RP-HPLC Systems Research Articles

Estimation of lipophilicity and design of new 17β-carboxamide glucocorticoids using RP-HPLC and quantitative structure-retention relationships analysis

Abstract Eight 17β-carboxamide glucocorticoids with local anti-inflammatory activity were selected and their retention behavior tested in six RP-HPLC systems (I–VI). logkw, a, and φ 0 parameters were calculated and correlation with previously determined logPo/w values was examined. RP-HPLC system IV, which consisted of cyano column and methanol–water mobile phases (50:50, 60:40, 70:30, and 80:20, v/v), was selected as the most reliable for lipophilicity prediction and used for the analysis of chromatographic behavior of remaining fourteen 17β-carboxamide glucocorticoids. Quantitative structure-retention relationships analysis was performed and PLS(logkw) model was selected as the most statistically significant. On the basis of selected model and interpretation of corresponding descriptors, new derivatives with higher logkw values and higher expected lipophilicity were designed.

Comparison of the Retention and Separation Selectivity of Aromatic Hydrocarbons with Polar Groups in RP-HPLC Systems with Different Stationary Phases and Eluents.

In this manuscript, the retention of aromatic hydrocarbons with polar groups has been compared for systems with various nonpolar columns of the types from C3 to C18 and different mobile phases composed of methanol, acetonitrile, or tetrahydrofuran as modifiers. The selectivity separation of the solutes in systems with different adsorbents, when one eluent modifier is swapped by another, has been explained, taking into account molecular interactions of the solutes with components of the stationary phase region (i.e., extracted modifier depending on the chain length of the stationary phase).

Characterization of bonded stationary phase performance as a function of qualitative and quantitative chromatographic factors in chaotropic chromatography with risperidone and its impurities as model substances.

Numerous stationary phases have been developed with the aim to provide desired performances during chromatographic analysis of the basic solutes in their protonated form. In this work, the procedure for the characterization of bonded stationary phase performance, when both qualitative and quantitative chromatographic factors were varied in chaotropic chromatography, was proposed. Risperidone and its three impurities were selected as model substances, while acetonitrile content in the mobile phase (20-30%), the pH of the aqueous phase (3.00-5.00), the content of chaotropic agents in the aqueous phase (10-100mM), type of chaotropic agent (NaClO4, CF3COONa), and stationary phase type (Zorbax Eclipse XDB, Zorbax Extend) were studied as chromatographic factors. The proposed procedure implies the combination of D-optimal experimental design, indirect modeling, and polynomial-modified Gaussian model, while grid point search method was selected for the final choice of the experimental conditions which lead to the best possible stationary phase performance for basic solutes. Good agreement between experimentally obtained chromatogram and simulated chromatogram for chosen experimental conditions (25% acetonitrile, 75mM of NaClO4, pH 4.00 on Zorbax Eclipse XDB column) confirmed the applicability of the proposed procedure. The additional point was selected for the verification of proposed procedure ability to distinguish changes in solutes' elution order. Simulated chromatogram for 21.5% acetonitrile, 85mM of NaClO4, pH 5.00 on Zorbax Eclipse XDB column was in line with experimental data. Furthermore, the values of left and right peak half-widths obtained from indirect modeling were used in order to evaluate performances of differently modified stationary phases applying a half-width plots approach. The results from half-width plot approach as well as from the proposed procedure indicate higher efficiency and better separation performance of the stationary phase extra densely bonded and double end-capped with trimethylsilyl group than the stationary phase with the combination of end-capping and bidentate silane bonding for chromatographic analysis of basic solutes in RP-HPLC systems with chaotropic agents. Graphical abstract ᅟ.

