Analysis of lycopene geometrical isomers in biological microsamples by liquid chromatography with coulometric array detection.

Methods of analysis for determining low quantities of lycopene cis-trans isomers in biological tissues are needed. Development of two liquid chromatography (LC) methods based on the polymeric C30 stationary phase equipped with coulometric electrochemical array detection (ED) is described. Separation of 13 lycopene isomers including prolycopene, (a novel tetra-cis-lycopene found in Tangerine tomatoes) was accomplished with both isocratic and gradient methods using different proportions of methanol, methyl tert.-butyl ether, water and 1 M ammonium acetate buffer. Carotenoids were detected at potential settings between 200 and 620 mV. Differences in generated current-voltage curves aided in tentative identification of trans carotenoid species and select cis isomers of lycopene. These methods were successfully applied in the analysis of small quantities of plasma, buccal mucosal cells, prostate and cervical tissues. Limits of detection for trans-lycopene by ED were found to be 50 fmol representing a 10- to 100-fold increase over conventional UV-Vis absorbance methods.     

Permeability of renal capillaries. I. Preparation of neutral and charged protein probes

This paper describes the preparation of charged and uncharged protein molecular probes for study of the permselectivity of renal capillaries. Horse heart myoglobin was used as a neutral myoglobin. Since it contained several fractions with different isoelectric points, it was purified by fast protein liquid chromatography (FPLC). To obtain a negatively charged myoglobin, the original horse heart myoglobin was treated with cyanate, resulting in net charge of -5.7 +/- 0.3 at physiological pH (mean +/- SEM). The charge was determined from the Donnan potential which develops over a semipermeable membrane separating the inside solution in which the protein was dissolved from a surrounding bath of equal ionic strength. Sperm whale myoglobin was similarly purified by FPLC and used as a positively (+1.7 +/- 0.2) charged isomer. Horseradish peroxidase (HRP) was purified by means of gel and ion-exchange chromatography and found to be neutral at physiological pH. Negatively charged (-14.0 +/- 0.5) HRP was obtained by succinylation. Two isomers of lactate dehyrogenase (LDH) were used, namely the slightly positive (+2) LDH-M4 and the strongly negative (-19) LDH-H4. These isomers, which occur naturally, did not require further purification. The Stokes-Einstein radii, as measured by gel chromatography, of inulin, myoglobin, HRP and LDH were 11, 17.5, 32 and 46 A, respectively. The chemical modifications did not alter the Stokes-Einstein radii. In biological studies on rat kidneys samples of both plasma and renal hilar lymph were found to contain radioactive low molecular weight degradation products in addition to the intact proteins. This necessitated separation of all individual samples on small Sephadex columns prior to analysis.     

Production and isolation of large quantities of monoclonal antibody using serum-free medium and fast protein liquid chromatography

A method for the production and purification of monoclonal antibody on a large laboratory scale is described. It involves the growth of monoclonal antibodies in serum-free medium, ultrafiltration through an Amicon XM100A filter and fast protein liquid chromatography on a mono Q column with an ionic strength and pH elution gradient. Up to 30 mg of antibody per litre of cell culture supernatant was purified in one day. The procedure can be carried out at 4 degrees.     

Force degradation behavior of glucocorticoid deflazacort by UPLC: isolation,identification and characterization of degradant by FTIR,NMR and mass analysis

In this investigation, sensitive and reproducible methods are described for quantitative determination of deflazacort in the presence of its degradation product. The method was based on high performance liquid chromatography of the drug from its degradation product on reverse phase using Acquity UPLC BEH C18 columns (1.7 µm, 2.1 mm × 150 mm) using acetonitrile and water (40:60 V/V) at a flow rate of 0.2 mL/minute in UPLC. UV detection was performed at 240.1 nm. Deflazacort was subjected to oxidative, acid, base, hydrolytic, thermal and photolytic degradation. The drug was found to be stable in water and thermal stress, as well as under neutral stress conditions. However, forced-degradation study performed on deflazacort showed that the drug degraded under alkaline, acid and photolytic stress. The degradation products were well resolved from the main peak, which proved the stability-indicating power of the method. The developed method was validated as per ICH guidelines with respect to accuracy, linearity, limit of detection, limit of quantification, accuracy, precision and robustness, selectivity and specificity. Apart from the aforementioned, the results of the present study also emphasize the importance of isolation characterization and identification of degradant. Hence, an attempt was made to identify the degradants in deflazacort. One of the degradation products of deflazacort was isolated and identified by the FTIR, NMR and LC-MS study.     

