Robust HPLC-MS/MS method for levofloxacin and ciprofloxacin determination in human prostate tissue.

Fluoroquinolones are the drugs of choice in the prevention of bacterial infections after transrectal ultrasound guided prostate biopsy. In order to improve assessment of antibacterial efficacy in the target tissue a simple, selective, rapid and robust HPLC-ESI-MS/MS method for the determination of levofloxacin and ciprofloxacin concentrations in human prostate bioptates was developed and validated. Preparation procedure for prostate samples (10mg) was carried out using homogenization and filtration steps. Analyses were performed within 3.5min using RP C18 column in the isocratic elution mode with mobile phase composed of a mixture of 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution (v/v; 79:21). The method was linear between 0.3µg/g and 15µg/g for levofloxacin and ciprofloxacin with coefficient of correlation (r) ≥0.999. The limit of detection and the limit of quantification for levofloxacin were 0.06µg/g and 0.2µg/g and for ciprofloxacin were 0.04µg/g and 0.13µg/g, respectively. Average concentrations (±SD) of levofloxacin and ciprofloxacin obtained from patients tissue were 5.4±2.2µg/g and 3.9±1.5µg/g, respectively. Additionally, during validation procedure a novel, experimental design approach was applied for the robustness study. For evaluation of analytical method robustness, Plackett-Burman design was employed and for sample preparation method robustness Fractional Factorial design was used. The developed and validated method was successfully applied to examine prostate tissue samples obtained from patients enrolled into a clinical study. Up to now, there has been no other HPLC-ESI-MS/MS method reported for the simultaneous determination of levofloxacin and ciprofloxacin in human prostatic tissue.

Ultra-high performance liquid chromatographic determination of levofloxacin in human plasma and prostate tissue with use of experimental design optimization procedures.

Fluoroquinolones are considered as gold standard for the prevention of bacterial infections after transrectal ultrasound guided prostate biopsy. However, recent studies reported that fluoroquinolone- resistant bacterial strains are responsible for gradually increasing number of infections after transrectal prostate biopsy. In daily clinical practice, antibacterial efficacy is evaluated only in vitro, by measuring the reaction of bacteria with an antimicrobial agent in culture media (i.e. calculation of minimal inhibitory concentration). Such approach, however, has no relation to the treated tissue characteristics and might be highly misleading. Thus, the objective of this study was to develop, with the use of Design of Experiments approach, a reliable, specific and sensitive ultra-high performance liquid chromatography- diode array detection method for the quantitative analysis of levofloxacin in plasma and prostate tissue samples obtained from patients undergoing prostate biopsy. Moreover, correlation study between concentrations observed in plasma samples vs prostatic tissue samples was performed, resulting in better understanding, evaluation and optimization of the fluoroquinolone-based antimicrobial prophylaxis during transrectal ultrasound guided prostate biopsy. Box-Behnken design was employed to optimize chromatographic conditions of the isocratic elution program in order to obtain desirable retention time, peak symmetry and resolution of levofloxacine and ciprofloxacine (internal standard) peaks. Fractional Factorial design 2(4-1) with four center points was used for screening of significant factors affecting levofloxacin extraction from the prostatic tissue. Due to the limited number of tissue samples the prostatic sample preparation procedure was further optimized using Central Composite design. Design of Experiments approach was also utilized for evaluation of parameter robustness. The method was found linear over the range of 0.030-10µg/mL for human plasma and 0.300-30µg/g for human prostate tissue samples. The intra-day and inter-day variability for levofloxacine from both plasma and prostate samples were less than 10%, with accuracies between 93 and 108% of the nominal values. The limit of detection and the limit of quantification for human plasma were 0.01µg/mL and 0.03µg/mL, respectively. For the prostate tissue, the limit of detection and the limit of quantification were 0.1µg/g and 0.3µg/g, respectively. The average recoveries of levofloxacin were in the range from 99 to 106%. Also, the method fulfills requirements of robustness what was determined and proved by Design of Experiments. The developed method was successfully applied to examine prostate tissue and plasma samples from 140 hospitalized patients enrolled into the clinical study, 12h after oral administration of LVF at a dose of 500mg. The mean (±SD) LVF concentration in prostate was 6.22±3.52µg/g and in plasma 2.54±1.14µg/mL. Due to simplicity of the method and relative small amount of sample needed for the assay, the method can be applied in clinical practice for monitoring of LVF concentrations in plasma and prostate gland.