New Insights into the Effects of Tetrahydrofuran and Dioxane on the Selectivity of Ternary Eluent Compositions in RP-HPLC Systems

The present paper discusses the tendencies in the retention changes of a diverse set of organic model compounds on HPLC C18 stationary phases in aqueous ternary eluent mixtures containing tetrahydrofuran or 1,4-dioxane, and one short chain aliphatic alcohol (ethanol, n-propanol or isopropanol). The set of compounds consisted of steroid and non-steroid molecules with different hydrogen bond donor and/or acceptor abilities. Every eluent mixture contained 75 V/V% water and 25 V/V% organic solvent(s). The composition of the mixture was changed in 5 V/V% steps, starting with the binary alcohol–water mixtures and finishing with the binary ether–water mixtures. The results show clearly the dependency of retention times on the eluent composition, the size of the molecules, and the occurence of the hydrogen bond donor/acceptor groups. The isopropanol–tetrahydrofurane–water mixtures resulted in selective changes in the retention times of the compounds with acidic groups or with other non-acidic OH or NH protons in the neighborhood of electron-withdrawing groups. Every compound has shown elevated retention times in the isopropanol–dioxane–water, n-propanol–tetrahydrofuran–water, or n-propanol–dioxane–water mixtures. Clear trends could not be observed in the eluents with ethanol. The probable reason for the retention enhancement is the adsorption of the organic components of the mobile phase on the surface of the stationary phase. The different effects of the alcohols may originate from the interaction of their varying aliphatic alkyl chains with the C18 chains. This phenomenon may result in different availability of the C18 chains for dioxane, tetrahydrofuran and the model compounds.

Comparison of the Retention of Aliphatic Hydrocarbons with Polar Groups in RP-HPLC Systems with Different Modifiers of the Binary Eluent.

The retention of aliphatic hydrocarbons with polar groups has been compared in respect to the separation selectivity changes in reversed-phase high-performance liquid chromatography with C18 stationary phase type and binary water eluent composed of methanol, acetonitrile, or tetrahydrofuran as modifiers. The changes in separation selectivity when one modifier is replaced by another in the eluent is explained, taking into consideration molecular interactions of the solutes with components of the stationary phase region, i.e., extracted modifier, and ordering of the stationary phase by the modifier.

Influence of LC Retention Data on Antitumor Acridinones’ Classification Evaluated by Factor Analysis Method

The application of factor analysis (FA) method in classification of the antitumor acridinones based on high-performance liquid chromatography (HPLC) retention data and calculated parameters of lipophilicity as well as some nonempirical structural parameters was studied. First, a group of 19 acridinone (imidazoacridinone and triazoloacridinone) derivatives was chromatographed in six RP-HPLC systems, and the values of their HPLC retention data as retention' times determined in both 10 min and 30 min gradient times were obtained as well as log kw (retention factor log k extrapolated to 0% organic modifier) parameters using DryLab program were calculated. Additionally, molecular modeling studies were performed based on the structural formula of considered acridinones and structural descriptors were derived as well as log P parameters using some commonly available software were calculated and subsequently used. A matrix of 19 x 32 HPLC data together with log P data and molecular properties parameters was subjected to factor analysis and led to extract three main factors with eigenvalues higher than 1. The first principal component (factor 1) accounted for, by 80.87% of the variance in data, the second principal component (factor 2) explained 7.77% and the third principal component (factor 3) was responsible by 4.46% of data variance. The total data variance was at the level 93.09% and was explained by the first three principal components. Moreover, one of the most significant influences on the values of factor 1 and factor 2 possessed HPLC retention data and calculated parameters describing lipophilicity, respectively. More importantly, distribution of individual drugs on the plane determined by two principal components produced patterns in good agreement with their chemical structures as well as with their antitumor activity.

