Modulated release of cyclosporine from soluble vinyl pyrrolidone--hydroxyethyl methacrylate copolymer hydrogels. A correlation of 'in vitro' and 'in vivo' experiments.

Soluble, uncrosslinked and high molecular weight copolymers of vinylpyrrolidone, VP, with 2-hydroxyethyl methacrylate, HEMA, prepared by free radical copolymerization, are proposed as supports for the modulated release of the immunosuppressor cyclosporine. Two copolymeric systems with copolymer compositions f(VP)=0.52 (namely VP--HEMA 60--40) and 0.42 (VP--HEMA 40--60) have been prepared and tested in vitro and in vivo using rats as animal model. Micellar electrokinetic capillary chromatography, MEKC, has been used for the simultaneous detection of the polymer reabsorption and the drug release for the in vitro experiments. The composition and microstructural distribution of the copolymer system controls the solubilization rate which modulates the in vitro release of the drug (with time profiles from a few days to several weeks for the VP--HEMA 60--40 and 40--60, respectively) and the in vivo response that correlates with the previous in vitro results: the more hydrophobic implant (VP--HEMA 40--60) reverts the immune response more slowly (2--4 weeks) compared to the more hydrophilic one (VP--HEMA 60--40, 1--2 weeks).

Controlled release of cyclosporine from VP-HEMA copolymer systems of adjustable resorption monitorized by MEKC.

Soluble, uncrosslinked and high molecular weight copolymers of vinylpyrrolidone, VP, with 2-hydroxyethyl methacrylate, HEMA, prepared by free radical copolymerization, are proposed as supports for the modulated release of drugs, taking cyclosporine as a model system. The copolymerization parameters described as reactivity ratios, rVP = 0.08 and rHEMA = 7.97, indicate that the copolymer systems prepared at high conversion have two main components with a microstructural arrangement which depends on the average composition, i.e., an initial HEMA-rich copolymer and a final PVP homopolymer or VP-rich copolymer. This microstructural distribution controls the resorption rate of the polymeric support and therefore the release process of cyclosporine which is demonstrated experimentally by the application of a modern technique known as micellar electrokinetic capillary chromatography (MEKC).

Recombinant growth hormone delivery systems based on vinylpyrrolidone-hydroxyethyl methacrylate copolymer matrices: monitoring optimization by capillary zone electrophoresis.

Experimental conditions for the fabrication of two new polymeric devices (i.e. films and slabs) useful for the controlled release of recombinant growth hormone (GH) are given. The release rate is controlled by the resorption profile of the vinylpyrrolidone-hydroxyethyl methacrylate (VP-HEMA) tested systems which is related to the copolymer composition. The suitability of capillary electrophoresis (CE) for following the complete preparation of the different VP-HEMA devices is shown. Moreover, CE allows simultaneous monitoring of the controlled release of GH and dissolved polymer during in vitro experiments. From these results, guidelines are given for the fabrication of polymeric devices containing protein as active drug as well as for the correct selection of conditions during in vitro experiments.

Detection of bovine whey proteins by on-column derivatization capillary electrophoresis with laser-induced fluorescence monitoring.

1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and 2-(p-toluidino)naphthalene-6-sulfonic acid (2,6-TNS) were evaluated as additives in different buffers for the detection of bovine whey proteins using laser-induced fluorescence (LIF) monitoring in capillary electrophoresis (CE). These N-arylaminonaphthalene sulfonates furnish a large fluorescence emission when associated to some proteins whereas their emission in aqueous buffers, such as those used in CE separations, is very small. To select the best detection conditions, the fluorescence of these probes was first compared using experiments carried out in a fluorescence spectrophotometer. Using bovine serum albumin (BSA) as a model protein, it was demonstrated that 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 8 and pH 10.2) and the fluorescent probe 2,6-TNS gave rise to the highest increase in fluorescence for BSA. When the composition of these separation buffers was optimized for the electrophoretic separations, CHES buffer, pH 10.2 was chosen as the most suitable buffer to detect bovine whey proteins. The limit of detection obtained for some whey proteins in CE separations was about 6.10(-8) M for BSA, 3.10(-7) M for beta-lactoglobulin A (beta-LGA), 3.10(-7) M for beta-lactoglobulin B (beta-LGB), and 3.10(-6) M for alpha-lactalbumin (alpha-LA). These detection limits were compared to those achieved using UV detection under the same separation conditions. The results showed that the detection limits of BSA, beta-LGA and beta-LGB were twice as good using LIF than with UV detection. However, the limit of detection for alpha-LA was better when UV was used. The applicability of LIF detection to CE separation of whey proteins in bovine milk samples was also demonstrated.

Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis.

The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78-4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.

Quantitation of active ingredients and excipients in nasal sprays by high-performance liquid chromatography, capillary electrophoresis and UV spectroscopy.

A study on the use of different analytical methodologies to determine active ingredients and excipients found in commercial nasal sprays is presented. Two of the developed methodologies consisted of separation techniques, i.e. high-performance liquid chromatography and capillary electrophoresis, and the third one involved a UV-spectroscopic multicomponent procedure. The samples studied are characterized by a high viscosity and the existence of a large number of particles in suspension; therefore, special emphasis is paid on the sample preparation required by each methodology. Advantages and drawbacks of each analytical technique are also discussed in terms of speed of analysis, sensitivity and reproducibility. From this work it is observed that although the UV method needs the most laborious sample preparation, the total time required per analysis is the shortest one. The best reproducibility in terms of analysis time and quantitation of the analyzed compounds is obtained using HPLC. CE allows the determination of more components in the same sample.

Use of detergents and high contents of organic solvents for simultaneous quantitation of ionic and nonionic drugs by electrokinetic chromatography.

Buffers containing high percentages of organic solvents, typically 50% of acetonitrile and/or methanol, together with sodium dodecyl sulfate (SDS) are employed for the separation and quantitation by electrokinetic chromatography (EKC) of analytes found in a nasal spray. Solutes consist of benzalkonium chloride, a family of highly positive compounds, and 2-phenylethanol and beclomethasone dipropionate, which are electrically neutral and poorly soluble in aqueous buffers. It is observed that the effect of both concentration of SDS and temperature on the separation depends on the organic solvent used and the solute nature. It is also observed that SDS-solute interaction for neutral and cationic compounds are weaker in the presence of high contents of acetonitrile than in methanol. Concentration of SDS, temperature, and organic solvent nature and content, allow one to modify the selectivity of the separation when neutral and ionic species have to be simultaneously determined. The optimization of EKC conditions enables the analysis of compounds in less than 5 min. A one-step sample treatment consisting of centrifugation of the nasal spray solved in acetonitrile, together with the referenced optimum separation conditions enable the reproducible quantitation of the analytes. Relative standard deviation values of inter-day migration times lower than 2.45% are obtained (R.S.D.n = 12), while R.S.D.n = 12 values for inter-day peak areas were lower than 6.32%.

Multiple peaks in high-performance liquid chromatography of proteins. beta-Lactoglobulins eluted in a hydrophobic interaction chromatography system.

The chromatographic behavior of beta-lactoglobulins when eluted in hydrophobic interaction chromatography systems is studied. By modifying some factors, such as pH and temperature, the relationship between shape of the chromatographic peak and protein structure is shown. At pH 4.5 and low temperature multiple peaks for beta-LG A and beta-LG B are observed and assigned to aggregates. The effects of other parameters, besides pH and temperature, such as volume and concentration of injected sample, contact time between protein and stationary and/or mobile phases, and nature and concentration of mobile phase upon aggregation are studied. Comparison of the chromatographic behavior of both variants of beta-lactoglobulin is made.

Separation of chiral polychlorinated biphenyls by micellar electrokinetic chromatography using beta- and gamma-cyclodextrin mixtures in the separation buffer.

Chiral polychlorinated biphenyls (PCBs) 45, 84, 88, 91, 95, 132, 136, 139, 149, 171, 183 and 196 were separated each in its two enantiomers by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC). Mixtures of beta- and gamma-cyclodextrins were used as chiral modifiers in a 2-(N-cyclohexylamino)ethanesulphonic acid (CHES) buffer containing urea and sodium dodecyl sulphate (SDS) micelles. Separations of multicomponent mixtures of PCBs into their enantiomers were also performed. A mixture of PCBs 45, 88, 91, 95, 136, 139, 149 and 196 was separated into all sixteen enantiomers in an analysis time of approx. 35 min.

Perfusion liquid chromatography of whey proteins.

