Enrofloxacin inclusion complexes with cyclodextrins

Inclusion complexes using α-, β-, γ-, and hydroxypropyl-β-CD (HP-β-CD) were produced with the antibiotic enrofloxacin, with the aim of increasing its solubility by complexation. Phase solubility diagrams were obtained, to confirm the formation of inclusion complexes, and to determine the solubility enhancement and stability constant of each complex. Enrofloxacin inclusion in β-CD showed the highest value of the complex stability constant (35.56?mmol?L^?1), but the greatest increase in solubility was obtained using HP-β-CD reaching a 1258% increase over enrofloxacin solubility in the absence of CD. The order of highest enrofloxacin solubility achieved was: HP-β-CD?>?α-CD?>?γ-CD?>?β-CD. In addition, formation of complexes was confirmed by differential scanning calorimetry and thermogravimetry, applied to the complexes obtained by the kneading technique. The influence of citric acid, alone or as an adjunct of β-CD, on the solubility of enrofloxacin was also determined. A solution of 15?mmol?L^?1 citric acid dissolved 10?g?L^?1 of enrofloxacin, but a gradual increase in β-CD concentration in the presence of citric acid did not increase the degree of solubilization of enrofloxacin.     

Liquid chromatography of cyclodextrins with indirect photometric detection of phenolphthalein inclusion complexes

Summary - and -cyclodextrin (CD) separated by liquid chromatography have been indirectly detected by depression of the background absorbance at a visible wavelength by means of inclusion complexation with phenolphthalein. The background signal was generated by phenolphthalein in an alkaline medium. Octadecyl-bonded poly(vinylalcohol) packings were employed as the stationary phase, which allowed the use of alkaline mobile phases, and eliminated post column mixing of reagents. The detection limits for - and -CD were 0.1–0.5 M at a signal to noise ratio of 3. -CD could not be measured by the present detection principle.     

Separation of steroids using temperature-dependent inclusion chromatography

The influence of temperature on retention and separation of estrogens, progesterone derivatives and beta-cyclodextrin in reversed-phase high-performance liquid chromatography has been studied. Steroids were detected using direct UV detection at 240 and 280 nm. Detection of beta-cyclodextrin was achieved using a post-column indirect photometric method. Chromatographic experiments were performed using an acetonitrile-water mobile phase (30%, v/v) and a wide range of column temperatures from 0 to 80 degrees C with 20 degrees C steps. Linear Van't Hoff plots were observed for steroids and beta-cyclodextrin when an unmodified binary mobile phase was applied. The retention of steroids was strongly influenced by temperature when the mobile phase was modified with beta-cyclodextrin at a concentration of 12 mM. Particularly, for 17beta-estradiol and 20alpha-hydroxyprogesterone a strong deviation from the linear Van't Hoff plots and a remarkable affinity for beta-cyclodextrin was observed. Polynomial regression calculations were performed to fit the set of experimental data points. Using third-order polynomial equations, minimum separation factor values (alphamin) were calculated for temperatures from -10 to + 100 degrees C with 1 degrees C steps. The best chromatographic conditions for separation of multicomponent samples were chosen. A possible retention mechanism for solutes in the presence of macrocyclic additives is discussed. The results presented describe the role of temperature in high-performance liquid chromatography systems in which the mobile phase is modified with an inclusion agent.     

Gas chromatography and high-performance liquid chromatography of natural steroids.

This review article underlines the importance of gas chromatography (GC), high-performance liquid chromatography (HPLC) and their hyphenated techniques using mass spectrometry (MS) for the determination of natural steroids, especially in human biological fluids. Steroids are divided into eight categories based on their structures and functions, and recent references using the above methodologies for the analysis of these steroids are cited. GC and GC-MS are commonly used for the determination of volatile steroids. Although HPLC is a widely used analytical method for the determination of steroids including the conjugated type in biological fluids, LC-MS is considered to be the most promising one for this purpose because of its sensitivity, specificity and versatility.     