SURFACTANT MEDIATED EXTRACTION OF TRITERPENES AND THEIR DIRECT HPLC ANALYSIS FROM THE MICELLAR SYSTEM

The pentacyclic triterpenes oleanolic acid (OA) and ursolic acid (UA) in Salvia triloba are attractive ingredients for cosmetic and pharmaceutical formulations. These triterpenoids are constitution isomers and differ only in the position of one methyl group in their chemical structures. Therefore it is difficult to achieve a baseline separation by standard RP-HPLC systems. Another challenge is that the complex plant contains a large variety of similar compounds. To achieve precise results for the target molecules, these compounds should be removed prior to analysis. Due to the large application of these two triterpenes, a fast quantification method is favored. If the triterpenes are extracted with aqueous two-phase systems, the target substances are enriched in a surfactant-rich phase. For this application, it is desirable to develop a method that is capable of quantifying both triterpenes directly by RP-HPLC without additional purification steps. The optimal chromatographic conditions for pure substances as well as for crude surfactant-containing extracts were accomplished on a Nucleodur C18 ISIS column by isocratic elution with methanol/water/acetic acid/triethanolamine (90:10:0.04:0.02 v/v) as the mobile phase and a column temperature of 10°C. The flow rate was 0.6 mL/min, and the detection wavelength was 210 nm. With these parameters, a baseline separation was achieved and the calibration curve showed a very good linearity (R2 > 0.999 for OA and UA) within the test range. The method is simple, rapid, and reliable for the quantification of crude extracts from plant material.

Separation selectivity of some phenolic acids in RP HPLC systems with binary mobile phase comprised various modifiers

Retention of phenolic acid has been correlated for reversed-phase high-performance liquid chromatography systems with different binary mobile phases containing methanol, acetonitrile or tetrahydrofuran as modifiers and buffer at pH 3.0, 4.6 and 5.0. The changes of separation selectivity of solutes, when one modifier is replaced by another in the eluent, has been explained taking into consideration molecular interactions of the solutes with components of the stationary phase region, i.e. extracted modifier, and ordering of the stationary phase by the modifier.

Evaluation of Orthogonal/Dissimilar RP-HPLC Systems Sets for Their Suitability as Method-Development Starting Points for Drug Impurity Profiles

Orthogonal or dissimilar separation systems provide different selectivities and their application can facilitate the development of methods to identify and quantify impurities in a drug substance. Two sets of chromatographic systems potentially applicable for method development were evaluated using four drug/impurity profiles. The sets consist of orthogonal or dissimilar systems and systems with good overall separation properties, selected in earlier studies. The aim of this study is to evaluate these systems for selectivity differences in the impurity profiles. These differences should allow determining the number of compounds occurring in an impurity profile. Then, one or a very limited number of systems is to be proposed for further method development. To examine the selectivity changes and separation quality for each impurity profile, both the normalized retention times tau and the resolutions between pairs of consecutively eluting peaks were plotted on parallel axes, representing the systems. For each profile, several systems of the studied sets can serve as potential starting points for further method development. All impurities could be separated from the active substance and from each other on at least one system. However, for the different profiles, different systems were selected as best, which makes that each system in a given set has its importance, depending on the properties of the profile.

The molecular descriptor log Sum AA and its alternatives in QSRR models to predict the retention of peptides

The use of the experimental molecular descriptor log Sum AA and some possible alternatives were evaluated in the QSRR analysis of peptides. To quantitatively characterize the structure of analytes in a previously proposed QSRR the following three structural descriptors were applied: the logarithm of the sum of gradient retention times of the amino acids composing the individual peptide, log Sum AA; the logarithm of the peptide’s van der Waals volume, log VDW Vol; and the logarithm of its theoretically calculated n-octanol–water partition coefficient, clog P. Taking into consideration that most amino acids were hardly retained in the different RP-HPLC systems on which the peptides retention was measured, the contribution of most amino acids to the log Sum AA descriptor is rather constant. Therefore, to enlarge the variability of the descriptor and the amino acids contributions for a given series of peptides, in a first instance, it was evaluated whether, by changing the chromatographic conditions, the retention differences between the amino acids could be increased, while maintaining their mutual selectivity. It was not evident to find such conditions. Secondly, it was also investigated whether the experimental descriptor log Sum AA can be replaced by a theoretical, either based on a simple or on a weighted counting of the amino acids composing the peptide. The weighting factor for the retained amino acids was determined by their experimental gradient retention times measured on different systems. The predictive abilities of the new QSRR models (applying the alternative descriptors) were assessed using the leave-one-out cross-validation procedure and compared to that of the initial model. Finally, a descriptor was defined for which the retention measurement of only a limited number of amino acids is required. It resulted in QSRR models with similar predictive properties as those with log Sum AA, but with a reduced workload.

