Enantiomeric HPLC resolution and absolute stereochemistry assignment of a new poligamain derivative.

A new aryltetralin lignan derivative, 1, was obtained by reacting dimethyl succinate and piperonal, furnishing the lactone 4-(3',4'-methylenedioxybenzyl)-4,5-dihydro-2(3H)-furanone, which was reacted once again with piperonal and LDA to give the dibenzylbutirolactone 7-hydroxyhinokinin. The cyclization of 7-hydroxyhinokinin into polygamain occurred in the presence of trifluoroacetic acid. The reduction of the furanic ring of polygamain was done by its reaction with DIBAL in THF, furnishing the diol functionalized lignin derivative 1 as single diastereomer. The enantiomeric fractions of 1 were obtained by preparative enantioselective HPLC. The absolute stereochemistry was assigned by electronic circular dichroism (ECD) and nuclear magnetic resonance (NMR) spectroscopy. An all-trans relative configuration was determined by NMR on the bases of ¹H coupling constants and nuclear Overhauser effect (n.O.e.) experiments. The absolute configuration at C1 was assigned on the basis of the ECD sign at 296 nm by comparison to the ECD spectra of structural analogues with defined stereochemistry. The assignment of the absolute configuration was confirmed by applying the exciton chirality method to the well-defined ECD couplets at 285 and 200 nm allied to the two electronic transitions L(b) and B(b) of the aromatic moieties, respectively. Rac-1 and its enantiomeric isomers were evaluated against important bacteria responsible for dental caries. The best results obtained for the (1R,2S,3S) isomer were against Streptococcus mutans (250 µM), Streptococcus salivarius (250 µM), Streptococcus sobrinus (280 µM) and Streptococcus mitis (280 µM). The (1S,2R,3R) isomer was active only against Streptococcus sanguinis (280 µM). The enantiomeric mixture was less active than the (1R,2S,3S) isomer.

A fast and validated mass spectrometry method for the evaluation of human serum albumin structural modifications in the clinical field.

A relatively fast analytical method for the identification and quantification of the post-transcriptional changes (PTCs) occurring in circulating human serum albumin (HSA) was developed. HSA is the most abundant protein in plasma and it represents the main determinant of plasma oncotic pressure, thus being the main modulator of fluid distribution between body compartments. Cirrhotic patients have low levels of HSA. Moreover, recent studies have demonstrated that during liver cirrhosis HSA presents PTCs affecting its properties. The HSA isoforms derived from these modifications could represent promising biomarkers for liver disease. Human plasma samples were collected from a cirrhotic patient (CH) and from an aged-matched non- cirrhotic subject (CT), purified by reverse-phase chromatography and analysed by an electrospray ionization quadrupole time-of-flight (ESI-Q-ToF) spectrometer. The deconvoluted ESI mass spectra from healthy subjects were all characterized by peaks attributed to mercaptoalbumin, nitrosylated, cysteinylated, glycated and N- terminal truncated HSA isoforms. The relative abundance of each isoform was derived and transformed into a relative per cent amount and the results were compared to those obtained analysing HSA from a CH plasma. The method was validated in terms of intra-day and inter-day reproducibility, both for quantitative results and PTCs molecular weight determination. The optimized method resulted in being effective in disclosing changes in HSA isoforms relative abundance and then it could be used for the systematic screening of cirrhotic patients to identify promising new biomarkers for liver diseases.

Effects of a new potent analog of tocainide on hNav1.7 sodium channels and in vivo neuropathic pain models.

The role of voltage-gated sodium channels in the transmission of neuropathic pain is well recognized. For instance, genetic evidence recently indicate that the human Nav1.7 sodium channel subtype plays a crucial role in the ability to perceive pain sensation and may represent an important target for analgesic/anti-hyperalgesic drugs. In this study a newly synthesized tocainide congener, named NeP1, was tested in vitro on recombinant hNav1.4 and hNav1.7 channels using patch-clamp technique and, in vivo, in two rat models of persistent neuropathic pain obtained either by chronic constriction injury of the sciatic nerve or by oxaliplatin treatment. NeP1 efficiently blocked hNav1.4 and hNav1.7 channels in a dose- and use-dependent manner, being by far more potent than tocainide. Importantly, the new compound displayed a remarkable use-dependent effect, which likely resulted from a very high affinity for inactivated compared to closed channels. In both models of neuropathic pain, NeP1 was greatly more potent than tocainide in reverting the reduction of pain threshold in vivo. In oxaliplatin-treated rats, NeP1 even produced greater and more durable anti-hyperalgesia than the reference drug tramadol. In addition, in vivo and in vitro studies suggest a better toxicological and pharmacokinetic profile for NeP1 compared to tocainide. Overall, these results indicate NeP1 as a new promising lead compound for further development in the treatment of chronic pain of neuropathic origin.

