Effect of β-cyclodextrins based nanosponges on the solubility of lipophilic pharmacological active substances (repaglinide)

Cyclodextrin based nanosponges (CD-NS) are nanostructured cross-linked polymers, usually obtained by reacting cyclodextrin with a cross-linker such as carbonyldiimidazole, organic carbonates or (±) epichlorohydrin. They have been used to increase the solubility and stability of poorly soluble pharmacological active substances, as they combine the complex forming properties of CDs and properties of polymers (such as the high molecular weight). The affinity of CDs for certain lipophilic molecules is characteristic to the polymeric nano-structured system and allows the development of specific drug delivery systems. Knowing that cyclodextrin capacity to form inclusion complexes is maintained and enhanced when the CD molecules form aggregates, cross-link together or copolymerize with other compounds, we have synthesized cyclodextrin based nanosponges (from β-cyclodextrin and sulfobutylether-β-cyclodextrin). The complexing properties of the polymers were investigated against repaglinide (a hypoglycemic agent, practically insoluble in water). Solubility studies were performed according to the method reported by Higuchi and Connors and the phase solubility diagrams were plotted. The repaglinide-nanosponges complexes were prepared, lyophilized and the resulted inclusion complexes were characterized by FT-IR and NMR. The solubility profile and the loading capacity of the cyclodextrin based polymers were also determined.     

Comparison of Micellar and Reversed-Phase Liquid Chromatography for Determination of Sulfamethoxazole and Trimethoprim

A simple, sensitive, and precise micellar liquid chromatographic method for simultaneous analysis of sulfamethoxazole and trimethoprim, with ultraviolet detection at 245 nm, has been developed, validated, and used for determination of the compounds in commercial pharmaceutical products. The compounds were well separated on a Hypersil ODS reversed-phase column at 35°C by use of a mobile phase consisting of 0.1M sodium dodecyl sulfate in a 2:98 (V/V) mixture of 1-butanol and pH 3.0 phosphate buffer solution at a flow rate of 1.0 mL min^?1. A comparative study of the performance of reversed-phase liquid chromatography with aqueous-organic or micellar-organic mobile phases for separation of sulfamethoxazole and trimethoprim is reported. The study showed that micellar liquid chromatography (MLC) and reversed-phase liquid chromatography (RP HPLC) are of similar efficiency, sensitivity, and selectivity for determination of sulfamethoxazole and trimethoprim.     

Micellar effect on hydrolysis of 4-methyl-2-nitroaniline phosphate

The effect of anionic surfactant sodium dodecyl sulfate (SDS) on the hydrolysis of a substrate (mono-4-methyl-2-nitroaniline phosphate) by HCl was studied at 303 K. The reaction followed the first-order kinetics with respect to both HCl and the substrate. SDS effectively catalyzes this reaction, which rate increases with the concentration of SDS due to an increase of dielectric constant of the medium. The kinetic data were fitted to Menger-Portnoy, Piszkiewicz and Berezin kinetic models to explain the observed micellar effects. The various activation parameters both in the presence and absence of SDS were evaluated; a reasonable mechanism was proposed. The rate constant in micellar phase, binding constant and index of cooperativity were calculated accordingly.     

Thermal behaviour of a modified encapsulation agent

Thermal behaviour of heptakis-6-iodo-6-deoxy-beta-cyclodextrin (HIDBCD) under inert and oxidative conditions was investigated by TG/DTG/DTA, FTIR, and using the hyphenate technique TG–FTIR. Due to the fact that thermal behaviour of HIDBCD was not studied before, we set our goal in the investigation of thermal degradation process in a dynamic air atmosphere vs. nitrogen atmosphere at a heating rate of 10 °C min^?1, up to 500 °C, respectively, 600 °C. It was found that the degradation process in air occurs in a single step, with a total mass loss of 99.9 %. The results of TG/DTG/DTA–FTIR indicated that the thermal behaviour of this cyclodextrin can be divided into three stages and more information was provided about the reaction sequences and the relevant products of reaction.     

