Comparison of reversed-phase enantioselective HPLC methods for determining the enantiomeric purity of (S)-omeprazole in the presence of its related substances.

A simple analytical high-performance liquid chromatography (HPLC) method was applied for the enantiomeric excess determination of esomeprazole ((S)-OME), the enantiopure active ingredient contained in drug products, in the presence of its potential organic impurities A-E. The enantioselective separation was accomplished on the immobilized-type Chiralpak ID-3 chiral stationary phase (CSP) under reversed-phase conditions. The results were evaluated and compared with those obtained by the official enantioselective method of European Pharmacopoeia used as the reference for checking the enantiomeric excess of (S)-OME. It has been established that the use of the Chiralpak ID-3 CSP allows the determination of the enantiomeric purity of (S)-OME without any interference coming from its chiral and achiral related substances. The analytical procedure of the drug regulatory agencies based on the AGP CSP suffered instead from poor specificity due to overlap of the peaks pertinent to the achiral impurity A and the chiral impurity (R)-OME (impurity F).

Direct separation of the enantiomers of oxaliplatin on a cellulose-based chiral stationary phase in hydrophilic interaction liquid chromatography mode.

(R,R)-oxaliplatin is an anticancer enantiopure active pharmaceutical ingredient. Little attention has been devoted to the analysis of its enantiomeric composition. The enantioselective HPLC method reported in the current Pharmacopoeias shows clear disadvantages with regard to the low resolution and long elution times. In this work, it has been proven the applicability of a last generation polysaccharide-based chiral stationary phase (CSP), i.e. the Chiralpak IC-3, in the enantioseparation of oxaliplatin. Experimental results demonstrated the benefits arising from the development of enantioselective hydrophilic interaction liquid chromatography (HILIC) based strategies. A baseline separation with resolution factor of 5.8 was achieved using a 100mm×4.6mm I.D. IC-3 column set at the temperature of 40°C and a mobile phase consisting of acetonitrile-water 100:5 mixture. At a flow rate of 1mLmin(-1) the separation was completed within 8min. The optimized method was proven to be sensitive with LOD and LOQ of the enantiomeric impurity of 0.07 and 0.21µgmL(-1), respectively.

Effect of the water content on the retention and enantioselectivity of albendazole and fenbendazole sulfoxides using amylose-based chiral stationary phases in organic-aqueous conditions.

Four commercially available immobilized amylose-derived CSPs (Chiralpak IA-3, Chiralpak ID-3, Chiralpak IE-3 and Chiralpak IF-3) were used in the HPLC analysis of the chiral sulfoxides albendazole (ABZ-SO) and fenbendazole (FBZ-SO) and their in vivo sulfide precursor (ABZ and FBZ) and sulfone metabolite (ABZ-SO2 and FBZ-SO2) under organic-aqueous mode. U-shape retention maps, established by varying the water content in the acetonitrile- and ethanol-water mobile phases, were indicative of two retention mechanisms operating on the same CSP. The dual retention behavior of polysaccharide-based CSPs was exploited to design greener enantioselective and chemoselective separations in a short time frame. The enantiomers of ABZ-SO and FBZ-SO were baseline resolved with water-rich mobile phases (with the main component usually being 50-65% water in acetonitrile) on the IF-3 CSP and ethanol-water 100:5 mixture on the IA-3 and IE-3 CSPs. A simultaneous separation of ABZ (or FBZ), enantiomers of the corresponding sulfoxide and sulfone was achieved on the IA-3 using ethanol-water 100:60 (acetonitrile-water 100:100 for FBZ) as a mobile phase.

Retention behavior of proton pump inhibitors using immobilized polysaccharide-derived chiral stationary phases with organic-aqueous mobile phases.