A perfusion reversed-phase (RP) HPLC method was developed for the rapid separation of the main bovine whey proteins: alpha-lactalbumin (alpha-LA), serum albumin (BSA) and the genetic variants of beta-lactoglobulin (A and B) (beta-LG A and beta-LG B). For the method development, the influence of factors favouring structural changes of proteins (temperature and organic acid concentration in the mobile phase), gradient and other chromatographic conditions and the mass of protein injected was examined. The optimized method allowed the separation of proteins in about 1.5 min (cycle time 3.5 min) with resolution around 1.0 for the beta-lactoglobulins. The method was applied to the determination of proteins in a whey from raw bovine milk. The precision of the determinations was < or = 3.75 mg per 100 ml (S.D.). With respect to the accuracy, errors < or = 7.0% in the determination of alpha-LA, beta-LG A and beta-LG B were obtained, compared with an RP-HPLC reference method. However, higher errors in the quantification of BSA were found owing to the lack of purity of the peak assigned. In addition, the proposed method has proved to be very useful in the detection of homologous whey proteins from different species (cow, sheep and goat) in milk mixtures.

Separation and analysis of 4'-epimeric UDP-sugars by ion-pair reversed-phase HPLC.

A simple and sensitive method for determination of 4'-epimeric UDP-sugars using ion-pair reversed-phase HPLC has been developed. The method presents advantages over existing ion-exchange HPLC procedures mainly concerning sensitivity and rapidity of analysis as well as efficiency and stability of the column. It is based on the ability of borate ions to react with cis-diols resulting in the formation of UDP-sugar-borate complexes with different charges. Good resolution and rapid separation (5-25 min) of all 4'-epimeric UDP-sugars tested was achieved with this method, was suitable for concentrations over 20 pmol. The applicability to biochemical analysis was demonstrated by the quantitative determination of the UDP-2-deoxyglucose and UDP-2-deoxygalactose formed in yeast cells upon incubation in the presence of 2-deoxygalactose.

Rapid analysis of whey proteins from different animal species by reversed-phase high-performance liquid chromatography.

This paper explores the possibilities of reversed-phase high performance liquid chromatography (RP-HPLC) for analysing whey proteins from the milk of cows and other animal species. An RP-HPLC method is proposed to separate and quantify bovine whey proteins. Using this method, bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin A and beta-lactoglobulin B were separated in less than 7 min. It is demonstrated that irreversible adsorption of bovine whey proteins occurs on unused columns (i.e. those not previously used to separate proteins). Therefore, prior conditioning of the column with whey proteins is required for valid protein quantification. Conditioning can be achieved by eluting a large amount of at least one of the bovine whey proteins through the unused column. The calibration curve (peak area vs. protein concentration) for the main bovine whey proteins was linear. This method also allowed good separation of caprine and ovine whey proteins and separation of some homologous whey proteins of different animal species. Detection of milk mixtures from different animal species was carried out using this method.

Arsenic mononucleotides. Separation by high-performance liquid chromatography and identification with myokinase and adenylate deaminase.

The spontaneous formation of arsenic mononucleotides has been detected in mixtures of arsenate and inosine or adenosine or its deoxy analogues. These compounds have been separated by high-performance liquid chromatography and identified by their behavior in the presence of myokinase and adenylate deaminase. The nucleoside 5'-arsenates are formed preferentially to the 2'- and 3'-arsenate analogues. All arsenic nucleotides detected showed similar kinetic and equilibrium constants of formation: about 8 X 10(-4) M-1 S-1 and 2 X 10(-3) M-1, respectively. These values are several orders of magnitude greater than those of their phosphoric analogues. The adenosine 5'-arsenate was able to substitute for 5'AMP in the reaction of myokinase and adenylate deaminase. The substitutions of the 2'- or 3'-hydrogen for hydroxyl groups in the ribose moiety of this compound slightly affected its suitability as substrate for myokinase but had drastic effect in the case of adenylate deaminase. The half-life of the arsenic nucleotides, at pH 7.0 and 25 degrees C, ranged from 30 to 45 min. The lability of these compounds is increased during catalysis with myokinase. Results on the reaction mechanism of myokinase with adenosine 5'-arsenate indicate that the mixed-anhydride analogue to ADP, adenosine 5'-(arsenate phosphate), is not detected either because it is not formed in the reaction with this enzyme or because it is rapidly hydrolyzed.