Normal-phase Silica Research Articles

Enhanced peak responses due to solvent interactions in high-performance liquid chromatography

The liquid chromatography of a variety of pure compounds gave larger or smaller peak areas and heights (responses) depending on the injection solvent. The high-performance liquid chromatographic system was unchanged, with the pump, flow-rate, column, column temperatures and detector type and settings invariant for each comparison. Only the solvent for the analyte varied. For reversed-phase columns, the responses of each compound increased with the polarity of the solvent, and only compounds capable of forming intramolecular hydrogen bonds exhibited this effect. The retention time was not affected. Using normal-phase silica columns with steroids and essentially non-aqueous mobile phase, an analogous dependence of response on solvent was also found.

High-performance liquid chromatography of water-soluble choline metabolites

We have developed a new method for the separation of [ 3H]choline metabolites by high-performance liquid chromatography. Using this method it is possible to separate, in one step, all of the known major water-soluble choline metabolites present in crude acid extracts of cells that have been incubated with [ 3H]choline, with baseline or near-baseline resolution. We use a gradient HPLC system with a normal-phase silica column as the stationary phase, and a linear gradient of increasing polarity and ionic strength as the mobile phase. The mobile phase is composed of two buffers: Buffer A, containing acetonitrile/water/ethyl alcohol/acetic acid/0.83 m sodium acetate (800/127/68/2/3), and buffer B (400/400/68/53/79), pH 3.6. A linear gradient from 0 to 100% buffer B, with a slope of 5%/min, is started 15 min after injection. At a flow rate of 2.7 ml/min and column temperature of 45°C, typical retention times for the following compounds are (in min): betaine, 10; acetylcholine, 18; choline, 22; glycerophosphocholine, 26; CDP-choline, 31; and phosphorylcholine, 40. This procedure has been applied in tracer studies of choline metabolism utilizing the neuronal NG 108-15 cell line and rat hippocampal slices as model systems. While the compounds labeled in the NG108-15 cells were primarily phosphorylcholine and glycerophosphocholine, reflecting high rates of phospholipid turnover, in the hippocampal slices choline and acetylcholine were the major labeled species. Identification of individual peaks was confirmed by comparing the elution profiles of untreated cell extracts with extracts that had been treated with hydrolyzing enzymes of differing specificities. This HPLC method may be useful in studies of acetylcholine and phosphatidylcholine metabolism, and of the possible interrelationships of these compounds in cholinergic cells.

A Routine Method for the Quantitative Determination of Polycyclic Aromatic Hydrocarbons (PAHs) in Urban Air

Abstract We have developed a method for the quantitative determination of polycyclic aromatic hydrocarbons (PAHs) present in urban air, which can be performed rather quickly, and which uses a minimal amount of solvents. Air samples were collected using a home-made low-volume air sampler equipped with glass fibre filter and polyurethane foam plugs. After Soxhlet extraction a liquid-liquid partition was carried out to isolate the PAH fraction. This liquid-liquid partition was performed in micro-scale, enabling us to use small quantities of the solvents and to separate the solution layers very rapidly using a centrifuge. Sample clean-up was accomplished on a high performance liquid chromatograph equipped with two normal phase silica columns. The losses of all investigated PAHs occurring during the various steps of sample clean-up have been determined. The qualitative and quantitative determination of the PAHs was carried out by capillary gas chromatography; the results were confirmed by GC/MS measurements. T...

Determination of pipemidic acid in plasma by normal-phase high-pressure liquid chromatography.

An improved high-pressure liquid chromatography procedure for determining the concentration of pipemidic acid in human plasma was developed, which uses a normal-phase silica gel column and aqueous mobile solvent system similar to that used in reverse-phase chromatography. Precolumn derivatization of pipemidic acid was achieved by a methylation reaction with boron trifluoride in methanol after extraction from plasma with chloroform containing 4% ethanol. The percent recovery of pipemidic acid was 88.3 +/- 7.7, the variation of which became negligible when quinacrine was used as an internal standard for the determination. High-pressure liquid chromatography analysis was performed by conventional silica gel and mobile solvent mixtures containing 3% of 0.14% HClO4 solution, 19% methanol, and 78% chloroform. The UV detector was set at 265 nm. The detection limit of pipemidic acid methyl ester was as low as 10 ng of the injection amounts or 0.5 micrograms of the plasma per ml, with 0.01 absorbance units (full scale) and a signal-to-noise ratio of 3.

Determination of the Antimalarial Primaquine in Whole Blood and Urine by Normal-Phase High-Performance Liquid Chromatography

Abstract A method has been developed to estimate primaquine in whole blood and urine by sensitive and selective high-performance liquid chromatography. Using the linear chain analogue of primaquine as the internal standard, a single-step extraction, normal-phase silica column with a basic mobile phase, levels down to 1 ng/ml of primaquine could be measured with good precision. Other anti-malarials like amodiaquine and pyrimethamine did not interfere in the assay. The major carboxylic acid metabolite of primaquine did not elute under the normal-phase chromatographic conditions. The method is suitable for use in clinical pharmacokinetic studies with primaquine.