Studies of the effect of grasshopper abdominal secretion on wound healing with the use of murine model.

ETHNOPHARMACOLOGICAL RELEVANCE: Grasshopper, belonging to Chorthippus sp., is a widespread insect inhabiting Polish territory. According to folk knowledge and folk tales, the grasshopper abdominal secretion was used by villagers of Central and South-West Poland as a natural drug accelerating the wound healing process. AIM OF THE STUDY: In the reported study the hypothesis about beneficial properties of grasshopper abdominal secretion on hard to heal wounds was verified. MATERIALS AND METHODS: The study was carried out with the use of a murine model (mice C57BL/6). In order to verify the beneficial properties of grasshopper abdominal secretion, the wounds of 8mm in diameter were formed on one side of each tested mouse. The influence of ethanolic extract of insects' secretion on healing process was evaluated in comparison to ethanolic solution of allantoin and 30% aqueous solution of ethanol (medium). The observation was carried out over a 14 day period. Finally the statistical analysis (ANOVA) was carried out to highlight the differences in wound healing rate between applied preparations. Moreover, qualitative composition of grasshoppers' secretion was studied with the use of GC/MS technique. RESULTS: During the first three days of observation, wounds treated with allantoin were healed with higher efficiency in comparison to ethanol and insect secretion preparations. The trend of healing changed from the 4th day of observation. Wounds treated with grasshoppers' abdominal secretion were closuring faster than wounds treated with allantoin or ethanol. In this part of observation, in the case of allantoin and ethanol application, the wound healing efficiency was similar. Since the 9th day of experiment the measurement of wounds size was problematic, due to crust formation. Finally at the 14th day of the study, wounds were totally healed. Morphological study enabled to observe all the phases of healing. In the 5th and 8th day, the infiltration of neutrophils and mononuclear cells in dermis was observed, which is characteristic for inflammatory phase of wound healing. On the 8th day of experiments, granulation of the tissue was clearly observed in the tested groups. Reepithelialization phase was observed from the 5th to 14th day, when the wound was totally healed. The analytical approach enabled to identify 38 compounds of hydrophobic or hydrophilic character. Among them, 6 amino acids, 14 organic acids and their derivatives, one sterol, 4 hydrocarbons, 5 carbohydrates, 2 inorganic acids, 4 alcohols, one diamine and one nucleoside were identified. CONCLUSION: The obtained results enabled to recognize the composition of grasshopper abdominal secretion. Some of the identified compounds possess therapeutic properties described in the literature. The performed in vivo study proved that application of insects secretion accelerates the healing process. The obtained results positively verified the scientific hypothesis based on ethnopharmacological premises about the beneficial properties of grasshopper abdominal secretion on wound healing process.

The simultaneous determination of hydrophobicity and dissociation constant by liquid chromatography-mass spectrometry.

Convenient methods for testing drug candidates' lipophilicity and acidity are highly requested in modern pharmaceutical research and drug development strategies. Reversed-phase high-performance liquid chromatography (RP HPLC) might be particularly useful for the determination of both dissociation constant and the (pH-dependent) partition coefficient related parameters, applicable in high-throughput analysis of multi-component mixtures. The general theory of combined pH/organic modifier gradient has recently provided equations relating gradient retention time and pH of the mobile phase. The purpose of this work was to facilitate the identification of analytes in this technique by its transfer to RP HPLC coupled with time-of-flight mass spectrometry with electrospray ionization source (ESI-TOF-MS). The accuracy of the proposed methodology was assessed by analyzing a set of known drugs. The ammonium formate, ammonium acetate or ammonium bicarbonate buffers were used to control pH during chromatographic analysis. In result, the pKa and hydrophobicity parameters were determined and the accuracy of the estimated values was assessed by comparing them with literature data. The gradient RP HPLC coupled with ESI-TOF-MS methods allowed for the rapid determination of dissociation constant and hydrophobicity and was shown to be especially applicable for complex mixtures. The use of ESI-TOF-MS detection allowed to achieve the medium-throughput screening rate (100 compounds/day) and provided a simple approach to assess pharmacokinetically important physicochemical properties of drugs.

Advanced QSRR modeling of peptides behavior in RPLC.