The magnetic memory of Titan's ionized atmosphere.

After 3 years and 31 close flybys of Titan by the Cassini Orbiter, Titan was finally observed in the shocked solar wind, outside of Saturn's magnetosphere. These observations revealed that Titan's flow-induced magnetosphere was populated by "fossil" fields originating from Saturn, to which the satellite was exposed before its excursion through the magnetopause. In addition, strong magnetic shear observed at the edge of Titan's induced magnetosphere suggests that reconnection may have been involved in the replacement of the fossil fields by the interplanetary magnetic field.

Methotrexate determination in pharmaceuticals by enantioselective HPLC.

A simple, sensitive and selective high performance liquid chromatographic method with UV detection for the chiral separation of racemic methotrexate (rac-Mtx) and enantiomeric purity of L-methotrexate in pharmaceutical formulations was developed and validated. The chiral separation was optimized studying both the nature of the stationary phase by using Chirobiotic T, Chiracel OJ and human serum albumin columns and the effect of the mobile phase composition. The best results in terms of enantioresolution and enantioselectivity were achieved with a polar organic mobile phase on Chirobiotic T stationary phase. Essential steps in method validation such as precision, accuracy, suitability and stability were studied according to ICH guidelines. At wavelength 303 nm, the limit of detection (S/N=3) was found to be 0.9 microg/ml for rac-Mtx. The separation of D-Mtx at 0.2% (w/w) level (as limit of quantitation) from the main drug L-Mtx was successfully obtained with 1.72 enantioresolution value. Enantiomeric purity of L-Mtx was determined in pharmaceutical formulations (tablets and injections) with inter- and intra-days relative standard deviation < or = 1.6%. Under the validated stereoselective HPLC conditions for methotrexate, folic acid was also analysed.

Cassini magnetometer observations during Saturn orbit insertion.

Cassini's successful orbit insertion has provided the first examination of Saturn's magnetosphere in 23 years, revealing a dynamic plasma and magnetic environment on short and long time scales. There has been no noticeable change in the internal magnetic field, either in its strength or its near-alignment with the rotation axis. However, the external magnetic field is different compared with past spacecraft observations. The current sheet within the magnetosphere is thinner and more extended, and we observed small diamagnetic cavities and ion cyclotron waves of types that were not reported before.

Stereoselective determination of allethrin by two-dimensional achiral/chiral liquid chromatography with ultraviolet/circular dichroism detection.

A two-dimensional achiral/chiral HPLC method with circular dichroism (CD) detection was optimized for the stereochemical resolution and determination of the elution order of the eight stereoisomers of synthetic allethrin. A monolithic silica HPLC column (Chromolith, Merck, 100 mm x 4.6 mm i.d.) was put orthogonally to an enantioselective OJ Daicel column (250 mm x 4.6 mm i.d.) by means of a switching valve. The resolution of cis and trans diastereoisomers on the silica column was obtained by using a mobile phase consisting of n-hexane:tert-butyl methyl ether (96:4) (v/v) at a flow rate of 1 ml min(-1). The cis and trans peaks were then switched to the enantioselective OJ column separately in two subsequent injections. The resolution of the four trans stereoisomers was accomplished by using n-hexane:tert-butyl methyl ether (90:10) (v/v), while the mobile phase composition for the four cis stereoisomers consisted of n-hexane:isopropanol (99.3:0.7) (v/v). The CD based detection system allowed the determination of the elution order on the basis of the CD signals of the single stereoisomers, together with the injection of pure stereoisomers. Under the final conditions, the validated method was applied to the determination of stereoisomeric composition and absolute configuration of the prevailing stereoisomers of real samples, i.e. commercial batches of different sources of d-allethrin.

Drug affinity to immobilized target bio-polymers by high-performance liquid chromatography and capillary electrophoresis.

