A collaborative study to investigate the retention reproducibility of barbiturates in HPLC with a view to establishing retention databases for drug identification

A collaborative study among 10 laboratories has been undertaken to investigate the interlaboratory reproducibility of retention measurements in a high performance liquid chromatographic (HPLC) system for separating barbiturates. The system involved an ODS-Hypersil column with an eluent of methanol:phosphate buffer (40:60, v/v) at pH 8.5; all laboratories used the same batch of packing material. The conventional methods of recording retention properties (retention times and capacity factors) gave poor interlaboratory reproducibility. Better results were obtained by expressing the retentions relative to a standard barbiturate (quinalbarbitone); relative adjusted retention times proved to be more effective than straightforward relative retention times. Retention indices based on the alkylarylketone scale were not as reproducible as the methods based on a single closely related compound. The best reproducibility was obtained using corrected capacity factors based on the retention of four barbiturates in a standard mixture. The results of the study are discussed with a view to selecting the best methods of recording retention in HPLC when considering the establishment of databases for drug identification.     

Copolymer of Di (methacryloyloxymethyl) naphthalene and divinylbenzene as a column packing for high-performance liquid chromatography

Summary A new porous polyaromatic ester packing was synthetized for high performance liquid chromatography. The relationship between retention and chain length of the members of homologous series of alkylbenzenes, N-alkylanilines, alkylarylethers, alkylbenzoates and alkylarylketones on this new stationary phase using different eluents was investigated. Using the alkylarylketone scale the retention indices of the homologues and test compounds were calculated. The results were compared with those obtained for poly (styrene-divinylbenzene) polymers. For both types of packing the first members of each homologous series gave non-linear behaviour. The methylene group index increments are different for the studied homologous series; thus there is no simple additivity of the retention indices. The efficiency of the porous polymeric columns is a function of the capacity factor of the solute and the organic component of the eluent.     

The application of retention indices using the alkylarylketone scale to the separation of the barbiturates by HPLC. I. The effect of the eluent

Summary In order to determine the applicability of retention indices, based on the alkylarylketone scale, as the basis of a reproducible method of reporting retentions, the separation of ten barbiturates and a set of column test compounds were examined on an octadecylsilyl bonded silica (ODS-Hypersil) column with methanol-buffer pH 8.5 as eluent. The effects on the capacity factors and retention indices, on changing the eluent composition, pH, ionic strength and temperature, showed that the retention indices of the barbiturates were much less susceptible to minor changes in the eluent than the capacity factors. For non-ionised compounds the retention indices were virtually independent of the experimental conditions. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Application of retention indices based on the alkylarylketone scale to the separation of the local anaesthetic drugs by high-performance liquid chromatography

The separation of the local anaesthetic drugs by reversed-phase high-performance liquid chromatography using a Hypersil ODS column has been examined in order to identify critical factors in the establishment of a retention database for interlaboratory comparisons. An eluent of methanol-aqueous orthophosphoric acid (15:85, v/v) (pH 2.5) containing 0.7% hexylamine has been used. The effects of small changes in the eluent composition, pH, temperature and the use of different column packing materials on the retentions of the analytes have been studied. The use of capacity factors, relative capacity factors and retention indices based on the alkylarylketone scale have been compared.     

The application of retention indices using the alkylarylketone scale to the separation of the barbiturates by HPLC. II The effect of the stationary phase

Summary The capacity factors and retention indices, based on the alkylarylketone scale, of ten barbiturates and a set of five column test compounds have been compared on a series of different octadecylsilyl bonded (ODS) silica columns under identical elution conditions of 40:60 methanol-buffer pH 8.5 at 30 °C. The retentions were highly reproducible on columns prepared from the same batch or different batches of ODS-Hypersil, but large differences in the capacity factors were found when column packings from different manufacturers were compared. Retention indices were more reproducible than capacity factors but they could not compensate for differences between the different packing materials. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

A fundamental study of the effects of modifiers in supercritical fluid chromatography