In QSRR the retention is modeled as a function of structural or molecular descriptors. Since the structural datasets can be very large a selection of informative variables is often required. But beside the question which subset of variables (descriptors) produces optimum predictions one should answer the question: can good prediction be used in the QSRR community even if the physical meaning of applied descriptors is hard to interpret? The main focus in this paper is put on different modeling methodologies applied and molecular descriptors used in the QSRR approaches. Besides the widely used multiple linear regression (MLR), these methodologies include partial least squares (PLS), uninformative variable elimination partial least squares (UVE-PLS), genetic algorithms (GA) prior to MLR or PLS. The comparison will focus on the predictive performance but also on the descriptors found to be most important for the chromatographic retention prediction of peptides. The results of this study showed that stepwise-MLR and UVE-PLS are producing better predictions than the rest of the studied methodologies. From the variables selected by various methodologies one can see that the important information for the retention mechanism of RPLC was given by 2D-, 3D-descriptors and descriptors from the empirical QSRR equations, which bring the information about hydrogen-bonding properties, molecular size, and complexity. Overall, for the considered data set the empirical QSRR models were predicting the peptides retention best.

Chromatographic behaviour of ionic liquid cations in view of quantitative structure-retention relationship.

The availability of ionic liquids (ILs) in wide areas of application often results in the requirement on their determination. The attention is also often focused on the knowledge of hydrophobicity as it plays a key role in the biological effects, in the assessment of environmental risk and in the prediction of the fate of chemicals in the environment and of its influence on retention in RP HPLC. One can get information regarding hydrophobicity and retention mechanism if quantitative structure-retention relationships (QSRRs) are identified. The QSRRs were derived for logarithms of retention factors extrapolated to a pure water (or aqueous buffer) eluent, log k(w), determined for the pyridinium and imidazolium ionic liquid (IL) cations on two C8 (Supelcosil LC-8-DB, Symmetry C8) and two C18 (ACE 5 C18, Symmetry C18) stationary phases with isocratic elution by a mobile phase consisting of acetonitrile/40 mM phosphate buffer. The analyses of ILs were performed at a flow rate of 1 mL min(-1) with UV detection at 218 nm. The QSRRs were derived based on the retention parameters determined experimentally and the structural descriptors of test analytes from molecular modeling. Separations of ILs were obtained with aqueous acetonitrile buffered at pH 3.55 mobile phases. The statistically most significant two-parameter QSRR regression equations related log k(w) to the solvent accessible surface (SAS) of the analytes and the differences in the energies of the highest occupied and the lowest unoccupied molecular orbitals (diffHL). These equations were especially good in case of columns with the highest carbon loads and larger specific surface areas, i.e. Symmetry C18 and Symmetry C8. On the other hand, the column ACE 5 C18 appeared to produce the best quality separations of the ILs studied. The QSRRs derived in the research shed light on the molecular mechanism of HPLC separation of ILs and helped to predict their relative separations.

Testing conception of engagement of imidazoline receptors in imidazoline drugs effects on isolated rat heart atria.

Recently, attention has been payed to the role of imidazolines in physiology of the heart. However, no systematic comparative studies were reported regarding the activity of a representative set of specific ligands towards imidazoline receptors in the heart preparations. The aim of this project was to test effects of a set of ligands on the pharmacological function of putative imidazoline receptors in isolated rat heart atria. Known imidazoline drugs with a postulated high affinity to imidazoline I(1) receptor: AGN192403, rilmenidine, moxonidine and clonidine were used. The specific ligands of imidazoline I(2) receptor: 2-BFI, BU239 and putative natural ligand for imidazoline I(1), I(2) and I(3) receptors, agmatine, were tested also. The spontaneously beating right and left atria, driven electrically, were studied. Dose-response curves for amplitude and rate of the contractions of the atria were produced by administration of increasing doses of the agents. Phentolamine as alpha(1)/alpha(2) adrenergic receptors blocker and idazoxan as I(2)/I(1)/alpha(2) receptors blocker were added in order to inhibit ino- and chronotropic effects of the compounds studied. The -log EC(50) parameters were calculated. The positive inotropic effect on left atria were evoked with the rank order of potency: agmatine >> clonidine > BU239 > rilmenidine > or = moxonidine and these effects were generally diminished by idazoxan. Moxonidine produced a weak positive inotropic effect potentiated by idazoxan. Rilmenidine and moxonidine were assumed to act as partial agonists of imidazoline I(1) receptor. AGN192403 did not change the amplitude of beating of left atria. The positive chronotropic effects on spontaneously beating right heart atria were with in the following order of potency: BU239 > or = agmatine >>> clonidine > AGN192403. Idazoxan markedly antagonized chronotropic effect of both BU239 and agmatine. 2-BFI weakly diminished the rate of beating of atria; moxonidine and rilmenidine had no effect. In conclusion, imidazoline receptors of the I(1) subtype may be involved in inotropic reaction of the agents studied, but this effect depends mainly on the alpha(2)/alpha(1) adrenergic receptors. Engagement of I(2) imidazoline receptors, along with the alpha(2) adrenergic ones, in chronotropic activity of isolated right atria of rat has been demonstrated.