In the present study, the chromatographic behavior of two immobilized polysaccharide-derived chiral stationary phases (CSPs), the Chiralpak ID-3 and Chiralpak IE-3, under aqueous mobile phases conditions is presented. Four proton pump inhibitors (PPIs) (omeprazole, lansoprazole, pentaprazole and rabeprazole) were selected as test compounds. The effect of the concentration of water in the mobile phase was investigated with respect to its contribution to enantioselectivity and retention. Under acetonitrile-water mobile phase conditions, retention behavior evidenced an interesting pattern. At lower water content, the retention factors decreased with increasing water and at higher water content a reversed trend was observed. These findings support the hypothesis that two retention mechanisms operated successively on the same CSP: the HILIC (with water-poor eluents) and RPLC (with water-rich eluents) modes. The retention factors were minimum in the intermediate region, corresponding to a water concentration of about 20%. Interestingly, the baseline separation of all PPIs investigated was optimized under organic-aqueous mobile phases containing a high water content (from about 50 to 65%). Thus, the dual retention behavior of the PPIs on the Chiralpak ID-3 and Chiralpak IE-3 made it possible to reach greener and harmless enantioselective conditions in a short analysis time.

A survey on illegal and counterfeit medicines for the treatment of erectile dysfunctions in Italy.

INTRODUCTION: In developed countries the phenomenon of pharmaceutical counterfeiting is steadily increasing through the illegal and the Internet market. Medicines for the treatment of erectile dysfunctions containing phosphodiesterase type 5 inhibitors (PDE5) are especially prone to falsification. AIMS: To obtain evidence of the health risks for patients taking these products and to provide useful information to general practitioners and specialists in sexual medicine. METHODS: First the samples were visually inspected and then analyzed to get information about their identity and quality. MAIN OUTCOME MEASURES: A survey on the PDE5 medicines analyzed by the Italian official medicines control laboratory between 2005 and 2011 was performed. All the analyzed medicines were gathered from the Italian illegal market (seizures by police forces) or were bought from illegal online pharmacies. Results. The study revealed that 24% of the analyzed samples were counterfeit and 54% were illegal medicines. In 12% of the cases an intermediate classification (illegal/counterfeit) was assigned. Only 7% of the samples were original. Moreover, the examination of the packaging evidenced potential risks: outer and immediate packaging missing; inconsistency between the carton box and the blister as regards the expiry date and/or the batch number; expiry date or manufacturer's name or country missing. CONCLUSIONS: In 19% of the samples a potential health risk for patients was identified due to either the presence in the sample of more than one undeclared PDE5(s) or an amount of the active ingredient higher than that declared (up to 190% of the maximum dose) or to the presence of potentially dangerous excipients of non-pharmaceutical origin or quality (e.g., gypsum or non-purified talc).

Carprofen analogues as sirtuin inhibitors: enzyme and cellular studies.

The best of both: SIRT1/2 inhibitors were developed by combining chemical features of selisistat (SIRT1-selective inhibitor; blue) and carprofen (anti-inflammatory drug; red). The most potent compound (shown) increased acetyl-p53 and acetyl-α-tubulin levels, and induced slight apoptosis at 50 µM in U937 cells, differently from selisistat and carprofen.

Application of 3 µm particle-based amylose-derived chiral stationary phases for the enantioseparation of potential histone deacetylase inhibitors.

In this work, we report on the difference in performance of the two 3 µm particle-based Chiralpak IA-3 and Chiralpak AD-3 chiral stationary phases (CSPs) in the direct resolution of four racemic cinnamyl 2-aminoanilides, endowed with histone deacetylase inhibitory activity. The 3 µm CSPs were explored to determine if they could provide an effective resolution of enantiomers in presence of alcoholic eluents such as pure methanol, ethanol and 2-propanol. Temperature variable enantioselective HPLC and subsequent van't Hoff analysis were performed. In most of cases the van't Hoff plots were found to show a non-linear behaviour. The knowledge of the enantiomeric elution order associated with the data coming from enantioselective HPLC permitted to advance some hypothesis about the groups involved in chiral recognition mechanism.

A chromatographic study on the exceptional enantioselectivity of cellulose tris(4-methylbenzoate) towards C5-chiral 4,5-dihydro-(1H)-pyrazole derivatives.