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.

Analytical methods for the determination of folic acid in a polymeric micellar carrier.

Amphiphilic copolymers have been the object of growing scientific interest due to their ability to form polymeric micelles in aqueous environments entrapping lipophilic drugs in their inner core. In this study, polyvinylalcohol substituted with oleic acid was employed as an amphiphilic micellar carrier for folic acid (FA), a model drug similar for its chemical-physical characteristics to methotrexate. In order to investigate the stability of the polymeric micelles, the drug incorporation and the kinetic aspects of drug release from these systems, selective analytical methods are required. The development of three analytical methods suitable for selectively identifying and reliably determining FA contained in the micelles and in the delivery systems is reported. UV derivative (first and second order) spectrophotometry was first applied to the aqueous solution of the FA containing micelles obtained at pH 9.0 and provided a characteristic spectral profiling with sharp peaks, related to the analyte, whose amplitude was used for quantitative application. A second approach involved a solid phase extraction (strong anion exchanger), which provided an effective clean up of the FA micelles solution, allowing accurate analysis to be performed also by a conventional spectrophotometric method. A RP-HPLC method, selectively supplying the FA separation from the micelles' components, was then used as a reference method to determine the accuracy of the spectrophotometric methods. These methods were applied to various micelle composition and to the delivery system study.

Determination of the dissociation constants (pKa) of basic acetylcholinesterase inhibitors by reversed-phase liquid chromatography.

An RP-HPLC study for the pKa determination of a series of basic compounds related to caproctamine, a dibenzylaminediamide reversible inhibitor of acetylcholinesterase, is reported. The 2-substituted analogues, bearing substituents with different electronegativity, were analysed by RP-HPLC by using C18 C4 stationary phases with a mobile phase consisting of mixture of acetonitrile and triethylamine phosphate buffer (pH range comprised between 4 and 10). Typical sigmoidal curves were obtained, showing the dependence of the capacity factors upon pH. In general, the retention of the investigated basic analytes increased with increasing of the pH. The inflection point of the pH sigmoidal dependence was used for the dissociation constant determination at a fixed acetonitrile percentage. When plotting pKa vs. percent of acetonitrile in the mobile phase for two representative compounds, linear regression were obtained: the y intercept gave the aqueous pKa(w). The pKa estimation by HPLC method was found to be useful to underline the difference of benzylamine basicity produced by the ortho aromatic substituents. The variation of pKa values (6.15-7.80) within the series of compounds was correlated with the electronic properties of the ortho-substituents through the Hammett sigma parameter, whereas the ability of substituents to accept H-bond was found to play a role in determining the conformational behavior of the molecules.

Cytotoxic and cytostatic effects induced by 4-hydroxynonenal in human osteosarcoma cells.

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Furthermore, numerous results demonstrate that lipid peroxidation products affect central biochemical pathways and intracellular signalling at physiological concentrations. 4-Hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. This work has focused on the evaluation of HNE nuclear content, so far never directly measured, by electrospray-ionization-mass-spectrometry (ESI/MS) and on the correlation between its concentration and the induced effects after exogenous administration. In a human osteosarcoma cell line (SaOS2), HNE exhibited an early cytotoxic effect characterized by apoptosis, cytostatic and differentiating effects characterized by slow growth, increase in alkaline phosphatase (ALP), and alpha5 integrin subunit content with decrease in tumorigenicity.

Stereoselective binding of 2-(4-biphenylyl)-3-substituted-3-hydroxypropionic acids on an immobilised human serum albumin chiral stationary phase.

A series of 2-(4-biphenylyl)-3,3'-hydroxy-substituted phenyl propionic acid, with anti-inflammatory properties, bearing two chiral centres, were studied by HPLC upon HSA-CSP (human serum albumin-based chiral stationary phase). The compounds were analysed in their stereoisomeric erythro and threo forms. The study involved the enantioselective analysis on HSA-CSP, the determination of the racemate lipophilicity (log k'(w)), a QSRR (quantitative structure-retention relationship) analysis and CD study for the assessment of the absolute configuration of the most retained enantiomer. Lipophilicity was found to be an important factor affecting the affinity of the compounds for the HSA stationary phase, but electronic properties seemed to play a role. The position of the substituent of the phenyl group on carbon 3 was found important to modulate stereoselective interaction, the highest value of enantioselectivities being found for the erythro ortho-substituted phenyl derivatives. The previously proposed two steps mechanism of enantiodiscrimination for cyclohexylphenyl substituted derivatives was confirmed for this series of derivatives bearing the biphenylyl moiety.

