B. del Castillo

Analytical applications of retinoid-cyclodextrin inclusion complexes. 1. Characterization of a retinal-β-cyclodextrin complex

In studies in these laboratories on the supramolecular chemistry of the retinoids, it has been recently confirmed that inclusion of these substances within the cavity of cyclodextrins protects their excited states, thus improving their photochemical stability. In the present paper, the isolation is described of a crystalline stable complex between retinal and beta-cyclodextrin, which has been characterized by means of several techniques including atomic force microscopy (AFM). The complex shows distinct spectroscopic differences from both retinal and beta-cyclodextrin. Thus, it absorbs at lambda(max) = 380 nm in water whereas retinal is insoluble; it shows room-temperature luminescence, which retinal does not; finally, it give 1H-NMR and 13C-NMR spectra in d6-DMSO with clear differences in chemical shifts with respect to those of beta-cyclodextrin. Besides these studies in solution, the behaviour of the complex in the solid state has been compared with that of physical mixtures of retinal and beta-cyclodextrin. IR spectroscopy shows clear differences, particularly a shift in the retinal carbonyl absorption (1644-1672 cm-1). AFM studies reveal the existence of aggregates; X-ray diffractometry also supports the formation of a cyclodextrin-retinal complex.

Spectrofluorimetric determination of stoichiometry and association constants of the complexes of harmane and harmine with β-cyclodextrin and chemically modified β-cyclodextrins ☆

The association characteristics of the inclusion complexes of the beta-carboline alkaloids harmane and harmine with beta-cyclodextrin (beta-CD) and chemically modified beta-cyclodextrins such as hydroxypropyl-beta-cyclodextrin (HPbeta-CD), 2,3-di-O-methyl-beta-cyclodextrin (DMbeta-CD) and 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbeta-CD) are described. The association constants vary from 112 for harmine/DMbeta-CD to 418 for harmane/HPbeta-CD. The magnitude of the interactions between the host and the guest molecules depends on the chemical and geometrical characteristics of the guest molecules and therefore the association constants vary for the different cyclodextrin complexes. The steric hindrance is higher in the case of harmine due to the presence of methoxy group on the beta-carboline ring. The association obtained for the harmane complexes is stronger than the one observed for harmine complexes except in the case of harmine/TMbeta-CD. Important differences in the association constants were observed depending on the experimental variable used in the calculations (absolute value of fluorescence intensity or the ratio between the fluorescence intensities corresponding to the neutral and cationic forms). When fluorescence intensity values were considered, the association constants were higher than when the ratio of the emission intensity for the cationic and neutral species was used. These differences are a consequence of the co-existence of acid-base equilibria in the ground and in excited states together with the complexation equilibria. The existence of a proton transfer reaction in the excited states of harmane or harmine implies the need for the experimental dialysis procedure for separation of the complexes from free harmane or harmine. Such methodology allows quantitative results for stoichiometry determinations to be obtained, which show the existence of both 1:1 and 1:2 beta-carboline alkaloid:CD complexes with different solubility properties.

Room temperature luminescence of a retinal complex of cyclodextrin

Abstract Retinal, which is normally insoluble in an aqueous medium and not fluorescent in solution at room temperature, emits a luminescence in the region of 450 nm, even in air-saturated aqueous solutions, when complexed to β or γ-cyclodextrin. The intensity decreases in the order β> γ and the quantum yields of luminescence are in the range 10−3–10−4. Further, retinal molecules in the inclusion complexes are subject to a bleaching process. Solubilization in micellar sodium dodecyl sulphate solutions also allows emission, albeit in smaller yield.

Modified β-cylclodextrins as enhancers of fluorescence emission of carbazole alkaloid derivatives

Experimental conditions for formation of inclusion complexes between carbazole and ellipticine and several cyclodextrins (CDs): β-cyclodextrin (β-CD), hydroxypropyl β-cyclodextrin (HPβ-CD), (2,6-di-O-methyl)-β-cyclodextrin (DMβ-CD), (2,3,6-tri-O-methyl)-β-cyclodextrin (TMβ-CD) and γ-cyclodextrin (γ-CD), are described. A notable increase of fluorescence intensity of the host molecules is observed upon complexation, attributed to protection of the excited state within the cyclodextrin cavity. This allows the quantitation of both compounds in aqueous media in concentration levels of 10−7 M to 10−6 M. Inclusion also affects the deprotonation equilibrium of the pyrrolic nitrogen of both compounds. Thus, β-CD facilitates the deprotonation of carbazole while HPβ-CD, DMβ-CD, TMβ-CD and γ-CD make it more difficult. Similar observations were made for ellipticine, although its behaviour was more complex because of the coexistence of two ionization equilibria owing to the presence of a basic pyridine-like nitrogen. Finally, the effect of cyclodextrins on fluorescence quenching caused by heavy halogen atoms was also studied. In the case of carbazole, a static quenching was observed, which is prevented by modified β-CDs. In the case of ellipticine, the quenching effect was related to acid-base equilibria, and therefore it has been studied under acidic (λex = 305 nm, λem = 520 nm) and basic (λex = 292 nm, λem = 430 nm) conditions.

Micellar electrokinetic capillary chromatography as an alternative method for the determination of ethinylestradiol and levo-norgestrel

A method for quantifying of ethinylestradiol (ETE) and levo-norgestrel (LEV) in pharmaceutical products by micellar electrokinetic chromatography (MEKC) is described. The separation was carried out at 25 degrees C and 25 kV, using a 20 mM borate buffer (pH 9.2), 15 mM sodium dodecylsulfate (SDS) in 30% acetonitrile/water (v/v). Under these conditions the analysis takes about 7 min. The method has been applied for quantifying both compounds in six different commercial contraceptives and the proposed method gave good results when compared with a reference liquid chromatographic (LC) method.