The molecular descriptor logSumAA and its alternatives in QSRR models to predict the retention of peptides.

The use of the experimental molecular descriptor logSum(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, logSum(AA); the logarithm of the peptide's van der Waals volume, logVDW(Vol); and the logarithm of its theoretically calculated n-octanol-water partition coefficient, clogP. 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 logSum(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 logSum(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 logSum(AA), but with a reduced workload.

Increasing conclusiveness of metabonomic studies by chem-informatic preprocessing of capillary electrophoretic data on urinary nucleoside profiles.

Nowadays, bioinformatics offers advanced tools and procedures of data mining aimed at finding consistent patterns or systematic relationships between variables. Numerous metabolites concentrations can readily be determined in a given biological system by high-throughput analytical methods. However, such row analytical data comprise noninformative components due to many disturbances normally occurring in analysis of biological samples. To eliminate those unwanted original analytical data components advanced chemometric data preprocessing methods might be of help. Here, such methods are applied to electrophoretic nucleoside profiles in urine samples of cancer patients and healthy volunteers. The electrophoretic nucleoside profiles were obtained under following conditions: 100 mM borate, 72.5 mM phosphate, 160 mM SDS, pH 6.7; 25 kV voltage, 30 degrees C temperature; untreated fused silica capillary 70 cm effective length, 50 microm I.D. Different most advanced preprocessing tools were applied for baseline correction, denoising and alignment of electrophoretic data. That approach was compared to standard procedure of electrophoretic peak integration. The best results of preprocessing were obtained after application of the so-called correlation optimized warping (COW) to align the data. The principal component analysis (PCA) of preprocessed data provides a clearly better consistency of the nucleoside electrophoretic profiles with health status of subjects than PCA of peak areas of original data (without preprocessing).

Antiaggregatory activity of hypoglycaemic sulphonylureas.

AIMS/HYPOTHESIS: Vascular complications observed in diabetes are often related to altered platelet functions. The most widely used hypoglycaemic drugs for treating Type II (non-insulin-dependent) diabetes mellitus are sulphonylurea derivatives. The purposes of this study were to evaluate the inhibitory effects of hypoglycaemic agents on platelet aggregation, to measure their lipophilicity and identify their structural parameters which assess their antiaggregatory activity. METHODS: An antiaggregatory test in vitro was carried out for 13 sulphonylurea derivatives. Aggregation of platelets, incubated with the agents at concentrations varying from 7.5 to 480 micromol/l, was induced by 10 micromol/l ADP. Drug lipophilicity parameter, log k(w), was measured by gradient HPLC and the agents were subjected to molecular modelling. RESULTS: The most pronounced inhibition of platelet aggregation was by glimepiride, gliclazide, gliquidone, glibenclamide and compound 2A. The IC(25) values were 15.9, 18.6, 20.4, 28.5 and 34.7 micromol/l, respectively. Quantitative structure-activity relationships indicate that antiaggregatory activity is mainly affected by electronic and not by lipophilic properties of the agents. CONCLUSION/INTERPRETATION: Glimepiride appeared to be a more potent ADP-induced platelet aggregation inhibitor in vitro than gliclazide. Antiaggregatory activity was shown for gliquidone and confirmed for glibenclamide. The QSAR analysis supports the hypothesis of a free radical mechanism of action of sulphonylurea derivatives previously suggested for gliclazide.

Quantitative structure/retention relationships in affinity chromatography.

Affinity chromatography (AC) followed by quantitative structure/retention relationships (QSRR) analysis provides information on both the analytes and the macromolecules forming the stationary phases. QSRR equations derived for test series of analytes (often drugs) are interpreted in terms of structural requirements of the specific binding sites on macromolecules. Chromatographically demonstrated differences in analyte/macromolecule interactions may be relevant to molecular pharmacology and rational drug design. Multiple regression analysis of appropriately designed sets of affinity-chromatographic data may help increase the speed and efficiency of search as for new drugs and reduce the need for in vivo screening. Specific high-performance affinity-chromatographic separations can be optimized by rational selection of chiral columns, the characteristics of which are provided by QSRR.

