 injections can be expected.

Aqueous normal-phase retention of nucleotides on silica hydride columns

The use of silica hydride-based stationary phases for the retention and analysis of nucleotides has been investigated. Both reversed-phase columns with a hydride surface underneath as well as those with an unmodified or a minimally modified hydride material were tested. With these systems, an aqueous normal-phase mode was used with high organic content mobile phases in combination with an additive to control pH for the retention of the hydrophilic nucleotides. Isocratic and gradient elution formats have been used to optimize separations for mixtures containing up to seven components. All conditions developed are suitable for methods that utilize mass spectrometry detection.

Aqueous normal phase retention of nucleotides on silica hydride‐based columns: Method development strategies for analytes revelant in clinical analysis

An aqueous normal phase HPLC method coupled with UV or ESI/MS detection was used for the determination of a wide variety of nucleotides, essential in metabolomics studies. Fifteen nucleotides were tested in clinically relevant mixtures at levels of 100 microg/mL for UV detection and 1 microg/mL for ESI-MS detection. Analysis times for all protocols developed were less than 20 min. The chromatographic conditions were changed to achieve optimized retention and separation of the nucleotides studied. The aqueous normal phase-HPLC methods were developed utilizing two columns, one having a minimally modified hydride surface another having an undecanoic acid moiety on a hydride surface. Volatile, low ionic strength mobile phases were used. Negative ion mode ESI-MS at near neutral pH mobile phase, combined with a TOF detector provided a highly sensitive and specific method, which is equally suitable for quadrupole and ion trap instruments.

Protein and peptide separations on high surface area capillaries

A 50 μm capillary that has been etched with ammonium hydrogen difluoride is evaluated as a separation medium for capillary electrochromatography. For a tryptic digest of transferrin, the etched capillary gave better resolution (more peaks in the overall peptide map) and longer retention than separations done under identical experimental conditions on an unetched fused‐silica capillary. Resolution on the etched capillary was improved by lowering the voltage from 300 to 267 V/cm. A four‐component protein sample also resulted in longer retention on an echted capillary than on an unetched fused‐silica capillary that were both coated with Polybrene. After correction for differences in electroosmotic flow between the two capillaries, the calculated electrophoretic mobilities for all four proteins were lower on the etched capillary than on the unetched fused‐silica capillary. The results of both the tryptic digest and protein experiments strongly indicate the presence of chromatographic effects on the etched capillary that contribute to the increased retention and improved resolution with respect to the unetched fused‐silica capillary.