C18 Silica Column Research Articles

Determination of Artemisinin in Artemisia annua L. by Reversed Phase HPLC

Artemisinin is a compound of current interest in the treatment of drug‐resistant malaria. An accurate, sensitive, and reproducible reversed‐phase HPLC with UV detection method is described for the determination of artemisinin in the herb of Artemisa annua L. Artemisinin in the extracts was converted into a UV‐absorbing compound, Q260 by being treated with 0.2% (by weight) NaOH solution and then 0.08 M acetic acid. A reversed‐phase C18 silica column (Discovery 250 × 4.6 mm, 5 µm) was selected as a fixed phase, and kept at 30°C. The mobile phase was 45/10/45 (by volume) methanol/acetonitrile/0.9' mM Na2HPO4‐3.6 mM NaH2PO4 buffer (pH 7.76). The flow rate was 0.5 mL/min. The detection wavelength was 260 nm. The standard calibration curve was linear with correlation coefficient 0.9996. The artemisinin content in the herb of Artemisa annua L. was 0.652%. The relative standard deviation and recovery were 1.1% and 98.9%, respectively.

Isolation and quantification of dinucleoside polyphosphates by using monolithic reversed phase chromatography columns

In former studies, dinucleoside polyphosphates were quantified using ion-pair reversed-phase perfusion chromatography columns, which allows a detection limit in the μmolar range. The aim of this study was both to describe a chromatographic assay with an increased efficiency of the dinucleoside separation, which enables the reduction of analytical run times, and to establish a chromatographic assay using conditions, which allow MALDI-mass spectrometric analysis of the resulting fractions. We compared the performance of conventional silica reversed phase chromatography columns, a perfusion chromatography column and a monolithic reversed-phase C18 chromatography column. The effects of different ion-pair reagents, flow-rates and gradients on the separation of synthetic diadenosine polyphosphates as well as of diadenosine polyphosphates isolated from human platelets were analysed. Sensitivity and resolution of the monolithic reversed-phase chromatography column were both higher than that of the perfusion chromatography and the conventional reversed phase chromatography columns. Using a monolithic reversed-phase C18 chromatography column, diadenosine polyphosphates were separable baseline not only in the presence of tetrabutylammonium hydrogensulfate (TBA) but also in the presence of triethylammonium acetate (TEAA) as ion-pair reagent. The later reagent is useful because, in contrast to TBA, it is compatible with MALDI mass-spectrometric methods. This makes TEAA particularly suitable for identification of unknown nucleoside polyphosphates. Furthermore, because of the lower backpressure of monolithic reversed-phase chromatography columns, we were able to significantly increase the flow rate, decreasing the amount of time for the analysis close to 50%, especially using TBA as ion-pair reagent. In summary, monolithic reversed phase C18 columns markedly increase the sensitivity and resolution of dinucleoside polyphosphate analysis in a time-efficient manner compared to reversed-phase perfusion chromatography columns or conventional reversed-phase columns. Therefore, further dinucleoside polyphosphate analytic assays should be based on monolithic silica C18 columns instead of perfusion chromatography or conventional silica reversed phase chromatography columns. In conclusion, the use of monolithic silica C18 columns will lead to isolation and quantification of up to now unknown dinucleoside polyphosphates. These chromatography columns may facilitate further research on the biological roles of dinucleoside polyphosphates.

Fast HPLC for quality control of Harpagophytum procumbens by using a monolithic silica column: method transfer from conventional particle-based silica column

The applicability of a monolithic C18-bonded silica column for the rapid HPLC separation of ingredients in medicinal plants and their phytopharmaceutical preparations has been evaluated in the author's laboratory. In this presentation, an existing method for the determination of the iridoid glycoside harpagoside in Harpagophytum procumbens (Devil's Claw) was successfully transferred from a conventional particle-based C18 silica column to a monolithic silica column. The very high porosity of the stationary phase allows chromatography with a much lower backpressure than on conventional columns. Therefore, the flow rate could be easily increased from 0.8 mL/min (particle-based column) to 5 mL/min (monolithic column) and the run-time reduced from 30 to 5 min (that is a reduction about 85%!), without losing any chromatographic resolution of the compound of interest. The amount of harpagoside was measured with the original method on a conventional particle-based silica column and on the adapted method on a monolithic silica column. The statistical mean t-test showed no significant differences of the variances and the means indicating that the fast HPLC method is an acceptable alternative. The shorter analysis time makes the method very valuable for commercial quality control of Harpagophytum extracts and its pharmaceutical preparations.