Derivatization of secondary amines with 2-naphthalenesulfonyl chloride for high-performance liquid chromatographic analysis of spectinomycin

A normal-phase high-performance liquid chromatographic (HPLC) method has been developed for the assay of spectinomycin hydrochloride and spectinomycin sulfate for detection at 254 nm. The method involves pre-column derivatization of secondary amines of spectinomycin with 2-naphthalenesulfonyl chloride (NSCl) using a catalyst. Lincomycin, 1-methylpyrrole, 2-acetyul-l-methylpyrrole, and 2-acetyl-pyrrole act as catalysts for sulfonylation of spectinomycin. Without a catalyst, the derivatization reaction forms a considerable amount of actinospectinoic acid, a degradation compound of spectinomycin, and peak area:weight ratio of the derivative is approximately 15% lower than those with the catalyst. Following derivatization the sample is extracted and chromatographed on a normal-phase silica column with detection at 254 nm. The method is applicable for the analysis of both the hydrochloride and sulfate salt forms of spectinomycin. All the known degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and the biosynthesis intermediates, dihydrospectinomycin diastereoisomers, are completely separated with this method. Mass spectrometric data confirms that spectinomycin is derivatized with NSCl at the secondary amines located at positions 6 and 8 of the ring structure. The standard curves for the HPLC assay of spectinomycin hydrochloride and sulfate are linear with correlation coefficients of 0.9997 and 0.9999, respectively over the range of 0.05 ng/ml to 0.3 mg/ml. The relative standard deviations (R.S.D.) of the HPLC assay methods for spectinomycin hydrochloride and sulfate are 0.67% and 0.86%, respectively. Spectinomycin hydrochloride and sulfate bulk drugs were assayed by the HPLC method and compared to gas—liquid chromatography and microbiological assay results. The HPLC method was used to assay spectinomycin in a veterinary formulation, Linco-Spectin® soluble powder. The sensitivity of the HPLC assay was determined to be approximately 4 ng sample load on the column, which suggests applicability in serum and residue level studies.

Direct Enzyme-Linked Immunosorbent Assay for Determining Aflatoxin M1 at Picogram Levels in Dairy Products

Protocols for detecting picogram quantities of aflatoxin M1 in dairy products were established. Milk samples were subjected to a reverse phase Sep-Pak C18 cartridge treatment before analysis by an enzyme-linked immunosorbent assay (ELISA) according to previously published procedures. M1 in yogurt, brick cheddar, and ripened Brie cheese was extracted by a modified Pons method, subjected to a normal phase silica cartridge treatment, and analyzed by ELISA. The detection limits for M1 in milk, yogurt, cheddar, and Brie were 10, 10, 50, and 25 ppt (ng/kg), respectively. Recovery for M1 added to these products was in the range 70-110%. Good agreement was found for M1 levels in several naturally contaminated milk samples analyzed by both ELISA and liquid chromatography.

Quantification and confirmation of four fusarium mycotoxins in corn by gas chromatography-mass spectrometry-selected ion monitoring

A rapid method for the simultaneous determination of T-2 toxin, HT-2 toxin, diacetoxyscirpenol and zearalenone has been developed. Corn samples (10 g) are extracted with methanol, defatted with hexane and subsequently cleaned-up using both reversed-phase (C, 18) and normal-phase (silica gel) Sep-Pak cartridges. Confirmation of identity is made by gas chromatography-mass spectrometry-selected ion monitoring of three ions characteristic of the trimethylsilyl derivatives of the myocotoxins. Use of deuterated internal standards makes the method quantitavely reliable and increases sensitivity. Confirmation of identity as well as quantitation can be achieved at levels of ca. 20–50 ppb★★, depending on the mycotoxin. Detection limits (without confirmation of identity) are estimated at 1–20 ppb. Recoveries at the 46–111 ppb level ranged from 80 to 103% with coefficients of variation ranging from 1.6 to 14.2%.

Separations of some coumarins of higher plants by liquid chromatography

The behaviour of 67 coumarins occurring in higher plants was examined by high-performance liquid chromatography. Resolutions of neutral coumarins were achieved on a normal-phase silica column (10-μm particles) in hexane—ethyl acetate solvent systems and on a C 18 reversed-phase column (5 μm) with aqueous acetonitrile or aqueous methanol. Phenolic coumarins were resolved by stepwise elution with increasing concentrations of aqueous methanol containing acetic acid, sometimes with the addition of tetrahydrofuran, on the reversed-phase column. A mixture of ten coumarins simulating the neutral coumarin fraction Citrus aurantifolia was fully resolved by the use of the two columns in sequence.

Liquid chromatographic separation of antidepressant drugs: I. Tricyclics.

