Unmodified Silica Column Research Articles

Amine Gradient Stationary Phases on In-House Built Monolithic Columns for Liquid Chromatography.

Stationary phase gradients on monolithic silica columns have been successfully and reproducibly prepared and characterized with comparisons made to uniformly modified stationary phases. Stationary phase gradients hold great potential for use in liquid chromatography (LC), both in terms of simplifying analysis as well as providing novel selectivity. In this work, we demonstrate the creation of a continuous stationary phase gradient on in-house synthesized monolithic columns by infusing an aminoalkoxysilane solution through the silica monoliths via controlled rate infusion. The presence of amine and its distribution along the length of gradient and uniformly modified columns were assessed via X-ray photoelectron spectroscopy (XPS). XPS showed a clear gradient in surface coverage along the length of the column for the gradient stationary phases while a near uniform distribution on the uniformly modified stationary phases. To demonstrate the application of these gradient stationary phases, the separations of both nucleobases and weak acids/weak bases on these gradient stationary phases have been compared to uniformly modified and unmodified silica columns. Of particular note, the retention characteristics of 11 gradient columns, 5 uniformly modified columns, and 5 unmodified columns have been tested to establish the reproducibility of the synthetic procedures. Standard deviations of the retention factors were in the range from 0.06 to 0.5, depending on the analyte species. We show that selectivity is achieved with the stationary phase gradients that are significantly different from either uniformly modified amine or unmodified columns. These results indicate the significant promise of this strategy for creating novel stationary phases for LC.

First detection and quantification of Nδ-monomethylarginine, a structural isomer of NG-monomethylarginine, in humans using MS3

The l-arginine metabolites methylated at the guanidino moiety, such as NG-monomethyl-l-arginine (LNMMA), asymmetric NG,NG-dimethyl-l-arginine (ADMA), and symmetric NG,NG'-dimethyl-l-arginine (SDMA), are long known to be present in human plasma. Far less is known about the structural isomer of LNMMA, Nδ-monomethyl-l-arginine (δ-MMA). In prior work, it has been detected in yeast proteins, but it has not been investigated in mammalian plasma or cells. In this work, we present a method for the simultaneous and unambiguous quantification of LNMMA and δ-MMA in human plasma that is capable of detecting δ-MMA separately from LNMMA. The method comprises a simple protein precipitation sample preparation, hydrophilic interaction liquid chromatography (HILIC) gradient elution on an unmodified silica column, and triple stage mass spectrometric detection. Stable isotope-labeled D6-SDMA was used as internal standard. The calibration ranges were 25–1000 nmol/L for LNMMA and 5–350 nmol/L for δ-MMA. The intra- and inter-batch precision determinations resulted in relative standard deviations of less than 12% for both compounds with accuracies of less than 6% deviation from the expected values. In a pilot study enrolling 10 healthy volunteers, mean concentrations of 48.0 ± 7.4 nmol/L for LNMMA and 27.4 ± 7.7 nmol/L for δ-MMA were found.

Rapid quantification of imidazolium-based ionic liquids by hydrophilic interaction liquid chromatography: Methodology and an investigation of the retention mechanisms

The separation of nine N,N′-dialkylimidazolium-based ionic liquids (ILs) by an isocratic hydrophilic interaction high-performance liquid chromatographic method using an unmodified silica column was investigated. The chosen analytical conditions using a 90:10 acetonitrile–ammonium formate buffer mobile phase on a high-purity, unmodified silica column were found to be efficient, robust, and sensitive for the determination of ILs in a variety of solutions. The retention window (k′=2–11) was narrower than that of previous methods, resulting in a 7-min runtime for the nine IL homologues. The lower limit of quantification of the method, 2–3μmolL−1, was significantly lower than those reported previously for HPLC-UV methods. The effects of systematically modifying the IL cation alkyl chain length, column temperature, and mobile-phase water and buffer content on solute retention were examined. Cation exchange was identified as the dominant retention mechanism for most of the solutes, with a distinct (single methylene group) transition to a dominant partitioning mode at the highest solute polarity.

HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY: A WORTHY TECHNIQUE FOR THE DETERMINATION OF LAMOTRIGINE IN TABLETS AND HUMAN PLASMA

A rapid, stability indicating hydrophilic interaction liquid chromatography (HILIC) method was developed for the determination of lamotrigine and its induced alkaline degradation product. The separation was performed on an unmodified silica column adopting UV detection at 290 nm with flow rate of 1.5 mL min−1. The mobile phase consisted of a mixture of acetonitrile:ammonium formate 0.01 M (85: 15, v/v) and the pH was adjusted at 5.0. The method showed good linearity in the range of 0.5-10 µg mL−1 with lower limit of detection (LOD) 0.1 µg mL−1 and limit of quantification (LOQ) 0.5 µg mL−1, respectively. The proposed method was applied to tablets assay and was further extended to the in vitro and in vivo determination of the drug in spiked and real human plasma. The mean percentage recoveries were 100.32 ± 2.59 (n = 3) and 95.63 ± 4.42 (n = 3), respectively. Kinetics for the stability study was performed.

Stable-Labeled Analogues and Reliable Quantification of Nonprotein Biomarkers by LC–MS/MS

The aim was to develop, and establish as suitable to begin assessment by full validation, a quantitative LC-MS/MS method for asparagine in human plasma. Therein, to utilize a stable-labeled analogue of asparagine to act as surrogate analyte, producing complete calibration curves and corresponding QC samples and another m/z distinct stable-labeled analogue to act as internal standard. From two candidates, the surrogate analyte was selected through statistical comparisons of concentration-response data and the resultant method employed protein precipitation and LC on an unmodified silica column with multiple reaction monitoring detection mode. The calibration range was 50-10,000 ng/ml. This method was successfully proven to meet the accuracy and precision acceptance criteria of current bioanalytical method validation guidelines.

A Stability-Indicating Assay of Brimonidine Tartrate Ophthalmic Solution and Stress Testing Using HILIC

A stability-indicating hydrophilic interaction liquid chromatography (HILIC) method has been developed and validated for the quantitative determination of Brimonidine tartrate (BT) formulated as an ophthalmic solution. Isocratic separation was achieved using an acetonitrile-buffer mixture (92:8, v/v) at pH 7.1 on an unmodified silica column (250 × 4.6 mm, 5 μm). The drug was subjected to oxidative, hydrolytic, photolytic and thermal stress conditions and complete separation was achieved for the parent compound and degradation products. The influence of acetonitrile, pH and ionic strength of the buffer was studied. Linearity range and recoveries for BT were 100–400 μg mL−1 and 100.12%, respectively. The method was validated for BT and indicated that the method was sufficiently sensitive with a limit of detection at 0.005 μg mL−1 and a limit of quantitation at 0.02 μg mL−1, respectively.

High performance liquid chromatography in pharmaceutical analyses

High performance liquid chromatography in pharmaceutical analyses

Investigation of chromatographic behaviour of ethoxylated alcohol surfactants in normal-phase and reversed-phase systems using high-performance liquid chromatography–mass spectrometry

Retention behaviour of ethoxylated alcohols was investigated on octadecyl silica, unmodified silica and aminosilica columns. Good separation according to the alcoholic alkyl length is achieved in reversed-phase systems. The elution order of the oligomers with different numbers of oxyethylene groups depends on the type of the organic solvent (methanol or acetonitrile) and on its concentration in the mobile phase. Different retention behaviour of lower and higher oligomers was observed. The distribution of the oligomers according to the number of oxyethylene units is suppressed, but it overlaps with the peak distribution according to the alkyl length in mobile phases containing high concentrations of acetonitrile. In normal-phase systems, the alkyl length affects the retention much less than the number of oxyethylene units. Better separation than on unmodified silica gel columns can be achieved on a chemically bonded aminosilica column in 2-propanol– n-hexane and especially in acetonitrile–water–dichloromethane mobile phases. Possible retention mechanisms in the systems studied are discussed. The retention is strongly affected by solvation of the oxyethylene groups by the mobile phase. The retention factors of higher oligomers with bimodal mass distribution can be described using a simple equation. HPLC–MS with atmospheric pressure chemical ionization allows sensitive detection, easy identification and reconstruction of the chromatographic peaks in chromatograms of complex mixtures of oligomers with different numbers of methylene and oxyethylene groups.