影响水溶性羧甲基壳聚糖稳定性的外界因素

以虾壳为原料,制备了水溶性羧甲基壳聚糖。通过对壳聚糖特性黏度的测定来表征其稳定性,具体研究了影响壳聚糖稳定性的因素温度、pH值、离子强度、紫外线和灭菌过程。结果表明溶液的酸碱度和离子强度对壳聚糖的特性黏度有很大的影响,在一定范围内,壳聚糖溶液的黏度随之变化敏感。紫外线照射和灭菌过程不但使特性黏度降低,更使壳聚糖的分子结构发生显著变化。特性黏度随时间而降低,在37℃时降低较快,在2℃～8℃冷藏条件下壳聚糖制品可保持一定的稳定性,为壳聚糖的存放条件提供了科学依据。     

Optimization of separation of a complex mixture of natural and synthetic corticoids by micellar liquid chromatography using sodium dodecyl sulphate. Application to urine samples

A systematic optimization of the separation of a mixture of corticoids by micellar liquid chromatography, using sodium dodecyl sulphate as surfactant, a Hypersil (250 mm x 3.2 mm I.D.) C18 column, a flow-rate of 0.5 ml min(-1), and UV absorbance detection at 245 nm has been carried out. Several mobile phases consisting of sodium dodecyl sulphate and different organic modifiers were tested of which tetrahydrofuran, PrOH and BuOH were finally selected. On the basis of analysis time, resolution and number of compounds separated, a mobile phase containing 36 mM sodium dodecyl sulphate and 1.91% butanol allowed the separation of thirteen corticoids out of sixteen in about 27 min. Under these conditions the optimal concentration of sodium dodecyl sulphate was found to be 36 mM. A bivariant optimization method for the mobile phase BuOH-sodium dodecyl sulphate corrobored these results. The effects of temperature, ionic strength and flow-rate effect have also been studied. The most important analytical figures of merit were assessed and compared with those obtained using conventional mobile phases. The optimized method was applied to human urine samples of subjects administered with Dezacor (tablets containing 30 mg of the active ingredient deflazacort) with and without sample preparation.     

Enantioselective metabolism of the designer drugs 3,4-methylenedioxymethamphetamine ('ecstasy') and 3,4-methylenedioxyethylamphetamine ('eve') isomers in rat brain and blood

The metabolism of MDA (3,4-methylenedioxyamphetamine), HMMA (3-hydroxy-4-methoxymethylamphetamine) and HME (3-hydroxy-4-methoxyethylamphetamin) of the popular designer drugs MDMA ('ecstasy', 3,4-methylenedioxymethamphetamine) and MDE ('eve', 3,4-methylenedioxyethylamphetamine) was determined in rat serum, whole blood and urine, as well as in whole brain structures (cortex and striatum) after subcutaneous administration of 20 mg/kg MDMA and MDE, respectively. MDMA and MDE were extracted from serum and homogenized brain structures using a solid-phase extraction procedure. The extracts were examined by a validated high-performance liquid chromatography procedure coupled with fluorimetric detection. Our results demonstrate that MDMA is metabolized to a higher degree than MDE, resulting in a higher concentration of neurotoxic dihydroxymetabolites and (S)-MDA. There was no difference between the metabolism of MDMA and MDE and its respective isomers. Different concentrations of the respective isomers of MDMA and MDE let us suggest an enantioselective metabolism for both MDMA and MDE.     

Enzymatic degradation of starch-based thermoplastic compounds used in protheses: identification of the degradation products in solution

Apart from favourable physico-chemical and mechanical properties, the most important requirement for a biodegradable polymer to be used in medical applications is its biocompatibility and the non-cytotoxicity of its degradation products. Their combined effect should assure the safe material degradation under controlled kinetics. The present work analyses the degradation behaviour of blends of corn starch with poly(ethylene-vinyl alcohol) copolymer (SEVA-C). The characterization included long-term degradation trials on simulated physiological solution with alpha-amylase up to 200 days. The degradation solutions were analysed by several techniques. High-performance liquid chromatography (HPLC) and colorimetric methods were used to monitor the liberation of carbohydrate as a consequence of starch hydrolysis by alpha-amylase. The hydration degree was followed by thermogravimetric analysis (TGA). Several degradation products such as carbohydrates ranging from C6 to C18 were identified. After alpha-amylase action, biodegradation was more pronounced in the first 100 days, after which the biodegradation rate decreased probably due to the structure and porosity of the material. The action of alpha-amylase solely led to the starch degradation, in contrast with other assays without enzymes where no carbohydrates were found in the degradation solutions.     