Novel worm-like amphiphilic micelles of folate-targeted cyclodextrin/retinoic acid for delivery of doxorubicin in KG-1 cells

Folate-targeted cyclodextrin/retinoic acid (CD/RA) conjugate was synthesized using carbonyldiimidazole (CDI) and dimethylaminopyridine (DMAP). The structure of the produced macromolecule was studied by FTIR and ¹HNMR. The developed macromolecule could self-aggregate to form micelles. Critical micelle concentration (CMC) of the macromolecule was determined by pyrene as a fluorescent probe. Doxorubicin (DOX)-loaded micelles were prepared by direct dissolution method. To optimize the effect of cyclodextrin type (α or β), the molar ratio of RA to CD and the drug content, a full factorial design was used and their effects on particle size, polydispersity index, zeta potential, loading efficiency (LE%), and release efficiency (RE₂₄%) in 24 h were studied. Orientation of folate ligand on the surface of the micelles was studied by X-ray photoelectron spectroscopy (XPS) technique. The cytotoxicity of DOX-loaded micelles was studied on KG-1 cells which overexpressed folate receptor (FR) and FR-negative HepG2 cells using MTT assay. FTIR and ¹HNMR spectra confirmed successful production of the micelles and XPS spectra showed surface orientation of folate. The best results obtained from β-cyclodextrin with molar ratio of 4 to RA and 15 % drug content. The optimized micelles showed the particle size of 103?±?4 nm, zeta potential of ?36 mV, polydispersity index of 0.28?±?0.05, LE% of 100 %, and RE₂₄% of 69.88?±?1.6 %. The IC₅₀ of targeted micelles was half of non-targeted micelles and free DOX.     

An Approach to Transfer Methods from HPLC to UHPLC Techniques in Some Carbapenems

Stability-indicating LC methods were developed and validated for the quantitative determination of doripenem, meropenem and tebipenem in the presence of their degradation products formed during forced degradation studies. Isocratic HPLC and UHPLC separations were performed with a core–shell Kinetex 1.7, 2.6 and 5 µm, all C18, 100A, 100 × 2.1 mm columns and the mobile phase composed of acetonitrile and 12 mmol L^?1 ammonium acetate in different ratios. The flow rates of the mobile phase were: 0.5 mL min^?1 for 1.7 µm column, and 1.0 mL min^?1 for 2.6 and 5 µm ones. Detection wavelength was 298 nm and temperature was set at 30 °C. All analysed drugs were exposed to stress conditions which caused their hydrolysis and thermal degradation. The methods were validated by evaluation of linearity, accuracy, precision, selectivity and robustness. Proposed methods were successfully applied for the determination of investigated antibiotics during kinetic studies in aqueous solutions and in the solid state. The advantages of chromatographic procedures which are based on the use of C18 stationary phases with different particle sizes in the analysis of selected carbapenems were discussed.     

Immobilization of proteins on plates of Dacron

Dacron (polyethylenetherephthalate) in the form of plates if proposed as a maxtrix to immobilized proteins. A three-step procedure is used to activate this support and to fix the protein on it. Amyloglucosidase was used as a model to test this method, and it showed advantages compared to the powder form of Dacron.     

Micellar solubilization of biopolymers in hydrocarbon solvents. ii. the case of horse liver alcohol dehydrogenase

The chemical characterization of horse liver alcohol dehydrogenase solubilized in isooctane via reverse micelles formed by the anionic surfactant di (2-ethyl-hexyl) sodium sulfosuccinate (AOT) and water (0.6 to 4% v/v) is presented. The enzyme’s catalytic activity toward acetaldehyde reduction is markedly dependent upon w0 = [H2O]/[AOT], and upon the pH of the stock aqueous solution (pHst), from which the hydrocarbon enzyme solution is prepared. Kinetically, the micellar solution appears to follow a normal Michaelis-Menten behavior, with a turnover number which, under the optimal conditions (w0 = 42, pHst = 8.8), appears to be higher than in bulk water. The affinity between enzyme and NADH, as judged from direct binding studies (quenching of the protein fluorescence), is much reduced with respect to water if concentrations refer to the water pool of the micelles, and comparable to water if concentrations refer to the overall volume (hydrocarbon plus water pool). Also, the Km values are much higher if concentrations refer to the water pool. Ultraviolet absorption studies show that the aromatic chromophores are not significantly perturbed on going from a water solution to the micellar solution. The essentially aqueous environment of the protein in the reverse micelles is confirmed by fluoresence studies. Circular dichroism studies show that the enzyme’s conformation in the micelles is similar to that in water; however, under certain conditions, small but significant changes of the main chain folding seem to occur, which do not impair enzymatic activity. The spectroscopic properties of NADH in the hydrocarbon phase (fluorescence and circular dichroism) are also investigated. The potential of the LADH-NADH system for technical applications (oxidoreduction of lipophylic substrates) is discussed.     