Iron(III) ion-modified chromatographic silica preparations and characterization

Stable, reproducible, iron(III) ion-modified silica stationary phases were prepared in aqueous media by a batch process. The catalytic activity of this modified stationary phase for oxidation of hydroquinone to benzoquinone was considerably greater than that of earlier iron(III) ion-modified silicas from non-aqueous media. Capacity factors of selected solutes were used to compare the retention properties of the aqueous to increase with the iron(III) ion-modified silica relative to those for the unmodified silica column, whereas the capacity factors for carbonyl-containing compounds (esters, ketones and aldehydes) became smaller or were unaffected.

Liquid—liquid extraction and high-performance liquid chromatography for the determination of a novel antidysrhythmic agent (UK-68,798) in human urine

A routine method for the determination of a novel class III antidysrhythmic agent, 1-(4-methane-sulphonamidophenoxy)-2-[N-(4-methanesulponamidophenethyl)-N-methylamino]ethane, in human urine has been developed. The method involves solvent extraction followed by high-performance liquid chromatography on an unmodified silica column with ultraviolet detection. Despite a low recovery of drug through the three-stage extraction procedure a reliable assay with high precision (coefficient of variation less than 6%) and a limit of determination of 2.5 ng/ml was achieved. The method has been applied to the analysis of samples following single oral and intravenous doses of 1–12.5 μg/kg of the drug to human volunteers.

Analysis of basic drugs by high-performance liquid chromatography using on-line solid-phase extraction and an unmodified silica column

A method is described for the high-performance liquid chromatographic analysis of a number of basic drugs on unmodified silica gel, which employs an aqueous methanol-rich eluent of pH 8. The analytical system was coupled to a column-switching assembly in order to facilitate on-line solid-phase extraction of the drugs from plasma. The method is reproducible, with a coefficient of variation of 3% or less for most compounds, even without internal standardisation. Drug recovery from plasma is efficient, being in excess of 90% in most instances. The drugs were detected by UV absorption at 254 nm.

Measurement of Cardioactive Drugs in Biological Samples by HPLC

Abstract Unmodified silica columns used with non-aqueous, usually methanolic, eluents containing an ionic modifier provide simple, flexible systems for the HPLC analysis of a wide range of basic cardioactive drugs and some quaternary ammonium compounds. Retention and peak shape are influenced primarily by eluent pH and ionic strength although changes in eluent composition and in the stationary phase may also prove useful. Recently it has been found that chemically-bonded strong cation exchange materials provide enhanced retention for analytes which are poorly retained on silica alone. Solvent extracts and elution fractions from bonded-phase extraction tubes may be analysed directly and, in addition to low-wavelength UV and fluorescence, electrochemical oxidation detection may be used routinely for a wide range of analytes including secondary and tertiary aliphatic amines.

Separation of aldicarb, aldicarb sulfoxide, and aldicarb sulfone on unmodified silica with reverse-phase eluents

Aldicarb, aldicarb sulfoxide, and aldicarb sulfone were chromatographed on an octyl-silica bonded-phase column and on an unmodified silica column using acetonitrile/water mobile phases. The elution order of the analytes from the silica column was different from that using the octyl-silica bonded phase and allowed confirmation of residues of aldicarb sulfoxide in citrus nectar. Isocratic elution of the unmodified silica column allowed rapid sample throughput.

Measurement of penbutolol and 4-hydroxypenbutolol in plasma or serum by HPLC.

A simple HPLC method for penbutolol and 4-hydroxypenbutolol assay has been developed. Plasma or serum (200 microliters) is vortex-mixed (30 s) with Tris solution (2 M, pH 10.6) containing an internal standard (50 microliters) and methyl t-butyl ether (200 microliters). After centrifugation, the extract (100 microliters) is analysed using an unmodified silica column (250 x 5 mm ID) and iso-octane-methanol-methyl t-butyl ether (55:25:20) containing ammonium perchlorate (10 mM, pH 5.7) as eluent and with fluorescence detection. No interference has been encountered and the limit of accurate measurement for both compounds is 5 micrograms/l.