The effects of organic modifiers on retention and peak shape in packed-column supercritical fluid chromatography were studied. The adsorption behavior of different modifiers was investigated on stationary phases consisting of hydrocarbon monolayers chemically bonded on silica. Adsorption isotherms for several modifiers were recorded using breakthrough measurements. The results were compared with those obtained by a simple method based on the injection of different sample sizes. Modifiers were selected to reflect various types of interactions with the silica support. Isotherms were found to be approximately Langmuirean. Experiments were performed to elucidate the influence of adsorbed molecules on the retention of selected test solutes. The introduction of a solvent modifier can lead to a substantial change in the mobile phase density, the effect of which is comparable with that of an increase in the density of pure carbon dioxide. Our results confirm that the effects of low concentrations of modifiers (between 0 and 2 percent) in packed-column SFC are largely due to deactivation of residual silanol groups on the silica support. The accessibility of the active sites was found to depend strongly on the size and structure of the modifier molecules. The decrease of retention due to the addition of low concentrations of modifiers could be described accurately by a model derived from Langmuir adsorption behavior. Some semi-quantitative rules for the selection of modifiers and the required concentrations for optimum deactivation of the support are discussed.     

The Effect of Binary Solvent Composition and Polarity on Separations in Reversed Phase Thin Layer and High Performance Liquid Chromatography

Abstract

The effect of the solvent's composition and polarity on separation in reversed phase thin layer and high performance liquid chromatography is discussed. These results show that retention times cannot be predicted merely from the polarity of the binary mobile phase. Although organic modifiers with the same physico-chemical properties and from the same solvent group were used, the retention times obtained using binary mobile phases having the same polarity, were different. It was also observed that normal chain carbon alcohols gave retention times different from those with a branched chain (n-propanol vs. iso-propanol), and the longer the alcohol chain the higher the R_f value. The results also show that not only the organic modifier used is important but the solute mixture used.     

Separation of anabolic steroids by micellar electrokinetic capillary chromatography

Summary The separation of seven analogous anabolic steroids was studied by micellar electrokinetic capillary chromatography (MECC). The retention order was found to be dependent on polarity. All of these steroids were well separated by the addition of organic modifiers to the separation buffer. Of the organic modifiers tested, 1-propanol gave the best separation, better than methanol or acetonitrile.     

Liquid chromatography of isomers of aromatic acids on microbore column packed with anion-exchange resin

Summary A low-capacity, anion-exchange, porous-polymer resin was used as the packing material in a microbore column. Using a phosphate buffer containing various organic modifiers, retention behavior of substituted benzoic acid isomers with amino, chloro, methyl, nitro and carboxylic groups was investigated. The effect of ethanol, acetonitrile and sodium dodecylsulfate used as organic modifiers on the retention behavior at various eluent pH values was studies and optimum separations of the positional isomers reported.     

An insight into the use of dimethylphenyl carbamate cyclofructan 7 chiral stationary phase in supercritical fluid chromatography: The basic comparison with HPLC

Cyclofructan‐based chiral stationary phases were previously shown as a promising possibility for separation of chiral compounds in high performance liquid chromatography. In this work retention and enantiodiscrimination properties of the 3,5‐dimethylphenyl carbamate cyclofructan 7 chiral stationary phase are described in supercritical fluid chromatography. The results obtained in both of the separation methods were compared. A set of compounds with axial or central chirality was used as analytes. The effect of mobile phase composition, that is, addition of different alcohol modifiers and/or trifluoroacetic acid to carbon dioxide, was examined in the supercritical system. Similarly, mobile phases composed of hexane modified with propan‐2‐ol and/or trifluoracetic acid were used in liquid chromatography. A linear free energy relationship model was utilized for characterization of interactions that are decisive for retention and separation in both techniques. Dispersion interactions showed similar negative values using both methods. The main contribution of hydrogen bond acidity was also comparable for both methods. The propensity to interact with n‐ and/or π‐electron pairs of solutes was significant only in the supercritical system.     

Separation of imidacloprid and its degradation products using reversed phase liquid chromatography with water rich mobile phases

Water rich mobile phases in RPLC are not generally used because of the longer retention times involved when organic modifiers such as methanol or acetonitrile are used. The problem of longer retention times can be addressed using hydrophobic alcohols such as pentanol in low quantities (less than 1%) as organic modifiers. The advantages of using these mobile phases in RPLC for the separation of water soluble and weakly retained congeners is demonstrated through the separation of imidacloprid and its degradation products using a 0.4% pentanol in water mobile phase with 0.2% acetic acid.     

