An improved HPLC method overcoming Beer's law deviations arising from supramolecular interactions in tolfenamic acid and cyclodextrins complexes

Inclusion complexes of tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, with methyl-βcyclodextrin and hydroxypropyl-β cyclodextrin were prepared and characterised. Spectrophotometric, chromatographic (RP-HPLC) and 1H NMR studies of the complexes were conducted. It was observed that cyclodextrins influence TA's molar absorptivity leading to Beer's law deviation. Consequently, the accuracy problem arose, urged for the application of specific chromatographic conditions for the determination of TA in the presence of CDs. A new HPLC method was developed and validated. TA was analysed on a C18 column 5 μm (150x4.6 mm), using a column thermostat regulated at 30°C. The mobile phase consisted of methanol-phosphate buffer solution (pH 3.2; 0.07 M) (90:10 v/v) and the flow rate was set at 2.0 ml min-1. The detector was operated at 286 nm. TA was successfully determined, overcoming the problems arising from the presence of cyclodextrins. Copyright (C) 1998 Elsevier Science B.V

Supramolecular interactions between tolfenamic acid and various cyclodextrins: Effects of complexation on physicochemical and spectroscopic data

Tolfenamic acid is a non-steroidal inflammatory drug with associated gastrointestinal side-effects and problems with bioavailability. Furthermore, it is extremely insoluble in water. Tolfenamic acid was complexed with various cyclodextrins (β-CyD, HP-β-CyD, methyl-β-CyD) in order to circumvent some of these problems. During the characterization of the inclusion complexes with spectroscopic methods, significant variations in the UV and NMR spectra were observed and attributed to the supramolecular interactions between the guest and host molecules. A validated HPLC method was applied to obtain accurate measurements. The complexation process was further examined; the time needed for the completion of the complexation was determined, the binding constants of the complexes were calculated and the energetics of the system were evaluated. The solubility of tolfenamic acid was 30-fold in the presence of HP-β-CyD and twofold in the presence of methyl-β-CyD

Study of structural features and thermodynamic parameters, determining the chromatographic behaviour of drug-cyclodextrin complexes

The chromatographic behaviour of host-guest inclusion complexes was studied, in order to predict the optimal conditions for their accurate analysis and overcome the significant analytical errors generated by the presence of cyclodextrins. Complexes of tolfenamic acid and ketoprofen with β-cyclodextrtin (βCD), 2-hydroxypropyl-βCD (HPβCD) and methyl-βCD (MeβCD) prepared in different molar ratios, were studied. Since the drug release from cyclodextrins' complexes is a prerequisite for its accurate quantitation, several parameters affecting the dissociation during the analysis were evaluated. In an attempt to explain the drug release mechanism from cyclodextrins, during HPLC analysis, the possible correlation of the NMR structural findings with the binding constants and the thermodynamic quantities of complexation were examined, in relation to their chromatographic behaviour. Finally, the presence of the solvation spheres around the supramolecules, which affect the complex stability, is suggested to be crucial for our chromatographic findings. Particularly, entropy change in the system is considered the most critical factor, determining the time required for dissociation of drug-cyclodextrin complexes, during drug quantitation. © 2005 Elsevier B.V. All rights reserved

Chromatographic behaviour of naproxen-cyclodextrin complexes: Stationary phase C8 alkyl chain as competitor for the drug release from cyclodextrin cavity

Cyclodextrins are known to alter the absorptivity of the guest molecules, therefore, analytical methods that are based on the spectrophotometric data present accuracy problems. In this work, using RP-HPLC methods for naproxen-cyclodextrins quantitation, extensive analytical inaccuracies are detected. Competitive complexation technique is utilised in an attempt to develop an analytical method enabling the determination of naproxen as a free drug. For this reason, stationary phases with silica ligands that can function as competing agents were used, thus contributing to the drug release. The release of the drug from cyclodextrins complexes is achieved by modification of the thermodynamic parameters that determine the stability constant, by changing: the interactions with the mobile phase components (e.g. pH, organic modifier, competitive agents) and the interactions with the stationary phase ligands (C8). After studying the parameters affecting the interaction between the alkyl-chain C8 and naproxen:cyclodextrin complexes, we developed and validated a new specific method for the accurate determination of the drug. Consecutive accumulation of the cyclodextrins molecules on the stationary phase was studied. © 2004 Elsevier B.V. All rights reserved