Genotoxicity and cytotoxicity assessment in lake drinking water produced in a treatment plant.

Chemical analyses and short-term mutagenicity bioassays have revealed the presence of genotoxic disinfection by-products in drinking water. In this study, the influence of the different steps of surface water treatment on drinking water mutagen content was evaluated. Four different samples were collected at a full-scale treatment plant: raw lake water (A), water after pre-disinfection with chlorine dioxide and coagulation (B), water after pre-disinfection, coagulation and granular activated carbon filtration (C) and tap water after post-disinfection with chlorine dioxide just before its distribution (D). Water samples, concentrated by solid phase adsorption on silica C18 columns, were tested in human leukocytes and HepG2 hepatoma cells using the comet assay and in HepG2 cells in the micronuclei test. A significant increase in DNA migration was observed in both cell types after 1 h treatment with filtered and tap water, and, to a lesser extent, chlorine dioxide pre-disinfected water. Similar findings were observed for the induction of "ghost" cells. Overloading of the carbon filter, with a consequent peak release, might explain the high genotoxicity found in water samples C and D. Cell toxicity and DNA damage increases were also detected in metabolically competent HepG2 cells treated with a lower concentration of tap water extract for a longer exposure time (24 h). None of the water extracts significantly increased micronuclei frequencies. Our monitoring approach appears to be able to detect contamination related to the different treatment stages before drinking water consumption and the results suggest the importance of improving the technologies for drinking water treatment to prevent human exposure to potential genotoxic compounds.

Chromatographic Properties of Monolithic Silica Capillary Columns for Polar and Nonpolar Compounds in Reversed-Phase HPLC

Retention properties of monolithic silica C18 columns were examined for polar and nonpolar compounds. Monolithic silica columns were prepared from two kinds of starting materials, tetramethoxysilane (abbreviated as TMOS) or a mixture of TMOS and methyltrimethoxysilane (abbreviated as Hybrid), and modified with dimethyloctadecylchlorosilane (C18). Retention factors of various compounds on Hybrid-C18 are about twice as much as that on TMOS-C18. The two types of columns showed very similar retention selectivity with few exceptions. TMOS-C18 showed slight preference toward planar polynuclear aromatic hydrocarbons (PAHs) compared to bulky aromatic hydrocarbons, while Hybrid-C18 showed opposite tendency. Some TMOS-C18 columns showed peak tailing for PAHs, and also for basic compounds in an acidic mobile phase, while Hybrid-C18 and majority of TMOS-C18 columns resulted in good peak shape for these compounds.

Simple 2D-HPLC using a monolithic silica column for peptide separation.

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.

Determination of the purity of ampicillin by micellar electrokinetic chromatography and reversed phase liquid chromatography on a monolithic silica column.

A micellar electrokinetic chromatographic (MEKC) method and a fast reversed-phase liquid chromatographic one have been developed for determining the purity of ampicillin. MEKC separation of ampicillin and its related substances was performed with the use of an untreated fused-silica capillary and 40 mM phosphate-borate buffer, pH 7.5 containing 75 mM SDS. The HPLC method employed a monolithic silica C18 column and a mobile phase composed of phosphate buffer, pH 5.2 and ACN, the flow rate being 4.0 mL/min. Both methods were successfully validated. Linearity, relative response factors, limits of quantitation, intermediate precision, and accuracy were evaluated. The methods proved to be fast, reliable, and sufficiently sensitive and, accordingly, well-suited for control of purity of ampicillin substance, injections, and capsules. A combination of both methods can be very useful in the confirmation of impurity profiles.

Separation of anions by ion-interaction chromatography with a novel cationic/zwitterionic eluent

Inorganic and organic anions can be separated on an ordinary silica C18 column using a mobile phase containing tetrabutylammonium hydroxide (TBAH) and an aminosulfonic acid zwitterion reagent (MOPS). The pH of this eluent is close to 7 and the background conductivity is about 50 μS, which is low enough to permit anion analyte detection by direct conductivity. Linear calibration curves were obtained for the six anions studied and detection limits ranged from 0.075 to 0.15 mg/l (ppm) for the five inorganic anions. The method was applied to the determination of water-soluble anions in aerosol samples at concentrations as low as 0.3 mg/l.

Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum.