Cationic cyclodextrins chemically-bonded chiral stationary phases for high-performance liquid chromatography

Two covalently bonded cationic β-CD chiral stationary phases (CSPs) prepared by graft polymerization of 6^A-(3-vinylimidazolium)-6-deoxyperphenylcarbamate-β-cyclodextrin chloride or 6^A-(N,N-allylmethylammonium)-6-deoxyperphenylcarbamoyl-β-cyclodextrin chloride onto silica gel were successfully applied in high-performance liquid chromatography (HPLC). Their enantioseparation capability was examined with 12 racemic pharmaceuticals and 6 carboxylic acids. The results indicated that imidazolium-containing β-CD CSP afforded more favorable enantioseparations than that containing ammonium moiety under normal-phase HPLC. The cationic moiety on β-CD CSPs could form strong hydrogen bonding with analytes in normal-phase liquid chromatography (NPLC) to enhance the analytes’ retention and enantioseparations. In reversed-phase liquid chromatography (RPLC), the analytes exhibited their maximum retention when the pH of mobile phase was close to their pK_a value. Inclusion complexation with CD cavity and columbic/ionic interactions with cationic substituent on the CD rim would afford accentuated retention and enantioseparations of the analytes.     

Determination of cyclodextrins in biological fluids by high-performance liquid chromatography with negative colorimetric detection using post-column complexation with phenolphthalein

A rapid and sensitive high-performance liquid chromatographic method for the analysis of beta- and gamma-cyclodextrin in aqueous biological fluids such as plasma, urine, or tissue homogenate is described. The chromatographic system consists of a microBondapak Phenyl column as stationary phase and a mobile phase of water with 10% methanol. After post-column addition of an alkaline solution of phenolphthalein, negative colorimetric detection is used. The elution solvent and post-column reagent were mixed in a capillary tubing of 1.5 m (1.0 mm I.D.). Two methods of sample treatment are given, one for large (1.0 ml) and one for small (0.1 ml) sample volumes. Both methods were shown to be linear and reproducible. The detection limit for beta-cyclodextrin was 1.0 microgram/ml (0.77 nmol/ml). The method was used in the determination of some pharmacokinetic parameters of beta-cyclodextrin in rats after intravenous injection.     

Solvent optimization of reversed-phase high-performance liquid chromatography for polar adrenal steroids using computer-predicted retentions

Computer-aided optimization of a mobile phase has been applied to the isocratic reversed-phase separation of ten polar adrenocortical steroids, including aldosterone and reduced metabolites of cortisol and cortisone. A method based on a seven-step procedure for calculation of the Chromatographic Optimization Function (COF) has been used. Logarithmically transformed retention indices were used for computing multiple polynomial regressions for the retention times of compounds as a function of solvent composition, with the resultant COF as a dependent parameter used for selection of the better mobile phase. Peak crossovers and overlaps are accommodated in this method and the maximum acceptable analysis time factor is incorporated as well as weighting factors for priority separations. The utility of this procedure for complex mixtures of closely eluting compounds is discussed with respect to the Overlapping Resolution Map method and with the COF method of Glajch and Kirkland as used for automated optimization. Its application to aldosterone-containing samples from human adrenocortical tumours is illustrated.     

Investigation of copper-azamacrocyclic complexes by high-performance liquid chromatography

The use of copper radioisotopes in imaging and therapy has prompted an increased interest in chelators which form stable copper complexes, such as Cu(II)-azamacrocyclic complexes. The effects of charge, stability and the size of the macrocyclic backbone of the Cu(II)-azamacrocyclic complexes on biological behavior have been evaluated. Here we report a reversed-phase high-performance liquid chromatography (HPLC) method to separate several Cu(II)-azamacrocyclic complexes, including Cu(II) complexes of 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) and 4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane (CB-DO2A). Absorbance at 280 nm was used to monitor the complexes as they eluted from the reversed-phase column. The effects of the concentration of the buffer, the pH of the buffered mobile phase and the concentration of the organic modifier, methanol, on the separation were investigated. Separation of these copper complexes by ion-pair HPLC with the use of a mass spectrometry-compatible ion-pair reagent, triethylammonium acetate, in the mobile phase at pH 6.3 is also presented. The reversed-phase chromatographic conditions utilized also allow the pK(a)s of Cu-TETA and the log(k'w) values of Cu-CB-TE2A, Cu-TETA and Cu-CB-DO2A to be estimated.     