Solvent effects on the fluorescent emission of some new benzimidazole derivatives

Abstract The benzimidazole moiety is present in a great variety of pharmaceutical compounds with anthelmintic action and other applications. These compounds exhibit native fluorescence which often depends on the substituents on the benzimidazole ring and the solvents. The fluorescence emission also changes with pH. In this article the solvatochromic effect is described in different solvents (polar protic and aprotic, alkanes, pyridine and acetic acid) of a series of new benzimidazole derivatives, namely 1-methyl-6-nitro-benzimidazole (6-NBI), 1-methyl-6-aminobenzimidazole (6-ABI) and 1-methyl-6-(p-tolylamino)benzimidazole (6-TABI). The fluorescence intensity is weaker for 6-NBI than for the other derivatives in all solvents tested due to quenching of fluorescence emission by the nitro group. The excitation and emission wavelengths are red shifted for 6-TABI with respect to those observed for 6-NBI and 6-ABI, due to the presence of the tolyl group. The fluorescence emission maximum is shifted for 6-ABI from λem = 362 nm in formic acid to λem = 470 nm in pyridine; this behaviour is explained in terms of the acid-base characteristics of these compounds. A remarkable shift in the emission wave-length can be also observed for 6-ABI with changing solvent polarity from 354 nm in alcohols to 420 nm in dimethyl sulphoxide. This behaviour could be related to the different equilibria that take place in the excited state, i.e., acid-base equilibria and also charge transfer processes, depending on the solvents.

Use of micellar media for the fluorimetric determination of ellipticine in aqueous solutions.

Ellipticine is a pyridocarbazole alkaloid with interesting antitumour activity. Use of neutral ellipticine is hampered by its very low water solubility and therefore this compound has been administered as a salt; however, nitrogen quaternization modifies the antitumour properties of ellipticine. Potential alternatives to quaternization include the use of cyclodextrins, and also the use of micellar media. The latter possibility is explored in this work as an analytical tool. The results obtained with model anionic (SDS), cationic (CTAB) and neutral (Brij-35) surfactants are described. Fluorimetric analysis shows that ellipticine solubilizes completely in the presence of all these compounds, as a result of its aromatic, planar structure. The use of micellar media considerably increases the slopes of the calibration curves with improved correlation coefficients (e.g. 0.8904 in water and 0.9982 with SDS). Micellar media also modify proton transfer processes, as a consequence of the apolar environment of the micellar phase. Deprotonation of ellipticine is hampered in SDS because of the relationship between this process and the surface charge of the micelles. Finally, fluorescence quenching in micellar media has been studied, it being found that surfactants provide protection towards this phenomenon.

Determination of thiols of biological and pharmacological interest by high-performance thin-layer chromatography and fluorescence scanning densitometry.

The application of high-performance thin-layer chromatography (HPTLC) with fluorescence scanning densitometry provides a simple, rapid and reliable system for the qualitative and quantitative determination of several thiols of biological and pharmacological interest. The determination of a mixture of thiols (captopril, coenzyme A, cysteamine, cysteine and glutathione), together with their disulphides may readily be performed by pre-chromatographic derivatization with the thiol-specific fluorobenzoxadiazole reagents SBD-F and ABD-F, followed by HPTLC separation on silica gel plates using isopropyl ether-methanol-water-acetic acid (9:8:2:1, v/v/v/v) as the developing solvent, and fluorodensitometric measurement of the fluorescing derivatives. Detection limits of about 30 pg (coenzyme A) to 6 pg (cysteamine) per spot were achieved; the relative standard deviation (RSD) of the complete procedure was 1.16-3.2%.

Use of enhancers in the HPTLC fluorescence analysis of thiols

Several thiols of biological and pharmacological interest, including glutathione, coenzyme A, acetylcysteine and captopril were derivatized with the fluorogenic reagents SBD-F and ABD-F and analysed by high-performance thin-layer chromatography (HPTLC)-fluorodensitometry on silica gel 60 plates, using isopropyl ether-methanol-water-acetic acid (9:8:2:1, v/v/v/v) as the developing solvent. The luminescence was considerably increased when several types of enhancers were applied as dipping reagents: Triton X-100, liquid paraffin and cyclodextrins; thus the detectability of the thiol fluorophores was improved. The influence of enhancer concentration, method of application, sample concentration, drying conditions and measuring time after plate dipping were investigated. The greatest enhancement was achieved using a 40% (v/v) solution of Triton X-100 in toluene as a dipping reagent for the determination of SBD-acetylcysteine; more than a 10-fold increase of the fluorescence signal was obtained, allowing low picogram detection limits.

Detection and characterization of cyclodextrin complexes with β-carboline derivatives by spectroscopic techniques

beta-Carboline alkaloids exhibit a great variety of pharmacological activities. The solid inclusion complexes of harmane and harmine with beta-cyclodextrin and also with hydroxypropyl-beta-cyclodextrin, have been prepared following different procedures. IR and NMR spectroscopies were employed to verify the interaction of the guest molecules with the cyclodextrin cavities. The differences observed in the IR and NMR spectra are in agreement with those described in the literature for other guest molecules. The shifts in the 13C- and 1H-NMR spectra confirm the existence of the inclusion complexes. The fluorescence emission spectra of these complexes dissolved in buffered aqueous solution (pH 7.3) exhibit the characteristic peaks of the cationic form for harmane alkaloids. The neutral bands are not present for the free alkaloids in aqueous solutions. Fluorescence quenching emission of the complexes is compared to that of the corresponding free alkaloids.