Reversed-phase Liquid Chromatographic System Research Articles

Role of stationary phase and eluent composition on the determination of log P values of N-hydroxyethylamide of aryloxyalkylen and pyridine carboxylic acids by reversed-phase high-performance liquid chromatography

The partition coefficients, P, between n-octanol and water of a number of growth stimulating substances, N-hydroxyethylamide of aryloxyalkylen- and pyridine carboxylic acids were obtained from Pomona College ( C log P), and Rekker's (log P Rekker) revised fragmental constant system was used to calculate log P data sets. Both of these data sets were correlated with two different substance lipophilicity parameters, log k w and ϕ 0. Log k w was obtained by extrapolation of log retention factor ( k) to 0% organic modifier measured in reversed-phase liquid chromatography (RPLC) systems. ϕ 0 values were obtained from the slopes and intercepts of these relationships. The RPLC experiments were performed on four commercially available reversed-phase columns. Binary mixtures of methanol–water, methanol–phosphate buffer (pH 7.0), methanol–tricine buffer (pH 7.0) and acetonitrile–water were used as mobile phases for the determination of log k w values. For the methanolic eluents linear regression provided satisfactory correlations ( r>0.99) for the relationships log k vs. organic modifier content in the eluent, while for the acetonitrile-containing eluents a second-degree polynominal regression was necessary. For all four RPLC columns, by linear regression satisfactory correlations ( r>0.99) were obtained between log k w and log P data using methanolic eluents. In such eluents ϕ 0 values were shown to be the second-best lipophilicity parameters. For acetonitrile-containing eluents the use of second-degree polynominal regression was necessary and, in contrast to methanol, significant influence of the applied column on regression results was observed. For acetonitrile-containing eluents the ϕ 0-index does not provide satisfactory results for our substances. No difference in regression results between the use of buffered and non-buffered eluents was observed.

High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C 18 column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μ M, 4% at 227 n M and 9% at the lower limit of quantitation, 4.5 n M. In urine, the coefficient of variation was 11% at the lower concentration, 227 n M and was 3% at 56.8 μ M. The between-assay coefficient of variation was 4% at the low concentration (5.1 n M) in plasma, 2% at 276.8 n M and 3% at 1.97 μ M. In urine, the between assay coefficient of variation was 4% at 204.6 n M, 3% at 5.12 μ M and 2% at 56.8 μ M.

Liquid Chromatographic and Mass Spectral Methods of Identification for Regioisomeric Dimethoxyamphetamines and Brominated Dimethoxyamphetamines

The six regioisomeric dimethoxyamphetamines are prepared from the commercially available dimethoxybenzaldehydes. The dimethoxyamphetamines show very similar mass spectra, and chromatographic methods must be used to differentiate the positional isomers. Bromination of the six isomeric dimethoxyamphetamines yields a monobromination product as the major component in all cases except for 3,5dimethoxyamphetamine, which yields the 2,6-dibrominated species as the major product. Mass spectrometric analysis readily divides the regioisomeric bromodimethoxyamphetamines into two groups of three compounds each. Only those isomers having a bromine substituent ortho- to the alkylamine side-chain show a major fragment at m/z from loss of bromine from the molecular ion. The major drug of abuse 4-bromo-2,5-dimethoxyamphetamine (DOB) is one of three compounds that do not yield the m/z ion. Though the mass spectra for the three non-m/z 194 isomers show some subtle differences, these compounds are best differentiated by a reversed-phase liquid chromatographic system.

Estimation of Surface Diffusion Coefficient in Reversed-Phase Liquid Chromatography Using Methanol/Water Mobile Phases.

An estimation procedure for the surface diffusion coefficient (Ds) is proposed for a reversed-phase liquid chromatographic system using an octadecylsilyl-silica gel and methanol/water mixtures. The procedure is based on a restricted molecular diffusion model for surface diffusion, in which Ds is correlated with molecular diffusivity by introducing restriction energy (Er) due to an adsorptive interaction between adsorbates and adsorbents. The ratio of Er to isosteric heat of adsorption (Qst) is linearly correlated with 1/(–Qst). It is indicated that the linear correlation between γ (=Er/(–Qst)) and 1/(–Qst) can be observed when a linear free-energy relation and an enthalpy-entropy compensation effect in adsorption equilibrium are established. Though both Er and γ are empirical parameters, Ds at given temperatures and mobile phase compositions can be consistently calculated with an error less than about 20% from only one datum of adsorption equilibrium constant.