In a recent survey of paralytic shellfish poisoning (PSP) toxins in Gymnodinium catenatum Graham extracts, using LC with postcolumn oxidation and fluorescence detection, three novel saxitoxin analogues were revealed in isolates from several locations, including Australian waters. We have named them as G. catenatum toxins, GC1 (1), GC2 (2), and GC3 (3). The compounds were isolated from a culture of the Australian strain by LC-MS-guided fractionation employing a C18-silica column and hydrophilic interaction chromatography. The unusual structures of these novel compounds were characterized by low- and high-resolution MS, MS/MS, and NMR spectroscopy. GC3 (3) was found to be the 4-hydroxybenzoate ester derivative of decarbamoylsaxitoxin, while GC1 (1) and GC2 (2) are the epimeric 11-hydroxysulfate derivatives of GC3 (3).

Preconcentration and adsorption of metal chelates with analysis by direct sample insertion inductively coupled plasma atomic emission spectrometry

An automated preconcentration system that utilizes adsorption of 8-hydroxyquinoline (8-HQ) metal chelates onto a silica-C18 column has been evaluated. The metal chelates are desorbed with methanol and sprayed into an inductively heated graphite direct sample insertion cup. In this manner the organic solvent and much of the 8-HQ is removed and does not appear to cause interferences in the plasma. The detection limits of the current system are 8, 30, 60, 10, 9 and 40 parts per trillion for Cd, Cu, Fe, Mn, Pb and Zn, respectively, utilizing the reagents as purchased. Instrumental detection limits of 1, 0.2, 90, 0.3, 2 and 10 ppt for the same elements were calculated assuming that blank contamination can be eliminated.

Capillary electrochromatography with monolithic silica column: I. Preparation of silica monoliths having surface-bound octadecyl moieties and their chromatographic characterization and applications to the separation of neutral and charged species.

Monolithic silica columns with surface-bound octadecyl (C18) moieties have been prepared by a sol-gel process in 100 microm ID fused-silica capillaries for reversed-phase capillary electrochromatography of neutral and charged species. The reaction conditions for the preparation of the C18-silica monoliths were optimized for maximum surface coverage with octadecyl moieties in order to maximize retention and selectivity toward neutral and charged solutes with a sufficiently strong electroosmotic flow (> 2 mm/s) to yield rapid analysis time. Furthermore, the effect of the pore-tailoring process on the silica monoliths was performed over a wide range of treatment time with 0.010 M ammonium hydroxide solution in order to determine the optimum time and conditions that yield mesopores of narrow pore size distribution that result in high separation efficiency. Under optimum column fabrication conditions and optimum mobile phase composition and flow velocity, the average separation efficiency reached 160 000 plates/m, a value comparable to that obtained on columns packed with 3 microm C18-silica particles with the advantages of high permeability and virtually no bubble formation. The optimized monolithic C18-silica columns were evaluated for their retention properties toward neutral and charged analytes over a wide range of mobile phase compositions. A series of dimensionless retention parameters were evaluated and correlated to solute polarity and electromigration property. A dimensionless mobility modulus was introduced to describe charged solute migration and interaction behavior with the monolithic C18-silica in a counterflow regime during capillary electrochromatography (CEC )separations. The mobility moduli correlated well with the solute hydrophobic character and its charge-to-mass ratio.

Dithizone derivatives as sensitive water soluble chromogenic reagents for the ion chromatographic determination of inorganic and organo-mercury in aqueous matrices

Water-soluble sulfonate and the novel carboxylate analogues of dithizone, combined with ion interaction chromatography on a Dionex Acclaim 120 C18 silica column (250 x 4.6 mm id) with an eluent consisting of 10 mM tetrabutylammonium bromide and 60:40 methanol:water, have been developed as highly sensitive chromogenic ligands for the quantitative isocratic determination of inorganic and organo-mercury compounds in aqueous matrices in under 12 min. Using an optimised post column reagent system containing 0.65 mM dye, 0.5% Triton X-100 and 50 mM sodium hydroxide, good linearity (0-7.5 mg L(-1) R2 > 0.999), reproducibility using peak area measurements (RSD 0.69-1.38%, n = 8), and limits of detection (4-12 microg L(-1)) were achieved for methyl mercury, inorganic mercury and phenyl mercury.