An experimental and theoretical study on the structure and photoactivity of XFe2O4 (X = Mn,Fe, Ni,Co, and Zn) structures

XFe₂O₄ magnetic nanoparticles (X = Mn, Fe, Co, Ni, and Zn) were prepared by using two methods: coprecipitation and hydrothermal. The synthesized nanoparticles were compared according to the separation in an external magnetic field and finally, the hydrothermal method was specified as a better synthesis method. The magnetic nanoparticles were characterized by physico-chemical analysis methods such as Vibrating Sample Magnetometer (VSM), X-ray diffraction (XRD), Fourier Transform Infrared spectroscopy (FTIR), nitrogen adsorption-adsorption isotherm and Transmission Electron Microscopy (TEM). Magnetic properties of synthesized nanoparticles were studied by ab-initio theoretical methods to confirm and compare with the experimental results. According to the VSM analysis, all of magnetic nanoparticles had good magnetization while CoFe₂O₄ nanoparticles showed the ferromagnetic behavior. The magnetic properties of XFe₂O₄ configurations were studied using Density Functional Theory ab-initio method. The theoretical results were consistent with experimental magnetizations in the absence of external field. Finally, the photocatalytic behavior of prepared samples was investigated in the presence of oxone as an accelerated agent for degradation of an azo dye.     

Stereoselective separation and pharmacokinetic dissipation of the chiral neonicotinoid sulfoxaflor in soil by ultraperformance convergence chromatography/tandem mass spectrometry

Ultraperformance convergence chromatography/tandem triple quadrupole mass spectrometry (UPC²-MS/MS) is a novel tool in separation science that combines the advantages of supercritical fluid chromatography with ultraperformance liquid chromatography/MS/MS technology. The use of nontoxic CO₂ fluid and a postcolumn additive to complement MS/MS allows better control of analyte retention for chiral separation and high-sensitivity determination with different chiral stationary phases. This paper reports the stereoselective separation and determination of the chiral neonicotinoid sulfoxaflor in vegetables and soil by UPC²-MS/MS. Baseline resolution (Rs?≥?1.56) of and high selectivity (LOQ?≤?1.83 μg/kg) for the four stereoisomers were achieved by postcolumn addition of 1 % formic acid–methanol to a Chiralpak IA-3 using CO₂/isopropanol/acetonitrile as the mobile phase at 40 °C, 2,500 psi, and for 6.5 min in electrospray ionization positive mode. Rearranged Van’t Hoff equations afforded the thermodynamic parameters ΔH ^ο and ΔS ^ο, which were analyzed to promote understanding of the enthalpy-driven separation of sulfoxaflor stereoisomers. The interday mean recovery, intraday repeatability, and interday reproducibility varied from 72.9 to 103.7 %, from 1.8 to 9.2 %, and from 3.1 to 9.4 %, respectively. The proposed method was used to study the pharmacokinetic dissipation of sulfoxaflor stereoisomers in soil under greenhouse conditions. The estimated half-life ranged from 5.59 to 6.03 d, and statistically nonsignificant enantioselective degradation was observed. This study not only demonstrates that the UPC²-MS/MS system is an efficient and sensitive method for sulfoxaflor stereoseparation, but also provides the first experimental evidence of the pharmacokinetic dissipation of sulfoxaflor stereoisomers in the environment. Graphical Abstract

Chemical structure and UPC²-MS/MS separation chromatogram of sulfoxaflor. (* stereogenic center)     

Quantitative analysis by high-performance liquid chromatography atmospheric pressure chemical ionization mass spectrometry: The determination of the renin inhibitor CP-80,794 in human serum

A specific method was developed for the quantitative determination of the renin inhibitor CP-BO,794. Serum extracts containing the drug and an internal standard were injected into a standard reverse-phase high-performance liquid chromatography (HPLC) column. The mobile phase, methanol/water (8/2), flowed at 1 ml/min through the column and then via a heated nebulizer interface into a corona discharge atmospheric pressure chemical ionization source. The assay minimum limit of quantification was 50 pg/mL. It exhibits satisfactory accuracy and precision over the range 50 pg/ml, to 10 ng/mL. A minor modification of the HPLC mobile phase was necessary to attain extremely low detection limits. The addition of a structural analogue contributed to enhancing the precision of the assay.     