Brief analysis of the retention process in RP‐HPLC systems with a C30 bonded stationary phase

The influence of the mobile-phase composition on the retention of eight model substances in different RP-HPLC systems with a C(30) alkyl bonded stationary phase has been studied. The aim of this study was to compare the performance of four valuable retention models assuming the partition and adsorption mechanism of retention. All the models were verified for different experimental data by four criteria: the sum of squared differences between the experimental and theoretical data; the approximation of the standard deviation; the Fisher test; and the F-test ratio.

The D-Diastereomer of ShK Toxin Selectively Blocks Voltage-gated K+ Channels and Inhibits T Lymphocyte Proliferation

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which are crucial in the activation of human effector memory T cells (T(EM)); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. The critical motif on the toxin for potassium channel blockade consists of neighboring lysine and tyrosine residues. Because this motif is sufficient for activity, an ShK analogue was designed based on D-amino acids. D-allo-ShK has a structure essentially identical with that of ShK and is resistant to proteolysis. It blocked Kv1.3 with K(d) 36 nm (2,800-fold lower affinity than ShK), was 2-fold selective for Kv1.3 over Kv1.1, and was inactive against other K(+) channels tested. D-allo-ShK inhibited human T(EM) cell proliferation at 100-fold higher concentration than ShK. Its circulating half-life was only slightly longer than that of ShK, implying that renal clearance is the major determinant of its plasma levels. D-allo-ShK did not bind to the closed state of the channel, unlike ShK. Models of D-allo-ShK bound to Kv1.3 show that it can block the pore as effectively as ShK but makes different interactions with the vestibule, some of which are less favorable than for native ShK. The finding that an all-D analogue of a polypeptide toxin retains biological activity and selectivity is highly unusual. Being resistant to proteolysis and nonantigenic, this analogue should be useful in K(+) channel studies; all-d analogues with improved Kv1.3 potency and specificity may have therapeutic advantages.

Reduction of silanophilic interactions in liquid chromatography with the use of ionic liquids

A suppression of silanophilic interactions by the selected ionic liquids added to the mobile phase in thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) is reported. Acetonitrile was used as the eluent, alone or with various concentrations of water and phosphoric buffer pH 3. Selectivity of the normal (NP) and the reversed (RP) stationary phase material was examined using a series of proton-acceptor basic drugs analytes. The ionic liquids studied appeared to significantly affect analyte retention in NP-TLC, RP-TLC and RP-HPLC systems tested. Consequently, the increased separation selectivity was attained. Due to ionic liquid additives to eluent even analytes could be chromatographed, which were not eluted from the silica-based stationary phase materials with 100% of acetonitrile in the mobile phase. Addition of ionic liquid already in very small concentration (0.5%, v/v) could reduce the amount of acetonitrile used during the optimization of basic analytes separations in TLC and HPLC systems. Moreover, the influence of temperature on the separation of basic analytes was demonstrated and considered in practical HPLC method development.

Macrocycleversus podant-type neutral ionophore in potentiometric detection of mucolytic agents following separation by various HPLC modes