Determination of Emamectin Benzoate in Freshwater and Seawater at Picogram-Per-Milliliter Levels by Liquid Chromatography with Fluorescence Detection

Abstract Emamectin benzoate is a very effective agricultural insecticide applied to various crops at very low rates. A method to analyze emamectin benzoate in seawater and freshwater was developed. The method yields good recoveries with limits of quantitation of 20 pg/mL for freshwater and 24 pg/mL for seawater (signal-to-noise ratio [S/N] ≥ 10). The limit of detection is 10 pg/mL (S/N ≥ 3). Emamectin benzoateis concentrated from a 50 mL sample on a C8 solid-phase extraction cartridge and eluted with 1% ammonium acetate in methanol. The eluate is evaporated to 1 mL, diluted with 1 % ammonium acetate in water, and partitioned into ethyl acetate. The ethyl acetate extract is concentrated to about 0.5 mL and diluted to about 1 mL with acetonitrile. 1-Methylimidazole is added to the diluted sample, and then trifluoroacetic anhydride is added to form a fluorescent derivative. The derivatized sample is injected onto a reversed-phase liquid chromatographic system equipped with a fluorescence detector. Overall method recoveries for concentrations ranging from 20 to 1010 pg/mL were 84 ± 9.5% for tap water and 89 ± 11% for seawater. These results are equivalent to recoveries previously obtained with a more elaborate method (90 ± 8.8% for freshwater). Controls were free of interferences or coeluting impurities. Fluorescence detection provides high sensitivity and selectivity, enabling trace-level analysis.

Determination of melarsoprol in biological fluids by high-performance liquid chromatography and characterisation of two stereoisomers by nuclear magnetic resonance spectroscopy.

The analysis of melarsoprol in whole blood, plasma, urine and cerebrospinal fluid is described. Extraction was made with a mixture of chloroform and acetonitrile followed by back-extraction into phosphoric acid. A reversed-phase liquid chromatography system with ultraviolet detection was used. The relative standard deviation was 1% at concentrations around 10 mumol/l and 3-6% at the lower limit of determination (9 nmol/l in plasma, 93 nmol/l in whole blood, 45 nmol/l in urine and 10 nmol/l in cerebrospinal fluid). Melarsoprol is not a stable compound and samples to be stored for longer periods of time should be kept at -70 degrees C. Plasma samples can be stored at -20 degrees C for up to 2 months. Chromatography showed that melarsoprol contains two components. Using nuclear magnetic resonance spectroscopy the two components were shown to be diastereomers which slowly equilibrate by inversion of the configuration at the As atom.

Evaluation of Commercial Chlorophyllin Copper Complex Preparations by Liquid Chromatography with Photodiode Array Detection

Abstract A reversed-phase liquid chromatographic (LC) system with photodiode array detection was used to analyze chlorophyllin copper complex (CCC). Analysis revealed significant differences in the porphyrin compositions of CCC samples from 5 industrial sources. Copper isochlorin e4 was identified as a major component in most commercial materials. Copper complexes of chlorin e6 and pheophorbide a and unidentified porphyrins with either chlorin- or non-chlorin-type spectra were found in some samples. The variability of porphyrin compositions in commercial preparations may affect the medicinal efficacy of CCC; therefore, analysis of porphyrin composition is important for CCC quality control.

High‐Resolution Separation of Amyloid β‐Peptides: Structural Variants Present in Alzheimer's Disease Amyloid

In Alzheimer's disease (AD), one of the cardinal neuropathological signs is deposition of amyloid, primarily consisting of the amyloid beta-peptide (Abeta). Structural variants of AD-associated Abeta peptides have been difficult to purify by high-resolution chromatographic techniques. We therefore developed a novel chromatographic protocol, enabling high-resolution reverse-phase liquid chromatography (RPLC) purification of Abeta variants displaying very small structural differences. By using a combination of size-exclusion chromatography and the novel RPLC protocol, Abeta peptides extracted from AD amyloid were purified and subsequently characterized. Structural analysis by microsequencing and electrospray-ionization mass spectrometry revealed that the RPLC system resolved a complex mixture of Abeta variants terminating at either residue 40 or 42. Abeta variants differing by as little as one amino acid residue could be purified rapidly to apparent homogeneity. The resolution of the system was further illustrated by its ability to separate the structural isomers of Abeta1-40. The present chromatography system might provide further insight into the role of N-terminally and posttranslationally modified Abeta variants, because each variant can now be studied individually.