The effect of pH dependent molecular conformation and dimerization phenomena of piroxicam on the drug:cyclodextrin complex stoichiometry and its chromatographic behaviour: A new specific HPLC method for piroxicam: cyclodextrin formulations

The complexation of piroxicam with various cyclodextrins was studied in the whole pH range, in an attempt to clarify the reported differences in stoichiometry of the resulting inclusion complexes. In this work, it was found that the pH dependent self-association phenomena, detected in both alkaline and acidic media, are the defining features for complex stoichiometry. Also, it was observed that the inclusion complexes of the piroxicam zwitterion with β-cyclodextrin (βCD), methyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin have a 2:2 stoichiometry in acidic pH, through inclusion of the benzyl ring moiety. As pH increased, it was determined that the different ionic state of the molecules creates new geometries, involving also the pyridyl-ring in the complexation. More stable inclusion complexes of 2:5 stoichiometry are observed at pH>4. A reversed phase liquid chromatography method, specific for the analysis of the included piroxicam, was developed and validated. The different stoichiometries and the dimers formation were investigated by interpretation of the two-dimensional 1H NMR spectroscopy. © 2004 Elsevier B.V. All rights reserved

Competitive complexation and SPE techniques combined with liquid chromatography for the separation and determination of cyclodextrin encapsulated drug substances

A new onalytical procedure, combining sample pretreatment with solid phase extroction ond high performonce liquid chromatography, was developed and opplied to ketoprofen:cyclodextrin (CD) complexes. The significont differences in molar absorptivity between free and CD bound drugs leod to analytical errors up to 30%. Procedures routinely used to remove most of the common excipients proved to be inadequate, creating the necessity of more specific (HPLC) onalytical methods including the step of dissociotion ond separation of drugs from CDs before the quontitation by UV detector. In the case of complexes such as Ketoprofen: CDs, strong binding to CDs inhibits the dissociation. Prior to the anolysis, the addition of molecules competing the binding in the CD cavity fairly decreased the strength of the complexation. When molecules, competing the binding to CD such os 1-odomantanol, were added to the samples the results were improved. The extent of the selective binding and the drug displocement were monitored with 1H-NMR spectroscopy and it was revealed that only partial dissociation of the complexes was obtained. The combination of the sample pre-treatment with a solid phase extraction technique was proved to be inevitable for the dissociation of the complex. When analyzing supramolecules spectral changes, arising from intramolecular and/or intermolecular interactions, are expected. Cansequently, analytical methods utilized should be carefully revised

HPLC analysis, isolation and identification of a new degradation product in carvedilol tablets

Carvedilol (CV) is an antagonist of α1 and β1,β2 membrane adrenoceptors and also a modulator of cardiac electrophysiological properties. It is widely prescribed for the treatment of cardiovascular diseases. During stability testing of CV solid dosage forms an unknown degradation product referred as UP, exceeded the identification thresholds of ICH Q3B guidelines. The HPLC analysis of the detected unknown product was performed by a newly, developed, specific and validated method, also suitable for the quantitative determination of the known CV impurities (imp B, C, E and F) and the other degradation products. The separation was achieved with an X-terra C18 column, using acetonitrile-phosphate buffer pH 2.5 as mobile phase. The isolation of UP was carried out by semi-preparative chromatography method, followed by deep freezing of the collected fractions until the organic and the aqueous phases were separated. Chromatographic behaviour of CV and UP was compared, in mobile phases of different pH and gave valuable information concerning the dissimilarities of their ionization. UP was further studied by MS and 1H NMR spectrometry, revealing structural similarities with the parent molecule. Finally, the unknown peak of degradation product was attributed to a new compound generated from the interaction of CV molecule and polyvinyl pyrrolidone (PVP) in the presence of water molecules. Moisture and temperature was proved to affect the formation of UP and its concentration in CV tablets. Appropriate modifications of the packaging of CV tablets can be made in order to reduce UP concentration down to the accepted levels, during the tablets' shelf life. © 2008 Elsevier B.V. All rights reserved