Rheological response and small-angle neutron-scattering study of diester-bonded cationic biodegradable gemini surfactants in presence of different additives

Morphological changes and internal packing arrangements of planar dicationic-ester-bonded biodegradable gemini surfactants ethane-1, 2-diyl-bis(N,N-dimethyl-N-alkylammonium acetoxy) dichlorides (m-E2-m) have been explored by exploiting small-angle neutron-scattering (SANS) measurements. The data have been analyzed on the basis of Hayter and Penfold model for macroion solutions to obtain information about the aggregation behavior at the molecular level. The extent of micellar growth and structural changes of the micelles formed by these surfactants have been found to depend on the number of methylene units in their tail length. The growth and variation of micellar shape are more pronounced for the surfactant with longer tail length (m?=?16), whereas the surfactants with shorter tail length showed less variation of these properties in aqueous solution. Semi-major axes of the micelles show flexibility while varying the concentration and temperature of the systems; however, semi-minor axes remain rigid. Changes in the structural parameters of the micelles with addition of different salts were also inferred from SANS measurements. The intensity of scattered neutrons at the low Q region was found to increase while varying the nature of salt from monovalent to trivalent. On the basis of rheological responses, the rich aggregation behavior resulting from the addition of sodium salicylate (NaSal) is attributed to the special molecular structure of the gemini surfactant and the appropriate interaction between the surfactant and NaSal. This is inferred on the basis of behavior observed by varying the chain length (m) of the gemini surfactant that resulted in the formation of different types of microstructures.     

Melt miscibility of HDPE/UHMWPE,LDPE/UHMWPE,and LLDPE/UHMWPE blends detected by dynamic rheometer

The dynamic rheological behavior of high density polyethylene (HDPE)/ultrahigh molecular weight polyethylene (UHMWPE) blends, low density polyethylene (LDPE)/UHMWPE blends and linear low density polyethylene (LLDPE)/ UHMWPE blends was measured in parallel plate rheometer at 200°C. The analysis of log-additivity rule, Cole-Cole plots and Han curves of the three series blends indicated that the LDPE/UHMWPE blends were miscible in the melt, while the HDPE/UHMWPE blends and LLDPE/UHMWPE blends showed phase separation. The DSC results of LLDPE/UHMWPE blends and HDPE/UHMWPE blends were consistent with the rheological properties, while for the thermal properties of LDPE/UHMWPE blends, results revealed three endothermic peaks, which indicated a liquid-solid phase separation in LDPE/UHMWPE blends.     

Gel-sol evolution of cyclodextrin-based nanosponges: role of the macrocycle size

The effect of the macrocycle size on the gel-to-sol evolution of cyclodextrin-based hydrogel is here investigated by using Fourier transform infrared absorption in attenuated total reflectance geometry (FTIR-ATR). Different types of nanosponges obtained by polymerization of α- and β-cyclodextrin (CDNS) with an activated derivative of ethylenediaminetetraacetic acid have been progressively hydrated in order to follow the evolution of these systems from a gel state to a liquid suspension. The in deep analysis of the high-frequency vibrational dynamics of the hydrogel during its gel-sol evolution revealed that the microscopic origin of this phenomenon is strictly connected to different hydrogen bond environments in which water molecules confined in the pores of nanosponges can arrange. By following a well consolidated approach, the OH stretching band of water, clearly observed in the high-frequency range of the vibrational spectra of nanosponges hydrogel, has been decomposed into sub-bands assigned to different arrangements of water molecules at various degrees of cooperativity. A comparison of the diagrams obtained for homologous CDNS prepared from α- and β-CD shows how the size of cyclodextrin macrocycle allows to efficiently modulate the gelation points at constant cyclodextrin/crosslinker molar ratio n.     

Chiral ligand-exchange high-performance liquid chromatography with copper (II)-L-phenylalanine complexes for separation of 3,4-dimethoxy-α-methylphenylalanine racemes

L-3, 4-dimethoxy-α-methylphenylalanine (L-DMMD) is an important intermediate for the synthesis of 3-hydroxy-α-methyl-L-tyrosine (L-methyldopa). This paper describes an efficient, accurate, and low-priced method of high-performance liquid chromatography (HPLC) using chiral mobile phase and conventional C₁₈ column to separate L-DMMD from its enantiomers. The effects of ligands, copper salts, organic modifiers, pHs of mobile phase, and temperatures on the retention factors (k') and selectivity (α) were evaluated to achieve optimal separation performance. Then, thermal analysis of the optimal separation conditions was investigated as well. It was confirmed that the optimal mobile phase was composed of 20 % (v/v) methanol, 8 mM L-phenylalanine (L-Phe), and 4 mM cupric sulfate in water of pH 3.2, and the column temperature was set at 20 °C. Baseline separation of two enantiomers could be obtained through the conventional C₁₈ column with a resolution (R) of 3.18 in less than 18 min. Thermodynamic data (??H and ??S) obtained by Van't Hoff plots revealed the chiral separation was an enthalpy-controlled process. To the best of our knowledge, this is the first report regarding the enantioseparation of DMMD by chiral ligand-exchange HPLC.     