Separation of strength and selectivity of mobile phase by spectral mapping technique.

The retention behaviour of seven monotetrazolium and nine ditetrazolium salts was studied in seven different mobile phases on alumina and impregnated alumina stationary phases. The strength and selectivity of the components of mobile phases were separately calculated by the spectral mapping technique. It was established that tetrahydrofuran (THF) has the highest solvent strength while the differences among ethanol, 1-propanol, 2-propanol and dioxane were relatively low. The selectivity of THF was also considerably different from those of other solvents. Hydrophobicity and electronic parameters were equally involved in the retention strength and selectivity of tetrazolium salts, indicating the mixed character of their interaction with the alumina stationary phase.

Quantitative structure-retention relationships with model analytes as a means of an objective evaluation of chromatographic columns.

The performance of several previously designed model series of test analytes has been tested to characterize in an objective, quantitative manner modern stationary phases for reversed-phase high-performance liquid chromatography (RP-HPLC) using quantitative structure-retention relationships (QSRRs). Three QSRR approaches and three respective series of test analytes recommended for studies of the molecular mechanism of chromatographic retention are employed: the reduced linear solvation energy relationship (LSER)-based model of Abraham, a model employing structural descriptors from molecular modeling, and a model relating retention to the n-octanol-water partition coefficient log P. All of the models and test analytes proposed provide reliable QSRR equations. Those equations discriminate in quantitative terms individual columns and chromatographic systems and can be interpreted in straightforward rational chemical categories. In view of QSRRs, the differences in the intermolecular interactions between a given stationary phase and a structurally defined analyte rationalize the observed differences in retention. The QSRR models (previously derived retrospectively) are demonstrated to work well on new sets of RP-HPLC data. At the same time, it has been confirmed that the three test series of analytes have properly been designed and can be recommended for comparative studies of analytical columns. QSRRs once derived on a given column for model analytes can be used to predict the retention of other analytes of a defined structure. That in turn can facilitate the procedure of the rational optimization of chromatographic separations.

Estimation of buspirone-bovine serum albumin binding by affinity capillary electrophoresis.

Drug-protein binding is an important process in pharmacokinetic phase of drug action. Capillary electrophoresis was employed, specifically the Hummel-Dreyer method and Scatchard analysis, to study the interactions of an anxiolytic drug, buspirone, with pure bovine serum albumin (BSA) and with BSA present in the human recombinant 5-HT(1A) serotonin receptor preparation. The binding constant of buspirone with BSA determined in free BSA solution was K = 5.55 x 10(4) M(-1) whereas its value with BSA present in the serotonin receptor preparation was K = 5.57 x 10(4) M(-1). The method was found to be inadequate for measuring the specific binding interactions between buspirone and the 5-HT(1A) receptor in the preparation employed.

Pharmacological classification of drugs based on neural network processing of molecular modeling data.

The performance of artificial neural network (ANN) models in predicting pharmacological classification of structurally diverse drugs based on their theoretical chemical parameters was demonstrated. The classification coefficients for psychotropic agents, beta-adrenolytic drugs, histamine H(1) receptor antagonists and drugs binding to alpha-adrenoceptors were 100, 100, 95 and 86%, respectively. A set of easily accessible non-empirical molecular parameters describing the structure of xenobiotics can provide information allowing the prediction of some pharmacological properties of drugs and drug candidates employing ANN models. Since ANN analysis can help cluster as well as segregate drugs and drug candidates according to their known and expected pharmacological properties, the number of routine biological assays might be reduced. The results presented here might be used to improve the efficiency of high throughput screening programs for new drug hits by demonstrating a promising procedure for diverse combinatorial library design and evaluation.

Synthesis and hypolipidemic and antiplatelet activities of alpha-asarone isomers in humans (in vitro), mice (in vivo), and rats (in vivo).