Retention behavior of cardiac steroids using cyclodextrin in the mobile phase in high-performance liquid chromatography

The retention behaviour of twenty cardiac steroids and four fluorescent derivatives was examined by the addition of cyclodextrin to the mobile phase in reversed-phase high-performance liquid chromatography. The addition of a suitable cyclodextrin improved the separation of isomeric cardiac steroids. The steroid A/B ring junction is the most important factor in the choice of the optimum cyclodextrin to be added; the C/D ring junction is less important. The hydroxyl group at the 3- or 12-position of the steroid enhanced the changes in retention times of these compounds. The effect of an unsaturated lactone ring at the 17 beta-position on the retention in the presence of cyclodextrin was also determined with cardenolide (five-membered ring) and bufadienolide (six-membered ring) but little difference was observed. Isomeric cardiac steroids, whose separation has not been done by the conventional method, were clearly separated by this method. The fluorescence intensity of 3-(1-anthroyl)-cardiac steroid was enhanced by the addition of cyclodextrin to the mobile phase.     

The association of inclusion complexes of cyclodextrins with azo dyes

OrangeII (4) and cyclodextrin (CD) form 2 : 1 and 2 : 2 complexes. The complexes self-associate and microscopy indicates the formation of a fibroid aggregate. In the induced c.d. spectrum, the ^* band of this complex appears at 500 nm in solution, but in the aggregate it changes to aJ-band due to the head-to-tail stacking of4 and aH-band due to its parallel stacking; this indicates that the aggregation expands not only in the direction of the symmetry axis of the CD, but also in the other two dimensions.²H-NMR spectroscopy from deuteron exchange and solvation between the aggregate and deuterium oxide exhibits quadrupole splitting in the region of 0–0.2 KHz. The orientation behavior obtained from this splitting suggests the formation of a liquid-crystaloid substance.¹³C-T ₁ NMR indicates that molecules4 and CD show the sameT ₁ values even at 333 K; this complex behaves like a single molecule. The behaviors of other azo dye-CD complexes are also discussed.DeceasedDedicated to Professor József Szejtli.     

Detection of Nepsilon-monomethyllysine using high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry

Nepsilon-Monomethyllysine was identified in the serum, urine, brain, and liver samples of rats treated per os with L-deprenyl. The identification procedure included reaction with Fmoc chloride, clean-up, and analysis using HPLC-UV-MS. Oral administration of (-)-N-14C-methyl-N-propynyl(2-phenyl-1-methyl)ethylammonium hydrochloride L-deprenyl) to rats resulted in transfer of the radiolabelled methyl group to the Nepsilon-amino group of the endogenous lysine. The radiolabelled Nepsilon-monomethyllysine was urinary eliminated together with the other radiolabelled deprenyl metabolites, such as deprenyl-N-oxide and methamphetamine. The presence of Nepsilon-monomethyllysine has also been traced, and its concentrations were compared in the serum, liver and brain of rats subjected to L-deprenyl treatment. Methyl group transfer from the L-deprenyl to endogenous compounds; and the urinary elimination of their products may offer a vital way to eliminate or to decrease the degree of drug transmethylation to the lysine constituents of blood vessels' proteins.     

The resolution of some chiral compounds in reversed-phase high-performance liquid chromatography by means of β-cyclodextrin inclusion complexes

Summary -Cyclodextrin is applied as the chiral component of the mobile phase in a systematic study of the resolution —into enantiomers — of mandelic acid and its derivatives by reversed-phase liquid chromatography. It is found that the stereoselectivity arising from inclusion in -cyclodextrin molecules is significant (=1.02÷1.05) only for the compounds showing at the asymmetric carbon atom the presence of first, an intact carboxylic group and second, a functional group capable of hydrogen bonding with the hydroxyl groups of -cyclodextrin. Results are discussed in the light of the three-point attachment model of stereoselectivity as well as of the structure of the inclusion complexes.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Thermodynamics of inclusion complexes between cyclodextrins and isoniazid