A set of ten C5-chiral 4,5-dihydro-(1H)-pyrazole derivatives was synthesized and analyzed by high-performance liquid chromatography (HPLC) on the polysaccharide-based Chiralcel OJ-H chiral stationary phase (CSP). The enantioseparations were carried out using pure ethanol as eluent. Different structural elements of the investigated compounds were recognized for obtaining a very high enantioselectivity. In order to clarify some aspects of the chiral discrimination process, the thermodynamic parameters associated to the enantiorecognition and the enantiomer elution order were established.

Synthesis, stereochemical separation, and biological evaluation of selective inhibitors of human MAO-B: 1-(4-arylthiazol-2-yl)-2-(3-methylcyclohexylidene)hydrazines.

Novel 1-(4-arylthiazol-2-yl)-2-(3-methylcyclohexylidene)hydrazine derivatives have been investigated for their ability to inhibit selectively the activity of the human B isoform of monoamine oxidase. These compounds were obtained as racemates and (R)-enantiomers by a stereoconservative synthetic pattern in high yield and enantiomeric excess. The (S)-enantiomers of the most active derivatives have been separated by enantioselective HPLC. All compounds showed selective activity against hMAO-B with IC(50) ranging between 21.90 and 0.018 microM.

Direct HPLC enantioseparation of omeprazole and its chiral impurities: application to the determination of enantiomeric purity of esomeprazole magnesium trihydrate.

Analytical and semipreparative high-performance liquid chromatography (HPLC) enantioseparation of the proton-pump inhibitor omeprazole (OME) and its potential organic chiral impurities were accomplished on the immobilised-type Chiralpak IA chiral stationary phase (CSP) under both polar organic and normal-phase conditions. The (S)-enantiomers were isolated with a purity of >99% ee and their absolute configuration was empirically assigned by circular dichroism (CD) spectroscopy. A chemo- and enantioselective HPLC method was validated to control the enantiomeric purity of the (S)-enantiomer of OME (ESO), an active ingredient contained in drug products, in the presence of chiral and achiral related substances. The precision, linearity and accuracy of the determination of the (R)-impurity as well as the recovery of ESO from a pharmaceutical preparation were determined. The proposed method uses the mixture methyl tert-butylether (MtBE)-ethyl acetate (EA)-ethanol (EtOH)-diethylamine (DEA) 60:40:5:0.1 (v/v/v/v) as a mobile phase. In these conditions, linearity over the concentration range 0.5-25 microg/ml for (R)-enantiomer was obtained. The limits of detection and quantification were 99 and 333 ng/ml, respectively. The intra and inter-day assay precision was less than 2% (RSD%).

Direct high-performance liquid chromatography enantioseparation of terazosin on an immobilised polysaccharide-based chiral stationary phase under polar organic and reversed-phase conditions.

High-performance liquid chromatography (HPLC) enantioseparation of terazosin (TER) was accomplished on the immobilised-type Chiralpak IC chiral stationary phase (CSP) under both polar organic and reversed-phase modes. A simple analytical method was validated using a mixture of methanol-water-DEA 95:5:0.1 (v/v/v) as a mobile phase. Under reversed-phase conditions good linearities were obtained over the concentration range 8.76-26.28 microg mL(-1) for both enantiomers. The limits of detection and quantification were 10 and 30 ng mL(-1), respectively. The intra- and inter-day assay precision was less than 1.66% (RSD%). The optimised conditions also allowed to resolve chiral and achiral impurities from the enantiomers of TER. The proposed HPLC method supports pharmacological studies on the biological effects of the both forms of TER and analytical investigations of potential drug formulations based on a single enantiomer. At the semipreparative scale, 5.3 mg of racemic sample were resolved with elution times less than 12 min using a mobile phase consisting of methanol-DEA 100:0.1 (v/v) and both enantiomers were isolated with a purity of > or = 99% enantiomeric excess (ee). The absolute configuration of TER enantiomers was assigned by comparison of the measured specific rotations with those reported in the literature.

High performance liquid chromatography-diode array and electrospray-mass spectrometry analysis of vardenafil, sildenafil, tadalafil, testosterone and local anesthetics in cosmetic creams sold on the Internet web sites.