A series of alpha-asarone isomers was synthesized and investigated for their hypolipidemic and antiplatelet activity. Considering the hypolipidemic activity in rats at a dose of 80 mg/kg/day, some isomers were more potent than clofibrate at 150 mg/kg. Compound 3 was one of the most active agents elevating the HDL cholesterol level by 56% and lowering the LDL cholesterol level by 46.8% in rats after 7 days of administration. The activities of the platelet aggregation test in vitro were significant but lower than those of the reference substances (indomethacine and acetylsalicylic acid). In the pulmonary thromboembolic in vivo test in mice, two compounds (alpha-asarone (6) and compound 4) produced significant antithrombotic effects at 100 mg/kg, namely 44% and 52% protection against lung microembolia, respectively. alpha-Asarone derivatives form a new group of potential hypolipidemic and/or antithrombotic agents. The compounds 3, 4, and 6 may serve as lead substances whose structural modifications may result in original drugs.

Computer simulation for the simultaneous optimization of any two variables and any chromatographic procedure.

Computer software that allows the simulation of any chromatographic separation as a function of simultaneous changes in any one or two variables that can affect sample separation order (selectivity) is described. For one example, an application is described for the simultaneous variation of the mobile phase pH and gradient time in reversed-phase liquid chromatography. The accuracy of such predictions is examined for a sample mixture of 17 substituted benzoic acids and anilines, and requirements for an acceptable predictive accuracy are summarized. In a second example, the separation of three peptides by capillary electrophoresis is optimized.

Retention of barbituric acid derivatives on immobilized artificial membrane stationary phase and its correlation with biological activity.

A series of 30 barbituric acid derivatives were subjected to high-performance liquid chromatography (HPLC) on the 'immobilized artificial membrane' (IAM) column with acetonitrile buffer mobile phase. The retention parameter log k(IAM) was related to the logarithms of partition coefficients determined in octanol-water partition system, log P, to a thin-layer chromatographic (TLC) parameter from partition TLC, R(m0), to an adsorption HPLC retention parameter, log k(0), and to a solubility parameter, delta. It was demonstrated that log k(IAM) correlated significantly to the other parameters of barbiturates determined in partition systems but not to delta. However, log k(IAM) appeared to be a distinctive descriptor of hydrophobicity of barbiturates as compared to the standard log P parameters. The parameter log k(IAM) was shown to correlate with bioactivity data of the agents studied.

Molecular mechanism of retention in reversed-phase high-performance liquid chromatography and classification of modern stationary phases by using quantitative structure-retention relationships.

Quantitative structure-retention relationships (QSRRs) were derived for logarithms of retention factors normalised to a hypothetical zero percent organic modifier eluent, log kw, determined on 18 reversed-phase high-performance liquid chromatography (RP-HPLC) columns for 25 carefully designed, structurally diverse test analytes. The study was aimed at elucidating molecular mechanism of retention and at finding an objective manner of quantitative comparison of retention properties and classification of modern stationary phases for RP-HPLC. Three QSRR approaches were employed: (i) relating log kw to logarithms of octanol-water partition coefficient (log P); (ii) describing log kw in terms of linear solvation-energy relationship-based parameters of Abraham; (iii) regressing log kw against simple structural descriptors acquired by calculation chemistry. All the approaches produced statistically significant and physically interpretable QSRRs. By means of QSRRs the stationary phase materials were classified according to the prevailing intermolecular interactions in the separation process. Hydrophobic properties of the columns tested were parametrized. Abilities of individual phases to provide contributions to the overall retention due to non-polar London-type intermolecular interactions were quantified. Measures of hydrogen-bond donor activity and dipolarity of stationary phases are proposed along with two other phase polarity parameters. The parameters proposed quantitatively characterize the RP-HPLC stationary phases and provide a rational explanation for the differences in retention patterns of individual columns observed when applying the conventional empirical testing methods.

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time III. Improving the accuracy of computer simulation.

Previous studies have shown that four experimental runs, where both temperature T and gradient time tG are varied, can be used for the reliable prediction of separation as a function of these two variables (two-dimensional optimization). Computer simulation (e.g., DryLab) can then be used to predict "optimized" conditions for maximum sample resolution using either isocratic or gradient elution. Samples that contain a large number of components (e.g., n>15-20) present a greater challenge. Resolution for these more complex samples is often quite sensitive to small changes in T or tG in turn requiring greater accuracy in predictions that result from computer simulation. In the present study of several samples, we have examined computer simulation errors that can arise from inexact expressions for retention time as a function of T, tG or isocratic %B. Resulting conclusions are applicable to both complex and simpler samples, in either one- or two-dimensional optimization. Means to anticipate and minimize the impact of these predictive errors are examined.