Temperature and solvation effects on homologous series selectivity in reversed phase liquid chromatography

Although the theoretical basis for temperature effects in reversed phase liquid chromatography (RPLC) has been well established, temperature is an under-utilized parameter for enhancing resolution in RPLC systems. However, the use of subambient operating temperatures can bring about significant increases in selectivity which are useful in separating solutes with subtle differences in their morphology. We have been examining the effects of temperature, particularly in the subambient range, and mobile phase solvation on RPLC solute selectivity for a homologous series of non-polar compounds on monomeric C 18 stationary phases. By using solute sets consisting of structural homologs, we have examined whether linear free energy relationships are operant, as well as measured enthalpic and entropic contributions to retention and selectivity over a wide temperature range. The solute homologous series sets include flexible chain solutes, such as the methylene series consisting of the C 1–C 6 n-alkylbenzenes rod-like solutes, such as the phenylene series consisting of benzene, biphenyl, p-terphenyl and p-quaterphenyl and plate-like solutes, such as the fused-ring series consisting of benzene, naphthalene, anthracene and 2,3-benzanthracene. This work is relevant in better understanding RPLC retention mechanisms as well as solute partitioning in other organized assemblies, and also is resulting in improved separation methods for many structurally similar compounds of environmental and pharmaceutical importance.

Experimental design for the rapid selection of separation conditions for methyl and propyl parahydroxybenzoate, phenylephrine hydrochloride and chlorphenamine maleate by ion-pair liquid chromatography

Methyl and propyl parahydroxybenzoate (MPHB, PPHB), phenylephrine hydrochloride (PE) and chlorphenamine maleate (CPM) are often combined as ingredients in cough-syrups. Due to distinct chemical structures, pKa values among other chemical properties are different. This may result in a particular chromatographic behaviour on ion-pair reversed-phase liquid chromatographic (LC) systems. A face-centred central composite design was applied to study the impact of four LC mobile phase parameters and parameter interactions on the retention of these four compounds. The mobile phase parameters studied were the concentration of methanol as organic modifier, the concentration of sodium dioctylsulphosuccinate (SDSS) as counter-ion, the concentration of dimethyloctylamine (DMOA) as competitive base and the pH. By means of the proposed design, mathematical regression models and response surface plots were calculated, which could predict the compounds' retention times with good statistical reliability. Adequate combination of the most relevant of these mobile phase parameters enabled complete chromatographic separations within short times of analysis.

Chromatographic Behavior of the Anthelmintic Fenbendazole and Its Major Metabolite Oxfendazole in Various Ion-Pair Liquid Chromatographic Systems

Abstract The chromatographic behavior of fenbendazole (FBZ) and oxfendazole (OFZ) in various reversed-phase liquid chromatographic (LC) systems has been investigated. The addition of negative and/or positive charged ion-pair reagents in the mobile phase has been examined, whereas the influence of mobile phase pH, mobile phase composition, and column temperature on retention and peak height has been evaluated. The observed behavior of the analytes during the various chromatographic processes has been discussed.

Comparison of reversed-phase chromatographic systems with principal component and cluster analysis

The separation of ethoxylated 1,1,3,3-tetramethylphenyl surfactants was investigated by reversed-phase liquid chromatography (RP-LC) using various supports (C 1, C 2, C 6, C 8, C 18, polyethylene-coated silica, and polyethylene-coated alumina) and with reversed-phase thin-layer chromatography (RP-TLC) using impregnated silica and alumina supports. The retention data matrix was evaluated both by principal component analysis and cluster analysis. The retention characteristics of both polyethylene-coated supports were similar to those of C 1, and the retention capacity of RP columns for surfactants increased with the increasing length of the covalently bonded hydrocarbon chain. Both multivariate mathematical statistical methods gave similar results and adequate separation was performed with the RP-LC and RP-TLC systems.