A simple high-performance liquid chromatography (HPLC) method with ultraviolet diode array (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) detection has been developed for the determination of vardenafil, sildenafil, tadalafil, testosterone, procaine, lidocaine, prilocaine, and benzocaine in cosmetic creams sold as promising remedies for male erectile dysfunction and female genitals stimulation. The presence of these substances in commercial cosmetic samples is prohibited. Aliquots (1 g) of the cosmetic creams under investigation were diluted 1:100 in methanol, subjected to ultrasonic treatment, added with benzoic acid as internal standard, and analyzed by HPLC-DAD and HPLC-ESI-MS after a further 1:1000 dilution. The compounds were separated by reversed phase chromatography with water (0.02% trifluoroacetic acid) and acetonitrile gradient elution and detected by UV-DAD at 228, 255 and 290 nm and by ESI-MS positive ionisation mode. Benzoic acid was used as internal standard. Linearity was studied with UV-DAD detection from 2.5-7.8 to 250 microg/g range, depending on the different compounds and with ESI-MS in the 3.3-8.9 to 250 ng/g range. Good determination coefficients (r(2) > or = 0.99) were found in both UV-DAD and ESI-MS. Limits of quantifications ranged between 2.5 and 7.8 microg/g for HPLC-UV-DAD assay and between 3.3 and 8.9 ng/g for HPLC-ESI-MS assay depending on different analyzed substances. At three concentrations spanning the linear dynamic ranges of both UV-DAD and ESI-MS assay, mean recoveries were always higher than 90% for the different analytes and intra-assay and inter-assay precision always better than 15% and 12%. This method was successfully applied to the analysis of substances under investigations present in cosmetic creams, freely sold on the Internet web-sites.

Development and validation of an enantioselective and chemoselective HPLC method using a Chiralpak IA column to simultaneously quantify (R)-(+)- and (S)-(-)-lansoprazole enantiomers and related impurities.

An accurate and reproducible high-performance liquid chromatographic (HPLC) method has been developed and validated for the direct separation of individual enantiomers of lansoprazole, a potent proton pump inhibitor belonging to the family of the substituted benzimidazoles. The enantiomers were resolved on a Chiralpak IA by using a mobile phase consisting of methyl-tert-butyl ether (MtBE)-ethyl acetate (EA)-ethanol (EtOH)-diethylamine (DEA) in the ratio 60:40:5:0.1 (v/v/v/v). Baseline separation of the enantiomers of lansoprazole was obtained with a resolution factor of 8.14. The standard curves for the two enantiomers were linear (r(2)>0.999) in the concentration range of 10-80microg/ml with a working concentration of about 60microg/ml for each enantiomer. Apparent recovery was 100.8% with a relative standard deviation less than 2%. The limit of quantization for each enantiomer of lansoprazole was 0.22microg/ml. The intra-day precisions were in the range of 0.21-0.36 and 0.59-0.66 while the inter-day precisions were in the range of 0.55-1.24 and 0.66-1.19% in terms of retention times and area response RSD% for (R)-(+)- and (S)-(-)-lansoprazole, respectively. The method was also able to resolve impurities from the enantiomers of lansoprazole.

Synthesis, in vitro, and in vivo biological evaluation and molecular docking simulations of chiral alcohol and ether derivatives of the 1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents.

Following our previous research on anti-inflammatory drugs (NSAIDs), we report here the synthesis of chiral 1,5-diarylpyrroles derivatives that were characterized for their in vitro inhibitory effects toward cyclooxygenase (COX) isozymes. Analysis of enzymatic affinity and COX-2 selectivity led us to the selection of one compound (+/-)-10b that was further tested in vitro in the human whole blood (HWB) and in vivo for its anti-inflammatory activity in mice. The affinity data have been rationalized through docking simulations.

Enantioseparation of kavain on Chiralpak IA under normal-phase, polar organic and reversed-phase conditions.