In vitro protein polymerization and nucleoprotein reconstitution of tobacco rattle virus

Investigation of the effects of pH, ionic strength, and temperature on coat protein polymerization of tobacco rattle virus (TRV) have been examined and correlated with conditions optimal for nucleoprotein rod reconstitution. In acidified aqueous solution, TRV protein existed predominantly in the subunit form (<10 S). A discrete 35 S aggregate formed when the pH was raised (pH 6.5–8.5) in low ionic strength buffers at 4 °C. The formation of larger aggregates (>50 S) occurred at higher temperature (21 °C) at low ionic strength. Reconstitution of acetic-acid-extracted protein and phenol-extracted RNA occurred optimally in 0.25 M glycine buffer (pH 8.0) at 4–10 °C. Reconstituted nucleoprotein and native virus sedimented at the same rate after sucrose density gradient centrifugation. The participation of 35 S protein aggregates in reconstitution is suggested.     

Novel and sensitive method for the detection of anandamide by the use of its dansyl derivative

The dansyl ester of anandamide was prepared and showed intense fluorescence on silica gel thin-layer chromatography when viewed under long-wavelength ultraviolet light (detection limit, 15 fmol). A high-performance liquid chromatography method for the quantitation of anandamide was developed using a C18 column (250 x 4.6 mm) with gradient elution (1% acetic acid-methanol) and detection at 255 nm. The method was applied to the measurement of anandamide in media from cultured hepatocytes. Sample preparation involved extraction with a C18 cartridge, derivatization with dansyl chloride, thin-layer chromatography, and quantitation. The detection limit in hepatocyte media is 4.3 nmol at a signal-to-noise ratio of three.     

Polymer characterization by temperature gradient interaction chromatography

Thermodynamic principles and some applications of the temperature gradient interaction chromatography (TGIC) recently developed for the characterization of synthetic polymers are described. TGIC is a form of high performance liquid chromatography (HPLC) that varies column temperature in a programmed manner to control the retention of polymeric species during isocratic elution. The retention of polymers strongly depends on their molecular weights, and the polymers are well separated by TGIC in terms of their molecular weights. TGIC is superior to size exclusion chromatography (SEC) in resolution and sample loading capacity, and has higher sensitivity to molecular weight in the analysis of nonlinear polymers. TGIC has an advantage over solvent gradient HPLC because it permits the use of refractive index sensitive detection method such as differential refractometry and light scattering due to the isocratic elution. In addition, temperature provides finer and more reproducible retention control than the solvent composition, which is important in determining the molecular weight distribution by secondary calibration methods. With TGIC analysis, we found that the molecular weight distribution of anionically polymerized polymers is much narrower than has been generally accepted from SEC analysis. We also found the TGIC separation conditions for polystyrene, polyisoprene, poly(methyl methacrylate), poly(vinyl chloride), and poly(vinyl acetate) over wide molecular weight range. Because of its sensitivity to the molecular weight alone, TGIC was successfully applied to the characterization of star shaped polystyrene, and the detailed linking kinetics between living polystyrene anions and a chlorosilane linking agent was investigated.     

Kinetics and equilibrium in insulin radioimmunoassay

The kinetics of insulin reaction has been studied with its specific antibody immobilized on the inner wall of the reaction tube; the radioimmunoanalytical determination of such a substance is based on the reaction. Independent variables were labelled and unlabelled insulin concentrations, temperature, viscosity, and the medium's ionic strength. Biexponential kinetics was found to be dependent on the concentrations fitted to the models discussed in the paper. The effect of temperature shows activation parameters similar to the viscous flow energy of water, which suggests that the reaction is diffusion-controlled. The results of the viscosity analysis points at the clearly negative influence of viscosity upon the direct reaction rate. Ionic strength has a noticeable, though not relevant, effect which seems to indicate that the variation resulting from the glycerol addition is not due to the influence of the dielectric constant in the solutions used.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation.