Potentiometric detection of lipophilic drugs such as clenbuterol, ambroxol and bromhexine in standard-bore cyano and narrow-bore hybrid, reversed-phase HPLC systems was developed to enable specific determination of these mucolytic agents in some marketed oral-formulations. The macrocycle and podant-type, neutral ionophores, respectively: hexakis(2,3,6-tri-O-octyl)-α-cyclodextrin (CDX) andN,N,N′,N′-tetracyclohexyl-oxybis(o-phenyleneoxy)diacetamide (TOPA), were used to construct PVC-based, liquid membrane electrodes. In acidic aqueous-acetonitrile, mobile phases both ionophores improved the sensitivity of these electrodes for mucolytic agents when compared to single-wavelength UV detection—as well as with the ionophore-free based electrode. In the case of bromhexine, indicating highest lipophilicity (logP6.40), detection limits of 2.6×1010 and 1.4×1010M (injected concentration) were achieved with a signal-to-noise (S/N) ratio of 3 with the TOPA-modified, liquid membrane electrode in the various RP-HPLC systems. The results of procedures validated for simultaneous potentiometric determination of clenbuterol and ambroxol in tablets marketed in different countries are also presented.

Potentiometric detection of exogenic beta-adrenergic substances in liquid chromatography

Potentiometric detection with poly(vinyl chloride) (PVC) based liquid membrane electrode coatings is presented for a series of 18 exogenic β-adrenergic substances in cation-exchange-HPLC and RP-HPLC systems. In both types of HPLC modes employing hybrid polymer-silica packings we observed that use of tetrakis( p-chlorophenyl)borate (TCPB) containing electrodes yielded limits of detection (DL) down to 10 −7–10 −8 M (injected concentrations). The use of eluents with high concentrations of acetonitrile (up to 55%) yielded detection limits down to 10 −9 M (injected concentrations). A quantitative structure-potentiometric response activity relationship (QSAR) was developed for the set of β-adrenergic substances and for a set of PVC-based electrodes using TCPB alone or in admixture with trioctylated α-cyclodextrin, dibenzo-18-crown-6, or calix[6]arene hexaethylester. A multiple linear regression model based on a computationally derived set of 14 molecular descriptors allowed prediction of the detection limits of β-adrenergic substances and other amine substances from their molecular structure.

Quantitative structure-retention relationships of xanthines in RP HPLC systems with the new Chromolith RP-18e stationary phases.

The structural descriptors with the best potential for predicting the reversed-phase retention of xanthines and their derivatives on the new Chromolith RP-18e columns have been examined, as has the molecular mechanism of the separation. QSRR analysis was performed by simultaneous solution of a set of linear equations. The logarithms of the capacity factors were the dependent variables. Structural descriptors calculated by means of semi-empirical methods of quantum chemistry were chosen as independent variables.

Integrated acquisition of analytical and biopharmaceutical screening data for beta-adrenergic-drugs employing diversified macrocycle supported potentiometric detection in HPLC systems.

Potentiometric detection with poly(vinyl chloride) (PVC) based liquid membrane electrode coatings is presented for a series of eighteen beta-adrenoceptor binding drugs (five agonists and thirteen antagonists) in cation exchange-HPLC and RP-HPLC systems. Incorporation of lipophilic cation-exchanger tetrakis(p-chlorophenyl)borate (TCPB) alone or in combination with trioctylated alpha-cyclodextrin into the polymeric liquid membrane gives very sensitive responses for racemic forms of bufuralol, propranolol, carazolol, clenbuterol, mabuterol, cimaterol, bisoprolol, oxprenolol, alprenolol, tertatolol, and bevantolol, especially in the cation-exchange HPLC system applying acetonitrile -- 40 mM phosphoric acid (15: 85, v/v, pH* = 2.35) as the mobile phase. In both applied orthogonal HPLC modes we observed that use of TCPB containing electrodes (no addition of neutral macrocyclic ionophores) gives more than five fold improvement in limit of detection down to 10(-7) M for mabuterol, bufuralol, alprenolol and tertatolol in comparison with UV detection. These results suggest that potentiometric detection, especially in RP-HPLC employing hybrid polymer-silica packings, can be considered as the promising alternative in the high-throughput drug abuse or doping control procedures of investigated beta-adrenergic agonists and beta-adrenolytics in humans and animals. The quantitative structure - potentiometric response relationships were developed for a set of eighteen beta-adrenenergic drugs and a set of PVC based electrodes using TCPB alone or in admixture with trioctylated alpha-cyclodextrin, dibenzo-18-crown-6 or calix[6]arene hexaethylacetate ester. A multiple linear regression model based on computationally derived set of molecular descriptors was used to predict detection limits of beta-blocking agents and beta-adrenergic agonists from their molecular structure in the developed potentiometric detectors. Principal components analysis (PCA) of data considering determined potentiometric detection limits revealed that it can be used to establish a reliable pharmacological classification of compounds with beta-adrenoceptor activity, especially for the differentiation of cardioselective and non-cardioselective beta1-antagonists.