Complex formation of α- and β-cyclodextrins with isoniazid, a antituberculous pharmaceutical, is studied using such methods as calorimetry and ¹H NMR at 298.15 K. On the basis of the obtained experimental data, it is shown that α- and β-cyclodextrins form 1 : 1 inclusion complexes with isoniazid, which are characterized by low stability in aqueous solution. Along with this, deeper penetration of isoniazid into the cavity of β-cyclodextrin, accompanied by more intensive dehydration of the reagents, is observed. The results are interpreted in terms of influence of structure of reagents and their state in solution on the binding mode, driving forces, and thermodynamic parameters of the complex formation.     

Characterization of in vitro metabolites of toad venom using high-performance liquid chromatography and liquid chromatography-mass spectrometry

The characterization of the in vitro metabolites of toad venom, which has been widely used as a traditional Chinese drug, Ch'an Su, has been completed. Toad venom contains bufotoxins (such as marinobufotoxin; marinobufagin 3-suberoylarginine ester) and bufogenins (such as marinobufagin and bufalin) as the main cardiac steroids. An in vitro experiment using the rat or human liver cytosolic fraction disclosed that marinobufotoxin produced marinobufagin, but not its 3-hemisuberate. Marinobufagin was subjected to the enzyme reaction using the rat or human liver microsomal fraction together with NADPH and NAD, which produced 3-dehydromarinobufagin and 3-epimarinobufagin. Marinobufagin produced its 3-sulfate upon treatment with the rat or human liver cytosolic fraction and 3'-phosphoadenosine 5'-phosphosulfate. Bufalin was also subjected to the above enzyme reactions and showed almost the same results except for the result that the hydroxylation occurred at the 5beta-position. On the other hand, small amounts of marinobufagin 3-glucuronide were obtained only by treatment with the human liver microsomal fraction and uridine 5'-diphosphoglucuronic acid. The structures of these metabolites were confirmed using authentic samples regarding their high-performance liquid chromatographic behavior and/or liquid chromatography-mass spectrometry analysis.     

Characterization of a novel diol column for high-performance liquid chromatography

For the investigation of a diol phase (Inertsil Diol column) in hydrophilic interaction chromatography, urea, sucrose and glycine were used as test compounds. The chromatographic conditions were investigated for optimal column efficiency. The column temperature used in common reversed-phase liquid chromatography could also be used for the separation and the flow-rate should be adjusted to 0.3-0.5 ml/min to optimize column efficiency. It is suggested that the velocity of the hydrophilic interaction is slower than the hydrophobic interaction in RPLC. The addition of trifluoroacetic acid is effective for the retention of glycine, but ineffective for urea and sucrose. The diol phase exhibited sufficient chemical stability even if exposed to water in high percentage, and could be applied with isocratic elution for the separation/analysis of amino acids and glucose.     

Characterization of chemically synthesized human relaxin by high-performance liquid chromatography

Highly purified human relaxin, produced by combining chemically synthesized A- and B-chains, was analyzed by reversed-phase high-performance liquid chromatography, ion-exchange chromatography and tryptic mapping in order to ascertain purity of the material, presence of uncleaved protecting groups, correctness of disulfide linkages and presence of deamidated or oxidized variants. It was shown by a variety of analytical methods that the product was of high purity; a minimum purity level as judged by the most discriminating assay was greater than 98%. Components of the relaxin preparation removed during the purification were identified to be variants containing deletions arising from incomplete coupling reactions in the solid phase peptide synthesis and/or oxidized methionine residues.     

Determination of triforine using high-performance liquid chromatography with tandem mass spectrometry

A method for determination of triforine using high-performance liquid chromatography with electrospray tandem mass spectrometry was developed. A simple ethyl acetate extraction with solvent exchange into water/methanol was used for sample preparation. The method was validated at 0.01 and 0.05 mg kg(-1) levels in apple, tomato and tinned blackcurrants. Recoveries were in the range 56.6-99.8% and no matrix suppression or enhancement effects were observed.