Use of an immobilised human serum albumin HPLC column as a probe of drug-protein interactions: the reversible binding of valproate.

The reversible binding of valproate to human serum albumin determines a decrease of the binding of ligands that selectively bind to site I, site II, and bilirubin binding site. The binding inhibition was followed by displacement chromatography methodology using increasing concentrations of the competitor, i.e. valproate, in the mobile phase. Significant binding inhibition was observed for drugs binding at site I and site II. The greater displacement was observed for the more retained enantiomer of benzodiazepines and profens. A reduction of the affinity was observed also in the case of phenol red, this compound being selected as representative of bilirubin binding site. Difference circular dichroism spectroscopy was also used to characterise the binding of valproate to human serum albumin. This antiepilectic drug was proved to affect the binding at site I, II, and bilirubin binding site. The data have physiological relevance because significant inhibition of the binding resulted at clinic concentrations of valproate.

Gas chromatography/mass spectrometric assay of endogenous cellular lipid peroxidation products: quantitative analysis of 9- and 10-hydroxystearic acids.

A sensitive, specific, accurate and reproducible gas chromatography/mass spectrometry method was developed for the assay of 9- and 10-hydroxystearic acids in samples obtained as cell extracts. The preparation of the samples required specific procedures to allow the analysis of both the free and the conjugated hydroxy acids as the corresponding methyl esters. The quantification used propyl-paraben as the internal standard and monitoring of a specific fragment of each isomeric hydroxy acid methyl ester, and allowed quantification of the conjugate and the free fractions of both 9- and 10-hydroxystearic acids. This method is suitable for identification and quantification (LOQ 1.8 and 4.4 ng, respectively) of these important metabolites of lipid peroxidation. In particular the development of an assay for the free 9-hydroxystearic acid methyl ester makes the method a reliable analytical tool for investigations of the role of this metabolite in the mechanisms of tumour cell proliferation.

Evidences of cyclodextrin-mediated enantioselective photodegradation of rac-nicardipine by capillary electrophoresis.

Capillary electrophoresis (CE) was applied to photostability studies on rac-nicardipine, a dihydropyridine chiral drug. CE methods were developed able to provide the enantioresolution of drug and its separation from the photodegradation products. Enantioresolution was achieved using 5% sulfated-beta-cyclodextrin (S-beta-CD) as chiral selector in 20 mM triethanolammonium phosphate solution (pH 3). The photostability studies were carried out on inclusion complexes of rac-nicardipine with beta-cyclodextrin (beta-CD) and (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CD) in aqueous solutions (pH 7.4 and 5). The CE analysis of the solutions exposed to UV-A and UV-B radiations showed a photoprotective effect by beta-CD; conversely, HP-beta-CD proved to favor the drug photodegradation. Moreover, evidences for CDs-mediated stereoselective photodegradation of rac-nicardipine were obtained. In fact, two distinct photodegradation profiles were observed for the nicardipine enantiomers in the presence of the CDs. The photodegradation was found to follow an apparent first-order kinetics and two different kinetic constants (k) were obtained for the two enantiomers. After exposure to UV-A and UV-B radiations, the solutions contained residual nicardipine with a significant change in the enantiomeric ratio; this effect was depending on the CD used for the inclusion complexation.

Enantioselective inhibition of the binding of rac-profens to human serum albumin induced by lithocholate.

The reversible binding of lithocholate to human serum albumin determines a decrease of the binding of rac-ketoprofen. The process was followed by displacement chromatography using increasing concentrations of the competitor, i.e., lithocholate, in the mobile phase. The inhibition of rac-ketoprofen binding resulting was enantioselective and greater displacement was observed for the (S) enantiomer. The displacement process resulting was competitive in nature, the two enantiomers of ketoprofen binding to the same binding site as the modifier. The investigation was extended to other nonsteroidal antiinflammatory drugs. The enantioselective binding inhibition was larger in the case of rac-naproxen and rac-suprofen with respect to the phenomenon observed in the case of rac-ketoprofen. The difference in circular dichroism spectroscopy was also used to characterize the binding of lithocholate to human serum albumin. This bile acid was proven to bind to site II on human serum albumin. The results, as obtained by displacement chromatography and difference circular dichroism spectroscopy, strongly support the hypothesized role of bile acids in inducing the enantioselective inhibition of ketoprofen binding to human serum albumin in patients suffering from liver diseases.