HPLC enantioseparation of amino acids and their protected derivatives used in peptide synthesis with pre-column chiral derivatization

Indirect chiral separation methods based on enantiomeric derivatizations were developed in order to monitor optical purity of uncoded amino acids and a new series of amino acid derivatives. Marfey's reagent was used for derivatization of amino groups: whilst boxyl groups were derivatised with (1R, 2R)-or (1S, 2S)-2-amino-1(4-nitrophenyl)-1,3-propanediol reagents were used, respectively. The separations of diastereomeric derivatives formed via derivatization were optimized in RP-HPLC and NP-HPLC systems.

Determination of solute lipophilicity, as log P(octanol) and log P(alkane) using poly(styrene–divinylbenzene) and immobilised artificial membrane stationary phases in reversed-phase high-performance liquid chromatography

A number of RP–HPLC systems have been characterized by the linear free energy relationship: (i) log SP=c+r.R 2+s.π H 2+a.∑α H 2+b.∑β 2+v.V x Here, SP is either log k′ or log k w for a series of solutes in a given system, where k′ is the capacity factor and k w is the capacity factor extrapolated to l00% water, and the solute descriptors are, R 2 an excess molar refraction, π 2 H the dipolarity/polarizability, ∑ α 2 H and ∑ β 2 the overall or effective hydrogen-bond acidity and basicity, and V x the McGowan characteristic volume. Comparison of the coefficients in Eq. (1)with those for water-solvent partitions confirms that the modified electrostatically coated C 18 phase of Pagliara et al. (J. Liq. Chromatogr., 18 (1995) 1721) can be used to obtain solute lipophilicities, as log P oct. For RP–HPLC systems based on poly(styrene–divinylbenzene), the coefficients in Eq. (i)are nearer those for the correlation of water–alkane partition coefficients, as log P alk, than for the correlation of log P oct, suggesting that the RP–HPLC systems with poly(styrene–divinylbenzene) phases could be used as a rapid method for determination of solute lipophilicity, as log P alk or as log P cyc, where the latter is the water–cyclohexane partition coefficient. Eq. (i)has also been applied to RP–HPLC log k′ values obtained with an immobilized artificial membrane (IAM) phase. A good regression equation was obtained, but the coefficients in this equation are substantially different to those for regressions with log P oct, log P alk, or log P cyc as the dependent variable. On the other hand, log k′ values from the RP–HPLC system of Miyake al. [J. Chromatogr., 389 (1987) 47], consisting of silica gel coated with dipalmitoyl phosphatidyl choline as a stationary phase, with aqueous acetonitrile mobile phases, yielded coefficients in Eq. (i)very similar to those for log P oct.

RPHPLC analysis of some nitrophenols

Problem of nitrophenols group analysis as well as separation to individuals is still attracting attention of research and practicing chemists, environmental chemists and the others. Chromatographic behaviour in RPHPLC systems expressed by such parameters as retention time, sorption capacity, selectivity and/or elution order was studied for group of 8 nitrophenols: 2,4,6-trinitrophenol, 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenol and 4-nitro-3-cresol. Condition for baseline separation of almost all of these nitrophenols on Separon SGX C18 were chosen. Sorption capacities were tested for 4 different RP materials. The data will be used for devising of Solid Phase Extraction (SPE) and/or column switching schema in RPHPLC sample pretreatment.