A simple normal-phase (silica), high-performance liquid chromatographic (HPLC) assay of amitriptyline (AMI), doxepin (DOX), imipramine (IMI), nortriptyline (NORT), desmethyldoxepin (DESDOX), desipramine (DESIP), and protriptyline (PRO) in serum with no coelution is described here. Trimipramine and promazine were used as internal standards. Extraction of the 1.0-ml serum samples (collected in plastic) was done with Bond-Elut C18 columns. The compounds of interest were eluted with 10 mM methanolic ammonium acetate. The eluates were evaporated at 56-58 degrees C and reconstituted with 200 microliters of the mobile phase. The mobile phase was absolute ethanol-acetonitrile-tert-butylamine (98:2:0.05, vol/vol/vol). Detection of eluted drugs was at 254 nm at 0.01 absorbance units full scale (AUFS), except for PRO, which was detected at 229 nm at 0.02 AUFS. Absolute recoveries were 87-97%. A 5-micron silica (4.6 X 250 mm) HPLC column was used; results with a 10-micron silica column (3.9 X 300 mm) are also presented. Peak height ratios with trimipramine were linear for each analyte between 25 and 1200 ng/ml. Peak height ratios with promazine as the internal standard were linear for each analyte between 25 and 600 ng/ml. Detection limits under the conditions described were 2 ng/ml for AMI, DOX, and IMI, 4 ng/ml for NORT, DESDOX, and DESIP, and 10 ng/ml for PRO. Coefficients of within-day and day-to-day variation at three concentration levels were less than 9.8% and less than 11.2%, respectively. The hydroxylated metabolites of IMI, DES, NORT, and the cis isomer of DOX are discussed. Steady-state daily dosages and corresponding serum levels are presented for 69 patients. The total assay time was less than 10 min for DESIP and 12 min for PRO. This assay can be used in correlating serum levels with clinical effects, compliancy, and pharmacokinetic studies.

High Performance Liquid Chromatography of Androgen Acetates

Abstract More than twenty C19O2 androgen acetates were chromatographed by both normal-phase (silica) and reversed-phase (C18) HPLC. These two HPLC systems together with normal-phase and reversed-phase HPLC of free androgens have made the separation of various C19O2 androgen isomers possible. 3,17-Diacetoxyandrostane derivatives, in general, are more polar in normalphase HPLC when the 3-acetoxyl group is equatorial than when it is axial. However, 3-acetoxyandrostan-17-one derivatives are more polar in normal-phase HPLC when the 3-acetoxyl group is axial than when it is equatorial. 17α-acetoxyandrostane derivatives in general are more polar than their 17α-analogs in both normal-phase and reversed-phase HPLC.

Mobility of oligopeptides on normal-phase silica: effect of positional isomerism.

AbstractIsomeric oligopeptides composed of five methionyl residues and one glycyl residue or of five γ‐methyl‐L‐glutamyl residues and one glycyl residue all exhibit marked differences in retention on normal‐phase silica. When the glycyl residue is at internal positions of hexa or heptapeptides, the peptide elutes most rapidly form the μPorasil column. Comparison of the effect of positional isomerism on retention in short oligopeptides with the effect on retention of hexamers and heptamers suggests that a change in peptide conformation may be responsible for the change in oligopeptide mobility.

The role of nitroaromatic compounds in the direct-acting mutagenicity of diesel particle extracts.

The Salmonella mutation assay has been adapted to a number of experimental procedures used to characterize the chemical components and molecular mechanisms involved in the direct-acting mutagenicity of diesel particle extracts. The mutagens were found to be unreactive towards purified DNA. Mutagenic activity was decreased in nitroreductase-deficient bacteria and increased in the absence of oxygen, suggesting that bacterial enzymes active the mutagens. The extract was fractionated by thin layer chromatography using both normal and reverse phase silica gel plates. On normal silica plates, a separation was made between unsubstituted PAH, nitro-PAH and the more polar aromatic compounds. About a third of the mutagenic activity in the particle extract was recovered in fractions containing monosubstituted nitro-PAH compounds. The absorption spectra and reverse phase chromatographic properties indicate 1-nitropyrene is a predominant component but not the only mutagen in these fractions. The remaining activity was in the more polar fractions, and mutagenicity assays using anaerobic conditions and nitro-reductase-deficient bacteria suggest they contain other nitro-substituted compounds.

Liquid-chromatographic measurement of endogenous and exogenous glucocorticoids in plasma.

The therapeutic response to and side effects of glucocorticoids will be better recognized and the recovery of the adrenals during the tapering of therapy with steroids better evaluated if endogenous and exogenous glucocorticoids are separately assessed. We describe a specific method for simultaneously measuring the concentrations of cortisone, cortisol, prednisone, and prednisolone in plasma by "high-pressure" liquid chromatography. The steroids, together with an internal standard, dexamethasone, are extracted from 1 mL of plasma with methylene chloride-ether, washed with acid and base, and separated isocratically on a normal-phase silica column with a mobile phase consisting of methylene chloride/tetrahydrofuran/methanol/glacial acetic acid (96.85/1/2.1/0.05 by vol) at a flow rate of 1.3 mL/min. The steroids are detected at 254 nm and quantitated by peak-height measurements; their retention times range from 6 to 20 min. The lower limits for routine detection of all four compounds is 10 microgram/L. The analytical recoveries are about 75%; the intra-day variability (CV) is 1 to 9%, and the inter-day variability 2 to 11%. Of 26 drugs and 20 steroids tested, only theophylline presents an interference problem.