Pentane-Methanol Mixture as a Model of the Stationary Phase in Reversed-Phase Liquid Chromatography Using Octadecyl-Bonded Silica Gel and Methanol

A pentane-methanol mixture was taken as a convenient model of practical stationary phase functioning in the reversed-phase liquid chromatography (RPLC) system consisting of octadecyl-bonded silica gel (ODS) and methanol. The capacity factor (k′) of tris(β-diketonato)chromium(IH) or bis(β-diketonato)palladium(II) observed in the ODS/methanol system was compared with the ratio of the solubility of the complex in a pentane-methanol mixture to that in methanol. A pentane-methanol mixture, containing methanol in a mole fraction range 0.42-0.66, was found to be a medium comparable to the stationary phase in the RPLC, because the distribution of the complex was similar. Furthermore, the incorporation of methanol into alkyl-moiety of ODS was confirmed experimentally. The above facts suggest that the stationary phase functioning in the RPLC consisting of ODS and methanol is a mixed phase of the alkyl-moiety of ODS and methanol incorporated from the mobile phase into the moiety for the complex tested.

Reversed-Phase High-Performance Liquid Chromatography Determination of Quinine in Plasma, Whole Blood, Urine, and Samples Dried on Filter Paper

The analysis of quinine in whole blood, plasma, urine, and samples dried on filter paper is described. Extraction was made with toluene followed by back-extraction into phosphate buffer. A reversed-phase liquid chromatography system with fluorescence detection was used. The within-day coefficient of variation of the method was 4-10% at the lower limit of determination (2 nM in plasma and 50 nM in whole blood, dried samples, and urine) and 2-4% at 10 microM. The quinine concentration was found to be lower in whole blood than in plasma (mean ratio, plasma-whole blood, 1.17). The concentration in capillary blood was lower than that in venous blood (mean ratio, capillary blood-venous blood, 0.93).

Electrocatalysis and determination of hydrazine compounds in liquid chromatography at a mixed-valent cobalt oxide/cyanocobaltate film electrode

A glassy carbon electrode coated with an electrodeposited film of mixed-valent cobalt oxide/cyanocobaltate (CoO/CNCo) enabled hydrazine compounds to be catalytically oxidized at the greatly reduced overpotential and in a wide operational pH range (pH 2.0–7.0). Electrocatalytic activity at the CoO/CNCo modified electrode was evaluated with respect to solution pH, film thickness, supporting electrolyte ions, potential scan rate, operating potential, concentration dependence and other variables. The CoO/CNCo film electrode was completely compatible with a conventional reversed-phase liquid chromatographic (RP-LC) system. Practical RP-LC amperometric detection (RP-LCEC) of hydrazines was performed. A dynamic linear response range over three orders of magnitude and a detection limit at the pmol level were readily obtained. The CoO/CNCO film electrode exhibited excellent electrocatalytic stability in the flowing streams.

Carbohydrate structures of human alpha-fetoprotein of patients with hepatocellular carcinoma: presence of fucosylated and non-fucosylated triantennary glycans.

Chemical structures of the sugar chains of various human alpha-fetoprotein (AFP) species with different affinity for Concanavalin A (Con A) and Lens culinaris agglutinin (LCA) were examined by pyridylamination of their oligosaccharides and stepwise exoglycosidase digestion. Using reversed-phase and size-fractionation high performance liquid chromatography systems we identified six pyridylamino-sugar chains. The Con A-reactive and LCA-nonreactive species of AFP from patients with hepatocellular carcinoma contained a biantennary sugar chain, and the Con A-reactive and LCA-reactive species had a biantennary one with a fucose residue at the innermost N-acetylglucosamine residue. The Con A-nonreactive and LCA-reactive species contained a biantennary sugar chain both with a bisecting-N-acetylglucosamine residue at the trimannosyl core and with a focus residue at the innermost N-acetylglucosamine residue. The Con A-nonreactive and LCA-nonreactive species contained a fucosylated triantennary sugar chain as a major component, and two minor components: a triantennary sugar chain and a biantennary sugar chain with a bisecting-N-acetylglucosamine residue at the trimannosyl core. Thus, the fucosylated and non-fucosylated triantennary sugar chains were newly identified in human AFP. Essentially identical results were obtained for AFP from the patient with gallbladder carcinoma which metastasizes to the liver. These results indicate that the increment in fucosylation and branching to form new antennae is a characteristic feature of the carbohydrate chains of AFP from patients with neoplastic diseases of the liver.