2,2′:6′,2″-Terpyridine[hydroxypropyl-β-cyclodextr in] 1:3 complex used as chelating agent for the determination of iron with a sensitive, selective and fast liquid chromatographic method

In the present work, 2,2′:6′,2″-Terpyridine (terpy), a substance with very poor aqueous solubility, was dissolved in water, after formation of its inclusion complex with hydroxypropyl-β-cyclodextrin (HPβCD), in a 1:3 stoichiometry. The obtained [terpy:(HPβCD)3] supramolecule, with enhanced aqueous solubility, enables its usage as a reagent at RP-LC methods. It was found that, terpy after inclusion complexation retains unaffected the ability of binding to Fe2+. It was also observed that, the stable, reddish-purple [Fe(terpy)]2+ complex was formed quantitatively in a wide pH range (2-9). Subsequently, iron as active substance or impurity in a drug product, can be determined through UV-vis measurements of [Fe(terpy)2]2+. Speed, sensitivity and selectivity are the most important features of the isocratic RP-LC method, developed to determine iron in pharmaceutical formulations. The duration of the chromatographic separation was less than 4.0 min. The method was linear, precise and accurate from 0.17 to 2.2 mg l-1 of iron and the detection limit was found to be 5 μg l-1. The absorbance at 318 and 552 nm allowed the quantitation of Fe (II) and Fe (III) after reduction, as well as of total Fe (II + III). Moreover, there were no interferences from Fe3+, Ni2+, Co2+ or Cu2+. © 2006 Elsevier B.V. All rights reserved

New SPE loading material for affinity-based separation of cyclodextrins from drug:CD complexes in order to overcome Beer's law deviations

Molecular inclusion of guest molecules within CDs is known to alter guest molecule spectrophotometric absorptivity, making their determination, based on spectrophotometric data, inaccurate. Therefore specific analytical methods capable of quantifying the drugs as free molecules must be developed and validated. SPE was selected to simplify sample and avoid more time-consuming alternatives. A new solid phase was synthesized and characterized by infrared spectrometry, differential scanning calorimetry and elemental analysis. The competitive complexation of adamantane groups immobilized on the silica substrate facilitates drug:CD complex dissociation and elimination of CD from samples. The drug molecules, now free from CD, can be easily analysed by an already available HPLC method. This new SPE loading material was employed in the determination of ketoprofen in its CD complex as a representative example of the utility of this novel material. The calculated analytical errors were reduced from a maximum of 20.79% (without SPE) to a minimum of 3.99%. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA

2,2′:6′,2″-Terpyridine[hydroxypropyl-β-cyclodextr in] 1:3 complex used as chelating agent for the determination of iron with a sensitive, selective and fast liquid chromatographic method

In the present work, 2,2′:6′,2″-Terpyridine (terpy), a substance with very poor aqueous solubility, was dissolved in water, after formation of its inclusion complex with hydroxypropyl-β-cyclodextrin (HPβCD), in a 1:3 stoichiometry. The obtained [terpy:(HPβCD)3] supramolecule, with enhanced aqueous solubility, enables its usage as a reagent at RP-LC methods. It was found that, terpy after inclusion complexation retains unaffected the ability of binding to Fe2+. It was also observed that, the stable, reddish-purple [Fe(terpy)]2+ complex was formed quantitatively in a wide pH range (2-9). Subsequently, iron as active substance or impurity in a drug product, can be determined through UV-vis measurements of [Fe(terpy)2]2+. Speed, sensitivity and selectivity are the most important features of the isocratic RP-LC method, developed to determine iron in pharmaceutical formulations. The duration of the chromatographic separation was less than 4.0 min. The method was linear, precise and accurate from 0.17 to 2.2 mg l-1 of iron and the detection limit was found to be 5 μg l-1. The absorbance at 318 and 552 nm allowed the quantitation of Fe (II) and Fe (III) after reduction, as well as of total Fe (II + III). Moreover, there were no interferences from Fe3+, Ni2+, Co2+ or Cu2+. © 2006 Elsevier B.V. All rights reserved