First baseline HPLC enantioseparation of kavain is described. Complete enantiodiscrimination was achieved on the immobilised-type Chiralpak IA chiral stationary phase (CSP) using pure methanol and simple methanol-water and ethanol-water mixtures as eluents. A water-dependent enantioselectivity was clearly demonstrated. Performance of the Chiralpak IA CSP in polar organic and RP conditions was compared with that of five coated polysaccharide-derived CSPs used in normal-phase mode.

High-performance liquid chromatography separation of enantiomers of flavanone and 2'-hydroxychalcone under reversed-phase conditions.

A reversed-phase high-performance liquid chromatography (HPLC) method was developed for evaluating the chiral discrimination ability of Chiralpak IA chiral stationary phase (CSP) towards flavanone. The effect of the nature and pH buffer as well as nature of alcohol modifier on enantioselectivity was investigated. Comparative study of enantioseparation in reversed-phase and polar organic conditions indicated a significative improvement in resolution when aqueous-based eluents were used. The developed reversed-phase chromatographic method was able to separate the enantiomers of flavanone from its isomeric form, the 2'-hydroxychalcone. The stereochemical stability of flavanone was studied by classical off-column HPLC kinetic procedures in aqueous and non-aqueous media. It was clearly demonstrated that the 2'-hydroxychalcone was involved as intermediate in the on-column and off-column enantiomerization process of flavanone.

HPLC enantioseparation and absolute configuration of novel anti-inflammatory pyrrole derivatives.

The assignment of the absolute configuration of novel anti-inflammatory pyrrole derivatives has been accomplished by a combined strategy based on independent physical methods. The key step of our stereochemical characterization approach is the production at mg-scale of enantiomerically pure forms by HPLC on Chiralpak IA stationary phase.

High-performance liquid chromatography enantioseparation of proton pump inhibitors using the immobilized amylose-based Chiralpak IA chiral stationary phase in normal-phase, polar organic and reversed-phase conditions.

The chiral resolving ability of the amylose-based Chiralpak IA chiral stationary phase towards omeprazole and other proton pump inhibitors under reversed-phase conditions was investigated. Organic modifier-buffer demonstrated to be a valid alternative elution mode with respect to conventional polar organic and normal-phases. No evidence of deterioration of performance of the enantioselective column after several multimodal cycles of elution was observed. Mobile phase composition was systematically changed in order to modulate the enantiomer elution order of set of compounds studied. A very simple method based on on-line detection of optical rotational sign during enantioselective HPLC was developed to assign the absolute configuration and enantiomeric elution order.

Rapid emergence of a viral resistant mutant in WHV chronically infected woodchucks treated with lamivudine and a pre-S/S CHO-derived hepatitis B virus vaccine.

To determine whether the addition of a pre-S/S human vaccine increases the antiviral activity of lamivudine, four woodchucks were treated with a daily dose of 100 mg/kg lamivudine and four 50 microg doses of CHO-derived pre-S/S human vaccine. WHV DNA titres decreased up to two logarithms in three woodchucks. At week 4, in three of the animals, the sequence analysis showed a predominant strain containing a nucleotide change from A to T at position 1696 of domain B of the WHV DNA polymerase. Vaccination did not further suppress WHV DNA, despite anti-HBs production in three animals. The woodchuck remains a useful model for characterising the biology and kinetics of the emergence of drug-resistant variants and could be used for pre-clinical studies of combinations of new antiviral drugs.

Analytical and semipreparative high performance liquid chromatography separation of stereoisomers of novel 3,4-dihydropyrimidin-4(3H)-one derivatives on the immobilised amylose-based Chiralpak IA chiral stationary phase.

Direct HPLC separation of stereoisomers of three novel 5-methyl-2-(alkylthio)-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones endowed with antiviral and potential antiproliferative and morphological differentiation activity against melanoma cells was performed by using the new immobilised amylose-based Chiralpak IA chiral stationary phase. Stereoselective conditions were achieved using normal phase eluents containing "non-standard" solvents such as ethyl acetate, methyl tertbutyl ether, or dichloromethane. In order to study the chiroptical properties of single stereoisomers, mg-scale separations were performed on analytical and semipreparative size Chiralpak IA columns in combination with ethyl acetate-based eluents.