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

Characterization, degradation, and mechanical strength of poly(D,L-lactide-co-epsilon-caprolactone)-poly(ethylene glycol)-poly(D,L-lactide-co-epsilon-caprolactone)

A series of three biocompatible P(CL-co-LA)-PEG-P(CL-co-LA) copolymers were synthesized using ring-opening polymerization and characterized by 1H-NMR, gel permeation chromatography, DSC, dynamic-mechanical analysis, and X-ray diffraction. The number of monomer units was kept constant, while the D,L-LA fraction was varied so as to constitute 0, 30, or 70% of the end segments. The molecular weights were sufficiently high to eventually permit 3D scaffold preparation. A degradation study was carried out over 26 weeks, and the effect of monomer composition on the rate of degradation as well as on changes in mechanical strength was investigated. Pure polycaprolactone (PCL)-poly(ethylene glycol) (PEG)-PCL copolymer, P(100/0), was a crystalline material displaying no measurable mass loss, a 30% reduction in mean molecular weight (Mn), and only very slight changes in tensile strength. The random incorporation of 30 and 70% D,L-LA into the end sections of the polymer chain, produced more and more amorphous materials, exhibiting increasingly high rates of degradation, mass loss, and loss of tensile strength. Compared with random P(CL-co-LA), the presence of the PEG block was found both to improve hydrophilicity and thus the rate of degradation and to infer a stabilizing quality, thereby pacing the decrease in tensile strength during degradation. The tested copolymers range from materials exhibiting low mechanical strength and high rate of degradation to slow-degrading materials with high mechanical strength suitable, e.g., for three-dimensional scaffolding.     

Kinetics of the heterogeneous acid hydrolysis of regenerated cellulose

The kinetics of the early stage of acid catalyzed heterogeneous hydrolysis of different samples of regenerated cellulose at low temperatures (0, 20, and 30°C) was studied. The degradation of macromolecules was followed by determining the decrease of the degree of polymerization by measuring the viscosity of cellulose solutions in cadoxen. Interpretation of the experimental results using equations for first order reactions showed that in the studied experimental range first order kinetics cannot be applied. It was found that the rate constant decreases during the course of the hydrolysis at constant temperature. However, the experimental results could be explained by kinetic equations for heterogeneous processes with a solid and a liquid phase in the heterogeneous system. Kinetic parameters calculated from these equations point out that the process could be described as a diffusion controlled process.     

The study of improved controlled release of vincristine sulfate from collagen-chitosan complex film

A water-in-oil-in-oil double-emulsion solvent/evaporation method was used to prepare vincristine sulfate (VCR) loaded poly(lactide-co-glycolide) microspheres, and then VCR microspheres were mixed with collagen and (or) chitosan swelling solution and lyophilized to form polymeric films. The films were cross-linked by 0.3% glutaraldehyde (GA). Encapsulation efficiency and release kinetics of VCR microspheres were determined, as well as release kinetics and in vitro degradation of the film. The rate of VCR release from the film submerged in PBS (pH 6.8) and the content were measured by high-performance liquid chromatography (HPLC). The physichemical properties of the film, such as surface morphology, mechanical function, and differential scanning calorimetry, were also measured. VCR was released from the film in a prolonged period and the initial burst release of the film was less significant. In the degradation experiment, the film containing chitosan degraded more slowly than that without chitosan. The films comprising collagen and chitosan could achieve the release kinectics of a relatively constant release. It has a promising future.     

Free-radical polymerization of methyl methacrylate at very high conversions, 2. Study of the kinetics of initiation by benzoyl peroxide

The initiation kinetics of the free-radical polymerization of methyl methacrylate at very high monomer conversions was investigated at 80, 100, and 120°C. Using a HPLC (high performance liquid chromatography) analytical technique with UV detection, the consumption of both monomer and initiator as well as the formation of reaction products of the latter could be followed. It was found that (besides very small amounts of biphenyl and benzoic acid) phenyl benzoate is the main decomposition product. The experimental results were analyzed quantitatively with the help of a reaction scheme which takes into account the most important pathways of that complex chemical reaction system. Thus, it was found that, due to the very strong cage effect in the present polymerization system, the experimentally accessible (apparent) first order rate constant of initiator decomposition is substantially lower than that for the primary cleavage of a benzoyl peroxide molecule. Efficiencies of chain initiation were calculated to be in the range of 0,1 to 0,2 in the (nearly) glassy polymer matrix.     

Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection

Positional and geometric isomers of mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms were separated on commercially available reversed-phase columns in gradient systems composed of acetonitrile and water, utilizing photodiode array detection. The biological samples were hydrolyzed with 2 M NaOH for 35-40 min at 85-90 degrees C. After cooling, the hydrolysates were acidified with 4 M HCl and the free fatty acids were extracted with dichloromethane. The organic solvent was removed in a gentle stream of argon. The fatty acids were determined after pre-column derivatization with dibromacetophenone in the presence of triethylamine. The reaction components were mixed and reacted for 2 h at 50 degrees C. Separations of derivatized fatty acids were performed on two C18 columns (Nova Pak C18, 4 microm, 250x4.6 mm, Waters) by binary or ternate gradient programs and UV detection at 254 and 235 nm. The geometric and positional isomers of some unsaturated fatty acids were substantially retained on the C18 columns and were distinct from some saturated fatty acids, endogenous substances in biological samples or background interference. Only slight separation of critical pairs of cis-9 C18:1/cis-11 C18:1 and cis-6 C18:1/trans-11 C18:1 was obtained. A ternate gradient program can be used for complete fractionation of a mixture of conjugated linoleic acid isomers (CLA) from cis-9, cis-12 and trans-9, trans-12 isomers of C18:2. The CLA isomers in the effluent were monitored at 235 nm. The CLA isomers were differentiated from saturated and unsaturated fatty acids using a photodiode array detector. The utility of the method was demonstrated by evaluating the fatty acid composition of duodenal digesta, rapeseed and maize oils.     

Effect of load and temperature on in vitro degradation of poly(glycolide-co-L-lactide) multifilament braids

The effects of load and temperature on in vitro degradation behaviors of poly(glycolide-co-L-lactide) 90/10 multifilament braids were investigated in phosphate buffer solution at pH 7.4. The property changes of the braids with time were monitored by tensile test, gel permeation chromatography analysis, and scanning electron microscopy. The interrelationships between material properties, time and experimental conditions were explored. The results showed that the polymer braids gradually lost their strength and molecular weight with the increasing in vitro time. While the load levels applied had no effect on the materials, raising temperatures significantly accelerated the degradation. It was found that for a given tensile breaking strength retention (BSR), the dependence of degradation time on temperature could be illustrated by an Arrhenius-type equation, from which the activation energy could be derived. Further analysis indicated that there are well-defined relationships between molecular weight, BSR and breaking strain retention, and these relationships can be illustrated mathematically. Finally, the surface morphology of the fiber showed visible change during the degradation process.     

Electro‐induced Cationic Polymerization of Vinyl Ethers by Using Ionic Liquid 1‐Butyl‐3‐methy­limidazolium Tetrafluoroborate as Initiator

Ionic liquids are used widely as solvents or electrolytes for electrochemical synthesis and applications due to their wide electrochemical windows, good electrical conductivity, and their excellent solubility to both organic and inorganic compounds. Until now, no papers have reported the use of ionic liquids as initiators in polymerization. In most cases, the kinetics of electro‐induced polymerization are studied by gas chromatography (GC), NMR, or the gravimetric method, which are complicated and time consuming. In this paper, the kinetics of electro‐induced polymerization of vinyl ethers using ionic liquid 1‐butyl‐3‐methylimidazolium tetrafluoroborate (BMIMBF₄) as initiator are investigated by real‐time Fourier transform near‐IR (FT‐NIR) spectroscopy. The results show that the ionic liquid BMIMBF₄ is an efficient initiator for electro‐induced polymerization of vinyl ethers. The final double‐bond conversion and polymerization rate can be adjusted by the electric‐field intensity and the concentration of the ionic liquid BMIMBF₄. Moreover, the post‐curing, as well as the obvious inhibition effects of KOH, indicate that the electro‐induced polymerization goes through a cationic process. The possible mechanism is also postulated.