Investigation of perchlorate, phosphate and ion-pairing eluent modifiers for the separation of cephalosporin epimers

The retention behavior of several pairs of 7 alpha- and 7 beta-cephalosporin epimers was investigated using perchlorate, phosphate and ion-pairing eluent modifiers. At pH 2.5, sodium perchlorate, sodium phosphate and sodium pentanesulfonate all provided separation of epimers with free 7-amino groups. When the 7-amino group was blocked, as in cephalexin and cefaclor, sodium perchlorate gave the best separation at pH 2.5. A tetrabutylammonium ion-pairing system at pH 7.0 provided separation of all epimer pairs containing a free carboxylic acid at the 3 position. Hydrophobic, residual silanol and ionic interactions were factors in the retention mechanism of the cephalosporins under the conditions investigated. An ionic interaction of perchlorate with the protonated amine of the cephalosporin was postulated as an explanation of the retention and selectivity effects observed with perchlorate as an eluent modifier.     

Enantiomeric impurity determination of levetiracetam using capillary electrochromatography

CEC was used to develop a method for the enantiomeric excess determination of levetiracetam, an antiepileptic drug. Different types of calibration curve were evaluated for use in the range between 0.01 and 1 mg/mL when aniracetam was used as an internal standard. The method gave comparable results when only the areas of the impurity were used in the calibration curve. The predicted detection and quantification limits from the S/N were 1.1 and 3.6 microg/mL, respectively. However, experimental results showed that LOD and LOQ were underestimated. Repeatability of injection was demonstrated by the RSD values obtained for retention time, resolution, ratios of the areas impurity/internal standard, and areas of impurity and internal standard individually, which were below or equal to 9.30%. The between-days variability experiments indicated that it is better to make a calibration curve daily. The finally selected calibration curves were used to test the accuracy of the developed method on bulk samples and Keppra tablets containing 250 mg levetiracetam. Both selected calibration curves performed similarly. The one using the internal standard information gave overall recoveries between 88 and 118%, while the one using areas gave results between 84 and 118%.     

Retention characteristics and practical applications of carbon sorbents

Reversed-phase separations provides a versatile technique in high-performance liquid chromatography. Porous graphitized carbon (PGC) support shows unique retention characteristics. Separations on PGC columns use typical reversed-phase eluents (water and organic modifiers miscible with water), however, the retention order of solutes generally does not follow their hydrophobicity order. Molecular hydrophobicity influences but not determines the elution order of any set of solutes. The properties of these supports, mechanisms of retention and application are discussed, along with correlations which can guide the choice of solvent combinations for typical separations.     

Direct determination of selenium in urine samples by electrothermal atomic absorption spectrometry using a Zr plus Rh-treated graphite tube and co-injection of Rh as chemical modifier

Different chemical modifiers for use with electrothermal atomic absorption spectrometry (ET AAS) were investigated in relation to determining the selenium in human urine samples. The samples were diluted in a solution containing 1% v/v HNO₃ and 0.02% m/v cetyltrimethylammonium chloride (CTAC). Studying the modifiers showed that the use of either Ru or Ir as the permanent modifier gave low sensitivity to Se and the peak shape was very noisy, while Zr or Rh gave no peak at all. The same occurred when Zr was used in solution. For mixtures of permanent modifiers, Ir plus Rh or Zr plus Rh gave very low sensitivity, Zr plus Rh with co-injection of Ir in solution was also not efficient, Zr plus Rh in solution gave good sensitivity, but the best results were obtained with a mixture of Zr and Rh as the permanent modifier and co-injection of Rh in solution. Using this last modifier, the following dilutions with the HNO₃ and CTAC were studied: 1:1, 1:2, 1:3 and 1:4. The best dilution was 1:1, which promoted good sensitivity and a more defined peak shape and made it possible to correct for the background using a deuterium arc lamp. Under these conditions, a characteristic mass of 26±0.2 pg was obtained for Se in aqueous solution. Six certified urine samples were analyzed using matrix matching calibration and the measured concentrations were in agreement with the certified values, according to a t-test at the 95% confidence level. Recovery tests were carried out and the recoveries were in the range 100–103%, with relative standard deviation better than 9%. The limit of detection (LOD, 3 sd, n=10) was 3.0 μg L⁻¹ in the sample. The treated graphite tube could be used for at least 600 atomization cycles without significant alteration of the analytical signal.     

Effects of organic modifiers on solute retention and electrokinetic migrations in micellar electrokinetic capillary chromatography.