Liquid Chromatography of Synthetic Polymers under Critical Conditions. The Case of Single Eluents and the Role of ϑ Conditions

As a rule, critical conditions in liquid chromatography of synthetic macromolecules are adjusted by both mixed mobile phase composition and temperature. The application of single component eluents is attempted in the present study. The enthalpic interactions in the column are controlled exclusively by temperature. Poly(methyl methacrylate)s (PMMA), poly(n-butyl methacrylate)s (PnBMA), and polystyrenes (PS) in various solvents are investigated with bare silica gel and silica C18 bonded phases. Using a series of single eluents based on various esters, PMMA probes, and bare silica gel, it is shown that minute variations in eluent nature strongly affect polymer retention, and temperature represents a too weak parameter to adjust critical conditions in thermodynamically good solvents. The idea is proposed and tested that the sensitivity of polymer retention toward temperature variations increases in the vicinity of ϑ conditions, where the structure of macromolecules in solution strongly depends on temperature. The critical conditions have been identified in some single eluents which are thermodynamicaly poor solvents for polymers investigated, namely, acetonitrile for PMMA at 66 °C and dimethylformamide for PnBMA at 154 °C and for PS at 95 °Call with C18 bonded silica gel. A “critical-like” elution was also observed in cyclohexane for PS at 9 °C with silica gel C18 packing, which is well below the ϑ temperature. It is demonstrated that poly(methyl methacrylate)s can be separated according to stereoregularity in the area of critical conditions using acetonitrile eluent and silica C18 column packing. The excessive polymer peak broadening and decreased recovery which were observed in some mixed mobile phases are diminished in the single component critical eluents studied, but they are not fully eliminated.

Simultaneous determination of all-trans, 9-cis, 13-cis retinoic acid and retinol in rat prostate using liquid chromatography-mass spectrometry.

Since retinoic acid (RA) and RA receptors are key developmental regulators during organogenesis, they might participate in the abnormal development of the prostate caused by early estrogen exposure. In order to test this assumption, a sensitive analytical method that can differentiate 9-cis, 13-cis, and all-trans RA in small tissue samples ( approximately 8 mg) is required. Since retinol is the metabolic precursor to RA, simultaneous quantification of retinol would also provide valuable information. Here, we report a liquid chromatography-mass spectrometry method for simultaneous determination of retinol and 9-cis, 13-cis, and all-trans RA in rat prostate. Mass spectrometric signal responses for RA were compared using positive ion atmospheric-pressure chemical ionization (APCI) and electrospray, as well as positive ion and negative ion APCI. Positive ion APCI was selected for all subsequent analysis for its better sensitivity, and to provide simultaneous determination of retinol and RA. Ventral prostate tissue samples were homogenized and extracted following simple protein precipitation without derivatization. Baseline separation of 9-cis, 13-cis, and all-trans RA standards was obtained by using a non-porous silica C18 column. Selected ion monitoring of the ions m/z 301 and m/z 269 was carried out for mass spectrometric quantitative analysis. The ion of m/z 301 corresponded to the protonated molecule of RA, whereas the ion of m/z 269 corresponded to loss of water or acetic acid from the protonated molecule of retinol or the internal standard retinyl acetate respectively. The method has a linear response over a concentration range of at least three orders of magnitude. The limit of quantitation was determined to be 702 fmol all-trans RA injected on-column. The method showed excellent intra- and inter-assay reproducibility and good recovery, and is suitable for analyzing RA and retinol in small tissue samples (approximately 8 mg).

Analysis of the Surface Diffusion of Alkylbenzenes and p-Alkylphenols in Reversed-Phase Liquid Chromatography Using the Surface-Restricted Molecular Diffusion Model

Surface diffusion of alkylbenzene and p-alkylphenol derivatives was measured in reversed-phase liquid chromatography on a C18-silica column with a methanol/water mixture (70/30, v/v) as the mobile phase. They were analyzed on the basis of the restricted molecular diffusion model for surface diffusion. The temperature dependence of the surface diffusion coefficient (Ds) arises probably from that of molecular diffusivity, suggesting a correlation between surface and molecular diffusion. Other correlations were also observed: (1) an enthalpy-entropy compensation of the retention equilibrium constant, (2) a linear free energy relationship between the retention and surface diffusion, and (3) a linear correlation between the restriction energy of surface diffusion and the isosteric heat of adsorption. The physical meaning of parameters involved in the surface-restricted molecular diffusion model is discussed on the basis of these results. A practical and convenient procedure for the estimation of Ds is also suggested. This study demonstrates how surface diffusion phenomena can be accounted for on the basis of the correlation between surface and molecular diffusion.