High-performance liquid chromatographic assay for morphine with electrochemical detection using an unmodified silica column with a non-aqueous ionic eluent

A quick, sensitive and accurate high-performance liquid chromatographic method for morphine measurement in plasma has been developed using an unmodified silica column with a non-aqueous ionic eluent. Small plasma samples (0.4 ml) were extracted with a liquid—liquid extraction column and dihydromorphine was used as internal standard. The limit of quantitation was 2 ng/ml. The electrochemical detector was capable of detecting 150 pg of morphine. A calibration line with a correlation of 0.9994 ± 0.00026 was produced over the range 2–80 ng/ml.

ChemInform Abstract: ANALYSIS OF COBALT COMPLEXES BY HIGH‐PERFORMANCE LIQUID CHROMATOGRAPHY USING ION‐PAIR REVERSED‐PHASE, SILICA, OR ALUMINA COLUMNS

High-performance liquid chromatography on unmodified silica or alumina columns either separately or in series has been compared with the ion-pair reversed-phase method for the analysis of pentacyanocobaltate(III) and bis(ethylenediamine)thiosulpnatocobalt(III) complexes.

High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents : II. Application of uv, fluorescence and electrochemical oxidation detection

Unmodified silica columns together with non-aqueous ionic eluents give stable yet flexible systems for the analysis of basic drugs by high-performance liquid chromatography. Low-wavelength UV and fluorescence detection may be used, and fluorescence may be optimised by, for example, post-column pH change or derivatisation of some primary aliphatic amines with o-phthaldialdehyde. A novel feature is that electrochemical oxidation can be used for the detection of most analytes and this detection mode is thus discussed in detail. Retention and relative response data (UV, 254 nm and electrochemical, + 1.2 V) have been generated for 462 compounds using a 125-mm Spherisorb S5W silica column and methanolic ammonium perchlorate 10 mM, pH 6.7) as eluent. This system can be used isocratically in qualitative analyses and also for quantitative work, when either the wavelength or the applied potential can be adjusted to optimise the response.

Rapid isocratic high-performance liquid chromatography of porphyrin esters on aminopropyl-bonded silica

A simple isocratic procedure for the separation of porphyrin esters according to the number of carboxyl groups using an aminopropyl-bonded silica column and a simple binary eluent is suggested. In this way, problems with the choice of a suitable unmodified silica column for isocratic separation are circumvented; entirely different aminopropyl-bonded silicas of various origins exhibit almost identical behaviour. The influence of end-capping of residual silanols and the chemical substitution of the primary amino group on the separation was studied. To achieve an isocratic separation of porphyrin esters on the bare silica used, the concentration of surface silanol groups must be decreased; suitable concentrations are tailored by means of partial trimethylsilylation. The isocratic separation of porphyrin esters can also be achieved on cyanopropyl-bonded silica. The suggested optimum procedure, using primary amino-bonded silica, gives the highest resolution and allows the baseline separation of porphyrin esters with two to eight carboxyl groups together with their Cu 2+ complexes and synthetic tetraphenylporphyrin as the internal standard.

Analysis of cobalt complexes by high-performance liquid chromatography using ion-pair reversed-phase, silica, or alumina columns

High-performance liquid chromatography on unmodified silica or alumina columns either separately or in series has been compared with the ion-pair reversed-phase method for the analysis of pentacyanocobaltate(III) and bis(ethylenediamine)thiosulpnatocobalt(III) complexes.

Liquid chromatography of sugars on silica-based stationary phases

A review is given of sugar analysis by liquid chromatography using silica columns. Aspects covered are column materials and preparation, chemically and physically modified amine columns, octadecy- and unmodified silica columns; eluent composition and elution mechanisms for the different types of columns used; detection methods, RI and UV detectors, visible lights, fluorescence, moving-wire, polarimetric and mass detection; and sample preparation and origin of samples.