Influences of seven organic modifiers, including urea, methanol (MeOH), dioxane (DIO), tetrahydrofuran (THF), acetonitrile (ACN), 1-propanol (1-PrOH) and 2-propanol (2-PrOH), on the solute retention and the electrokinetic migrations in micellar electrokinetic capillary chromatography (MEKC) are investigated with sodium dodecyl sulfate (SDS) micelle as pseudostationary phase. It is observed that in the limited concentration ranges used in the MEKC systems the effect of organic modifier concentration on the retention can be described by the equation logk1=logk1w-SC for most binary aqueous-organic buffer, but deviations from this retention equation are observed at ACN and particularly THF as organic modifiers. With parameter S as a measure of the elutropic strength, the elutropic strength of the organic modifiers is found to follow a general order urea 相似文献   

Effect of class I and II organic modifiers on retention and selectivity in vesicle electrokinetic chromatography

Vesicles are large aggregates of surfactant monomers consisting of a spherical bilayer surrounding an internal cavity of solvent. The bilayer structure allows vesicles to be attractive models for the study of various transmembrane and binding processes. The use of thermodynamically stable vesicles (TSV) formed from oppositely charged surfactants for use as a pseudostationary phase in electrokinetic chromatography (EKC) was first accomplished using dodecyltrimethylammonium bromide and sodium dodecyl sulfate (DTAB/SDS). Surfactant vesicles have demonstrated enhanced separation characteristics compared to conventional micelles in EKC, although only investigated in aqueous media. Organic modifiers have been widely studied and used in EKC to enhance separation conditions. In this study, vesicles formed from cetyltrimethylammonium bromide and sodium octyl sulfate (CTAB/SOS) were investigated in the presence of "class I and II" organic modifiers. Electrophoretic and chromatographic parameters were examined as well as linear solvation energy relationship analysis (LSER) to characterize the effects of the modifiers on retention and selectivity in EKC. LSER analysis is a useful way to quantitatively investigate solute/solvent interactions responsible for retention and selectivity.     

Comparison of the enantioseparation of racemic uridine analogs on Whelk-O 1 and ChiralPak-AD columns

The commercially available, brush-type (S,S)-Whelk-O 1 chiral stationary phase (CSP) has been used to separate 10 racemates of structurally related uridine analogs, potentially anti-viral agents, under various mobile phase compositions, using various temperatures. The enantioseparation was evaluated by comparing the Whelk-O 1 column performance with that of ChiralPak-AD column, reported previously. The comparison involved the role of some distinctive structural features of the racemates, type and composition of the solvent modifiers, as well as effect of temperature on the chiral discrimination. Despite the fact that both columns separate almost all the uridine analogs, significant differences were observed in their chiral recognition, as revealed from their retention, selectivity, resolution and elution order. The chiral recognition processes, responsible for enantioseparation on the Whelk-O 1 column, were relatively more systematic and easier to manipulate than on ChiralPak-AD column. Enantioseparation on the latter are of more complex nature and frequently gave results that were contradictory to the expectations. On the other hand, the performance in the ChiralPak-AD column was superior to that of the Whelk-O 1 column. Limitations in column handling and maintenance (pressure and temperatures) as well as limited solvent choice lead to the preference of the Whelk-O 1 column, in spite of its lower (but adequate) performance.     

High throughput screening or drug impurity profiles using gradients in normal phase chromatography

Summary Developmental drug substances and a variety of precursors and process-related impurities were separated using mobile phase gradients under normal phase conditions. These separation conditions produced efficient separations in a fraction of the time necessary for comparable reversed-phase separations. The use of elevated temperatures, polar modifiers and moderate levels of acidic and basic additives allowed highly polar analytes to be eluted with high efficiency and selectivity using fast flow rates. Reproducibility for retention time and peak areas was demonstrated to be very good despite very short equilibration times.     

Synthesis of an N-butylpyrrolidine phase using the allyl bonded stationary phase as an intermediate

Summary The versatility of the allyl phase is exploited by using it as an intermediate in the production of a weak anion exchange material that would be difficult to synthesize directly using organochlorosilane reagents. The stability of the allyl phase under the severe reaction conditions (170° and 140 atm of CO) of aminomethylation also is demonstrated. The product N-butylpyrrolidine phase is characterized spectroscopically by ESCA and DRIFT which also indicate a high conversion (greater than 90 %) of the allyl phase. In addition, the carbon-nitrogen ratio of the product phase supports a high yield. Chromatographic characterization is done by the retention indices of the alkylarylketone homologous series and by capacity factor (k′) determinations for inorganic anions and amino acids.