Biochemistry and Physiology of Hopanoids in Bacteria

Publisher Summary Hopanoids are amphipathic components of bacterial cell membranes and maintain membrane stability by increasing rigidity in the lipid matrix. Thus, the function of hopanoids in bacteria is analogous to that of membrane sterols in eukaryotes. Biosynthesis of hopanoids does not require oxygen during squalene cyclization and other reactions. This chapter describes the structures, distribution, and physiological role of hopanoids as membrane stabilizers. Structural variations in hopanoids result from linkage between a hydrophobic triterpenic moiety and a polar side-chain. The simplest hopanoids, diplopterol and diploptene, are readily extractable from freeze-dried cells by organic solvents and can be characterized by gas-liquid chromatography (GLC) and by combined GLC-mass spectroscopy. Another methods for analysis of periodate-oxidized and borohydride-reduced hopanoids, where hopanoid alcohols are converted into benzoate or naphthoate derivatives, yielding chromogenic compounds that can be separated with a reversed-phase high-pressure liquid chromatography (HPLC) system using ultraviolet-radiation detection are presented. Hopanoid-containing fractions can usually be detected only by proton nuclear magnetic-resonance (NMR) spectroscopy exploiting the characteristic pattern of methyl singlets in the hopane skeleton. The occurrence of hopanoids in the specialized organisms exemplifies their general role as membrane reinforcers in eubacteria. The presence of hopanoids is essential for survival of the bacteria that contain them. The chapter accounts for the biosynthesis and principles of genetics for the hopanoids. Hopanoids are abundant natural products of eubacteria. The lipid family of hopanoids consists of numerous structural variants that lead to their detection and analysis.

A selective HPLC/RIA for dexamethasone and its prodrug dexamethasone-21-sulphobenzoate sodium in biological fluids.

A combined high performance liquid chromatography/radioimmunoassay procedure is described for the simultaneous determination of dexamethasone (DEX) and its prodrug dexamethasone-21-sulphobenzoate sodium (DSS) in plasma. After precipitation of the plasma proteins by acetonitrile, the protein-free supernatant was injected onto a C18 reversed phase liquid chromatographic system and DSS- and DEX-containing fractions were collected. Hydrolysis of DSS by 0.01 N NaOH, followed by fractions extraction of both hydrolysed DSS and DEX fractions with ethyl acetate allowed the use of a dexamethasone-specific radioimmunoassay for the selective determination of both compounds. The method is accurate and reproducible (intraday variability for DSS and DEX < 6%, interday variability for DEX 14%), allowing quantification of DEX and DSS as low as 0.3 ng/mL and 0.7 ng/mL, respectively.

Relationships between the reversed-phase liquid chromatographic retention characteristics and physico-chemical parameters of some β-casomorphin peptides

Log k′ w and slope values for ten β-casomorphin peptides were calculated in ten different reversed-phase liquid chromatographic systems using methanol as organic modifier. The differences between the retention behaviours of C 8, C 18 and C 30 columns were fairly small the eluent composition had a greater impact than the length of the hydrophotic ligand on the retention of peptides. A quaternary amino column showed considerably different retention characteristics. The results suggest that each substructure in the peptides makes a similar contribution to the overall retention of the peptide. The retention parameters of peptides mainly correlated with their electronic and steric characteristics. This finding emphasizes the importance of the electrostatic interactions between the free silanol groups on the surface of the reversed-phase support and the polar substructures of peptides.

Temperature dependence of retention in reversed-phase liquid chromatography. 2. Mobile-phase considerations.

The retention mechanism in reversed-phase liquid chromatography (RPLC) has been examined over a wide temperature range with emphasis on the role of the mobile phase. van't Hoff plot shapes were used to assess the retention mechanism, and the data showed evidence of the hydrophobic effect when water-rich and/or hydrogen-bonded mobile phases such as methanol/water were used. However, different van't Hoff plot shape was observed with acetonitrile/water mobile phases, indicating a change in the retention mechanism. These data showed that the hydrophobic effect, which had previously been proposed as the driving force for retention, is not a satisfactory explanation for the retention process in all RPLC systems.