Binding properties of human albumin modified by covalent binding of penicillin.

Derivatisation of lysine residues in human albumin was performed in vitro by reaction with penicillin G. This modification reaction has been reported to occur in patients treated with high dosages of the antibiotic. The structure of the modified protein was characterised by mass spectrometry and circular dichroism. The number of the lysine residues involved depends on the time of incubation and on the drug/protein molar ratio. The secondary structure of the modified protein does not change significantly with respect to the native protein. Furthermore, the binding properties of the modified albumin were characterised by CD spectroscopy. Phenylbutazone, diazepam and bilirubin, known to bind to specific binding areas, were used as markers. A decrease of the affinity to the high-affinity binding sites was observed after the modification.

Determination of 5-aminosalicylic acid related impurities by micellar electrokinetic chromatography with an ion-pair reagent.

A micellar electrokinetic chromatographic (MEKC) method was developed for the quantification of mesalazine or 5-aminosalicylic acid (5-ASA) and its major impurities 3-aminosalicylic acid, salicylic acid, sulfanilic acid and 4-aminophenol. The optimisation of the experimental conditions was carried out considering some important requirements: resolution, reproducibility, detection limits of 0.1% (m/m) or less, short total analysis time. Preliminary investigations employing sodium dodecyl sulfate (SDS) as surfactant did not lead to the necessary resolution of the studied compounds; the addition of tetrabutylammonium bromide (TBAB) to the SDS micellar system resulted in the complete separation of all the compounds. The effects on the separation by several parameters such as TBAB concentration, SDS concentration, background electrolyte pH and concentration, were evaluated. Using a fused-silica capillary (8.5 cm effective length) complete analysis was obtained in a very short time. Under the optimised final conditions [120 mM 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer, pH 10.20, 65 mM SDS in the presence of 55 mM TBAB and 5% methanol] the method was validated for specificity, precision, linearity, limits of detection and quantitation, and robustness: the 5-ASA related impurities can be quantified at least at the 0.1% (m/m) level.

Properties of a stationary phase based on immobilised chicken liver basic fatty acid-binding protein.

The fatty acid-binding proteins (FABPs) are a class of low-molecular-mass proteins that bind fatty acids and are thought to be involved in their intracellular transport. FABPs have been isolated and studied from several tissues, but their precise function and mechanism of action are still not clear. Chicken liver (basic) fatty acid-binding protein (bFABP) was immobilised on aminopropyl silica and the developed stationary phase was used to examine the enantioselective properties of this protein and to study the binding of drugs to bFABP. The retention and enantioselectivity of the new column for a large number of chiral drugs was investigated. The enantiomers of basic and neutral compounds were poorly retained and not resolved by the bFABP column. On the contrary the resolution of the enantiomers of some acidic compounds was obtained. Therefore the influence of the mobile phase pH and organic modifier on the chromatographic performance of acidic compounds was studied. In order to clarify the retention mechanism, competitive displacement studies were also carried out by adding short-chain fatty acids to the mobile phase as displacing agents and preliminary quantitative structure-retention relationship correlations were developed to describe the nature of the interactions between the chemical structures of the analytes and the observed chromatographic results.

Addition reactions of aldehydes to lithium enolates of 1,3-dioxolan-4-ones: a configurational reassessment

The results for the addition reactions of chiral lithium (2S)-enolates of 1,3-dioxolan-4-ones to aldehydes and to acetophenone, yielding the corresponding dioxolanone alcohols have been revised. The results reported herein differ from those reported in the literature, both in product distribution and in the stereochemical assignment of the products. In fact, in several cases no stereocontrol was observed at the C5 carbon atom of the lithium enolate. The (2S,5R,1'S)/(2S,5R,1'R) stereochemistry was also reassessed for several dioxolanone alcohols. The major conformers are considered to have an intramolecular hydrogen-bonded five-membered ring structure instead of the six-membered ring structure previously suggested for cyclic dioxolanone alcohols.