Micellar solubilization of biopolymers in hydrocarbon solvents. I. a structural model for protein-containing reverse micelles

Three proteins (horse liver alcohol dehydrogenase, ribonuclease, lysozyme) were solubilized in hydrocarbon with the help of reverse micelles formed by aqueous di(2-ethyl-hexyl) sodium sulfosuccinate (AOT). Sedimentation and diffusion coefficients of the micellar aggregates were measured with an analytical ultracentrifuge. Partial specific volumes were also evaluated from density measurements. The molecular weight of the protein-containing reverse micelles (M _t ) could thus be determined for each protein system at various w₀ values (w₀ - [H₂O]/[AOT]). For horse liver alcohol dehydrogenase at w₀ = 46.4, for example,M _t is ca. 2,670,000 Daltons; for lysozyme at wo = 22.5,M _t is ca. 323,000 Daltons and increases by increasing w₀. On the basis of these experimentally determined molecular weights, a structural model for the protein-containing reverse micelle is proposed. The model is based upon the assumption that the protein is confined in the water pool of a spherical micelle, and that the inner core volume is the sum of the protein volume and the volume of all water molecules present in a micelle. It is possible then to calculate the micellar structure at each w₀ value. For example, in the case of ribonuclease at w₀ = 20, the inner core radius is ca. 37.5 A, and a layer of water of ca. 22 A separates the protein surface from the surfactant layer. The possible implications of this model for the reactivity of enzymes solubilized in hydrocarbons by reverse micelles are discussed.     

Comparison of the performance of butanol and pentanol as modifiers in the micellar chromatographic determination of some phenethylamines

A procedure was developed for the determination of several phenethylamines (amphetamine, arterenol, ephedrine, phenylephrine, phenylpropanolamine, mephentermine, methoxyphenamine, pseudoephedrine and tyramine), using micellar mobile phases of sodium dodecyl sulfate (SDS), a C18 column and UV detection. The drugs were eluted at short retention times with conventional acetonitrile-water or methanol-water mobile phases. In contrast, in the micellar system, they were strongly retained due to association with the surfactant adsorbed on the stationary phase, and needed the addition of butanol or pentanol to be eluted from the column. These modifiers allowed a simple way of controlling the retention. The chromatographic efficiencies obtained with the hybrid mobile phases of SDS-butanol and SDS-pentanol were also very high, mostly in the N=3000-7000 range, significantly greater than those achieved with a conventional acetonitrile-methanol-water mobile phase. Butanol and pentanol yielded similar selectivities, but the latter modifier permitted significantly shorter retention times than butanol, and was preferred to expedite the analysis of the pharmaceuticals. Most binary combinations of the nine phenethylamines can be resolved with these mobile phases. A mobile phase of 0.15 M SDS-5% pentanol was used to assay five of the phenethylamines (amphetamine, ephedrine, phenylephrine, phenylpropanolamine and pseudoephedrine) in 22 pharmaceutical preparations, which contained diverse accompanying compounds. The results agreed with the declared compositions and with those obtained with a mobile phase of methanol-acetonitrile-0.05 M phosphate buffer (pH 3) 10:5:85, with no interferences and relative errors usually below 2%. However, with the aqueous-organic mobile phase, the retention time for phenylephrine was too low and could not be usually evaluated.     

Investigation of enhanced ion abundances from a carrier process in high-performance liquid chromatography particle beam mass spectrometry

Enhancements in ion abundances in high-performance liquid chromatography (HPLC) particle beam (PB) mass spectrometry were observed during coelution or when certain substances (e.g., ammonium acetate) were added to the mobile phase during a reverse-phase gradient elution HPLC separation. These enhancements were observed with two commercial PB interfaces and a variety of compounds generally not amenable to separation by gas chromatography. The enhancements are attributed to both improved chromatographic efficiency and a PB carrier process.