Rice alpha-mannosidase digesting the high mannose glycopeptide of glutelin.

alpha-Mannosidase (EC 3.2.1.24) from rice dry seeds was purified to homogeneity. Optimum pH and Km for pNP-alpha-Man hydrolysis were pH 4.3-4.5 and 1.04 mM, respectively. The enzyme digested mannobioses such as Manalpha-1,2Man, Manalpha-1,6Man, Manalpha-1,3Man but Manalpha-1,4Man. Zn2+ ion was required for the activity, whereas EDTA and swainsonine inhibited the activity by 80 and 96%, respectively. The rice storage protein, glutelin was prepared and its basic subunits were shown to have high mannose-type sugar chains by two-dimensional mapping using NH2-P and C18 silica columns. They were Man9GlcNAc2, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2 and Man5GlcNAc2. All these oligosaccharides were digested by the purified alpha-mannosidase, and Man-GlcNAc2 and mannose were formed. Glycopeptides, having these high mannose-type sugar chains, could also be digested by the alpha-mannosidase. Subunits were prepared from glutelin basic subunit and the richest subunit among them, subunit 2 (isoform 2), was digested by the alpha-mannosidase. Isoform 2 was digested by V8 protease only partially and slowly. However, isoform 2, pre-treated with the alpha-mannosidase, was rapidly and completely digested by V8 protease.

Uniform-sized molecularly imprinted polymers for 2-arylpropionic acid derivatives selectively modified with hydrophilic external layer and their applications to direct serum injection analysis.

Uniform-sized molecularly imprinted polymers (MIPs) for (S)-naproxen and -ibuprofen selectively modified with hydrophilic external layer, restricted access media (RAM)-MIPs, have been prepared. First, the MIP for (S)-naproxen or -ibuprofen was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multistep swelling and thermal polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and it was added directly to the MIP for (S)-naproxen or -ibuprofen 4 h after the start of molecular imprinting. The obtained RAM-MIP material for (S)-naproxen or -ibuprofen was applied for direct serum injection assays of the drug by a column-switching system, consisting of a RAM-MIP material and conventional C18-silica column. However, leakage of the imprint molecule prevented accurate and precise assays of the drug. This problem has been overcome by using the RAM-MIP for (S)-naproxen for the assays of ibuprofen in rat plasma. The optimized column-switching system was applied successfully to the assay of ibuprofen in rat plasma after oral administration.

Basic Subunit of Glutelin, Rice Major Storage Protein, has N-Linked Sugar Side Chain.

The basic subunit of glutelin from rice dry seeds were shown to have N-linked sugar chains by lectin staining with PHA-E4, WGA and Con A. NaIO4 and N-glycanase treatments also provided the evidences of N-linked sugar chains. The purified basic subunit was applied to NEPHGE and eight isoforms of it were separated. The fractionated eight isoforms were shown to be nearly homogeneous by C18-silica column chromatography. Each was subject to lectin staining. The eight isoforms were hybridized with PNA, PHA-E4 and Con A. This lectin staining indicated that each of the isoforms of the basic subunit had N-linked sugar chain. This is the first report showing the occurrence of N-linked sugar chain in isoforms of glutelin basic subunit.

Simultaneous Determination of Pyridoxine Hydrochloride and Meclizine Hydrochloride in Tablet Formulations by HPLC

A method for the simultaneous determination of pyridoxine hydrochloride and meclizine hydrochloride in Vominore tablets using HPLC is described. The mobile phase consisted of dodecyl sulphate sodium salt dissolved in a mixture of water, acetonitrile, methanol, glacial acetic acid and tetrahydrofuran. A Symmetry C18 silica column (250 times 4.6 mm) gave the best resolution with minimal tailing factors. The method was suitable for the analysis of the two components in tablet dosage forms and as a stability indicating method. Matrix components did not interfere with the HPLC chromatograms of the two active ingredients. The method was accurate, repeatable and reproducible. The percentage recovery, bias and relative standard deviations for the HPLC measurements were within US Pharmacopeial limits.

Influence of mobile phase conditions on the clean-up effect of restricted-access media precolumns for plasma samples injected in a column-switching system

Various mobile phases including phosphate buffer, pure water and five kinds of biological buffers (pH around 7) were systematically studied in terms of their ability to clean-up plasma matrix on precolumns of restrictedaccess media in a column-switching system. The necessary washing time, buffer pH, type and content of organic modifier were evaluated with respect to plasma elution profiles on restricted-access media precolumns. The influence of different mobile phases on the recovery of plasma matrix from alkyl-diol silica precolumns was studied by means of a scanning spectrophotometer. Our results show that phosphate buffer near physiological pH with small amounts of 2-propanol or acetonitrile was prefereble for direct injection of large plasma volumes (500 μL). More than 93% of the proteins in a plasma matrix can be recovered within 3 min from the alkyl-diol silica C18 column (25 mx 4 mm I.D.) as measured at 280 nm for all selected mobile phases except for tris (hydroxymethyl) aminomethane buffer, from which only about 88% was obtained.